U.S. patent application number 15/470531 was filed with the patent office on 2017-07-13 for automated devices, systems, and methods for the fabrication of tissue.
The applicant listed for this patent is Organovo, Inc.. Invention is credited to Samir Damle, Vivian Gorgen, Vaidehi Joshi, Frank Lin, Keith MURPHY, Stephen Pentoney, Clay Platt, Sharon C. Presnell, Harry Scott Rapoport.
Application Number | 20170199507 15/470531 |
Document ID | / |
Family ID | 52427884 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170199507 |
Kind Code |
A1 |
MURPHY; Keith ; et
al. |
July 13, 2017 |
AUTOMATED DEVICES, SYSTEMS, AND METHODS FOR THE FABRICATION OF
TISSUE
Abstract
Described herein are improvements to bioprinting technology that
facilitate automation of tissue and organ fabrication
processes.
Inventors: |
MURPHY; Keith; (Los Angeles,
CA) ; Pentoney; Stephen; (San Diego, CA) ;
Lin; Frank; (San Diego, CA) ; Gorgen; Vivian;
(San Diego, CA) ; Platt; Clay; (Lake Forest,
CA) ; Rapoport; Harry Scott; (San Diego, CA) ;
Damle; Samir; (North Tustin, CA) ; Joshi;
Vaidehi; (San Diego, CA) ; Presnell; Sharon C.;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Organovo, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
52427884 |
Appl. No.: |
15/470531 |
Filed: |
March 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14447412 |
Jul 30, 2014 |
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15470531 |
|
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61860644 |
Jul 31, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B29L 2031/40 20130101;
C12M 33/00 20130101; A61F 2/00 20130101; B29K 2071/02 20130101;
B33Y 30/00 20141201; B29K 2033/08 20130101; B29C 64/106 20170801;
G05B 19/27 20130101; B33Y 80/00 20141201; B41J 3/407 20130101; C12M
33/12 20130101; B29C 64/393 20170801; C12M 41/00 20130101; B29C
64/182 20170801; B33Y 50/02 20141201; C12M 21/08 20130101; B29C
64/112 20170801; B29K 2995/0056 20130101; B33Y 70/00 20141201; C12M
25/00 20130101; A61F 2240/00 20130101; B29L 2031/7532 20130101;
G05B 2219/49023 20130101; B33Y 10/00 20141201; B41J 2/04
20130101 |
International
Class: |
G05B 19/27 20060101
G05B019/27; B29C 67/00 20060101 B29C067/00 |
Claims
1.-20. (canceled)
21. A bioprinter comprising: (a) a printer head comprising at least
one cartridge and at least one deposition orifice, the cartridge
comprising contents from a bio-ink, a support material, or a
combination thereof; (b) a receiving surface for receiving the
contents from the printer head; (c) a three-dimensional calibration
system comprising: (1) a tip triangulation sensor, fixed to the
receiving surface, for determining a position of the deposition
orifice, the tip triangulation sensor comprising a first laser and
a first sensor configured to detect light from the first laser; (2)
a surface triangulation sensor, fixed to the printer head, for
determining a position of a target print surface, the surface
triangulation sensor comprising a second laser and a second sensor
configured to detect light from the second laser; and (d) a
processor coupled to the three-dimensional calibration system and
configured to: (1) calculate one or more print height changes
during bioprinting based on (a) the position of the deposition
orifice determined from the tip triangulation sensor and (b) the
position of the target print surface determined from the surface
triangulation sensor, the print height is a distance between the
deposition orifice and the target print surface; and (2) adjust for
any print height changes during bioprinting by adjusting one or
more bioprinting parameters.
22. The bioprinter of claim 21, wherein the bioprinting parameters
are selected from the group consisting of a deposition rate, a
relative travel speed of the printer head, the print height, a cell
type, a deposition order, a deposition location, and combinations
thereof.
23. The bioprinter of claim 21, wherein the print height changes
are determined before, after, or before and after the contents are
dispensed from the printer head.
24. The bioprinter of claim 21, wherein the three-dimension
calibration system is configured to dynamically map the target
print surface during bioprinting, wherein a thickness of each track
of deposited bio-ink is measured.
25. The bioprinter of claim 21, wherein the three-dimensional
calibration system is configured to monitor for any structural
changes of a tissue construct during bio-printing.
26. The bioprinter of claim 21, wherein the three-dimensional
calibration system creates a topographical map of the receiving
surface.
27. The bioprinter of claim 26, wherein the receiving surface
comprises an assay plate.
28. The bioprinter of claim 26, wherein the receiving surface
comprises a transwell insert.
29. The bioprinter of claim 27, wherein the three-dimensional
calibration system is configured to check for any plate positioning
errors.
30. The bioprinter of claim 27, wherein the three-dimensional
calibration system is configured to determine a center of a well in
the assay plate.
31. The bioprinter of claim 21, wherein the three-dimensional
calibration system creates a topographical map of the deposition
orifice.
32. The bioprinter of claim 21, wherein the printer head comprises
an independent z-axis positioning mechanism configured to adjust,
independently for each deposition orifice, a location of the
deposition orifice relative to the receiving surface.
33. The bioprinter of claim 32, wherein the independent z-axis
positioning mechanism further comprises a brake mechanism to
prevent the printer head from inadvertent contact with the
receiving surface.
34. The bioprinter of claim 21, wherein the printer head comprises
at least two cartridges, wherein at least one cartridge comprises a
capillary tube, and at least one cartridge comprises a needle.
35. The bioprinter of claim 34, wherein the printer head comprises
at least three cartridges, wherein at least one cartridge comprises
a capillary tube, at least one cartridge comprises a needle, and at
least one cartridge is configured for ink-jet printing.
36. The bioprinter of claim 21, further comprising one or more
coaxial nozzles, each coaxial nozzle has at least two independent
inputs and at least two independent outputs.
37. A method for automating a bioprinting process comprising: (a)
varying a position of a tip triangulation sensor relative to a
deposition orifice during a plurality of measurements to produce a
first distance versus position data set, the tip triangulation
sensor comprises a first laser and a first sensor configured to
detect light from the first laser; (b) calculating, based on the
first distance versus position data set, a center X coordinate of
the deposition orifice, a center Y coordinate of the deposition
orifice, and an average deposition orifice height; (c) varying a
position of a surface triangulation sensor relative to a target
print surface during a plurality of measurements to produce a
second distance versus position data set, the surface triangulation
sensor comprises a second laser and a second sensor configured to
detect light from the second laser; (d) constructing, based on the
second distance versus position data set, one or more surface maps
of the target print surface during bioprinting of a
three-dimensional tissue construct; and (e) adjusting, based on the
first and the second distance versus position data sets, one or
more bioprinting parameters to correct for any print height
changes, the print height is a distance between the deposition
orifice and the target print surface.
38. The method of claim 37, wherein the bioprinting parameters are
selected from the group consisting of a deposition rate, a relative
travel speed of the printer head, the print height, a cell type, a
deposition order, a deposition location, and combinations
thereof.
39. The method of claim 37, further comprising creating a
topographical map of the receiving surface.
40. The method of claim 39, wherein the receiving surface comprises
an assay plate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. application Ser.
No. 61/860,644, filed Jul. 31, 2013, which is hereby incorporated
by reference in its entirety.
BACKGROUND OF INVENTION
[0002] A number of pressing problems confront the healthcare
industry. As of June 2013, there were approximately 118,000
patients registered by United Network for Organ Sharing (UNOS) as
needing an organ transplant. Between January and March 2013, only
6,891 transplants were performed. Each year more patients are added
to the UNOS list than transplants are performed, resulting in a net
increase in the number of patients waiting for a transplant. For
example, as of 2011, over 15,000 people were registered as: needing
a liver graft/transplant; however only about 5,800 liver
transplants were performed that year. In 2010, the median wait time
for a liver was over 12 months.
[0003] Additionally, the research and development cost of a new
pharmaceutical compound is approximately $1.8 billion. See Paul, et
al. (2010). How to improve R&D productivity: the pharmaceutical
industry's grand challenge. Nature Reviews Drug Discovery
9(3):203-214. Drug discovery is the process by which dings are
discovered and/or designed. The process of drug discovery generally
involves at least the steps of: identification of candidates,
synthesis, characterization, screening, and assays for therapeutic
efficacy. Despite advances in technology and understanding of
biological systems, drug discovery is still a lengthy, expensive,
and inefficient process with low rate of new therapeutic
discovery.
SUMMARY OF THE INVENTION
[0004] There is a need for tools and techniques that facilitate
application of regenerative medicine and tissue engineering
technologies to relieving the urgent need for tissues and organs.
There is also a need for tools and techniques that substantially
increase the number and quality of innovative, cost-effective new
medicines, without incurring unsustainable R&D costs. The
inventors describe herein improvements to devices, systems, and
methods for fabricating tissues and organs that allow for enhanced
speed, accuracy, and scalability. Specifically, the devices,
systems, and methods described offer advantages including, but not
limited to, improved scalability of production while maintaining
control of the spatial orientation of materials, improved
three-dimensional calibration of deposition equipment, and improved
utilization of multiple distinct materials without significant
interruption of a fabrication process.
[0005] In one aspect, disclosed herein are automated bioprinters
comprising: a printer head comprising one or more cartridges, each
cartridge comprising contents selected from: bio-ink, support
material, and a combination thereof; and for each cartridge: an
actuation means that vertically positions the cartridge relative to
a receiving surface to produce a particular three-dimensional
geometry in the dispensed contents of the cartridge, the actuation
means operating independently from other actuation means; and a
deposition orifice; a receiving surface for receiving dispensed
contents of a cartridge; and a calibration means comprising a
three-dimensional calibration system, the system comprising at
least two lasers, a sensor fixed to the receiving surface for
determining the position of the deposition orifice; and a sensor
fixed to the printer head for determining the position of the
receiving surface; whereby the system calculates a print height,
the print height comprising the distance between the deposition
orifice and the receiving surface; whereby a construct is
bioprinted without stopping the bioprinter to manually replace one
or more cartridges or to manually adjust the positioning of the
printer head or the positioning of one or more of the cartridges.
In some embodiments, the three-dimensional calibration system
creates a topographical map of the receiving surface. In some
embodiments, the three-dimensional calibration system creates a
topographical map of the deposition orifice. In some embodiments,
the three-dimensional calibration system monitors the print height
during deposition of the contents of a cartridge. In some
embodiments, the automated bioprinter further comprises at least
one die, each die controlling simultaneous deposition of a
plurality of constructs in parallel and arranged in a pattern, each
construct having a particular three-dimensional geometry;
deposition of a single construct having a particular
three-dimensional geometry; or a combination thereof. In some
embodiments, the automated bioprinter further comprises one or more
multiaxial nozzles for controlling the deposition of one or more
constructs having a particular three-dimensional geometry, wherein
each multiaxial nozzle has dual or greater concentric flow
capability with at least two independent inputs for at least two
different materials and at least two independent outputs for the
preparation of a multiaxial tube with a core layer and a mantle
layer and optionally one or more intermediate layers in between the
core and mantle layers, any two adjacent layers having different
composition of materials with respect to each other and the
materials being selected from bio-ink, support material, and a
combination thereof. In further embodiments, each of the one or
more multiaxial nozzles further comprises a means to independently
regulate the flow of each of the at least two different materials
through the at least two independent outputs. In still further
embodiments, the means to independently regulate the flow of each
of the at least two different materials through the at least two
independent outputs allows for continuous extrusion or sputter
extrusion. In some embodiments, one of the at least two different
materials is removed after bioprinting to create one or more voids.
In some embodiments, at least one cartridge further comprises a
means to adjust and/or maintain the temperature of the
cartridge.
[0006] In another aspect, disclosed herein are automated
bioprinters comprising: a printer head comprising at least three
cartridges, wherein (i) at least one cartridge comprises a
capillary tube containing bio-ink; (ii) at least one cartridge
comprises a needle containing bio-ink; (iii) at least one cartridge
is configured for ink-jet printing; and (iv) each cartridge
comprises contents selected from bio-ink, support material, and a
combination thereof; and a receiving surface for receiving
dispensed contents of a cartridge; whereby a construct is
bioprinted without stopping the bioprinter to manually replace one
or more cartridges or to manually adjust the positioning of the
printer head or the positioning of one or more of the cartridges.
In some embodiments, the needle is in communication with a bio-ink
reservoir, bio-ink deposition is by pneumatic displacement, and the
bio-ink is a liquid or semi-solid composition. In some embodiments,
the capillary tube deposits bio-ink through a positive displacement
mechanism, and the bio-ink is a solid or semi-solid composition. In
some embodiments, the automated bioprinter further comprises a
calibration means comprising a three-dimensional calibration
system, the system comprising at least two lasers, a sensor fixed
to the receiving surface for determining the position of a
deposition orifice; and a sensor fixed to the printer head for
determining the position of the receiving surface; whereby the
system calculates a print height, the print height comprising the
distance between the deposition orifice and receiving surface. In
some embodiments, the automated bioprinter further comprises at
least one die, each die controlling simultaneous deposition of a
plurality of constructs in parallel and arranged in a pattern, each
construct having a particular three-dimensional geometry;
deposition of a single construct having a particular
three-dimensional geometry; or a combination thereof. In some
embodiments, the automated bioprinter further comprises one or more
multiaxial nozzles for controlling the deposition of one or more
constructs having a particular three-dimensional geometry, wherein
each multiaxial nozzle has dual or greater concentric flow
capability with at least two independent inputs for at least two
different materials and at least two independent outputs for the
preparation of a multiaxial tube with a core layer and a mantle
layer and optionally one or more intermediate layers in between the
core and mantle layers, any two adjacent layers having different
composition of materials with respect to each other and the
materials being selected from bio-ink, support material, and a
combination thereof. In some embodiments, at least one cartridge
further comprises a means to adjust and/or maintain the temperature
of the cartridge.
[0007] In another aspect, disclosed herein are automated
bioprinters comprising: at least one die, each die controlling
simultaneous deposition of a plurality of constructs in parallel
and arranged in a pattern, each construct having a particular
three-dimensional geometry; deposition of a single construct having
a particular three-dimensional patterned geometry; or a combination
thereof; whereby the single construct or plurality of constructs
are bioprinted without stopping the bioprinter to manually replace
or to manually adjust the positioning of one or more components of
the bioprinter. In some embodiments, the die is permanently fixed.
In some embodiments, the die is reversibly fixed. In some
embodiments, the die controls simultaneous deposition of 2-384
constructs in parallel and arranged in a pattern. In some
embodiments, the die is connected to one or more chambers for
containing a uniform layer of bio-ink, support material, or a
combination thereof. In some embodiments, the die controls
simultaneous deposition of a plurality of materials in parallel. In
further embodiments, the die comprises an input port for each
material. In some embodiments, each construct in the plurality of
constructs has the same three-dimensional geometry. In some
embodiments, each of the constructs in the plurality of constructs
is in contact with one another to form a single construct.
[0008] In another aspect, disclosed herein are automated
bioprinters comprising one or more multiaxial nozzles for
controlling the deposition of one or more constructs having a
particular three-dimensional geometry, wherein each multiaxial
nozzle has dual or greater concentric flow capability with at least
two independent inputs for at least two different materials and at
least two independent outputs for the preparation of a multiaxial
tube with a core layer and a mantle layer and optionally one or
more intermediate layers in between the core and mantle layers, any
two adjacent layers having different composition of materials with
respect to each other and the materials being selected from
bio-ink, support material, and a combination thereof; whereby the
one or more constructs are bioprinted without stopping the
bioprinter to manually replace or to manually adjust the
positioning of one or more components of the bioprinter. In some
embodiments, each of the one or more multiaxial nozzles further
comprises a means to independently regulate the flow of each of the
at least two different materials through the at least two
independent outputs. In further embodiments, the means to
independently regulate the flow of each of the at least two
different materials through the at least two independent outputs
allows for continuous extrusion or sputter extrusion. In some
embodiments, one of the at least two different materials is removed
after bioprinting to create one or more voids. In some embodiments,
one or more of the multiaxial nozzles are coaxial nozzles. In some
embodiments, one or more of the multiaxial nozzles are triaxial
nozzles.
[0009] In another aspect, disclosed herein are bioprinters
comprising: one or more printer heads, wherein each printer head
comprises a cartridge carrier for receiving and holding a plurality
of cartridges, the cartridge carrier rotatable to align a selected
cartridge with a drive pathway, each cartridge comprising contents
selected from one or more of: bio-ink and support material; a drive
means for dispensing the contents of the selected cartridge; a
receiving surface for receiving dispensed contents of the selected
cartridge; an actuation means for positioning the one or more
printer heads relative to the receiving surface to produce a
particular three-dimensional geometry in the dispensed contents of
the selected cartridge; and a calibration means for determining the
position of: 1) a deposition orifice associated with the selected
cartridge, and 2) a print target surface supported by the receiving
surface. In some embodiments, the drive means utilizes positive
displacement, pneumatic displacement, hydraulic displacement,
acoustic resonance, or a combination thereof to dispense the
contents of a cartridge. In some embodiments, the cartridge carrier
receives and holds 2-10 cartridges. In some embodiments, each
printer head comprises a deposition orifice for each cartridge. In
some embodiments, each printer head comprises a common deposition
orifice for the plurality of cartridges. In some embodiments, the
plurality of cartridges comprises a wash cartridge to decontaminate
a dispensing path of the bioprinter. In some embodiments, the
receiving surface is moved relative to the one or more printer
heads to produce a particular three-dimensional geometry in the
dispensed contents of the plurality of cartridges. In some
embodiments, at least one of the cartridges is connected to a
remote reservoir of contents. In some embodiments, at least one of
the cartridges is a disposable, single-use cartridge. In some
embodiments, the calibration means calculates a print height, the
print height comprising the distance between a deposition orifice
and the print target surface. In further embodiments, the
calibration means monitors the print height during deposition of
materials. In some embodiments, the calibration means utilizes one
or more sensors selected from: triangulation sensors, ultrasonic
distance sensing probes, and digital cameras. In some embodiments,
the bio-ink comprises mammalian cells.
[0010] In another aspect, disclosed herein are cartridge carriers
for a bioprinter, the cartridge carrier attached to a positionable
printer head of the bioprinter and adapted to receive and hold a
plurality of cartridges, each cartridge comprising contents
selected from one or more of: bio-ink and support material, the
cartridge carrier rotatable to align a selected cartridge with a
drive pathway of the bioprinter such that a drive mechanism engages
the selected cartridge to dispense said contents. In some
embodiments, the cartridge carrier is adapted to receive and hold
2-10 cartridges.
[0011] In another aspect, disclosed herein are three-dimensional
calibration systems for an automated bioprinter, the system
comprising: a sensor fixed to a receiving surface of the bioprinter
for determining the position of a deposition orifice of the
bioprinter; and a sensor fixed to a printer head of the bioprinter
for determining the position of a print target surface associated
with the receiving surface; whereby the system calculates a print
height, the print height comprising the distance between the
deposition orifice and a print target surface. In some embodiments,
the sensors are selected from: triangulation sensors, ultrasonic
distance sensing probes, and digital cameras.
[0012] In another aspect, disclosed herein are bioprinters
comprising: a printer head, the printer head comprising a means for
receiving and holding a cartridge, the cartridge comprising
contents selected from one or more of: bio-ink and support
material; a drive means for dispensing the contents of the
cartridge; a receiving surface for receiving dispensed contents of
the cartridge; an actuation means for positioning the printer head
relative to the receiving surface; and a die for controlling
simultaneous deposition of a plurality of constructs in parallel,
each construct having a particular three-dimensional geometry. In
some embodiments, the drive means utilizes positive displacement,
pneumatic displacement, hydraulic displacement, acoustic resonance,
or a combination thereof to dispense the contents of a cartridge.
In some embodiments, the printer head comprises a deposition
orifice for the cartridge. In some embodiments, the receiving
surface is moved relative to the one or more printer heads to
produce a particular three-dimensional geometry in the dispensed
contents of the cartridge. In some embodiments, the cartridge is
connected to a remote reservoir of contents. In some embodiments,
the cartridge is a disposable, single-use cartridge. In some
embodiments, the bioprinter further comprises a calibration means
for determining the position of: 1) a deposition orifice associated
with the cartridge, and 2) a print target surface supported by the
receiving surface. In further embodiments, the calibration means
calculates a print height, the print height comprising the distance
between a deposition orifice and the print target surface. In still
further embodiments, the calibration means monitors the print
height during deposition of materials. In further embodiments, the
calibration means utilizes one or more sensors selected from:
triangulation sensors, ultrasonic distance sensing probes, and
digital cameras. In some embodiments, the die is permanently fixed.
In some embodiments, the die is reversibly fixed. In some
embodiments, the die controls simultaneous deposition of 2-384
constructs in parallel. In further embodiments, the die controls
simultaneous deposition of 96 constructs in parallel. In still
further embodiments, the die controls simultaneous deposition of 24
constructs in parallel. In still further embodiments, the die
controls simultaneous deposition of 12 constructs in parallel. In
some embodiments, the die is connected to a chamber for containing
a uniform layer of bio-ink or support material. In some
embodiments, the die controls simultaneous deposition of a
plurality of materials in parallel. In further embodiments, the die
comprises an input poll for each material. In some embodiments,
each construct in the plurality of constructs has the same
three-dimensional geometry. In some embodiments, the bio-ink
comprises mammalian cells.
[0013] In another aspect, disclosed herein are tissue constructs
fabricated by the bioprinters described herein.
[0014] In another aspect, disclosed herein are dies for controlling
materials deposited from a bioprinter, the die comprising: one or
more input ports for receiving bio-ink or support material
deposited from the bioprinter; and a plurality of output molds,
each output mold comprising a well associated with each input port
for shaping the bio-ink or support material; whereby the die allows
simultaneous deposition of a plurality of constructs in parallel,
each construct having a particular three-dimensional geometry. In
some embodiments, the die controls simultaneous deposition of 2-384
constructs in parallel. In further embodiments, the die controls
simultaneous deposition of 96 constructs in parallel. In still
further embodiments, the die controls simultaneous deposition of 24
constructs in parallel. In still further embodiments, the die
controls simultaneous deposition of 12 constructs in parallel. In
some embodiments, the die is connected to a chamber for containing
a uniform layer of bio-ink or support material, the chamber
positioned between the die and a drive mechanism of the bioprinter.
In some embodiments, the die controls simultaneous deposition of a
plurality of materials in parallel.
[0015] In another aspect, disclosed herein are dies comprising one
or more molds for stamping or cutting to shape one or more
three-dimensional bioprinted tissues into a desired geometry.
[0016] In another aspect, disclosed herein are printer heads
comprising: a plurality of cartridges, each cartridge comprising
contents selected from: bio-ink, support material, and a
combination thereof; a calibration means for determining the
position of a deposition orifice associated with each cartridge;
and for each cartridge: a means for receiving and holding the
cartridge that aligns the cartridge with a drive pathway; a drive
means that dispenses the contents of the cartridge, the drive means
operating independently from other drive means; and an actuation
means that vertically positions the cartridge relative to a
receiving surface to produce a particular three-dimensional
geometry in the dispensed contents of the cartridge, the actuation
means operating independently from other actuation means; provided
that the printer head is for a bioprinter. In various embodiments,
each drive means utilizes positive displacement, pneumatic
displacement, hydraulic displacement, acoustic resonance, or a
combination thereof to dispense the contents of the cartridge.
[0017] In another aspect, disclosed herein are bioprinters
comprising: a printer head comprising: a cartridge carrier for
receiving and holding a plurality of cartridges, each cartridge
comprising contents selected from: bio-ink, support material, and a
combination thereof; and for each cartridge: a means for receiving
and holding the cartridge that aligns the cartridge with a drive
pathway; a drive means that dispenses the contents of the
cartridge, the drive means operating independently from other drive
means; an actuation means that vertically positions the cartridge
relative to a receiving surface to produce a particular
three-dimensional geometry in the dispensed contents of the
cartridge, the actuation means operating independently from other
actuation means; a receiving surface for receiving dispensed
contents of a cartridge; and a calibration means for determining
the position of: 1) a deposition orifice associated with each
cartridge, and 2) a print target surface supported by the receiving
surface. In various embodiments, each drive means utilizes positive
displacement, pneumatic displacement, hydraulic displacement,
acoustic resonance, or a combination thereof to dispense the
contents of the cartridge.
BRIEF DESCRIPTION OF FIGURES
[0018] FIG. 1 illustrates a non-limiting example of a laser
distance sensor mounted in the horizontal position.
[0019] FIG. 2 illustrates a non-limiting example of a laser
distance sensor mounted in the vertical position detecting range to
capillary.
[0020] FIG. 3 illustrates a non-limiting example of a laser
distance sensor in the horizontal position detecting range to
capillary with wire.
[0021] FIG. 4 illustrates a non-limiting schematic example of a
fully automated calibration configuration with dual sensors; in
this case, a schematic depicting a tip triangulation sensor mounted
to a print surface and a surface distance sensor mounted to a pump
head, wherein information from the two sensors is used to automate
a bioprinting process.
[0022] FIG. 5 illustrates a non-limiting example of a
two-dimensional representation of a bio-printed tissue
construct.
[0023] FIG. 6 illustrates a non-limiting example of a
three-dimensional construct generated by continuous deposition of
PF-127 using a NovoGen MMX.TM. bioprinter connected to a syringe
with a 510 .mu.m needle; in this case, a pyramid-shaped
construct.
[0024] FIG. 7 illustrates a non-limiting example of a
three-dimensional construct generated by continuous deposition of
PF-127 using a NovoGen MMX.TM. bioprinter connected to a syringe
with a 510 .mu.m needle; in this case, cube-shaped (left) and
hollow cube-shaped (right) constructs.
[0025] FIG. 8-1 illustrates a non-limiting schematic example of a
two-part die fixed to a deposition mechanism; in this case, a die
that controls the geometry of deposited material to facilitate
deposition of multiple constructs in parallel.
[0026] FIG. 8-2 illustrates a non-limiting schematic example of the
two-part die of FIG. 8-1; in this case, a schematic depicting the
die attached to a chamber filled with a cell suspension forming a
uniform layer of bio-ink, which is extruded though the die to form
contiguous, hollow, cylindrical structures.
[0027] FIG. 9-1 illustrates a non-limiting schematic example of a
centrifugal apparatus intended for spinning cartridges containing
bio-ink to increase cell concentration from a suspension and a
compatible cartridge; in this case, (a) a perspective view of a
centrifugal apparatus; (b). a perspective view of a compatible
cartridge; and (c) a schematic view along A-A of the compatible
cartridge are shown.
[0028] FIG. 9-2 illustrates a non-limiting schematic example of a
cartridge loaded onto an extrusion device.
[0029] FIG. 9-3 illustrates a non-limiting example of a die
installed on a cartridge, for which bio-ink extruded through this
die will form six contiguous, hollow, cylindrical structures; in
this case, (a) a perspective view of a cartridge with installed
die; (b) a schematic view along B-B of the cartridge with installed
die; and (c) three different perspective views of the die itself
are shown.
[0030] FIG. 10 illustrates a non-limiting schematic example of a
two material extrusion plate, or mold lid, which is compatible with
a standard 24-well plate.
[0031] FIG. 11 illustrates a non-limiting schematic example of the
extrusion plate of FIG. 10; in this case, a schematic depicting the
individual geometric molds, each adapted to deposit materials into
a well of the standard 24-well plate, as well as the channels
connected to each geometric mold and to the material input
ports.
[0032] FIG. 12 illustrates a non-limiting schematic example of the
mold lid of FIG. 10; in this case, a schematic depicting the
individual geometric molds, each adapted to deposit two distinct
materials into a specific geometry.
[0033] FIG. 13 illustrates a non-limiting example of a bioprinter
with two printer heads including multiple print cartridges, each
cartridge containing a distinct material for deposition; in this
case, each printer head utilizes a separate deposition tip for each
cartridge.
[0034] FIG. 14 illustrates a non-limiting example of a bioprinter
with two printer heads including multiple print cartridges, each
cartridge containing a distinct material for deposition; in this
case, each printer head utilizes a common deposition tip for the
multiple cartridges.
[0035] FIG. 15 illustrates a non-limiting example of a bioprinter
with two printer heads, one of which is connected to a remote
reservoir in the form of a single container; in this case, the
printer head utilizes a single deposition tip.
[0036] FIG. 16 illustrates a non-limiting example of a disposable,
single use bio-ink cartridge in the form of a plastic syringe; in
this case, an exploded view is shown.
[0037] FIG. 17 illustrates a non-limiting example of a cartridge
configured as part of an ink-jet head; in this case, in an exploded
view, the ink-jet head comprises a syringe body with syringe
plunger, a syringe adapter, a support for the ink-jet head, an
ink-jet valve coupled to a high precision dispense tip, a union
element between the syringe adapter and the ink-jet valve, and an
ink-jet valve holder.
[0038] FIG. 18 illustrates a non-limiting example of a bioprinter
with two printer heads; in this case, each print head receives
bio-ink from a reservoir.
[0039] FIG. 19 illustrates a non-limiting schematic diagram
depicting exemplary parameters for exposing a UV cross-linkable
material to a UV light source in the context of a bioprinter.
[0040] FIG. 20 illustrates a non-limiting example of a NovoGen
MMX.TM. bioprinter including a UV module; in this case, a printer
head is positioned such that a capillary tube is partially
introduced to a UV module.
[0041] FIG. 21 illustrates a non-limiting example of a NovoGen
MMX.TM. bioprinter including a UV module; in this case, a printer
head is positioned such that a capillary tube is entirely
introduced to a UV module.
[0042] FIG. 22 illustrates a non-limiting exemplary printer head;
in this case, a printer head with four cartridges, wherein each
cartridge includes its own deposition tip and has independent
z-axis (e.g., vertical) motion relative to the other cartridges as
well as an independent drive mechanism to dispense the contents of
the cartridge.
[0043] FIG. 23a illustrates a non-limiting example of a printer
head with independent z-axis motion; in this case, the printer head
has a z-axis positioning mechanism to adjust the location of an
attached cartridge relative to the target printing surface and a
drive mechanism to dispense the contents of the attached
cartridge.
[0044] FIG. 23b illustrates a non-limiting example of the z-axis
positioning mechanism to adjust the location of an attached
cartridge relative to the target printing surface for the printer
head shown in FIG. 23a.
[0045] FIG. 24a illustrates the non-limiting example of FIGS. 23a
and 23b, without a cartridge and dispense tip.
[0046] FIG. 24b illustrates the non-limiting example of FIGS. 23a
and 23b; in this case, an internal view of the custom fluidic stage
and the high precision linear stage are shown.
[0047] FIG. 25 illustrates a non-limiting example of the z-axis
positioning mechanism of FIG. 23b; in this case, the mounting plate
is shown along with the optical interrupt sensor and custom sensor
bracket.
[0048] FIG. 26 illustrates a non-limiting example of a bioprinter
with the printer head of FIG. 22.
[0049] FIG. 27 illustrates a non-limiting example of a printer head
comprising a means to control syringe temperature; in this example,
(a) a printer head is shown with a heat exchange jacket around a
syringe; (b) the heat exchange jacket around the syringe is shown
in the absence of the print head; and (c) a schematic view of the
heat exchange jacket around the syringe is shown.
[0050] FIG. 28 illustrates a non-limiting example of extrusion
through a coaxial nozzle; in this example, sputter-extrusion of a
core layer with simultaneous continuous extrusion of a mantle layer
leads to a segmented coaxial tube, which is subsequently severed
between the segments to form spherical "organoids".
[0051] FIG. 29 illustrates a non-limiting example of the
specifications for a coaxial nozzle.
[0052] FIG. 30 illustrates a non-limiting example of a coaxial tube
with a cell-containing core layer and cell-free mantle layer
extruded from a coaxial nozzle; in this case, histology of a cross
section illustrates compact cell core structure.
[0053] FIG. 31 illustrates a non-limiting example of six-day old
bio-printed vascular vessels using 50:50 normal human lung
fibroblasts:human pulmonary endothelial cells; in this example, two
separate views of CD31 stained cross-sections of the vessel show
migration of almost all the endothelial cells to the lumen of the
vessel. All scale bars represent 200 microns.
[0054] FIG. 32 illustrates a non-limiting example of coaxial
structures using I-bio-ink; in this case, histology of embedded
tubular structure and surrounding hydrogel is observed.
[0055] FIG. 33 illustrates a non-limiting example of a patch
created with I-bio-ink; in this case, histology is shown using H
& E staining.
[0056] FIG. 34 illustrates non-limiting examples of bio-printing
constructs, some of which comprise I-bio-ink (shown in darker
shading); in this case, (a) spheres or cylinders are shown; (b)
coaxial cylinders are shown; (c) coaxial spheres are shown; and (d)
complex structures derived from I-bio-ink and normal bio-ink
(bio-ink without immunomodulatory cells).
DETAILED DESCRIPTION OF INVENTION
[0057] The invention relates to the fields of regenerative
medicine, tissue/organ engineering, biologic and medical research,
and drug discovery. More particularly, the invention relates to
improved devices for fabricating tissues and organs, systems and
methods for calibrating and using such devices, and tissues and
organs fabricated by the devices, systems, and methods disclosed
herein. By way of example, the devices, systems, and methods
described herein offer significant improvements to scalability of
production while maintaining accuracy in the spatial positioning of
materials through use of die and mold apparatus attached to a
bioprinter that allows for bioprinting multiple constructs in
parallel. By way of further example, the devices, systems, and
methods described herein offer significant improvements to
three-dimensional calibration of deposition equipment through use
of sensors to rapidly and accurately determine the relative
positions of a material deposition orifice and a target print
surface including the height of a deposition orifice above a print
surface. By way of still further example, the devices, systems, and
methods described herein offer significant improvements to tissue
fabrication with multiple distinct materials through use of a means
to exchange print cartridges without performing a changeover
operation and through the introduction of disposable, single-use
print cartridges.
[0058] Disclosed herein, in certain embodiments, are automated
bioprinters comprising: a printer head comprising one or more
cartridges, each cartridge comprising contents selected from:
bio-ink, support material, and a combination thereof; and for each
cartridge: an actuation means that vertically positions the
cartridge relative to a receiving surface to produce a particular
three-dimensional geometry in the dispensed contents of the
cartridge, the actuation means operating independently from other
actuation means; and a deposition orifice; a receiving surface for
receiving dispensed contents of a cartridge; and a calibration
means comprising a three-dimensional calibration system, the system
comprising at least two lasers, a sensor fixed to the receiving
surface for determining the position of the deposition orifice; and
a sensor fixed to the printer head for determining the position of
the receiving surface; whereby the system calculates a print
height, the print height comprising the distance between the
deposition orifice and the receiving surface; whereby a construct
is bioprinted without stopping the bioprinter to manually replace
one or more cartridges or to manually adjust the positioning of the
printer head or the positioning of one or more of the
cartridges.
[0059] Also disclosed herein, in certain embodiments, are automated
bioprinters comprising: a printer head comprising at least three
cartridges, wherein (i) at least one cartridge comprises a
capillary tube containing bio-ink; (ii) at least one cartridge
comprises a needle containing bio-ink; (iii) at least one cartridge
is configured for ink-jet printing; and (iv) each cartridge
comprises contents selected from bio-ink, support material, and a
combination thereof; and a receiving surface for receiving
dispensed contents of a cartridge; whereby a construct is
bioprinted without stopping the bioprinter to manually replace one
or more cartridges or to manually adjust the positioning of the
printer head or the positioning of one or more of the
cartridges.
[0060] Also disclosed herein, in certain embodiments, are automated
bioprinters comprising: at least one die, each die controlling
simultaneous deposition of a plurality of constructs in parallel
and arranged in a pattern, each construct having a particular
three-dimensional geometry; deposition of a single construct having
a particular three-dimensional patterned geometry; or a combination
thereof; whereby the single construct or plurality of constructs
are bioprinted without stopping the bioprinter to manually replace
or to manually adjust the positioning of one or more components of
the bioprinter.
[0061] Also disclosed herein, in certain embodiments, are automated
bioprinters comprising one or more multiaxial nozzles for
controlling the deposition of one or more constructs having a
particular three-dimensional geometry, wherein each multiaxial
nozzle has dual or greater concentric flow capability with at least
two independent inputs for at least two different materials and at
least two independent outputs for the preparation of a multiaxial
tube with a core layer and a mantle layer and optionally one or
more intermediate layers in between the core and mantle layers, any
two adjacent layers having different composition of materials with
respect to each other and the materials being selected from
bio-ink, support material, and a combination thereof; whereby the
one or more constructs are bioprinted without stopping the
bioprinter to manually replace or to manually adjust the
positioning of one or more components of the bioprinter.
[0062] Also disclosed herein, in certain embodiments, are
bioprinters comprising: one or more printer heads, wherein each
printer head comprises a cartridge earner for receiving and holding
a plurality of cartridges, the cartridge carrier rotatable to align
a selected cartridge with a drive pathway, each cartridge
comprising contents selected from one or more of: bio-ink and
support material; a drive means for dispensing the contents of the
selected cartridge; a receiving surface for receiving dispensed
contents of the selected cartridge; an actuation means for
positioning the one or more printer heads relative to the receiving
surface to produce a particular three-dimensional geometry in the
dispensed contents of the selected cartridge; and a calibration
means for determining the position of: 1) a deposition orifice
associated with the selected cartridge, and 2) a print target
surface supported by the receiving surface.
[0063] Also disclosed herein, in certain embodiments, are cartridge
carriers for a bioprinter, the cartridge carrier attached to a
positionable printer head of the bioprinter and adapted to receive
and hold a plurality of cartridges, each cartridge comprising
contents selected from one or more of: bio-ink and support
material, the cartridge carrier rotatable to align a selected
cartridge with a drive pathway of the bioprinter such that a drive
mechanism engages the selected cartridge to dispense said
contents.
[0064] Also disclosed herein, in certain embodiments, are
three-dimensional calibration systems for an automated bioprinter,
the system comprising: a sensor fixed to a receiving surface of the
bioprinter for determining the position of a deposition orifice of
the bioprinter; and a sensor fixed to a printer head of the
bioprinter for determining the position of a print target surface
associated with the receiving surface; whereby the system
calculates a print height, the print height comprising the distance
between the deposition orifice and a print target surface.
[0065] Also disclosed herein, in certain embodiments, are
bioprinters comprising: a printer head, the printer head comprising
a means for receiving and holding a cartridge, the cartridge
comprising contents selected from one or more of: bio-ink and
support material; a drive means for dispensing the contents of the
cartridge; a receiving surface for receiving dispensed contents of
the cartridge; an actuation means for positioning the printer head
relative to the receiving surface; and a die for controlling
simultaneous deposition of a plurality of constructs in parallel,
each construct having a particular three-dimensional geometry.
[0066] Also disclosed herein, in certain embodiments, are tissue
constructs fabricated by the bioprinters described herein.
[0067] Also disclosed herein, in certain embodiments, are dies for
controlling materials deposited from a bioprinter, the die
comprising: one or more input ports for receiving bio-ink or
support material deposited from the bioprinter; and a plurality of
output molds, each output mold comprising a well associated with
each input port for shaping the bio-ink or support material;
whereby the die allows simultaneous deposition of a plurality of
constructs in parallel, each construct having a particular
three-dimensional geometry.
[0068] Also disclosed herein, in certain embodiments, are printer
heads comprising: a plurality of cartridges, each cartridge
comprising contents selected from: bio-ink, support material, and a
combination thereof; a calibration means for determining the
position of a deposition orifice associated with each cartridge;
and for each cartridge: a means for receiving and holding the
cartridge that aligns the cartridge with a drive pathway; a drive
means that dispenses the contents of the cartridge, the drive means
operating independently from other drive means; and an actuation
means that vertically positions the cartridge relative to a
receiving surface to produce a particular three-dimensional
geometry in the dispensed contents of the cartridge, the actuation
means operating independently from other actuation means; provided
that the printer head is for a bioprinter.
[0069] Also disclosed herein, in certain embodiments, are
bioprinters comprising: a printer head comprising: a cartridge
carrier for receiving and holding a plurality of cartridges, each
cartridge comprising contents selected from: bio-ink, support
material, and a combination thereof; and for each cartridge: a
means for receiving and holding the cartridge that aligns the
cartridge with a drive pathway; a drive means that dispenses the
contents of the cartridge, the drive means operating independently
from other drive means; an actuation means that vertically
positions the cartridge relative to a receiving surface to produce
a particular three-dimensional geometry in the dispensed contents
of the cartridge, the actuation means operating independently from
other actuation means; a receiving surface for receiving dispensed
contents of a cartridge; and a calibration means for determining
the position of: 1) a deposition orifice associated with each
cartridge, and 2) a print target surface supported by the receiving
surface.
Certain Definitions
[0070] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their
entirety.
[0071] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "a nucleic acid" includes one or more nucleic acids,
and/or compositions of the type described herein which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth. Any reference to "or" herein is intended
to encompass "and/or" unless otherwise stated.
[0072] As used herein, "allograft" means an organ or tissue derived
from a genetically non-identical member of the same species as the
recipient.
[0073] As used herein, "bio-ink" means a liquid, semi-solid, or
solid composition comprising a plurality of cells. Bio-ink has high
cell density, or native-like cell density. In some embodiments,
bio-ink consists essentially of cells. In some embodiments, bio-ink
comprises cell solutions, ceil aggregates, cell-comprising gels,
multicellular bodies, or tissues. In some embodiments, the bio-ink
additionally comprises support material. In some embodiments, the
bio-ink additionally comprises non-cellular materials that provide
specific bi (c)mechanical properties that enable bioprinting.
[0074] As used herein, "bioprinting" means utilizing
three-dimensional, precise deposition of cells (e.g., cell
solutions, cell-containing gels, cell suspensions, cell
concentrations, multicellular aggregates, multicellular bodies,
etc.) via methodology that is compatible with an automated,
computer-aided, three-dimensional prototyping device (e.g., a
bioprinter).
[0075] As used herein, "cartridge" means any object that is capable
of receiving (and holding) a bio-ink or a support material.
[0076] As used herein, a "computer module" means a software
component (including a section of code) that interacts with a
larger computer system. In some embodiments, a software module (or
program module) comes in the form of a file and typically handles a
specific task within a larger software system. In some embodiments,
a module is included in one or more software systems. In other
embodiments, a module is seamlessly integrated with one or more
other modules into one or more software systems. A computer module
is optionally a stand-alone section of code or, optionally, code
that is not separately identifiable. A key feature of a computer
module is that it allows an end user to use a computer to perform
the identified functions.
[0077] As used herein, "implantable" means biocompatible and
capable of being inserted or grafted into or affixed onto a living
organism either temporarily or substantially permanently.
[0078] As used herein, "organ" means a collection of tissues joined
into structural unit to serve a common function. Examples of organs
include, but are not limited to, skin, sweat glands, sebaceous
glands, mammary glands, bone, brain, hypothalamus, pituitary gland,
pineal body, heart, blood vessels, larynx, trachea, bronchus, lung,
lymphatic vessel, salivary glands, mucous glands, esophagus,
stomach, gallbladder, liver, pancreas, small intestine, large
intestine, colon, urethra, kidney, adrenal gland, conduit, ureter,
bladder, fallopian tube, uterus, ovaries, testes, prostate,
thyroid, parathyroid, meibomian gland, parotid gland, tonsil,
adenoid, thymus, and spleen.
[0079] As used herein, "patient" means any individual. The term is
interchangeable with "subject," "recipient," and "donor." None of
the terms should be construed as requiring the supervision
(constant or otherwise) of a medical professional (e.g., physician,
nurse, nurse practitioner, physician's assistant, orderly, hospice
worker, social worker, clinical research associate, etc.) or a
scientific researcher.
[0080] As used herein, "stem cell" means a cell that exhibits
potency and self-renewal. Stem cells include, but are not limited
to, totipotent cells, pluripotent cells, multipotent cells,
oligopotent cells, unipotent cells, and progenitor cells. Stem
cells are optionally embryonic stem cells, peri-natal stem cells,
adult stem cells, amniotic stem cells, and induced pluripotent stem
cells.
[0081] As used herein, "tissue" means an aggregate of cells.
Examples of tissues include, but are not limited to, connective
tissue (e.g., areolar connective tissue, dense connective tissue,
elastic tissue, reticular connective tissue, and adipose tissue),
muscle tissue (e.g., skeletal muscle, smooth muscle and cardiac
muscle), genitourinary tissue, gastrointestinal tissue, pulmonary
tissue, bone tissue, nervous tissue, and epithelial tissue (e.g.,
simple epithelium and stratified epithelium), endoderm-derived
tissue, mesoderm-derived tissue, and ectoderm-derived tissue.
[0082] As used herein, "xenograft" means an organ or tissue derived
from a different species as the recipient.
Current Methods of Organ Transplants
[0083] Currently, there is no reliable method for de novo organ
synthesis. Organs are only derived from living donors (e.g., for
kidney and liver donations), deceased donors (e.g., for lung and
heart donations) and, in a few cases, animals (e.g., porcine heart
valves). Thus, patients needing an organ transplant must wait for a
donor organ to become available. This results in a shortage of
available organs. Additionally, reliance on organs harvested from a
living organism increases the chance of transplant rejection.
Transplant Rejections
[0084] In certain instances, a patient receiving an organ
transplant experience hyperacute rejection. As used herein,
"hyperacute rejection" means a complement-mediated immune response
resulting from the recipient's having pre-existing antibodies to
the donor organ. Hyperacute rejection occurs within minutes and is
characterized by blood agglutination. If the transplanted organ is
not immediately removed, the patient may become septic. Xenografts
will produce hyperacute rejection unless the recipient is first
administered immunosuppressants. In some embodiments, a tissue or
organ fabricated de novo will not comprise any antigens and thus
cannot be recognized by any antibodies of the recipient.
[0085] In certain instances, a patient receiving an organ
transplant experiences acute rejection. As used herein, "acute
rejection" means an immune response that begins about one week
after transplantation to about one year after transplantation.
Acute rejection results from the presence of foreign HLA molecules
on the donor organ. In certain instances, APCs recognize the
foreign HLAs and activate helper T cells. In certain instances,
helper T cells activate cytotoxic T cells and macrophages. In
certain instances, the presence of cytotoxic T cells and
macrophages results in the death of cells with the foreign HLAs and
thus damage (or death) of the transplanted organ. Acute rejection
occurs in about 60-75% of kidney transplants, and 50-60% of liver
transplants. In some embodiments, a tissue or organ fabricated de
novo will not comprise any HLAs and thus will not result in the
activation of helper T cells.
[0086] In certain instances, a patient receiving an organ
transplant experiences chronic rejection. As used herein, "chronic
rejection" means transplant rejection resulting from chronic
inflammatory and immune responses against the transplanted tissue.
In some embodiments, a tissue or organ fabricated de novo will not
comprise any antigens or foreign HLAs and thus will not induce
inflammatory or immune responses.
[0087] In certain instances, a patient receiving an organ
transplant experiences chronic allograft vasculopathy (CAV). As
used herein, "chronic allograft vasculopathy" means loss of
function in transplanted organs resulting from fibrosis of the
internal blood vessels of the transplanted organ. In certain
instances, CAV is the result of long-term immune responses to a
transplanted organ. In some embodiments, a tissue or organ
fabricated de novo will not comprise any antigens or foreign HLAs
and thus will not result in an immune response.
[0088] In order to avoid transplant rejection, organ recipients are
administered immunosuppressant drugs. Immunosuppressants include,
but are not limited to, corticosteroids (e.g., prednisone and
hydrocortisone), calcineurin inhibitors (e.g., cyclosporine and
tacrolimus), anti-proliferative agents (e.g., azathioprine and
mycophenolic acid), antibodies against specific components of the
immune system (e.g., basiliximab, dacluzimab, anti-thymocyte
globulin (ATG) and anti-lymphocyte globulin (ALG) and mTOR
inhibitors (e.g., sirolimus and everolimus)). However,
immunosuppressants have several negative side-effects including,
but not limited to, susceptibility to infection (e.g., infection by
pneumocystis carinii pneumonia (PCP), cytomegalovirus pneumonia
(CMV), herpes simplex virus, and herpes zoster virus) and the
spread of malignant cells, hypertension, dyslipidaemia,
hyperglycemia, peptic ulcers, liver and kidney injury, and
interactions with other medicines. In some embodiments, a tissue or
organ fabricated de novo will not result in an immune response and
thus will not require the administration of an
immunosuppressant.
Infections
[0089] In certain instances, a donor organ may be infected with an
infectious agent. Following the transplant of the infected organ,
the infectious agent is able to spread throughout the donor (due in
part to the use of immunosuppressant drugs). By way of non-limiting
example, recipients have contracted HIV, West Nile Virus, rabies,
hepatitis C, lymphocytic choriomeningitis virus (LCMV),
tuberculosis, Chagas disease, and Creutzfeldt-Jakob disease from
transplanted organs. While such infections are rare, they can
nevertheless occur--social histories for deceased donors are often
inaccurate as they are necessarily derived from next-of-kin,
serological tests may produce false-negative results if
seroconversion has not occurred, or serological tests may also
produce false-negatives due to hemodilution following blood
transfusion. Further, many uncommon infectious agents are not
screened for due to the limited time a harvested organ is viable.
In some embodiments, a tissue or organ fabricated de novo will not
comprise any infectious agents.
Donor Complications
[0090] A living donor may also experience complications as a result
of donating an organ. These complications include nosocomial
infections, allergic reactions to the anesthesia, and surgical
errors. Further, an organ donor may one day find themselves in need
of the organ they donated. For example, the remaining kidney of a
kidney donor or the remaining lobe of a liver donor may become
damaged. In some embodiments, a tissue or organ fabricated de novo
obviates the need for donor organs and thus will avoid negative
side-effects to the donor.
[0091] In light of the shortage of available organs and all the
complications that can follow a donor organ transplant, there is a
need for a method of de novo fabrication of tissues and organs.
Tissue Engineering
[0092] Tissue engineering is an interdisciplinary field that
applies and combines the principles of engineering and life
sciences toward the development of biological substitutes that
restore, maintain, or improve tissue function through augmentation,
repair, or replacement of ail organ or tissue. The basic approach
to classical tissue engineering is to seed living cells into a
biocompatible and eventually biodegradable environment (e.g., a
scaffold), and then culture this construct in a bioreactor so that
the initial cell population can expand further and mature to
generate the target tissue upon implantation. With an appropriate
scaffold that mimics the biological extracellular matrix (ECM), the
developing tissue may adopt both the form and function of the
desired organ after in vitro and in vivo maturation. However,
achieving high enough cell density with a native tissue-like
architecture is challenging due to the limited ability to control
the distribution and spatial arrangement of the cells throughout
the scaffold. These limitations may result in tissues or organs
with poor mechanical properties and/or insufficient function.
Additional challenges exist with regard to biodegradation of the
scaffold, entrapment of residual polymer, and industrial scale-up
of manufacturing processes. Scaffoldless approaches have been
attempted. Current scaffoldless approaches are subject to several
limitations: [0093] Complex geometries, such as multi-layered
structures wherein each layer comprises a different cell type, may
require definitive, high-resolution placement of cell types within
a specific architecture to reproducibly achieve a native
tissue-like outcome. [0094] Scale and geometry are limited by
diffusion and/or the requirement for functional vascular networks
for nutrient supply. [0095] The viability of the tissues may be
compromised by confinement material that limits diffusion and
restricts the cells' access to nutrients.
[0096] Disclosed herein, in certain embodiments, are devices,
systems, and methods that generate a three-dimensional tissue
construct. The devices, systems, and methods disclosed herein
utilize a three-phase process: (i) pre-processing, or bio-ink
preparation, (ii) processing, i.e. the actual automated
delivery/printing of the bio-ink particles into the bio-paper by
the bioprinter, and (iii) post-processing, i.e., the
maturation/incubation of the printed construct in the bioreactor.
Final structure formation takes place during post-processing via
the fusion of the bio-ink particles. The devices, systems, and
methods disclosed herein have the following advantages: [0097] They
are capable of producing cell-comprising tissues and/or organs.
[0098] They mimic the environmental conditions of the natural
tissue-forming processes by exploiting principles of developmental
biology. [0099] They can achieve a broad array of complex
topologies (e.g., multilayered structures, repeating geometrical
patterns, segments, sheets, tubes, sacs, etc.). [0100] They are
compatible with automated means of manufacturing and are
scalable.
[0101] Bioprinting enables improved methods of generating
cell-comprising implantable tissues that are useful in tissue
repair, tissue augmentation, tissue replacement, and organ
replacement. Additionally, bioprinting enables improved methods of
generating micro-scale tissue analogs including those useful for in
vitro assays.
Bioprinting
[0102] Disclosed herein, in certain embodiments, are devices,
systems, and methods for fabricating tissues and organs. In some
embodiments, the devices are bioprinters. In some embodiments, the
methods comprise the use of bioprinting techniques. In further
embodiments, the tissues and organs fabricated by use of the
devices, systems, and methods described herein are bioprinted.
[0103] In some embodiments, bioprinted cellular constructs,
tissues, and organs are made with a method that utilizes a rapid
prototyping technology based on three-dimensional, automated,
computer-aided deposition of cells, including cell solutions, cell
suspensions, cell-comprising gels or pastes, cell concentrations,
multicellular bodies (e.g., cylinders, spheroids, ribbons, etc.),
and support material onto a biocompatible surface (e.g., composed
of hydrogel and/or a porous membrane) by a three-dimensional
delivery device (e.g., a bioprinter). As used herein, in some
embodiments, the term "engineered," when used to refer to tissues
and/or organs means that cells, cell solutions, cell suspensions,
cell-comprising gels or pastes, cell concentrates, multicellular
aggregates, and layers thereof are positioned to form
three-dimensional structures by a computer-aided device (e.g., a
bioprinter) according to computer code. In further embodiments, the
computer script is, for example, one or more computer programs,
computer applications, or computer modules. In still further
embodiments, three-dimensional tissue structures form through the
post-printing fusion of cells or multicellular bodies similar to
self-assembly phenomena in early morphogenesis.
[0104] While a number of methods are available to arrange cells,
multicellular aggregates, and/or layers thereof on a biocompatible
surface to produce a three-dimensional structure including manual
placement, positioning by an automated, computer-aided machine such
as a bioprinter is advantageous. Advantages of delivery of cells or
multicellular bodies with this technology include rapid, accurate,
and reproducible placement of cells or multicellular bodies to
produce constructs exhibiting planned or pre-determined
orientations or patterns of cells, multicellular aggregates and/or
layers thereof with various compositions. Advantages also include
assured high cell density, while minimizing cell damage.
[0105] In some embodiments, methods of bioprinting are continuous
and/or substantially continuous. A non-limiting example of a
continuous bioprinting method is to dispense bio-ink from a
bioprinter via a dispense tip (e.g., a syringe, capillary tube,
etc.) connected to a reservoir of bio-ink. In further non-limiting
embodiments, a continuous bioprinting method is to dispense bio-ink
in a repeating pattern of functional units. In various embodiments,
a repeating functional unit has any suitable geometry, including,
for example, circles, squares, rectangles, triangles, polygons, and
irregular geometries. In further embodiments, a repeating pattern
of bioprinted function units comprises a layer and a plurality of
layers are bioprinted adjacently (e.g., stacked) to form an
engineered tissue or organ. In various embodiments, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, or more layers are bioprinted
adjacently (e.g., stacked) to form an engineered tissue or
organ.
[0106] In some embodiments, a bioprinted functional unit repeats in
a tessellated pattern. A "tessellated pattern" is a plane of
figures that fills the plane with no overlaps and no gaps. An
advantage of continuous and/or tessellated bioprinting can include
an increased production of bioprinted tissue. Increased production
can include achieving increased scale, increased complexity, or
reduced time or cost of production. Another non-limiting potential
advantage can be reducing the number of calibration events that are
required to complete the bioprinting of a three-dimensional
construct. Continuous bioprinting also facilitates printing larger
tissues from a large reservoir of bio-ink, optionally using a
syringe mechanism.
[0107] Methods in continuous bioprinting optionally involve
optimizing and/or balancing parameters such as print height, pump
speed, robot speed, or combinations thereof independently or
relative to each other. In one example, the bioprinter head speed
for deposition was 3 mm/s, with a dispense height of 0.5 mm for the
first layer and dispense height was increased 0.4 mm for each
subsequent layer. In some embodiments, the dispense height is
approximately equal to the diameter of the bioprinter dispense tip.
Without limitation a suitable and/or optimal dispense distance does
not result in material flattening or adhering to the dispensing
needle. In various embodiments, the bioprinter dispense tip has an
inner diameter of about, 20, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 .mu.m,
or more, including increments therein. In various embodiments, the
bio-ink reservoir of the bioprinter has a volume of about 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100 cubic centimeters, or more,
including increments therein. The pump speed may be suitable and/or
optimal when the residual pressure build-up in the system is low.
Favorable pump speeds may depend on the ratio between the
cross-sectional areas of the reservoir and dispense needle with
larger ratios requiring lower pump speeds. In some embodiments, a
suitable and/or optimal print speed enables the deposition of a
uniform line without affecting the mechanical integrity of the
material.
[0108] In some embodiments, a bioprinted functional unit is a
multiaxial tube comprising a core layer, a mantle layer, and
optionally one or more intermediate layers between the core and
mantle layers. In some embodiments, a bioprinted functional unit is
a triaxial tube.
[0109] In some embodiments, a bioprinted functional unit is a
coaxial tube. In certain embodiments, the coaxial tube comprises a
core layer (inner layer) and a mantle layer (outer layer). In
certain embodiments, the bio-ink of the core layer differs from the
bio-ink of the mantle layer and the resultant coaxial tube is a
cylinder with bilayer morphology. In certain embodiments, the
bio-ink of the core layer comprises only non-cellular components
and can be removed after bioprinting and the resultant coaxial tube
is a hollow cylinder. In other embodiments, the core layer is
non-continuously bioprinted in a "sputter core feed" pattern while
the mantle layer is continuously bioprinted (see FIG. 28). The
sputter-extrusion of a core layer with concomitant continuous
extrusion of a mantle layer creates a segmented coaxial tube.
Subsequent manipulation of the segmented coaxial tube may result in
the mantle layer collapsing onto itself along a region where the
core layer is absent. Severing each segment at these collapsed
regions creates spherical spheres, or organoids, with distinct
mantle and core structure.
[0110] In some embodiments, methods of bioprinting are
non-continuous and/or substantially non-continuous. A non-limiting
example of a non-continuous bioprinting method is to dispense
bio-ink from a bioprinter via a cartridge configured as part of an
ink-jet head. In some embodiments, the ink-jet head comprises a
high precision dispense tip and an ink-jet solenoid valve closely
coupled to a syringe pump. In some embodiments, dispense volume is
controlled by syringe pressure, valve actuation time, or a high
precision dispense tip, or any combination thereof. In some
embodiments, dispense volume is controlled by syringe pressure,
valve actuation time, and a high precision dispense tip. In various
embodiments, the ink-jet dispense volume is 10, 20, 30,40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or
200 picoliters per droplet, or more, including increments therein.
In some embodiments, bulk dispensing is performed by using an
external pressurized reservoir.
[0111] The inventions disclosed herein include business methods. In
some embodiments, the speed and scalability of the devices and
methods disclosed herein are utilized to design, build, and operate
industrial and/or commercial facilities for production of
engineered tissues and/or organs. In further embodiments, the
engineered tissues and/or organs are produced, stored, distributed,
marketed, advertised, and sold as, for example, materials, tools,
and kits for medical treatment of tissue damage, tissue disease,
and/or organ failure or materials, tools, and kits to conduct
biological assays and/or ding screening as a service.
Bioprinter
[0112] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
bioprinter is any instrument that automates a bioprinting process.
In certain embodiments, a bioprinter disclosed herein comprises: a
printer head comprising a cartridge carrier for receiving and
holding a plurality of cartridges, each cartridge comprising
contents selected from: bio-ink, support material, and a
combination thereof; and for each cartridge: a means for receiving
and holding the cartridge that aligns the cartridge with a drive
pathway; a drive means that dispenses the contents of the
cartridge, the drive means operating independently from other drive
means; and an actuation means that vertically positions the
cartridge relative to a receiving surface to produce a particular
three-dimensional geometry in the dispensed contents of the
cartridge, the actuation means operating independently from other
actuation means; a receiving surface for receiving dispensed
contents of a cartridge; and a calibration means for determining
the position of: 1) a deposition orifice associated with each
cartridge, and 2) a print target surface supported by the receiving
surface. In certain embodiments, a bioprinter disclosed herein
comprises: one or more printer heads (also called print heads),
wherein a printer head comprises a means for receiving and holding
at least one cartridge, and wherein said cartridge comprises
contents selected from one or more of: bio-ink and support
material; a means for calibrating the position of at least one
cartridge; and a means for dispensing the contents of at least one
cartridge. In certain embodiments, a bioprinter disclosed herein
comprises; one or more printer heads, wherein each printer head
comprises a cartridge carrier for receiving and holding a plurality
of cartridges, the cartridge carrier rotatable to align a selected
cartridge with a drive pathway, each cartridge comprising contents
selected from one or more of: bio-ink and support material; a drive
means for dispensing the contents of the selected cartridge; an
actuation means for positioning the one or more printer heads
relative to the receiving surface to produce a particular
three-dimensional geometry in the dispensed contents of the
selected cartridge; and a calibration means for determining the
position of: 1) a deposition orifice associated with the selected
cartridge, and 2) a print target surface supported by the receiving
surface. In certain embodiments, a bioprinter disclosed herein
comprises a printer head, the printer head comprising a means for
receiving and holding a cartridge, the cartridge comprising
contents selected from one or more of: bio-ink and support
material; a drive means for dispensing the contents of the
cartridge; a receiving surface for receiving dispensed contents of
the cartridge; an actuation means for positioning the printer head
relative to the receiving surface; and a die for controlling
simultaneous deposition of a plurality of constructs in parallel,
each construct having a particular three-dimensional geometry.
[0113] In various embodiments, a bioprinter dispenses bio-ink
and/or support material in pre-determined geometries (e.g.,
positions, patterns, etc.) in two or three dimensions. In some
embodiments, a bioprinter achieves a particular geometry by moving
a printer head relative to a printer stage or receiving surface
adapted to receive bioprinted materials. In other embodiments, a
bioprinter achieves a particular geometry by moving a printer stage
or receiving surface relative to a printer head. In further
embodiments, a bioprinter achieves a particular three-dimensional
geometry by moving the receiving surface relative to one or more
printer heads. In some embodiments, a bioprinter achieves a
particular geometry by moving a printer head relative to a printer
stage or receiving surface adapted to receive bioprinted materials
and by moving the printer stage or receiving stage relative to the
printer head. In certain embodiments, the bioprinter is maintained
in a sterile environment.
[0114] In some embodiments, a bioprinter disclosed herein comprises
one or more printer heads. In further embodiments, a printer head
comprises a means for receiving and holding at least one cartridge.
In some embodiments, a printer head comprises a deposition orifice
for each cartridge. In some embodiments, the cartridge is connected
to a remote reservoir of contents. In some embodiments, the
cartridge is a disposable, single-use cartridge. In some
embodiments, a printer head comprises a means for receiving and
holding more than one cartridge. In some embodiments, the means for
receiving and holding at least one cartridge is selected from:
magnetic attraction, a collet chuck grip, a ferrule, a nut, a
barrel adapter, or a combination thereof. In some embodiments, the
means for receiving and holding at least one cartridge is a collet
chuck grip. In some embodiments, a printer head comprises an
independent actuation means for z-axis positioning of the printer
head relative to a receiving surface. In some embodiments, a
printer head further comprises an independent means to adjust
and/or maintain the temperature of the printer head, the cartridge,
and/or the deposition orifice. In further embodiments, a printer
head comprises a heat exchanger jacket around the cartridge to
allow coolant flow, wherein an external device regulates the
temperature and flow of coolant to the printer head. In other
embodiments, a printer head comprises a heat exchanger jacket
around the cartridge to allow heated liquid flow, wherein an
external device regulates the temperature and flow of the heated
liquid to the printer head.
[0115] In some embodiments, a bioprinter disclosed herein comprises
one or more printer heads, wherein each printer head comprises a
cartridge carrier for receiving and holding a plurality of
cartridges. In further embodiments, the cartridge carrier receives
and holds any suitable number of cartridges. In various
embodiments, the cartridge carrier suitably receives and holds
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more cartridges. In some embodiments, the cartridge
carrier receives and holds about 2 to about 10 cartridges. In
further embodiments, each printer head comprises a deposition
orifice for each cartridge. In other embodiments, each printer head
comprises a common deposition orifice for the plurality of
cartridges. In some embodiments, the plurality of cartridges
comprises a wash cartridge to decontaminate a dispensing path of
the bioprinter. In certain embodiments, a bioprinter further
comprises a corresponding wash station to provide a place for spent
wash medium to be stored. In some embodiments, at least one of the
cartridges is connected to a remote reservoir of contents. In some
embodiments, at least one of the cartridges is a disposable,
single-use cartridge. In some embodiments, a printer head comprises
an independent actuation means for z-axis positioning of the
printer head relative to a receiving surface. In some embodiments,
a printer head further comprises an independent means to adjust
and/or maintain the temperature of the printer head, the cartridge,
and/or the deposition orifice.
[0116] Referring to FIG. 13, in a particular embodiment, the
bioprinter has two printer heads, each of which comprises a
deposition orifice for each of the attached cartridges.
[0117] Referring to FIG. 14, in a particular embodiment, the
bioprinter has two printer heads, comprises a common deposition
orifice for the attached plurality of cartridges.
[0118] Referring to FIG. 15, in a particular embodiment, the
bioprinter has two printer heads, and a remote reservoir of
material is connected to one of the two printer heads. Use of a
reservoir is optionally controlled by electronic, pneumatic, or
mechanical signaling.
[0119] Referring to FIG. 18, in a particular embodiment, the
bioprinter has two printer heads, each of which is connected to an
internal reservoir system.
[0120] Referring to FIG. 26, in a particular embodiment, the
bioprinter has one printer head with four cartridges. Each
cartridge has independent z-axis motion with respect to the other
cartridges. Each cartridge is also associated with its own
deposition orifice. In certain further embodiments, only one
cartridge at a time is lowered to interact with the printer stage
or receiving surface adapted to receive bioprinted materials. In
other further embodiments, one or more cartridges are lowered to
interact with the printer stage or receiving surface adapted to
receive bioprinted materials.
[0121] In some embodiments, a bioprinter disclosed herein comprises
a means for calibrating the position of at least one cartridge. In
some embodiments, the means for calibrating the position of at
least one cartridge of is selected from: laser alignment, optical
alignment, mechanical alignment, piezoelectric alignment, magnetic
alignment, electrical field or capacitance alignment, ultrasound
alignment, image-based alignment, or a combination thereof. In some
embodiments, the means for calibrating the position of at least one
cartridge is laser alignment. In some embodiments, the means for
calibrating the position of at least one cartridge is image-based
alignment. In some embodiments, the means for calibrating the
position of at least one cartridge is a combination of laser
alignment and image-based alignment.
[0122] In some embodiments, a bioprinter disclosed herein comprises
a calibration means for determining the position of; 1) a
deposition orifice associated with the selected cartridge, and 2) a
print target surface supported by the receiving surface. In further
embodiments, the calibration means calculates a print height, the
print height comprising the distance between a deposition orifice
and the print target surface. In still further embodiments, the
calibration means monitors the print height during deposition of
materials. In some embodiments, the calibration means utilizes one
or more sensors selected from: triangulation sensors, ultrasonic
distance sensing probes, and digital cameras. In some embodiments,
the calibration means is a three-dimensional calibration system for
an automated bioprinter. In various embodiments, the
three-dimensional calibration system comprises a sensor fixed to a
receiving surface of the bioprinter for determining the position of
a deposition orifice of the bioprinter; and a sensor fixed to a
printer head of the bioprinter for determining the position of a
print target surface associated with the receiving surface; whereby
the system calculates a print height, the print height comprising
the distance between the deposition orifice and a print target
surface. In some embodiments, the sensors are selected from:
triangulation sensors, ultrasonic distance sensing probes, and
digital cameras.
[0123] In some embodiments, a bioprinter disclosed herein comprises
a means for dispensing the contents of at least one cartridge. In
some embodiments, the means for dispensing the contents of at least
one cartridge is application of a piston, application of pressure,
application of compressed gas, application of hydraulics, or
application of a combination thereof. In some embodiments, the
means for dispensing the contents of at least one cartridge is
application of a piston. In some embodiments, the diameter of the
piston is less than the diameter of a cartridge.
[0124] In some embodiments, a bioprinter disclosed herein comprises
a drive means for dispensing the contents of a selected cartridge.
In some embodiments, the drive means for dispensing the contents of
a selected cartridge utilizes positive displacement, pneumatic
displacement, hydraulic displacement, acoustic resonance, or a
combination thereof. In some embodiments, a printer head has its
own independent drive means for dispensing the contents of a
selected cartridge.
[0125] Referring to FIGS. 23a, 24a, and 24b, in a particular
embodiment, the drive means for dispensing the contents of a
cartridge is a drive mechanism that comprises dual linear guide
rails, a stepper motor and an integral lead screw and motor shaft.
In this particular embodiment, the drive mechanism is attached to a
z-axis positioning mechanism to adjust the location of the attached
cartridge relative to the target printing surface.
[0126] In some embodiments, a bioprinter disclosed herein further
comprises a receiving surface. In further embodiments, a receiving
surface is a non-cytotoxic surface onto which a bioprinter
dispenses bio-ink and/or support material. In some embodiments, a
bioprinter disclosed herein further comprises a printer stage. In
further embodiments, a receiving surface is a surface of a printer
stage. In other embodiments, a receiving surface is component
separate from a printer stage, but is affixed to or supported by a
stage. In some embodiments the receiving surface is flat or
substantially flat. In some embodiments the surface is smooth or
substantially smooth. In other embodiments, the surface is both
substantially flat and substantially smooth, In still further
embodiments the receiving surface is designed specifically to
accommodate the shape, size, texture, or geometry of the bioprinted
structure. In still further embodiments, the receiving surface
controls or influences the size, shape, texture, or geometry of a
bioprinted construct. In some embodiments, the receiving surface is
moved relative to the one or more printer heads to produce a
particular three-dimensional geometry in the dispensed contents of
a cartridge or a plurality of cartridges. In some embodiments, the
receiving surface is a standard assay plate. In some embodiments,
the receiving surface is a transwell insert fitted into a 24-well
transwell carrier.
[0127] In some embodiments, a bioprinter disclosed herein comprises
an actuation means for positioning the printer head relative to the
receiving surface. In other embodiments, a bioprinter disclosed
herein comprises an actuation means for positioning the one or more
printer heads relative to the receiving surface to produce a
particular three-dimensional geometry in the dispensed contents of
the selected cartridge. In some embodiments, the actuation means
comprises a ball or lead screw. In some embodiments, the actuation
means comprises an optical encoder. In some embodiments, the
actuation means comprises a brake mechanism to prevent the printer
head from inadvertent contact with the receiving surface. In other
embodiments, the actuation means does not comprise a brake
mechanism to prevent the printer head from inadvertent contact with
the receiving surface. In some embodiments, a printer head has its
own independent actuation means for z-axis positioning to adjust
the location of the cartridge relative to the target printing
surface. In some embodiments, a bioprinter disclosed herein further
comprises a second x-axis and/or y-axis positioning mechanism.
[0128] Referring to FIGS. 23a, 23b, and 25, in a particular
embodiment, a printer head has its own independent actuation means
for z-axis positioning to adjust the location of the cartridge
relative to the target printing surface.
[0129] Referring to FIG. 22, in a particular embodiment, a printer
head comprises four independent cartridges, each with its own
z-axis positioning mechanism for independent z-axis motion relative
to the other cartridges.
[0130] In some embodiments, a bioprinter disclosed herein comprises
a die for controlling simultaneous deposition of a plurality of
constructs in parallel and arranged in a pattern, each construct
having a particular three-dimensional geometry; deposition of a
single construct having a particular three-dimensional geometry; or
a combination thereof. In further embodiments, the die is
permanently fixed. In other embodiments, the die is reversibly
fixed. In some embodiments, the die controls simultaneous
deposition of any suitable number of constructs in parallel. In
various embodiments, the die suitably controls simultaneous
deposition of about 2, 6, 12, 24, 48, 64, 96, 384, 1536, 3456, or
9600 constructs in parallel. In some embodiments, the die controls
simultaneous deposition of about 2 to about 384 constructs in
parallel. In some embodiments, the die is configured to be
complementary to a standard assay plate. In some embodiments, each
construct in the plurality of constructs has the same
three-dimensional geometry. In other embodiments, the plurality of
constructs does not have the same three-dimensional geometry.
Non-limiting examples of the geometries are cylinders, hollow
cylinders, polygons, sheets, and circles. In some embodiments, the
die controls simultaneous deposition of a plurality of materials in
parallel. In further embodiments, the die comprises an input port
for each material. In some embodiments, the die is connected to a
chamber for containing a uniform layer of bio-ink or support
material. In some embodiments, each of the constructs in the
plurality of constructs is in contact with one another to form a
single construct.
[0131] In some embodiments, a bioprinter disclosed herein comprises
a die comprising one or more molds for stamping or cutting to shape
one or more three-dimensional bioprinted tissues into a desired
geometry. In some embodiments, a bioprinter disclosed herein
comprises a coaxial or triaxial nozzle for extrusion of one or more
three-dimensional bioprinted tissues into a desired geometry.
Non-limiting examples of the geometries are cylinders, hollow
cylinders, polygons, sheets, and spheres. In some embodiments, an
outer diameter of the cylinder is 50, 60, 70, 80, 90, 100, 150,
200, 250, 300, 350, 400, 450, or 500 .mu.m, or more, including
increments therein. In some embodiments, an outer diameter of the
cylinder is 250 .mu.m or 500 .mu.m. In further embodiments, the
coaxial nozzle has dual flow capability with at least two
independent inputs for at least two different materials and at
least two independent outputs for the preparation of a coaxial tube
with a core layer and a mantle layer, the core layer and mantle
layer having different composition of materials with respect to
each other. In still further embodiments, each of the one or more
coaxial nozzles further comprises a means to independently regulate
the flow of each of the at least two different materials through
the at least two independent outputs. In some embodiments, the
means to independently regulate the flow of each of the at least
two different materials through the at least two independent
outputs allows for continuous extrusion or sputter extrusion.
[0132] Referring to FIGS. 8-1 and 8-2, in a particular embodiment,
a two-part die is connected to a chamber for containing a uniform
layer of bio-ink. This chamber comprises a removable lid and an
adjustable bottom plate. In order to obtain a uniform layer of
bio-ink within the chamber, the chamber is charged with a cell
suspension and centrifuged. After the lid and supernatant are
removed, the chamber is inverted and attached either permanently or
temporarily to an extrusion mechanism. This extrusion mechanism
pushes the adjustable bottom plate towards the die, driving the
bio-ink into the two-part die. The two-part die comprises a first
die with a C-shape mold and a second die that compresses the
C-shaped bio-ink to form a contiguous, hollow cylindrical structure
as bio-ink exits the second die.
[0133] Referring to FIG. 9-3, a die with molds to simultaneously
create six hollow tubes is installed onto a cartridge. In other
embodiments, the die has molds to extrude one or more tubes in the
same or different shapes. In some embodiments, the cartridge is
pre-treated prior to installation of the die to increase the cell
concentration. A centrifugal apparatus, such as that shown in FIG.
9-1, is used with a cartridge having a removable plug at one end
and a self-sealing port at the other end. The self-sealing port
provides the entry of bio-ink suspension and later removal of
supernatant. The cartridge contains a piston with seals to prevent
leakage. Once a die is installed onto a cartridge containing
bio-ink, the cartridge may be attached to a bioprinter disclosed
herein or to an extrusion device (see FIG. 9-2).
[0134] Referring to FIGS. 10, 11, and 12, in a particular
embodiment, a die is configured to be an extrusion plate, or mold
lid, compatible for a 24-well plate. The die has 24 individual
geometric molds and input ports for materials #1 and #2. In
general, such geometric molds can produce a single geometry or a
mixture of geometries. In this particular embodiment, all 24
individual geometric molds will produce the same geometry for all
24 wells of the receiving surface. Once material is injected
through the input ports, the material flows through pre-defined
pathways in the form of the geometric molds that control the
deposition or extrusion location and geometry of the material into
each individual well of the standard 24-well assay plate, which
serves as the receiving surface. In this way, material is deposited
or extruded into a single well, multiple wells simultaneously or
all wells simultaneously. In this particular embodiment, the
resultant tissue in an individual well is optionally composed of
one or two materials.
[0135] In some embodiments, a bioprinter disclosed herein further
comprises a means for adjusting temperature. In some embodiments,
the means for adjusting temperature adjusts and/or maintains the
ambient temperature. In other various embodiments, the means for
adjusting temperature adjusts and/or maintains the temperature of,
by way of non-limiting example, the print head, cartridge, contents
of the cartridge (e.g., bio-ink, support material, etc.), the
printer stage, and the receiving surface.
[0136] In some embodiments, the means for adjusting temperature is
a heating element. In some embodiments, the means for adjusting
temperature is a heater. In some embodiments, the means for
adjusting temperature is a radiant heater, a convection heater, a
conductive heater, a fan heater, a heat exchanger, or a
combination, thereof. In some embodiments, the means for adjusting
temperature is a cooling element. In some embodiments, the means
for adjusting temperature is a container of coolant, a chilled
liquid, ice, or a combination thereof. In some embodiments, the
means for adjusting temperature is a radiant cooler, convection
cooler, a conductive cooler, a fan cooler, or a combination
thereof.
[0137] In various embodiments, the means for adjusting temperature
adjusts a temperature to about 0, 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, or 90.degree. C. including
increments therein. In some embodiments, temperature is adjusted to
between about 40.degree. C. and about 90.degree. C. In some
embodiments, temperature is adjusted to between about 37.degree. C.
and about 90.degree. C. In other embodiments, temperature is
adjusted to between about 0.degree. C. and about 10.degree. C.
[0138] In some embodiments, the means for adjusting temperature
adjusts the temperature of the deposition tip and/or part or all of
the cartridge. In some embodiments, the means for adjusting
temperature is located on the printer head. In further embodiments,
the means for adjusting temperature is a heat exchanger jacket
located around at least part of the cartridge to allow flow of
temperature-regulated liquid, wherein an external device regulates
the temperature and flow of the temperature-regulated liquid to the
printer head. In some embodiments, the temperature range of the
temperature-regulated liquid is 4.degree. C. to 40.degree. C. In
some embodiments, a cooling or heating device is not located on the
printer head and the printer head must be moved to the location of
the cooling or heating device within the bioprinter.
[0139] Referring to FIG. 27, in a particular embodiment, a printer
head comprises a heat exchanger jacket located around a syringe,
wherein an external device regulates the temperature and flow of
coolant to the printer head. The outer jacket is made of PET.
Coolant flows into the jacket at a maximum of 27 psi. The coolant
flows out of the jacket at about 1 atm.
[0140] In some embodiments, a bioprinter disclosed herein, further
comprises a means for applying a wetting agent to one or more of:
the printer stage; the receiving surface, the deposition orifice,
bio-ink, support material, or the printed construct. In some
embodiments, the means for applying the wetting agent is any
suitable method of applying a fluid (e.g., a sprayer, a pipette, an
inkjet, etc.). In some embodiments, the wetting agent is water,
tissue culture media, buffered salt solutions, serum, or a
combination thereof. In further embodiments, a wetting agent is
applied after the bio-ink or supporting material is dispensed by
the bioprinter. In some embodiments, a wetting agent is applied
simultaneously or substantially simultaneously with the bio-ink or
supporting material being dispensed by the bioprinter. In some
embodiments, a wetting agent is applied prior to the bio-ink or
supporting material being dispensed by the bioprinter.
Printer Head
[0141] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
bioprinter disclosed herein comprises one or more printer heads
(also called print heads). In further embodiments, a printer head
comprises a means for receiving and holding at least one cartridge.
In still further embodiments, a printer head attaches at least one
cartridge to a bioprinter.
[0142] Many means for receiving and holding at least one cartridge
are suitable. Suitable means for receiving and holding at least one
cartridge include those that reliably, precisely, and securely
attach at least one cartridge to a bioprinter. In various
embodiments, the means for receiving and holding at least one
cartridge is, by way of non-limiting example, magnetic attraction,
a collet chuck grip, a ferrule, a nut, a barrel adapter, or a
combination thereof.
[0143] In some embodiments, a printer head disclosed herein
receives and holds one cartridge. In various other embodiments, a
printer head disclosed herein receives and holds 2, 3, 4, 5, 6, 7,
8, 9, 10, or more cartridges simultaneously. In further
embodiments, a printer head disclosed herein further comprises a
means to select a cartridge to be employed in bioprinting from
among a plurality of cartridges received and held.
[0144] In some embodiments, a printer head disclosed herein further
comprises (or is in fluid communication with) a reservoir to
contain bio-ink and/or support materials beyond the capacity of the
one or more cartridges. In further embodiments, a reservoir
supplies bio-ink and/or support materials to one or more cartridges
for dispensing via a dispensing orifice. Printer head
configurations including a reservoir are particularly useful in
continuous or substantially continuous bioprinting applications.
Many volumes are suitable for a reservoir disclosed herein. In
various embodiments, a reservoir has an internal volume of, for
example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,
350, 400, 450, 500 ml or more, including increments therein.
[0145] In some embodiments, bioprinting involves using a computer
to configure parameters such as cell type, print height, pump
speed, robot speed, deposition order, deposition location, or
combinations thereof independently or relative to each other. In
further embodiments, computer code specifies the positioning of a
printer head to configure printer head height above a receiving
surface. In further embodiments, computer code specifies the
direction and speed of the motion of a printer head to configure
dispensing characteristics for bio-ink and/or support material.
[0146] In some embodiments, a printer head disclosed herein is
connected to an independent z-axis positioning mechanism. In some
embodiments, a printer head disclosed herein is connected to an
independent means to adjust and/or maintain the temperature of the
printer head, the attached cartridge, and/or the deposition
orifice. In some embodiments, a printer head disclosed herein is
connected to an independent z-axis positioning mechanism and an
independent means to adjust and/or maintain the temperature of the
printer head, the attached cartridge, and/or the deposition
orifice.
[0147] Referring to FIG. 22, in particular embodiments, a printer
head comprises four independent print cartridges, each with its own
deposition tip and attached to an independent drive mechanism to
dispense the contents of the cartridge. Each cartridge also has
independent z-axis motion relative to the other three cartridges
via its own z-axis positioning mechanism. Although shown in FIG. 22
with each cartridge associated with a syringe needle, each
cartridge independently has a deposition tip selected from syringe
needle, capillary tube, or inkjet tip. In certain embodiments, only
one cartridge at a time is lowered to interact with the printer
stage or receiving surface adapted to receive bioprinted materials.
In other further embodiments, one or more cartridges are lowered to
interact with the printer stage or receiving surface adapted to
receive bioprinted materials. Optionally, each cartridge has an
independent means to adjust and/or maintain the temperature of the
cartridge, the deposition orifice, and/or the contents. In certain
embodiments, one or more cartridges are able to reach a gel heater
and cooler located on the bioprinter. In further embodiments, only
two cartridges, either the two right-most or the two left-most, are
able to reach a gel heater and cooler located on the
bioprinter.
[0148] Referring to FIG. 26, in a particular embodiment, a
bioprinter comprises the printer head as described in FIG. 22. This
bioprinter allows for the use of one or two multi-well plates or
Petri dishes as the receiving surface. This bioprinter also
comprises a calibration means to determine the position of each
deposition orifice and each print target surface supported by the
receiving surface. Optionally, the bioprinter may further comprise
a second x-axis and/or y-axis positioning mechanism to enhance the
capabilities of the bioprinter.
[0149] Referring to FIGS. 23a, 23b, 24a, 24b, and 25, in a
particular embodiment, a printer head has a custom fluidic stage
and a high precision linear stage. The printer head has independent
z-axis motion relative to other components within the bioprinter
through the use of an attached z-axis positioning mechanism to
rapidly adjust the location of the attached cartridge relative to
the target printing surface with high precision and resolution. The
positioning mechanism comprises a lead screw and an optical
encoder. In addition, the printer head is attached to a drive
mechanism to dispense the contents of the attached cartridge. In
this particular embodiment, the printer head is connected to an
integrated positioning mechanism and drive mechanism. This
particular positioning mechanism also does not comprise a braking
mechanism to prevent the printer head from inadvertent contact with
the receiving surface. A hybrid linear actuator drives the plunger
attached to a carriage assembly (see FIG. 24b). Lead screw pitch
and motor specifications are optimized for speed and precision. The
carriage assembly is mounted to the linear guide system (see FIG.
25), which includes a custom sensor bracket attached to the linear
stage, an optical interrupt sensor, and an optical flag integrated
on the mounting plate.
Cartridge Carriers
[0150] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
bioprinter disclosed herein comprises one or more printer heads. In
further embodiments, a printer head comprises a cartridge carrier
for receiving and holding a plurality of cartridges, each cartridge
comprising contents selected from: bio-ink, support material, and a
combination thereof; and for each cartridge: a means for receiving
and holding the cartridge that aligns the cartridge with a drive
pathway; a drive means that dispenses the contents of the
cartridge, the drive means operating independently from other drive
means; and an actuation means that vertically positions the
cartridge relative to a receiving surface to produce a particular
three-dimensional geometry in the dispensed contents of the
cartridge, the actuation means operating independently from other
actuation means. In other embodiments, a printer head comprises a
cartridge carrier for receiving and holding a plurality of
cartridges, the cartridge carrier rotatable to align a selected
cartridge with a drive pathway.
[0151] In some embodiments, the cartridge earner is attached to a
positionable printer head of the bioprinter and adapted to receive
and hold a plurality of cartridges. In further embodiments, the
cartridge carrier is rotatable to align a selected cartridge with a
drive pathway of the bioprinter such that a drive mechanism engages
the selected cartridge to dispense said contents.
[0152] In some embodiments, each cartridge comprises contents
selected from one or more of: bio-ink and support material.
[0153] In some embodiments, the cartridge carrier is adapted to
receive and holds a plurality of cartridges. In various
embodiments, a cartridge carrier is adapted to receive and hold
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more cartridges. In some embodiments, a cartridge carrier
is adapted to receive and hold about 2 to about 10 cartridges. In
some embodiments, a cartridge carrier is adapted to receive and
hold 4 cartridges.
[0154] Many means for receiving and holding at least one cartridge
are suitable. Suitable means for receiving and holding at least one
cartridge include those that reliably, precisely, and securely
attach at least one cartridge to a bioprinter. In various
embodiments, the means for receiving and holding at least one
cartridge is, by way of non-limiting example, magnetic attraction,
a collet chuck grip, a ferrule, a nut, a barrel adapter, or a
combination thereof.
[0155] In some embodiments, the cartridge carrier is
cylindrical.
Dies
[0156] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
bioprinter disclosed herein comprises a die for controlling
simultaneous deposition of a plurality of constructs in parallel,
each construct having a particular three-dimensional geometry.
[0157] In some embodiments, the die comprises one or more input
ports for receiving bio-ink or support material deposited from the
bioprinter; and a plurality of output molds, each output mold
comprising a well associated with each input port for shaping the
bio-ink or support material; whereby the die allows simultaneous
deposition of a plurality of constructs in parallel, each construct
having a particular three-dimensional geometry.
[0158] In some embodiments, the die is permanently fixed. In other
embodiments, the die is reversibly fixed. In some embodiments, each
construct in the plurality of constructs has the same
three-dimensional geometry. In some embodiments, the plurality of
constructs is of homogeneous geometries. In other embodiments, the
plurality of constructs is of heterogeneous geometries. In some
embodiments, the plurality of constructs is bioprinted into
standard assay plates.
[0159] In some embodiments, the die controls simultaneous
deposition of 2-384 constructs in parallel. In some embodiments,
the die controls simultaneous deposition of 96 constructs in
parallel. In some embodiments, the die controls simultaneous
deposition of 24 constructs in parallel. In some embodiments, the
die controls simultaneous deposition of 12 constructs in parallel.
In various embodiments, the die controls simultaneous deposition of
about 2, 4, 6, 8, 12, 24, 36, 48, 96, or 384 constructs in
parallel.
[0160] In some embodiments, the die is connected to a chamber for
containing a uniform layer of bio-ink or support material, the
chamber positioned between the die and a drive mechanism of the
bioprinter.
[0161] In some embodiments, the die controls simultaneous
deposition of a plurality of materials in parallel. In further
embodiments, the die comprises an input port for each material.
Multiaxial Nozzles
[0162] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
bioprinter disclosed herein comprises one or more multiaxial
nozzles for-extrusion of one or more three-dimensional bioprinted
tissues into a desired geometry.
[0163] In some embodiments, the one or more multiaxial nozzles are
one or more specifically shaped nozzles such that bio-ink passing
through the nozzles is shaped into a specific geometry.
Non-limiting examples of the geometries are cylinders, hollow
cylinders, polygons, sheets, and spheres. In some embodiments, the
bioprinter has one or more coaxial nozzles. In some embodiments,
the bioprinter has one or more triaxial nozzles. In some
embodiments, the bioprinter has one or more coaxial or triaxial
nozzles.
[0164] The multiaxial nozzle has dual or greater concentric flow
capability to prepare a multiaxial tube with multiple-layer
morphology consisting of a core layer (inner layer), a mantle layer
(outer layer), and optionally one or more intermediate layers
between the core and mantle layers. Dimensions of the nozzle vary
based on design modifications.
[0165] In some embodiments of a nozzle, the outer diameter of the
nozzle is 0.5 to 4 mm. In some embodiments of a nozzle, the outer
diameter of the nozzle is 0.5 to 3 mm. In some embodiments of a
nozzle, the outer diameter of the nozzle is 0.5 to 2 mm. In some
embodiments of a nozzle, the outer diameter of the nozzle is 1 to 4
mm. In some embodiments of a nozzle, the outer diameter of the
nozzle is 2 to 4 mm. In some embodiments of a nozzle, the outer
diameter of the nozzle is 3 to 4 mm.
[0166] In some embodiments of a coaxial nozzle, the inner diameter
of the nozzle is 0.1 to 1.5 mm. In some embodiments of a coaxial
nozzle, the inner diameter of the nozzle is 0.1 to 1.3 mm. In some
embodiments of a coaxial nozzle, the inner diameter of the nozzle
is 0.1 to 1.0 mm. In some embodiments of a coaxial nozzle, the
inner diameter of the nozzle is 0.1 to 0.8 mm. In some embodiments
of a coaxial nozzle, the inner diameter of the nozzle is 0.1 to 0.6
mm. In some embodiments of a coaxial nozzle, the inner diameter of
the nozzle is 0.1 to 0.4 mm. In some embodiments of a coaxial
nozzle, the inner diameter of the nozzle is 0.6 to 1.5 mm. In some
embodiments of a coaxial nozzle, the inner diameter of the nozzle
is 0.6 to 1.3 mm. In some embodiments of a coaxial nozzle, the
inner diameter of the nozzle is 0.6 to 1.0 mm.
[0167] In some embodiments, the multiaxial nozzle further comprises
a means to independently regulate the flow of each of at least two
different materials through at least two independent outputs to
prepare the coaxial tube, wherein any two adjacent layers have
different composition of materials with respect to each other. In
various embodiments, the flow rate of extrusion through the nozzle
is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mL/min, or more, including
increments therein.
[0168] In some embodiments of a coaxial nozzle, the flow rate of
the inner layer bio-ink is equivalent to the flow rate of the outer
layer bio-ink. In some embodiments of a coaxial nozzle, the flow
rate of the inner layer bio-ink is different from the flow rate of
the outer layer bio-ink. In some embodiments of a coaxial nozzle,
the flow rate of the inner layer bio-ink is 50% faster than the
flow rate of the outer layer bio-ink. In some embodiments of a
coaxial nozzle, the flow rate of the inner layer bio-ink is twice
as fast as the flow rate of the outer layer bio-ink. In some
embodiments, the flow rate of any one of the layers is slower than
an adjacent layer.
[0169] In some embodiments, the multiaxial nozzle is capable of
continuous extrusion and/or sputter extrusion. In some embodiments
of a coaxial nozzle, the core layer is prepared by sputter
extrusion as the mantle layer is prepared by continuous extrusion.
In other embodiments of a coaxial nozzle, the core layer is
prepared by continuous extrusion as the mantle layer is prepared by
sputter extrusion.
[0170] Spheres and cylinders (e.g., FIG. 34a) represent fundamental
geometries using the methods described herein, In various
embodiments of a bioprinted structure, I-bio-ink (shown in dark
shading in FIG. 34a) is the sole component, is present in a
gradient with normal bio-ink (i.e., bio-ink without
immunomodulatory cells), or is absent altogether. Coaxial or
triaxial extrusion nozzles facilitate multi-laminated structures of
a cylindrical--(e.g., FIG. 34b: I-bio-ink is placed in the core
layer or mantle layer) or spherical-nature (e.g., FIG. 34c:
I-bio-ink is placed in the core layer or mantle layer). Triaxial
deposition involves an additional symmetrically nestled cylinder
when viewed in cross-section (not shown). These geometries
represent variable building blocks that are utilized to construct
much larger and more complex structures with the goal of
recapitulating a degree of tissue function in vitro and at
tissue-like cellular densities with or without the inclusion of
biomaterials. Thus some representative assemblages utilizing the
basic building blocks are presented in FIG. 34d: (A) patterning
example with tubular construct, (B) patches generated from spheres,
where patterning can be varied with precision on the resolution of
the spherical bio-ink, (C) use of gradient I-bio-ink cylinders in
patches and tubular constructs, and (D) use, of homogeneous
I-bio-ink and normal bio-ink cylinders in patches with simple
pattern.
Cartridges
[0171] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
cartridge attached to the bioprinter comprises bio-ink or support
material. In some embodiments, the bioprinter dispenses bio-ink or
support material in a specific pattern and at specific positions in
order to form a specific cellular construct, tissue, or organ. In
order to fabricate complex tissue constructs, the bioprinter
deposits the bio-ink or support material at precise speeds and in
uniform amounts. Thus, there is a need for a cartridge with (a) a
dispensing orifice that is smooth or substantially smooth, and (b)
an internal surface that is smooth or substantially smooth. As used
herein, "cartridge" means any object that is capable of receiving
(and holding) a bio-ink and/or support material.
[0172] In some embodiments, a cartridge disclosed herein comprises
bio-ink. In some embodiments, a cartridge disclosed herein
comprises support material. In some embodiments, a cartridge
disclosed herein comprises a combination of bio-ink and support
material. In some embodiments, a cartridge disclosed herein
comprises bio-ink comprising mammalian cells.
[0173] Disclosed herein, in certain embodiments, are cartridges for
use with a bioprinter disclosed herein, comprising at least one
dispensing orifice. In some embodiments, a cartridge comprises one
dispensing orifice. In various other embodiments, a cartridge
comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30,
40, 50, 60, 70, 80, 90, 100, or more dispensing orifices. In
further embodiment, the edges of a dispensing orifice are smooth or
substantially smooth.
[0174] Many shapes are suitable for the dispensing orifices
disclosed herein, In various embodiments, suitable shapes for
dispensing orifices include, by way of non-limiting examples,
circular, ovoid, triangular, square, rectangular, polygonal, and
irregular. In some embodiments, the orifice is circular. In other
embodiments, the orifice is square. In yet other embodiments, the
orifice is oval, oblong, or rectangular and dispenses solid or
semi-solid bio-ink and/or support materials in a ribbon-like
form.
[0175] In some embodiments, the internal surface of the cartridge
is smooth or substantially smooth. In some embodiments, the
cartridge is comprised of a rigid structure to facilitate
calibration. In some embodiments, the walls of the cartridge are
comprised of a material that resists attachment of cells. In some
embodiments, the cartridges are comprised of a material that is
biocompatible.
[0176] In some embodiments, the cartridge is a disposable,
single-use cartridge. The disposable, single-use cartridge is
installed into an appropriate means for receiving and holding the
cartridge on the bioprinter that ultimately aligns the cartridge
with a drive pathway. The cartridge is preferably filled in a
streamlined manufacturing process and is easily installed into and
removed from the print head of the bioprinter. In some embodiments,
the cartridge is a plastic syringe.
[0177] Referring to FIG. 16, in a particular embodiment, a
disposable, single-use cartridge is a plastic syringe, shown here
with a syringe needle as the dispensing orifice.
[0178] Many types of cartridges are suitable for use with
bioprinters disclosed herein and the methods of using the same. In
some embodiments, a cartridge is compatible with bioprinting that
involves extruding a semi-solid or solid bio-ink or a support
material through one or more dispensing orifices. In some
embodiments, a cartridge is compatible with bioprinting that
involves dispensing a liquid or semi-solid cell solution, cell
suspension, or cell concentration through one or more dispensing
orifices. In some embodiments, a cartridge is compatible with
non-continuous bioprinting. In some embodiments, a cartridge is
compatible with continuous and/or substantially continuous
bioprinting. In some embodiments, the cartridge is connected to a
remote reservoir of contents. A remote reservoir suitably contains
a wide range of volumes. In various embodiments, a remote reservoir
suitably contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mL of
contents or more. In various further embodiments, a remote
reservoir suitably contains about 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 mL of contents or more. In still further embodiments, a
remote reservoir suitably contains about 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000 mL of contents or more.
[0179] In some embodiments, a cartridge is a capillary tube or a
micropipette. In some embodiments, a cartridge is a syringe or a
needle, or a combination thereof. Many internal diameters are
suitable for substantially round or cylindrical cartridges. In
various embodiments, suitable internal diameters include, by way of
non-limiting examples, 1, 10, 50, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000 or more .mu.m, including increments therein. In
other various embodiments, suitable internal diameters include, by
way of non-limiting examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100 or more mm, including increments
therein. In some embodiments, a cartridge has an internal diameter
of about 1 .mu.m to about 1000 .mu.m. In a particular embodiment, a
cartridge has an internal diameter of about 500 .mu.m. In another
particular embodiment, a cartridge has an internal diameter of
about 250 .mu.m. Many internal volumes are suitable for the
cartridges disclosed herein. In various embodiments, suitable
internal volumes include, by way of non-limiting examples, 1, 10,
20, 30, 40, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 or
more .mu.l, including increments therein. In other various
embodiments, suitable internal volumes include, by way of
non-limiting examples, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 200, 300, 400, 500 or more ml, including increments
therein. In some embodiments, a cartridge has a volume of about 1
.mu.l to about 50 .mu.l. In a particular embodiment, a cartridge
has a volume of about 5 .mu.l.
[0180] In some embodiments, a cartridge is compatible with ink-jet
printing of bio-ink and/or support material onto a receiving
surface such as that described in U.S. Pat. No. 7,051,654. In
further embodiments, a cartridge includes dispensing orifices in
the form of voltage-gated nozzles or needles under the control of
the computer code described herein.
[0181] In some embodiments, the ink-jet printing requires a syringe
pump closely coupled to a specialized high speed ink-jet solenoid
valve. In some embodiments, dispense volume is controlled by
syringe pressure, valve actuation time and a precision dispense
tip. In some embodiments, the precision dispense control is
controlled through a two stage electronic valve, wherein a high
voltage pulse is followed by a lower voltage hold current. In
further embodiments, the high voltage pulse is 12 to 40 VDC, and
the lower voltage hold current is 12 to 24 VDC. In some
embodiments, the valve timing is controlled by TTL control voltage.
In some embodiments, the pulse is 0.35 to 2.0 milliseconds. In some
embodiments, the syringe pressure is 0 to 120 psig.
[0182] In some embodiments, the cartridge is primed. In some
embodiments, priming the cartridge increases the accuracy of the
dispensing, deposition, or extrusion process. As used herein,
"primed" means the contents of the cartridge are made ready for
dispensing, deposition, or extrusion by compacting and advancing
the contents of the cartridge until the material to be dispensed
(bio-ink or supporting material) is located in a position in
contact with the dispensing orifice. See, e.g., FIG. 3. In some
embodiments, the cartridge is primed when the contents are compact
or substantially compact, and the contents are in physical contact
with the orifice of the cartridge.
[0183] In some embodiments, a cartridge is marked to indicate the
composition of its contents. In further embodiments, a cartridge is
marked to indicate the composition of a bio-ink and/or support
material contained therein. In some embodiments, the surface of the
cartridge is colored. In some embodiments, the outer surface of the
cartridge is dyed, painted, marked with a pen, marked by a sticker,
or a combination thereof.
[0184] In some embodiments, the outer surface of a cartridge is
marked to increase the opacity of the surface of the cartridge
(e.g., to increase the amount of a laser beam that is reflected off
the surface of the cartridge). In some embodiments, the surface of
a cartridge is colored. In some embodiments, the outer surface of a
cartridge is scored. As used herein, "scored" means marking the
surface of a cartridge to reduce the smoothness of the surface. Any
suitable method is used to score the surface of a cartridge (e.g.,
application of an acidic substance, application of a caustic
substance, application of an abrasive substance, etc.). In some
embodiments, the outer surface of a cartridge is painted, polished
(e.g., fire polished), etched (e.g., laser etched), marked with a
pen, marked by a sticker, or a combination thereof.
Grip
[0185] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
cartridge attached to a bioprinter comprises bio-ink and/or support
material. In some embodiments, the bioprinter dispenses the bio-ink
and/or support material in a specific pattern and at specific
positions in order to form a specific cellular construct, tissue,
or organ. In some embodiments, a cartridge comprising bio-ink is
disposable. In some embodiments, the cartridge is ejected from the
bioprinter following extrusion of the contents. In some
embodiments, a new cartridge is subsequently attached to the
bioprinter.
[0186] In order to fabricate complex structures, the bioprinters
disclosed herein dispense bio-ink and/or support material from a
cartridge with a suitable repeatable accuracy. In various
embodiments, suitable repeatable accuracies include those of about
.+-.5, 10, 20, 30, 40, or 50 .mu.m on any axis. In some
embodiments, the bioprinters disclosed herein dispense bio-ink
and/or support material from a cartridge with a repeatable accuracy
of about .+-.20 .mu.m. However, uncontrolled removal and insertion
of cartridges can result in alterations of the position of the
printer head (and thus the cartridges) with respect to the tissue
construct, such that precision of the placement of the first
bio-ink particle deposited from a new cartridge optionally varies
relative to the last bio-ink particle deposited from the previous
cartridge. Thus, there is a need for a method of attaching and
securing a cartridge to a printer head, wherein said attaching and
securing produce minimal alterations in the position of the printer
head.
[0187] Disclosed herein, in certain embodiments, are methods of
attaching a cartridge to a bioprinter, comprising: (a) inserting
the cartridge into a collet chuck, wherein the collet chuck is
attached to a printer head of the bioprinter; and (b) adjusting the
outer collar of the collet chuck; wherein the inserting and
adjusting do not substantially alter the position of the printer
head.
[0188] Disclosed herein, in certain embodiments, are systems for
attaching a cartridge to a bioprinter, comprising: a means for
receiving and securing a cartridge to a printer head of a
bioprinter; wherein use of the means for receiving and securing the
cartridge do not substantially alter the position of the printer
head. In some embodiments, the means for receiving and securing the
cartridge to a printer head is a chuck or ferrule. As used herein,
"chuck" means a holding device consisting of adjustable jaws. In
some embodiments, the means for receiving and securing the
cartridge to a printer head is a collet. As used herein, "collet"
means a subtype of chuck- that forms a collar around the object to
be held and exerts a strong clamping. As used herein, "ferrule"
means a band (e.g., a metal band) used to secure one object to
another. In some embodiments, the means for receiving and securing
the cartridge to a printer head is a barrel adaptor. As used
herein, "barrel adaptor" means a threaded tube used to secure one
object to another.
UV Module
[0189] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
bioprinter comprises an ultraviolet (UV) light module. In further
embodiments, a UV module enables a bioprinter disclosed herein to
automatically cure and print UV cross-linkable materials. In some
embodiments, a UV module comprises a light chamber for exposing the
contents of a cartridge (e.g., a glass capillary tube) to UV light.
In some embodiments, a UV module comprises a UV light line to
expose the contents of a cartridge to UV light evenly along its
length. In some embodiments, a UV module comprises a slot to accept
an optional attenuation filter that reduces the intensity of UV
light to which the contents of a cartridge is exposed. In some
embodiments, a UV module is integrated with a bioprinter. In
various further embodiments, a UV module is permanently,
semi-permanently, or reversibly attached to a bioprinter. In other
embodiments, a UV module is not attached to a bioprinter.
[0190] In some embodiments, a UV module described herein is used by
first aspirating uncured material into a glass capillary tube. In
further embodiments, the glass capillary then is lowered into the
light chamber, where a high power fiber optic light line transmits
UV light from a UV light source across the length of the capillary.
In still further embodiments, once cured (e.g., cross-linked), the
glass capillary is lifted out of the light chamber and the material
is ready to print (e.g., be extruded or deposited, etc.). For
applications where the contents of a cartridge include bio-ink
and/or support material that comprises cells, an optional
attenuation filter is placed between the capillary and the light
guide to achieve lower UV intensities.
[0191] Many UV light sources are suitable for use with the UV
module described herein. UV light is electromagnetic radiation with
a wavelength in the range 10 nm to 400 nm and energies from 3 eV to
124 eV. UV light, in some cases, alters chemical bonds in molecules
and can cause chemical reactions. UV light from various sources is
characterized by having many suitable wavelengths and many suitable
associated energies. See Table 1, infra.
TABLE-US-00001 TABLE 1 Wavelength range Energy per photon UV light
type (nanometers) (electronvolts) Ultraviolet A (UVA) 400-315 nm
3.10-3.94 eV or long wave Near UV 400-300 nm 3.10-4.13 eV
Ultraviolet B (UVB) 315-280 nm 3.94-4.43 eV or medium wave Middle
UV 300-200 nm 4.13-6.20 eV Ultraviolet C (UVC) 280-100 nm 4.43-12.4
eV or short wave Far UV 200-122 nm 6.20-10.2 eV Vacuum UV 200-100
nm 6.20-12.4 eV Low UV 100-88 nm 12.4-14.1 eV Super UV 150-10 nm
8.28-124 eV Extreme UV 121-10 nm 10.25-124 eV
[0192] In some embodiments, suitable sources of UV light include,
by way of non-limiting examples, UV lamps, UV fluorescent lamps, UV
LEDs, UV lasers, and the like. A UV lamp (e.g., black light, black
light blue or BLB lamps, etc.) emits long-wave UV radiation and
very little visible light. Fluorescent black lights are typically
made using a UV-emitting phosphor. The phosphor typically used for
a near 368-371 nm emission peak is either europium-doped strontium
fluoroborate (SrB.sub.4O.sub.7F:Eu.sup.2+) or europium-doped
strontium borate (SrB.sub.4O.sub.7:Eu.sup.2+), whereas the phosphor
used to produce a peak around 350-353 nm is lead-doped barium
silicate (BaSi.sub.2O.sub.5:Pb.sup.+). Fluorescent black lights are
also typically made using Wood's glass, a nickel-oxide-doped glass,
which blocks almost all visible light above 400 nm. A black light
may also be formed, very inefficiently, by simply using Wood's
glass instead of clear glass as the envelope for a common
incandescent bulb. UV fluorescent lamps without a phosphorescent
coating to convert UV to visible light, emit ultraviolet light with
two peaks at 253.7 nm and 185 nm due to the peak emission of the
mercury within the bulb. With the addition of a suitable phosphor
(phosphorescent coating), they can be modified to produce UVA or
UVB. Light-emitting diodes (LEDs) can be manufactured to emit light
in the UV range, although practical LED arrays are limited below
365 nm. UV laser diodes and UV solid-state lasers can be
manufactured to emit light in the UV range. Direct UV-emitting
laser diodes are available at 375 nm. UV diode lasers have been
demonstrated using crystals of cerium doped with lithium strontium
aluminum fluoride (Ce:LiSAF), Wavelengths shorter than 375 nm are
generated from diodes in solid-state modules that use frequency
doubling or tripling diode-pumped solid state (DPSS) technology.
Wavelengths available include 262, 266, 349, 351, 355, and 375
nm.
[0193] Referring to FIG. 19, in a particular embodiment, a UV
module comprises a UV light source that is a UV lamp. Further in
this embodiment, a cartridge includes a glass capillary tube into
which a UV cross-linkable material is aspirated. The cartridge is
positioned within the light chamber of the UV module such that it
is about 3 inches from the UV lamp. The UV lamp in this embodiment
emits UV light with a wavelength of about 365 nm.
[0194] Referring to FIG. 20, in a particular embodiment, a
bioprinter includes a printer head 800, which holds a cartridge
810. In this embodiment, a cartridge 810 includes a glass capillary
tube which is partially lowered into a UV light module 820 attached
to the bioprinter. The UV module 820 in this case, comprises a
housing, a cover, and an opening to allow introduction of the
cartridge 810.
[0195] Referring to FIG. 21, in a further particular embodiment, a
printer head 800, which holds a cartridge 810 is completely lowered
into a UV light module 820.
[0196] In some embodiments, a UV module enables a bioprinter
disclosed herein to automatically cure and print UV cross-linkable
materials by exposing them to UV light for a pre-determined period
of time. Many durations of exposure to a UV light source are
suitable. In light of the disclosure provided herein, those of
skill in the art will recognize that particular UV cross-linkable
materials are suited to particular exposure times. In some
embodiments, exposure time is selected to completely UV cross-link
a material, resulting in a more solid structure. In other
embodiments, exposure time is selected to partially UV cross-link a
material, resulting in a more semi-solid structure. In various
embodiments, suitable exposure times include, by way of
non-limiting examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more seconds.
In other various embodiments, suitable exposure times include, by
way of non-limiting examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more
minutes,
[0197] In some embodiments, exposure time is about 5 seconds to
about 15 minutes. In further embodiments, exposure time is about 10
seconds to about 10 minutes, In still further embodiments, exposure
time is about 15 seconds to about 5 minutes. In some cases,
exposure time is adjusted for the presence of cells in a UV
cross-linkable material.
Receiving Surface
[0198] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, the
bioprinter dispenses a plurality of elements, sections, and/or
areas of bio-ink and/or support material onto a receiving surface.
In further embodiments, dispensing occurs in a specific pattern and
at specific positions. In still further embodiments, the locations
at which the bioprinter deposits bio-ink and/or support material
onto a receiving surface are defined by user input and translated
into computer code.
[0199] In some embodiments, each of the elements, sections, and/or
areas of bio-ink and/or support material has dimensions of less
than 300 mm.times.300 mm.times.160 mm. By way of example only, the
dimensions of a section of bio-ink or support material are
optionally 75 mm.times.5.0 mm.times.5.0 mm; 0.3 mm.times.2.5
mm.times.2.5 mm; 1 mm.times.1 mm.times.50 mm; or 150 mm.times.150
mm.times.80 mm. Due to the generally small size of each section,
and in some cases, the high degree of precision required, minute
imperfections in the receiving surface may result in imperfections
(and possibly, failure) of a cellular construct, tissue, or organ.
Thus, there is a need for a substantially smooth and substantially
flat receiving surface, or a defined or substantially defined
receiving surface, that is able to receive sections of bio-ink
and/or support material.
[0200] Disclosed herein, in certain embodiments, are receiving
surfaces for receiving one or more structures generated by the
bioprinter disclosed herein. In some embodiments, the receiving
surface is flat or substantially flat. In some embodiments, the
receiving surface is smooth or substantially smooth. In some
embodiments, the receiving surface is flat or substantially flat.
In some embodiments, the receiving surface is defined or
substantially defined. In other embodiments the receiving surface
is designed specifically to accommodate the shape, size, texture,
or geometry of a specific bioprinted structure. In further
embodiments, the receiving surface controls or influences the size,
shape, texture, or geometry of a bioprinted construct.
[0201] In some embodiments, the receiving surface comprises a solid
material, a semi-solid material, or a combination thereof. In some
embodiments, the receiving surface comprises glass, coated glass,
plastic, coated plastic, metal, a metal alloy, or a combination
thereof. In some embodiments, the receiving surface comprises a
gel. In some embodiments, the receiving surface and any coatings
thereon are biocompatible. In various embodiments, the receiving
surface comprises any of the support materials and/or confinement
materials disclosed herein. In specific embodiments, the receiving
surface comprises polymerized NovoGel.TM. or polymerized agarose,
polymerized gelatin, extracellular matrix (Or components thereof),
collagen, or a combination thereof. In some embodiments, the
receiving surface is a standard assay plate. In some embodiments,
the receiving surface is a transwell insert fitted into a 24-well
transwell carrier.
Software
[0202] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, one or
more cartridges attached to the bioprinter comprise bio-ink and/or
support material. In some embodiments, the bioprinter dispenses
bio-ink or support material in a specific pattern and at specific
positions in order to form a specific cellular construct, tissue,
or organ.
[0203] In order to fabricate complex tissue constructs, the
bioprinter deposits the bio-ink or support material at precise
locations (in two or three dimensions) on a receiving surface. In
some embodiments, the locations at which the bioprinter deposits
bio-ink and/or support material are defined by user input and
translated into computer code. In further embodiments, the computer
code includes a sequence of instructions, executable in the central
processing unit (CPU) of a digital processing device, written to
perform a specified task. In some embodiments, additional
bioprinting parameters including, by way of non-limiting examples,
print height, pump speed, robot speed, control of variable
dispensing orifices, UV intensity, and UV exposure time are defined
by user input and translated into computer code. In other
embodiments, such bioprinting parameters are not directly defined
by user input, but are derived from other parameters and conditions
by the computer code described herein.
[0204] Disclosed herein, in certain embodiments, are methods for
fabricating tissue constructs, tissues, and organs, comprising: a
computer module receiving input of a visual representation of a
desired tissue construct; a computer module generating a series of
commands, wherein the commands are based on the visual
representation and are readable by a bioprinter; a computer module
providing the series of commands to a bioprinter; and the
bioprinter depositing bio-ink and/or support material according to
the commands to form a construct with a defined geometry.
Non-Transitory Computer Readable Storage Medium
[0205] In some embodiments, the locations at which the bioprinter
deposits the bio-ink and/or support material are defined by user
input and translated into computer code. In some embodiments, the
devices, systems, and methods disclosed herein further comprise
non-transitory computer readable storage media or storage media
encoded with computer readable program code. In further
embodiments, a computer readable storage medium is a tangible
component of a digital processing device such as a bioprinter (or a
component thereof) or a computer connected to a bioprinter (or a
component thereof). In still further embodiments, a computer
readable storage medium is optionally removable from a digital
processing device. In some embodiments, a computer readable storage
medium includes, by way of non-limiting examples, CD-ROMs, DVDs,
flash memory devices, solid state memory, magnetic disk drives,
magnetic tape drives, optical disk drives, cloud computing systems
and services, and the like. In some cases, the program and
instructions are permanently, substantially permanently,
semi-permanently, or non-transitorily encoded on the storage
media.
Computer Modules
[0206] In some embodiments, the devices, systems, and methods
described herein comprise software, server, and database modules.
In some embodiments, a "computer module" is a software component
(including a section of code) that interacts with a larger computer
system. In further embodiments, a software module (or program
module) comes in the form of one or more files and typically
handles a specific task within a larger software system.
[0207] In some embodiments, a module is included in one or more
software systems. In other embodiments, a module is integrated with
one or more other modules into one or more software systems. A
computer module is optionally a stand-alone section of code or,
optionally, code that is not separately identifiable. In some
embodiments, the modules are in a single application. In other
embodiments, the modules are in a plurality of applications. In
some embodiments, the modules are hosted on one machine. In other
embodiments, the modules are hosted on a plurality of machines. In
some embodiments, the modules are hosted on a plurality of machines
in one location. In other embodiments, the modules are hosted a
plurality of machines in more than one location. Further described
herein is the formatting of location and positioning data. In some
embodiments, the data files described herein are formatted in any
suitable data serialization format including, by way of
non-limiting examples, tab-separated values, comma-separated
values, character-separated values, delimiter-separated values,
XML, JSON, BSON, and YAML. A key feature of a computer module is
that it allows an end user to use a computer to perform the
identified functions.
Graphic User Interface
[0208] In some embodiments, a computer module comprises a graphic
user interface (GUI). As used herein, "graphic user interface"
means a user environment that uses pictorial as well as textual
representations of the input and output of applications and the
hierarchical or other data structure in which information is
stored. In some embodiments, a computer module comprises a display
screen. In further embodiments, a computer module presents, via a
display screen, a two-dimensional GUI. In other embodiments, a
computer module presents, via a display screen, a three-dimensional
GUI such as a virtual reality environment. In some embodiments, the
display screen is a touchscreen or multitouchscreen and presents an
interactive GUI.
[0209] In some embodiments, the display screen presents a GUI that
consists essentially of a grid comprising regularly spaced objects
of substantially the same shape and substantially equal size. The
objects presented have any suitable shape. In some embodiments,
suitable shapes for objects include, by way of non-limiting
examples, circle, oval, square, rectangle, triangle, diamond,
polygon, or a combination thereof.
[0210] In some embodiments, a user defines the content of one or
more objects to form a two-dimensional or three-dimensional visual
representation of a desired tissue construct. See, e.g., FIG. 5. In
some embodiments, the user-defined content of an object is, by way
of non-limiting examples, a bio-ink with various compositions or
support material with various compositions. In some embodiments,
the user defines the content of an object by modifying the color of
the cell or the shape of the object.
Bio-Ink
[0211] Disclosed herein, in certain embodiments, are devices,
systems, and methods for fabricating tissues and organs. In some
embodiments, the devices comprise one or more printer heads for
receiving and holding at least one cartridge that optionally
contains bio-ink. In some embodiments, the methods comprise the use
of bio-ink. In further embodiments, the tissues and organs
fabricated by use of the devices, systems, and methods described
herein comprise bio-ink at the time of fabrication or
thereafter.
[0212] Bio-ink has high cell density or native-like cell density.
In various embodiments, the cell density of bio-ink is 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 percent, or more,
including increments therein. In some embodiments, bio-ink consists
essentially of cells.
[0213] In some embodiments, "bio-ink" includes liquid, semi-solid,
or solid compositions comprising a plurality of cells. In some
embodiments, bio-ink comprises liquid or semi-solid cell solutions,
cell suspensions, or cell concentrations. In further embodiments, a
cell solution, suspension, or concentration comprises a liquid or
semi-solid (e.g., viscous) carrier and a plurality of cells. In
still further embodiments, the carrier is a suitable cell nutrient
media, such as those described herein. In some embodiments, bio-ink
comprises semi-solid or solid multicellular aggregates or
multicellular bodies. In further embodiments, the bio-ink is
produced by 1) mixing a plurality of cells or cell aggregates and a
biocompatible liquid or gel in a pre-determined ratio to result in
bio-ink, and 2) compacting the bio-ink to produce the bio-ink with
a desired cell density and viscosity. In some embodiments, the
compacting of the bio-ink is achieved by centrifugation, tangential
flow filtration ("TFF"), or a combination thereof. In some
embodiments, the compacting of the bio-ink results in a composition
that is extrudable, allowing formation of multicellular aggregates
or multicellular bodies. In some embodiments, "extrudable" means
able to be shaped by forcing (e.g., under pressure) through a
nozzle or orifice (e.g., one or more holes or tubes). In some
embodiments, the compacting of the bio-ink results from growing the
cells to a suitable density. The cell density necessary for the
bio-ink will vary with the cells being used and the tissue or organ
being produced. In some embodiments, the cells of the bio-ink are
cohered and/or adhered. In some embodiments, "cohere," "cohered,"
and "cohesion" refer to cell-cell adhesion properties that bind
cells, multicellular aggregates, multicellular bodies, and/or
layers thereof. In further embodiments, the terms are used
interchangeably with "fuse," "fused," and "fusion." In some
embodiments, the bio-ink additionally comprises support material,
cell culture medium, extracellular matrix (or components thereof),
cell adhesion agents, cell death inhibitors, anti-apoptotic agents,
anti-oxidants, extrusion compounds, and combinations thereof.
Cells
[0214] Disclosed herein, in various embodiments, are bio-inks that
include liquid, semi-solid, or solid compositions comprising a
plurality of cells. In some embodiments, bio-ink comprises liquid
or semi-solid cell solutions, cell suspensions, or cell
concentrations. In some embodiments, any mammalian cell is suitable
for use in bio-ink and in the fabrication of tissues and organs
using the devices, systems, and methods described herein. In
various embodiments, the cells are any suitable cell. In further
various embodiments, the cells are vertebrate cells, mammalian
cells, human cells, or combinations thereof. In some embodiments,
the type of cell used in a method disclosed herein depends on the
type of cellular construct, tissue, or organ being produced. In
some embodiments, the bio-ink comprises one type of cell (also
referred to as a "homogeneous ink"). In some embodiments, the
bio-ink comprises more than one type of cell (also referred to as a
"heterogeneous ink").
[0215] In further embodiments, the cells are, by way of
non-limiting examples, contractile or muscle cells (e.g., skeletal
muscle cells, cardiomyocytes, smooth muscle cells, and myoblasts),
connective tissue cells (e.g., bone cells, cartilage cells,
fibroblasts, and cells differentiating into bone forming cells,
chondrocytes, or lymph tissues), bone marrow cells, endothelial
cells, skin cells, epithelial cells, breast cells, vascular cells,
blood cells, lymph cells, neural cells, Schwann cells,
gastrointestinal cells, liver cells, pancreatic cells, lung cells,
tracheal cells, corneal cells, genitourinary cells, kidney cells,
reproductive cells, adipose cells, parenchymal cells, pericytes,
mesothelial cells, stromal cells, undifferentiated cells (e.g.,
embryonic cells, stem cells, and progenitor cells),
endoderm-derived cells, mesoderm-derived cells, ectoderm-derived
cells, and combinations thereof.
[0216] In some embodiments, the cells are adult, differentiated
cells. In further embodiments, "differentiated cells" are cells
with a tissue-specific phenotype consistent with, for example, a
smooth muscle cell, a fibroblast, or an endothelial cell at the
time of isolation, wherein tissue-specific phenotype (or the
potential to display the phenotype) is maintained from the time of
isolation to the time of use. In other embodiments, the cells are
adult, non-differentiated cells. In further embodiments,
"non-differentiated cells" are cells that do not have, or have
lost, the definitive tissue-specific traits of for example, smooth
muscle cells, fibroblasts, or endothelial cells. In some
embodiments, non-differentiated cells include stem cells. In
further embodiments, "stem cells" are cells that exhibit potency
and self-renewal. Stem cells include, but are not limited to,
totipotent cells, pluripotent cells, multipotent cells, oligopotent
cells, unipotent cells, and progenitor cells. Stem cells are
optionally embryonic stem cells, adult stem cells, amniotic stem
cells, and induced pluripotent stem cells. In yet other
embodiments, the cells are a mixture of adult, differentiated cells
and adult, non-differentiated cells.
[0217] In some embodiments, the cells are immunomodulatory cells.
In further embodiments, the immunomodulatory cells are selected
from mesenchymal stem cells (MSCs) or macrophages, or a combination
of both. In some embodiments, the mesenchymal stem cells are bone
and/or adipose derived. In some embodiments, the macrophages are
sourced from tissue and/or blood). In some embodiments, the
macrophages are M2-activated macrophages. In some embodiments, the
M2-activated macrophages, isolated from spleen, blood, and/or other
tissue locations, are added to a bio-ink admixture. In some
embodiments, neutral macrophages are activated in vitro prior to
being added to a bio-ink admixture. In some embodiments, neutral
macrophages are added to a bio-ink admixture that incorporates the
necessary cytokines for M2-activation of the macrophages. In some
embodiments, neutral macrophages are added to a bio-ink admixture
that contains cells that produce the necessary cytokines for
M2-activation of the macrophages. In some embodiments, mixtures of
macrophages and MSCs are combined in bio-ink admixtures of cells.
In some embodiments, parenchymal support cells such as, but not
limited to, fibroblasts, are added to the bio-ink admixtures to
enhance the structural properties of the bio-ink. As used herein,
"I-bio-ink" means a bio-ink admixture that contains some proportion
of immunomodulatory cells as described herein, in addition to the
possible, but not mandatory presence of non-immunomodulatory cells
and/or biomaterial support. In some embodiments, I-bio-ink
comprises (1) parenchymal cells; and (2) MSCs, macrophages, or a
combination thereof. In some embodiments, I-bio-ink comprises (1)
parenchymal cells; (2) MSCs, macrophages, or a combination thereof;
and (3) biomaterial support, wherein the biomaterial support is one
or more extrusion compounds as defined herein. In some embodiments,
the biomaterial support is a hydrogel.
Cell Culture Media
[0218] In some embodiments, the bio-ink comprises a cell culture
medium. The cell culture medium is any suitable medium. In various
embodiments, suitable cell culture media include, by way of
non-limiting examples, Dulbecco's Phosphate Buffered Saline,
Earle's Balanced Salts, Hanks' Balanced Salts, Tyrode's Salts,
Alsever's Solution, Gey's Balanced Salt Solution, Kreb's-Henseleit
Buffer Modified, Kreb's-Ringer Bicarbonate Buffer, Puck's Saline,
Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Eagle's
Medium/Nutrient F-12 Ham, Nutrient Mixture F-10 Ham (Ham's F-10),
Medium 199, Minimum Essential Medium Eagle, RPMI-1640 Medium, Ames'
Media, BGJb Medium (Fitton-Jackson Modification), Click's Medium,
CMRL-1066 Medium, Fischer's Medium, Glascow Minimum Essential
Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15
Medium (Leibovitz), McCoy's 5A Modified Medium, NCTC Medium, Swim's
S-77 Medium, Waymouth Medium, William's Medium E, or combinations
thereof. In some embodiments, the cell culture medium is modified
or supplemented. In some embodiments, the cell culture medium
further comprises albumin, selenium, transferrins, fetuins, sugars,
amino acids, vitamins, growth factors, cytokines, hormones,
antibiotics, lipids, lipid carriers, cyclodextrins, or a
combination thereof.
Extracellular Matrix
[0219] In some embodiments, the bio-ink further comprises one or
more components of an extracellular matrix or derivatives thereof.
In some embodiments, "extracellular matrix" includes proteins that
are produced by cells and transported out of the cells into the
extracellular space, where they may serve as a support to hold
tissues together, to provide tensile strength, and/or to facilitate
cell signaling. Examples, of extracellular matrix components
include, but are not limited to, collagen, fibronectin, laminin,
hyaluronates, elastin, and proteoglycans. For example,
multicellular aggregates optionally contain various ECM proteins
(e.g., gelatin, fibrinogen, fibrin, collagen, fibronectin, laminin,
elastin, and/or proteoglycans). The ECM components or derivatives
of ECM components can be added to the cell paste used to form the
multicellular aggregate. The ECM components or derivatives of ECM
components added to the cell paste can be purified from a human or
animal source, or produced by recombinant methods known in the art.
Alternatively, the ECM components or derivatives of ECM components
can be naturally secreted by the cells in the elongate cellular
body, or the cells used to make the elongate cellular body can be
genetically manipulated by any suitable method known in the art to
vary the expression level of one or more ECM components or
derivatives of ECM components and/or one or more cell adhesion
molecules or cell-substrate adhesion molecules (e.g., selectins,
integrins, immunoglobulins, and adherins). The ECM components or
derivatives of ECM components may promote cohesion of the cells in
the multicellular aggregates. For example, gelatin and/or
fibrinogen can suitably be added to the cell paste, which is used
to-form multicellular aggregates. The fibrinogen can then be
converted to fibrin by the addition of thrombin.
[0220] In some embodiments, the bio-ink further comprises an agent
that encourages cell adhesion.
[0221] In some embodiments, the bio-ink further comprises an agent
that inhibits cell death (e.g., necrosis, apoptosis, or
autophagocytosis). In some embodiments, the bio-ink further
comprises an anti-apoptotic agent. Agents that inhibit cell death
include, but are not limited to, small molecules, antibodies,
peptides, peptibodies, or combination thereof. In some embodiments,
the agent that inhibits cell death is selected from: anti-TNF
agents, agents that inhibit the activity of an interleukin, agents
that inhibit the activity of an interferon, agents that inhibit the
activity of an GCSF (granulocyte colony-stimulating factor), agents
that inhibit the activity of a macrophage inflammatory protein,
agents that inhibit the activity of TGF-B (transforming growth
factor B), agents that inhibit the activity of an MMP (matrix
metalloproteinase), agents that inhibit the activity of a caspase,
agents that inhibit the activity of the MAPK/JNK signaling cascade,
agents that inhibit the activity of a Src kinase, agents that
inhibit the activity of a JAK (Janus kinase), or a combination
thereof. In some embodiments, the bio-ink comprises an
anti-oxidant.
Extrusion Compounds
[0222] In some embodiments, the bio-ink further comprises an
extrusion compound (i.e., a compound that modifies the extrusion
properties of the bio-ink). Examples of extrusion compounds
include, but are not limited to gels, hydrogels (including UV
cross-linkable and thermoreversible hydrogels described herein),
surfactant polyols (e.g., Pluronic F-127 or PF-127),
thermo-responsive polymers, hyaluronates, alginates, extracellular
matrix components (and derivatives thereof), collagens, other
biocompatible natural or synthetic polymers, nanofibers, and
self-assembling nanofibers.
[0223] Gels, sometimes referred to as jellies, have been defined in
various ways. For example, the United States Pharmacopoeia defines
gels as semisolid systems consisting of either suspensions made up
of small inorganic particles or large organic molecules
interpenetrated by a liquid. Gels include a single-phase or a
two-phase system. A single-phase gel consists of organic
macromolecules distributed uniformly throughout a liquid in such a
manner that no apparent boundaries exist between the dispersed
macromolecules and the liquid. Some single-phase gels are prepared
from synthetic macromolecules (e.g., carbomer) or from natural gums
(e.g., tragacanth). In some embodiments, single-phase gels are
generally aqueous, but will also be made using alcohols and oils.
Two-phase gels consist of a network of small discrete
particles.
[0224] Gels can also be classified as being hydrophobic or
hydrophilic. In certain embodiments, the base of a hydrophobic gel
consists of a liquid paraffin with polyethylene or fatty oils
gelled with colloidal silica, or aluminum or zinc soaps. In
contrast, the base of hydrophobic gels usually consists of water,
glycerol, or propylene glycol gelled with a suitable gelling agent
(e.g., tragacanth, starch, cellulose derivatives,
carboxyvinylpolymers, and magnesium-aluminum silicates). In certain
embodiments, the rheology of the compositions or devices disclosed
herein is pseudo plastic, plastic, thixotropic, or dilatant.
[0225] Suitable hydrogels include those derived from collagen,
hyaluronate, fibrin, alginate, agarose, chitosan, and combinations
thereof. In other embodiments, suitable hydrogels are synthetic
polymers. In further embodiments, suitable hydrogels include those
derived from poIy(acrylic acid) and derivatives thereof,
poly(ethylene oxide) and copolymers thereof, poly(vinyl alcohol),
polyphosphazene, and combinations thereof. In various specific
embodiments, the support material is selected from: hydrogel,
NovoGel.TM., agarose, alginate, gelatin, Matrigel.TM., hyaluronan,
poloxamer, peptide hydrogel, poly(isopropyl n-polyacrylamide),
polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate,
polydimethylsiloxane, polyacrylamide, poly(lactic acid), silicon,
silk, or combinations thereof.
UV Cross-Linkable Hydrogels
[0226] Suitable hydrogels include methacrylated hydrogels, such as
Polyethylene (glycol) diacrylate-based (PEG-DA) hydrogels, which
are used in cell biology due to their ability to cross-link in
presence of UV light and due to their inertness to cells, PEG-DA is
commonly used as scaffold in tissue engineering since
polymerization occurs rapidly at room temperature and requires low
energy input, has high water content, is elastic, and can be
customized to include a variety of biological molecules.
Photoinitiators
[0227] In some embodiments, an extrusion compound comprises a
photoinitiator, which is a molecule that upon absorption of light
at a specific wavelength produces reactive species capable of
catalyzing polymerization or polycondensation reactions. These
reactions area also called photopolymerization or radiation curing.
Photoinitiators are typically ketones which contain both aromatic
and carbonyl groups.
[0228] There are two types of photoinitiators: cationic and radical
photoinitiators. Radical photoinitiators are water-compatible and
act on molecules containing an acrylate or styrene group. The range
of wavelengths used is typically near UV (300 nm-400 nm) but recent
progress in initiator chemistry is expanding the ranges of
wavelengths that can be used. Photoinitiators used in biology such
as Irgacure 2959, 184, and 651 fall into this class.
[0229] A suitable photoinitiator for use with the bio-inks and/or
support materials described herein is Irgacure 2959
(4-(2-hydroxyethoxy) phenyl-(2-propyl) ketone (Glycosan BioSystems,
Inc.; Salt Lake City, Utah) due to its high solubility in water and
its minimal toxicity compared to other Irgacure species. Upon
absorption of UV light, Irgacure 2959 dissociates into 2 primary
radicals which then react with the vinyl (C.dbd.C) groups of PEG-DA
to initiate polymerization. There are three phases in
photopolymerization: photoinitiation, propagation, and termination.
Rate of reaction during the first step is dependent on the nature
of the photoinitiator (quantum yield, photoinitiator efficiency)
and intensity of light while the later steps (propagation and
termination) are a function of vinyl bond concentration and the
rate constants for propagation and termination.
Thermoreversible Gels
[0230] In some embodiments, hydrogel-based extrusion compounds are
thermoreversible gels (also known as thermo-responsive gels or
thermogels). In some embodiments, a suitable thermoreversible
hydrogel is not a liquid at room temperature. In specific
embodiments, the gelation temperature (Tgel) of a suitable hydrogel
is about 10.degree. C., about 15.degree. C., about 20.degree. C.,
about 25.degree. C., about 30.degree. C., about 35.degree. C., and
about 40.degree. C., including increments therein. In certain
embodiments, the Tgel of a suitable hydrogel is about 10.degree. C.
to about 25.degree. C. In some embodiments, the bio-ink (e.g.,
comprising hydrogel, one or more cell types, and other additives,
etc.) described herein is not a liquid at room temperature. In
specific embodiments, the gelation temperature (Tgel) of a bio-ink
described herein is about 10.degree. C., about 15.degree. C., about
20.degree. C., about 25.degree. C., about 30.degree. C., about
35.degree. C., and about 40.degree. C., including increments
therein. In certain embodiments, the Tgel of a bio-ink described
herein is about 10.degree. C. to about 25.degree. C.
[0231] Polymers composed of polyoxypropylene and polyoxyethylene
form thermoreversible gels when incorporated into aqueous
solutions. These polymers have the ability to change from the
liquid state to the gel state at temperatures that can be
maintained in a bioprinter apparatus. The liquid state-to-gel state
phase transition is dependent on the polymer concentration and the
ingredients in the solution.
[0232] Poloxamer 407 (Pluronic F-127 or PF-127) is a nonionic
surfactant composed of polyoxyethylene-polyoxypropylene copolymers.
Other poloxamers include 188 (F-68 grade), 237 (F-87 grade), 338
(F-108 grade). Aqueous solutions of poloxamers are stable in the
presence of acids, alkalis, and metal ions. PF-127 is a
commercially available polyoxyethylene-polyoxypropylene triblock
copolymer of general formula E106 P70 E106, with an average molar
mass of 13,000. The polymer can be further purified by suitable
methods that will enhance gelation properties of the polymer. It
contains approximately 70% ethylene oxide, which accounts for its
hydrophilicity. It is one of the series of poloxamer ABA block
copolymers. PF-127 has good solubilizing capacity, low toxicity and
is, therefore, considered a suitable extrusion compound.
[0233] In some embodiments, the viscosity of the hydrogels and
bio-inks presented herein is measured by any means described. For
example, in some embodiments, an LVDV-II+CP Cone Plate Viscometer
and a Cone Spindle CPE-40 is used to calculate the viscosity of the
hydrogels and bio-inks. In other embodiments, a Brookfield (spindle
and cup) viscometer is used to calculate the viscosity of the
hydrogels and bio-inks. In some embodiments, the viscosity ranges
referred to herein are measured at room temperature. In other
embodiments, the viscosity ranges referred to herein are measured
at body temperature (e.g., at the average body temperature of a
healthy human).
[0234] In further embodiments, the hydrogels and/or bio-inks are
characterized by having a viscosity of between about 500 and
1,000,000 centipoise, between about 750 and 1,000,000 centipoise;
between about 1000 and 1,000,000 centipoise; between about 1000 and
400,000 centipoise; between about 2000 and 100,000 centipoise;
between about 3000 and 50,000 centipoise; between about 4000 and
25,000 centipoise; between about 5000 and 20,000 centipoise; or
between about 6000 and 15,000 centipoise.
[0235] In some embodiments, the bio-ink comprises cells and
extrusion compounds suitable for continuous bioprinting. In
specific embodiments, the bio-ink has a viscosity of about 1500
mPas. A mixture of Pluronic F-127 and cellular material may be
suitable for continuous bioprinting. Such a bio-ink is optionally
prepared by dissolving Pluronic F-127 powder by continuous mixing
in cold (4.degree. C.) phosphate buffered saline (PBS) over 48
hours to 30% (w/v). Pluronic F-127 may also be dissolved in water.
Cells are cultivated and expanded using standard sterile cell
culture techniques. The cells may be pelleted at 200 g for example,
and re-suspended in the 30% Pluronic F-127 and aspirated into a
reservoir affixed to a bioprinter where it can be allowed to
solidify at a gelation temperature from about 10 to about
25.degree. C. Gelation of the bio-ink prior to bioprinting is
optional. The bio-ink, including bio-ink comprising Pluronic F-127
can be dispensed as a liquid.
[0236] In various embodiments, the concentration of Pluronic F-127
can be any value with suitable viscosity and/or cytotoxicity
properties. A suitable concentration of Pluronic F-127 may also be
able to support weight while retaining its shape when bioprinted.
In some embodiments, the concentration of Pluronic F-127 is about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, or about 50%. In some embodiments, the
concentration of Pluronic F-127 is between about 30% and about 40%,
or between about 30% and about 35%.
[0237] Referring to FIG. 6, in a particular embodiment, a
three-dimensional, pyramid-shaped construct is generated by
continuous deposition of PF-127 using a NovoGen MMX.TM. bioprinter
connected to a syringe with a 510 .mu.m needle.
[0238] Referring to FIG. 7, in a particular embodiment, a
three-dimensional, cube-shaped (left) and hollow cube-shaped
(right) constructs generated by continuous deposition of PF-127
using a NovoGen MMX.TM. bioprinter connected to a syringe with a
510 .mu.m needle.
[0239] In some embodiments, the non-cellular components of the
bio-ink (e.g., extrusion compounds, etc.) are removed prior to use.
In further embodiments, the non-cellular components are, for
example, hydrogels, surfactant polyols, thermo-responsive polymers,
hyaluronates, alginates, collagens, or other biocompatible natural
or synthetic polymers. In still further embodiments, the
non-cellular components are removed by physical, chemical, or
enzymatic means. In some embodiments, a proportion of the
non-cellular components remain associated with the cellular
components at the time of use.
[0240] In some embodiments, the cells are pre-treated to increase
cellular interaction. For example, cells are optionally incubated
inside a centrifuge tube after centrifugation in order to enhance
cell-cell interactions prior to shaping the bio-ink.
Support Material
[0241] Disclosed herein, in certain embodiments, are devices,
systems, and methods for fabricating tissues and organs. In some
embodiments, the devices comprise one or more printer heads for
receiving and holding at least one cartridge that optionally
contains support material. In some embodiments, the methods
comprise the use of support material. In further embodiments, the
tissues and organs fabricated by use of the devices, systems, and
methods described herein comprise support material at the time of
fabrication or thereafter.
[0242] In some embodiments, the support material is capable of
excluding cells growing or migrating into or adhering to it. In
some embodiments, the support material is permeable for nutrient
media.
[0243] In some embodiments, the viscosity of the support material
is changeable. In some embodiments, the viscosity of the support
material is changed by modifying the temperature of the support
material. In some embodiments, the viscosity of the support
material is changed by modifying the pressure of the support
material. In some embodiments, the viscosity of the support
material is changed by modifying the concentration of the support
material. In some embodiments, the viscosity of the support
material is changed by cross-linking (e.g., by use of a chemical
cross-linker), or photocrosslinking (e.g., using ultraviolet light
exposure).
[0244] In some embodiments, the permeability of the support
material is changeable. In some embodiments, the permeability of
the support material is modified by varying the temperature of the
support material or the temperature surrounding the support
material. In some embodiments, the permeability of the support
material is modified by contacting the support material with an
agent that modifies permeability.
[0245] In some embodiments, the compliance (i.e., elasticity or
hardness) of the support material is modified. In some embodiments,
the compliance of the support material is modified by varying the
temperature of the support material or the temperature surrounding
the support material. In some embodiments, the compliance of the
support material is modified by contacting the support material
with an agent that modifies compliance.
[0246] Many support materials are suitable for use in the methods
described herein. In some embodiments, hydrogels (including UV
cross-linkable and thermoreversible hydrogels described herein) are
exemplary support materials possessing one or more advantageous
properties including: non-adherent, biocompatible, extrudable,
bioprintable, non-cellular, and of suitable strength. In some
embodiments, suitable hydrogels are natural polymers. In one
embodiment, the confinement material is comprised of NovoGel.TM..
In further embodiments, suitable hydrogels include those derived
from surfactant polyols (e.g., Pluronic F-127), collagen,
hyaluronate, fibrin, alginate, agarose, chitosan, derivatives or
combinations thereof. In other embodiments, suitable hydrogels are
synthetic polymers. In further embodiments, suitable hydrogels
include those derived from poly(acrylic acid) and derivatives
thereof, poly(ethylene oxide) and copolymers thereof, poly(vinyl
alcohol), polyphosphazene, and combinations thereof, In various
specific embodiments, the confinement material is selected from:
hydrogel, NovoGel.TM., agarose, alginate, gelatin, Matrigel.TM.,
hyaluronan, poloxamer, peptide hydrogel, poly(isopropyl
n-polyacrylamide), polyethylene glycol diacrylate (PEG-DA),
hydroxyethyl methacrylate, polydimethylsiloxane, polyacrylamide,
poly(lactic acid), silicon, silk, or combinations thereof.
[0247] In some embodiments, the support material contains cells
prior to being present in the bioprinter. In some embodiments, the
support material is a hydrogel containing a suspension of cells. In
some embodiments, the support material is a hydrogel containing a
mixture of more than one cell type.
Exemplary Uses of Support Materials
[0248] In some embodiments, the support material is used as
building units for constructing a biological construct (e.g.,
cellular construct, tissue, organ, etc.). In further embodiments,
the support material unit is used to define and maintain the
domains void of cellular material (i.e., the intermediate cellular
units) of a desired construct. In some embodiments, the support
material is capable of assuming any shape or size.
[0249] For example, according to one embodiment, NovoGel.TM.
solution (originally in powder phase mixed with buffer and water)
may be heated to reduce its viscosity and taken up in a
micropipette with a desired dimension (or in a chamber of a desired
shape by negative displacement of a piston). The NovoGel.TM.
solution in the pipette (or the chamber) may be cooled to room
temperature, for example by forced air on the exterior of the
pipette (or the chamber) or plunging the micropipette into a
container with cold liquid, so that it can solidify into a gel with
the desired shape, i.e., a support material. The resulting support
material is optionally dispensed from the pipette or chamber during
the construction of a particular cellular construct, tissue, or
organ. See e.g., FIG. 5.
[0250] In some embodiments, the support material is used for
increasing the viability of the engineered tissue or organ after
bioprinting. In further embodiments, support material provides
direct contact between the tissue or organ and a nutrient medium
through a temporary or semi-permanent lattice of confinement
material (e.g., support material). In some embodiments, the tissue
is constrained in a porous or gapped material. Direct access of at
least some of the cells of the tissue or organ to nutrients
increases the viability of the tissue or organ.
[0251] In further embodiments, the methods disclosed herein
comprise additional and optional steps for increasing the viability
of an engineered tissue or organ including: 1) optionally
dispensing base layer of confinement material (e.g., support
material) prior to placing cohered multicellular aggregates; 2)
optionally dispensing a perimeter of confinement material; 3)
bioprinting cells of the tissue within a defined geometry; and 4)
dispensing elongate bodies (e.g., cylinders, ribbons, etc.) of
confinement material overlaying the nascent tissue in a pattern
that introduces gaps in the confinement material, such as a
lattice, mesh, or grid.
[0252] In some embodiments, the gaps overlaying pattern are
distributed uniformly or substantially uniformly around the surface
of the tissue or organ. In other embodiments, the gaps are
distributed non-uniformly, whereby the cells of the tissue or organ
are exposed to nutrients non-uniformly. In non-uniform embodiments,
the differential access to nutrients is optionally exploited to
influence one or more properties of the tissue or organ. For
instance, it may be desirable to have cells on one surface of a
bioprinted, cellular construct, tissue, or organ proliferate faster
than cells on another surface. In some embodiments, the exposure of
various parts of the tissue or organ to nutrients can be changed at
various times to influence the development of the tissue or organ
toward a desired endpoint.
[0253] In some embodiments, the confinement material is removed at
any suitable time, including but not limited to, immediately after
bioprinting (e.g., within 10 minutes), after bioprinting (e.g.,
after 10 minutes), before the cells are substantially cohered to
each other, after the cells are substantially cohered to each
other, before the cells produce an extracellular matrix, after the
cells produce an extracellular matrix, just prior to use, and the
like. In various embodiments, confinement material is removed by
any suitable method. For example, in some embodiments, the
confinement material is excised, pulled off the cells, digested, or
dissolved.
Methods and Systems for Calibrating the Position of a Bioprinter
Cartridge
[0254] Disclosed herein, in certain embodiments, are bioprinters
for fabricating tissues and organs. In some embodiments, a
cartridge attached to the bioprinter comprises a bio-ink and/or a
support material. In some embodiments, the bioprinter deposits the
bio-ink or support material in a specific pattern and at specific
positions in order to form a specific tissue construct. In some
embodiments, a cartridge comprising bio-ink is disposable. In some
embodiments, the cartridge is ejected from the bioprinter following
extrusion, dispensing, or deposition of the contents. In some
embodiments, a new cartridge is attached to the bioprinter.
[0255] In order to fabricate complex structures, the bioprinters
disclosed herein dispense bio-ink and/or support material from a
cartridge with a suitable repeatable accuracy. In various
embodiments, suitable repeatable accuracies include those of about
.+-.5, 10, 20, 30, 40, or 50 .mu.m on any axis. In some
embodiments, the bioprinters disclosed herein dispense bio-ink
and/or support material from a cartridge with a repeatable accuracy
of about .+-.20 .mu.m. However, in some embodiments, due to the
removal and insertion of cartridges, the position of the printer
head (and thus the cartridges) with respect to the tissue construct
varies. Thus, there is a need for a method of precisely calibrating
the position of the printer head, cartridge, and dispensing orifice
with respect to the printer stage, receiving surface, tissue, or
organ.
[0256] In some embodiments, the method of calibrating the position
of a printer head comprises use of at least one laser. In further
embodiments, the method of calibrating the position of a printer
head comprises use of a first and second laser. In still further
embodiments, the method of calibrating the position of a printer
head comprises use of a first and second laser and one or more
cameras. In some embodiments, the method of calibrating the
position of a printer head comprises at least one laser and at
least one camera. In some embodiments, the method of calibrating
the position of a printer head comprises at least camera.
[0257] In some embodiments, the method of calibrating the position
of a printer head comprises manual (e.g., visual) calibration.
[0258] In some embodiments, the method of calibrating the position
of a printer head comprises image-based calibration. In some
embodiments, the method of calibrating the position of a printer
head comprises one or more cameras.
[0259] In some embodiments, the method of calibrating the position
of a printer head comprises manual calibration and laser
calibration. In some embodiments, the method of calibrating the
position of a printer head comprises manual calibration,
image-based calibration, and laser calibration.
[0260] In some embodiments, the position of the printer head is
calibrated along one axis, wherein the axis is selected from the
x-axis, the y-axis, and the z-axis. In some embodiments, the
position of the printer head is calibrated along two axes, wherein
the axes are selected from the x-axis, the y-axis, and the z-axis.
In some embodiments, the position of the printer head is calibrated
along three axes, wherein the axes are selected from the x-axis,
the y-axis, and the z-axis.
[0261] In some embodiments, calibration is made by use of at least
one laser. In further embodiments, calibration is made by use of a
first and a second laser. In still further embodiments, calibration
is made by use of a first and a second laser and one or more
cameras. In some embodiments, calibration is made by use of at
least one camera.
Method for Calibrating Using a Horizontal Laser
[0262] Disclosed herein, in certain embodiments, are methods of
calibrating the position of a printer head comprising a dispensing
orifice. In some embodiments, a method disclosed herein further
comprises activating a laser and generating at least one
substantially stable and/or substantially stationary laser beam,
and where said laser beam is horizontal to the ground. See, e.g.,
FIG. 1.
[0263] In some embodiments, the methods comprise, calibrating the
position of a printer head along at least one axis, wherein the
axis is selected from the x-axis, y-axis, and z-axis. In some
embodiments, the methods comprise calibrating the position of the
printer head along at least two axes, wherein the axis is selected
from the x-axis, y-axis, and z-axis. In some embodiments, the
methods comprise calibrating the position of the printer head along
at least three axes, wherein the axis is selected from the x-axis,
y-axis, and z-axis. In some embodiments, the methods comprise (a)
calibrating the position of the printer head along the y-axis; (b)
calibrating the position of the printer head along the x-axis;
and/or (c) calibrating the position of the printer head along the
z-axis; wherein each axis corresponds to the axis of the same name
in the Cartesian coordinate system. In some embodiments,
calibration is made by use of at least one laser. In some
embodiments, calibration is made by use of a first and a second
laser. In some embodiments, calibration is made by the additional
use of at least one camera.
[0264] In some embodiments, calibrating the position of a printer
head along the y-axis comprises: (a) positioning the printer head
so that the printer head is (i) located in a first y octant and
(ii) the dispensing orifice is below the upper threshold of the
laser beam; (b) moving the printer head towards the laser beam and
stopping said movement as soon as the laser beam is interrupted by
the printer head, wherein the position at which the laser beam is
interrupted by the printer head is the first y position; (c)
re-positioning the printer head so that the printer head is located
in the second y octant and the dispensing orifice is below the
upper threshold of the laser beam; (d) moving the printer head
towards the laser beam and stopping said movement as soon as the
laser beam is interrupted by the printer head, wherein the position
at which the laser beam is interrupted is the second y position;
(e) and calculating the mid-point between the first y position and
the second y position.
[0265] In some embodiments, calibrating the position of a printer
head along the x-axis comprises: (a) positioning the printer head
(i) at the mid-point between the first y position and the second y
position, and (ii) outside the sensor threshold of the laser; and
(b) moving the printer head towards the sensor threshold and
stopping said movement as soon as the printer head contacts the
sensor threshold; wherein the position at which the printer head
contacts the sensor increased by half the printer head width is the
x position.
[0266] In some embodiments, calibrating the position of a printer
head along the y-axis comprises: (a) positioning the printer head
so that the laser beam can measure the precise location of one side
of the printer head; (b) positioning the printer head so that the
laser beam can measure the precise location of the opposing side of
the printer head; (c) and calculating the midpoint location of the
printer head to be relative to the laser location during each
measurement and the measured distances.
[0267] In some embodiments, calibrating the position of a printer
head along the x-axis comprises: (a) positioning the printer head
so that the laser beam can measure the precise location of one side
of the printer head; (b) positioning the printer head so that the
laser beam can measure the precise location of the opposing side of
the printer head; (c) and calculating the midpoint location of the
printer head to be relative to the laser location during each
measurement and the measured distances.
[0268] In some embodiments, calibrating the position of a printer
head along the z-axis comprises: (a) positioning the printer head
so that the dispensing orifice is located above the laser beam; and
(b) moving the printer head towards the laser beam and stopping
said movement as soon as the laser beam is interrupted by the
printer head, wherein the position at which the laser beam is
interrupted is the z position.
Method for Calibrating Using a Vertical Laser
[0269] Disclosed herein, in certain embodiments, are methods of
calibrating the position of a printer head comprising a dispensing
orifice. In some embodiments, a method disclosed herein further
comprises activating the laser and generating at least one
substantially stable and/or substantially stationary laser beam,
and where said laser beam is vertical to the ground. See, e.g.,
FIG. 2. In some embodiments, a method disclosed herein further
comprises the use of at least one camera.
[0270] In some embodiments, the methods comprise, calibrating the
position of a printer head along at least one axis, wherein the
axis is selected from the x-axis, y-axis, and z-axis. In some
embodiments, the methods comprise calibrating the position of a
printer head along at least two axes, wherein the axis is selected
from the x-axis, y-axis, and z-axis. In some embodiments, the
methods comprise calibrating the position of a printer head along
at least three axes, wherein the axis is selected from the x-axis,
y-axis, and z-axis.
[0271] In some embodiments, the methods comprise (a) calibrating
the position of the printer head along the y-axis; (b) calibrating
the position of the printer head along the x-axis; and (c)
calibrating the position of the printer head along the z-axis;
wherein each axis corresponds to the axis of the same name in the
Cartesian coordinate system.
[0272] In some embodiments, calibrating the position of a printer
head along the y-axis comprises: (a) positioning the printer head
so that the printer head is (i) located in a first y octant and
(ii) the dispensing orifice is outside the sensor threshold of the
laser; (b) moving the printer head towards the laser beam and
stopping said movement as soon as the laser beam is interrupted by
the printer head, wherein the position at which the laser beam is
interrupted by the printer head is the first y position; (c)
re-positioning the printer head so that the printer head is located
in the second y octant and the dispensing orifice is outside the
sensor threshold of the laser; (d) moving the printer head towards
the laser beam and stopping said movement as soon as the laser beam
is interrupted by the printer head, wherein the position at which
the laser beam is interrupted is the second y position; (e) and
calculating the mid-point between the first y position and the
second y position.
[0273] In some embodiments, calibrating the position of a printer
head along the x-axis comprises: (a) positioning the printer head
(i) at the mid-point between the first y position and the second y
position, and (ii) outside the sensor threshold of the laser; and
(b) moving the printer head towards the sensor threshold and
stopping said movement as soon as the printer head contacts the
sensor threshold; wherein the position at which the printer head
contacts the sensor increased by half the printer head width is the
x position.
[0274] In some embodiments, calibrating the position of a printer
head along the z-axis comprises: (a) positioning the printer head
so that the dispensing orifice is located above the laser beam so
that it is just outside of the laser sensor range threshold; and
(b) lowering the printer head until the sensor threshold is
reached, wherein the position at which the laser sensor threshold
is reached is the z position. In some embodiments, steps (a) and
(b) are repeated at multiple points of the printer head and
measured heights are averaged to determine the z position.
[0275] In some embodiments, calibrating the position of a printer
head along the z-axis comprises: (a) positioning the printer head
so that the laser beam can measure the precise location of one or
more points on the bottom of the printer head; (b) calculating the
absolute or average location of the printer head based on the laser
position and known measured distance.
Method for Calibrating Using a Vertical and Horizontal Laser
[0276] Disclosed herein, in certain embodiments, are methods of
calibrating the position of a printer head comprising a dispensing
orifice, wherein the printer head is attached to a bioprinter,
comprising calibrating the position of the printer head along at
least one axis, wherein the axis is selected from the x-axis,
y-axis, and z-axis. In some embodiments, the method comprises
calibrating the position of a printer head along at least two axes,
wherein the axis is selected from the x-axis, y-axis, and z-axis.
In some embodiments, the method comprises calibrating the position
of a printer head along at least three axes, wherein the axis is
selected from the x-axis, y-axis, and z-axis.
[0277] In some embodiments, the methods comprise (a) calibrating
the position of the printer head along the y-axis; (b) calibrating
the position of the printer head along the x-axis; and (c)
calibrating the position of the printer head along the z-axis;
wherein each axis corresponds to the axis of the same name in the
Cartesian coordinate system.
[0278] In some embodiments, calibration comprises use of a first
laser and a second laser. In some embodiments, the first laser is a
vertical laser and the second laser is a horizontal laser. In some
embodiments, calibration further comprises use of at least one
camera.
System for Calibrating Using a Laser
[0279] Disclosed herein, in certain embodiments, are systems for
calibrating the position of a cartridge comprising a deposition
orifice, wherein the cartridge is attached to a bioprinter, said
system comprising: a means for calibrating the position of the
cartridge along at least one axis, wherein the axis is selected
from the y-axis, x-axis, and z-axis.
[0280] Also disclosed herein, in certain embodiments, are systems
for calibrating the position of a printer head comprising a
dispensing orifice, wherein the printer head is attached to a
bioprinter, said system comprising: a means for calibrating the
position of the printer head along an x-axis; a means for
calibrating the position of the printer head along a y-axis; and a
means for calibrating the position of the printer head along a
z-axis.
[0281] In some embodiments, a system for calibrating the position
of a printer head comprises a means for calibrating the printer
head along the x-axis, y-axis, and z-axis. In some embodiments, the
means for calibrating a printer head along the x-axis, y-axis, and
z-axis is laser alignment, optical alignment, mechanical alignment,
piezoelectric alignment, magnetic alignment, electrical field or
capacitance alignment, ultrasound alignment, image-based alignment,
or a combination thereof.
[0282] In some embodiments, a system for calibrating the position
of a printer head comprises a means for calibrating the printer
head along the x-axis, y-axis, and z-axis. In some embodiments, the
means for calibrating a printer head along the x-axis, y-axis, and
z-axis is laser alignment. In some embodiments, the laser alignment
means comprises at least one laser. In some embodiments, the laser
alignment means comprises a plurality of lasers. In some
embodiments, the system for calibrating the position of a printer
head further comprises an additional means for calibrating the
printer head along the x-axis, y-axis, and z-axis. In some
embodiments, the means for calibrating the printer head along the
x-axis, y-axis, and z-axis is image-based alignment using at least
one camera.
[0283] In some embodiments, the laser alignment means it has any
suitable accuracy. In various embodiments, suitable accuracies
include those of about .+-.5, 10, 20, 30, 40, or 50 .mu.m on any
axis. In some embodiments, the laser alignment means is accurate to
.+-.40 .mu.m on the vertical axis and .+-.20 .mu.m on the
horizontal axis.
[0284] In some embodiments, the laser path is uninterrupted between
the laser source and the measurement point. In some embodiments,
the laser path is altered by up to 179.degree. by use of a
reflective surface or optical lens. In some embodiments, the laser
path is altered by 90.degree.. In some embodiments, a horizontal
laser beam is used to measure in a vertical path by deflection
using a reflective surface. In some embodiments, a vertical laser
beam is used to measure in a horizontal path by deflection using a
reflective surface.
Three-Dimensional Calibration System for Full Automation
[0285] Disclosed herein, in certain embodiments, are bioprinters
comprising the means to automatically determine the x, y, z
coordinates of the deposition orifice and the print target surface
before and during the bioprinting process. The automatic
three-dimensional determination of such coordinates both before and
during the bioprinting process ensures that biomaterials are
printed at the target surface at the proper positions and using the
optimum dispensing orifice to target surface separation distance.
The frequent replacement of deposition orifices and receiving
surfaces prior to a bioprinting job necessitates the accurate
determination of the positioning of these two elements before
initiation of bioprinting in order to control and maximize
bioprinting quality. Determination of the positions of the
deposition orifice and the printing target surface during the
bioprinting process enables dynamic mapping of the target surface.
Such positional determination during the bioprinting process is
important as the surface height may not be as uniform as required
due to product tolerances or shrinkage of the surface material and
the surface height is continuously changing as layer upon layer of
bio-ink is deposited onto the surface. Moreover, dynamic
measurement of the thickness of each track of deposited bio-ink is
optionally used as feedback to adjust bioprinting parameters such
as deposition rate, dispensing orifice/receiving surface relative
travel speed and print height (distance between the deposition
orifice and print target surface). The three-dimensional positional
determination optionally allows for printer deposition error
checking, plate positioning error checking, monitoring for
structural changes such as construct shrinkage during or after a
bioprinting job, automated well center determination for transwell
and microtiter plates, and automated quality control monitoring,
especially with the incorporation digital camera technology.
[0286] Disclosed herein, in certain embodiments, are
three-dimensional calibration systems for an automated bioprinter
comprising a sensor fixed to a receiving surface of the bioprinter
for determining the position of a deposition orifice of the
bioprinter; and a sensor fixed to a printer head of the bioprinter
for determining the position of a print target surface associated
with the receiving surface; whereby the system calculates a print
height, the print height comprising the distance between the
deposition orifice and a print target surface. In some embodiments,
a three-dimensional calibration system comprises at least two
lasers, a sensor fixed to the receiving surface for determining the
position of a deposition orifice; and a sensor fixed to the printer
head for determining the position of the receiving surface; whereby
the system calculates a print height, the print height comprising
the distance between the deposition orifice and receiving
surface.
[0287] In some embodiments, the sensors are selected from:
triangulation sensors, ultrasonic distance sensing probes, and
digital cameras.
[0288] In some embodiments, the calibration system has any suitable
accuracy. In various embodiments, suitable accuracies include those
of about .+-.5, 10, 20, 30, 40, or 50 .mu.m on any axis.
[0289] Referring to FIG. 4, in a particular embodiment, a
three-dimensional calibration system with triangulation sensors is
depicted. The left-hand side of FIG. 4 shows a printer head with
attached capillary held above the tip sensing triangulation sensor.
The relative position of the capillary tip (deposition orifice) and
the sensor beam is varied in order to produce a distance vs.
position data file from which both the capillary center X, Y
coordinate and the average tip height can be determined
automatically. The right-hand side of FIG. 4 shows the print head
at a later time with attached capillary held above the target print
surface. The relative position of the surface sensing triangulation
sensor beam and the target printing surface is varied in order to
produce a distance vs. position data file from which a surface map
of the print surface can be constructed. The information obtained
from these two sensors can be combined and used to automate the
bioprinting process, with the deposition of materials occurring
with the optimum separation between the deposition orifice and
target printing surface. This separation can be adjusted to correct
for surface height changes throughout the bioprinting process using
the information recorded as described above.
EXAMPLES
[0290] The following specific examples are to be construed as
merely illustrative, and not limitative of the remainder of the
disclosure in any way whatsoever. Without further elaboration, it
is believed that one skilled in the art can, based on the
description herein, utilize the present invention to its fullest
extent.
Example 1
HASMC-HAEC Mixed Cellular Cylinders
Cell Culture
[0291] Smooth muscle cells: Primary human aortic smooth muscle
cells (HASMC) were maintained and expanded in low glucose
Dulbecco's modified eagle medium (DMEM; Invitrogen Corp., Carlsbad,
Calif.) supplemented with 10% fetal bovine serum (FBS), 100 U/ml
Penicillin, 0.1 mg/ml streptomycin, 0.25 .mu.g/ml of amphotericin
B, 0.01M of HEPES (all from Invitrogen Corp., Carlsbad, Calif.), 50
mg/L of proline, 50 mg/L of glycine, 20 mg/L of alanine, 50 mg/L of
ascorbic acid, and 3 .mu.g/L of Q1SO4 (all from Sigma, St. Louis,
Mo.) at 37.degree. C. and 5% CO.sub.2. Confluent cultures of HASMCs
between passage 4 and 8 were used in all studies.
[0292] Endothelial cells: Primary human aortic endothelial cells
(HAEC) were maintained and expanded in Medium 200 supplemented with
2% FBS, 1 .mu.g/ml of hydrocortisone, 10 ng/ml of human epidermal
growth factor, 3 ng/ml of basic fibroblast growth factor, 10
.mu.g/ml of heparin, 100 U/ml Penicillin, 0.1 mg/ml streptomycin,
and 0.25 .mu.g/ml of amphotericin B (all from Invitrogen Corp.,
Carlsbad, Calif.). The cells were grown on gelatin (from porcine
serum; Sigma, St. Louis, Mo.) coated tissue culture treated flasks
at 37.degree. C. and 5% CO.sub.2. Confluent cultures of HAEC's
between passage 4 and 8 were used in all studies.
NovoGel.TM. Mold
[0293] Preparation of 2% w/v NovoGel.TM. solution: 1 g of low
melting point NovoGel.TM. was dissolved in 50 ml of Dulbecco's
phosphate buffered saline (DPBS). Briefly, the DPBS and NovoGel.TM.
were heated to 85.degree. C. on a hot plate with constant stirring
until the NovoGel.TM. dissolved completely. NovoGel.TM. solution
was sterilized by steam sterilization at 125.degree. C. for 25
minutes. The NovoGel.TM. solution remained in liquid phase as long
as the temperature is maintained above 66.5.degree. C. Below this
temperature a phase transition occurs, the viscosity of the
NovoGel.TM. solution increases and the NovoGel.TM. forms a solid
gel.
[0294] Preparation of NovoGel.TM. mold: A NovoGel.TM. mold was
fabricated for the incubation of cellular cylinders using a
Teflon.RTM. mold that fits a 10 cm Petri dish. Briefly, the
Teflon.RTM. mold was pre-sterilized using 70% ethanol solution and
subjecting the mold to UV light for 45 minutes. The sterilized mold
was placed on top of the 10 cm Petri dish (VWR International LLC,
West Chester, Pa.) and securely attached. This assembly
(Teflon.RTM. mold+Petri dish) was maintained vertically and 45 ml
of pre-warmed, sterile 2% NovoGel.TM. solution was poured in the
space between the Teflon.RTM. mold and the Petri dish. The assembly
was then placed horizontally at room temperature for 1 hour to
allow complete gelation of the NovoGel.TM.. After gelation, the
Teflon.RTM. print was removed and the NovoGel.TM. mold was washed
twice using DPBS. 17.5 ml of HASMC culture medium was then added to
the NovoGel.TM. mold.
HASMC-HAEC Cylinders
[0295] Fabrication of HASMC-HAEC mixed cellular cylinders: To
prepare mixed cellular cylinders HASMC and HAEC were individually
collected and then mixed at pre-determined ratios. Briefly, the
culture medium was removed from confluent culture flasks and the
cells were washed with DPBS (1 ml/5 cm.sup.2 of growth area). Cells
were detached from the surface of the culture flasks by incubation
in the presence of trypsin (1 ml/15 cm.sup.2 of growth area) for 10
minutes. HASMC were detached using 0.15% trypsin while HAEC were
detached using 0.1 % trypsin. Following the incubation appropriate
culture medium was added to the flasks (2.times. volume with
respect to trypsin volume). The cell suspension was centrifuged at
200 g for 6 minutes followed by complete removal of supernatant
solution. Cell pellets were resuspended in respective culture
medium and counted using a hemacytometer. Appropriate volumes of
HASMC and HAEC were combined to yield mixed cell suspensions
containing 5, 7.5, 10, 12.5, and 15% HAEC (as a % of total cell
population). The mixed cell suspensions were centrifuged at 200 g
for 5 minutes followed by complete removal of supernatant solution.
Mixed cell pellets were resuspended in 6 ml of HASMC culture medium
and transferred to 20 ml glass vials, followed by incubation on an
orbital shaker at 150 rpm for 60 minutes, and at 37.degree. C. and
5% CO.sub.2. This allows the cells to aggregate with one another
and initiate cell-cell adhesions. Post-incubation, the cell
suspension was transferred to a 15 ml centrifuge tube and
centrifuged at 200 g for 5 minutes. After removal of the
supernatant medium, the cell pellet was resuspended in 400 .mu.l of
HASMC culture medium and pipetted up and down several times to
ensure all cell clusters were broken. The cell suspension was
transferred into a 0.5 ml microfuge tube placed inside a 15 ml
centrifuge tube followed by centrifugation at 2000 g for 4 minutes
to form a highly dense and compact cell pellet. The supernatant
medium was aspirated and the cells were transferred into capillary
tubes (OD 1.0 mm, ID 0.5 mm, L 75 mm; Drummond Scientific Co.,
Broomall, Pa.) by aspiration so as to yield cell cylinders 50 mm in
length. The cell paste inside the capillaries was incubated in
HASMC medium for 20 minutes at 37.degree. C. and 5% CO.sub.2. The
cellular cylinders were then deposited from the capillary tubes
into the grooves of the NovoGel.TM. mold (covered with HASMC
medium) using the plunger supplied with the capillaries. The
cellular cylinders were incubated for 24 and 48 hours at 37.degree.
C. and 5% CO.sub.2.
Example 2
Multi-Layered Vascular Tubes
Cell Culture
[0296] Smooth muscle cells: Primary human aortic smooth muscle
cells (HASMC; GIBCO) were maintained and expanded in low glucose
Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 100 U/ml Penicillin, 0.1 mg/ml streptomycin,
0.25 .mu.g/ml of amphotericin B, 0.01M of HEPES (all from
Invitrogen Corp., Carlsbad, Calif.), 50 mg/L of proline, 50 mg/L of
glycine, 20 mg/L of alanine, 50 mg/L of ascorbic acid, and 3
.mu.g/L of CuSO.sub.4 (all from Sigma, St. Louis, Mo.) at
37.degree. C. and 5% CO.sub.2. Confluent cultures of HASMC between
passage 4 and 8 were used in all studies.
[0297] Endothelial cells: Primary human aortic endothelial cells
(HAEC) were maintained and expanded in Medium 200 supplemented with
2% FBS, 1 .mu.g/ml of hydrocortisone, 10 ng/ml of human epidermal
growth factor, 3 ng/ml of basic fibroblast growth factor, 10
.mu.g/ml of heparin, 100 U/ml Penicillin, 0.1 mg/ml streptomycin,
and 0.25 .mu.g/ml of amphotericin B (all from Invitrogen Corp.,
Carlsbad, Calif.). The cells were grown on gelatin (from porcine
serum) coated tissue culture treated flasks at 37.degree. C. and 5%
CO.sub.2. Confluent cultures of HAEC between passage 4 and 8 were
used in all studies.
[0298] Fibroblasts: Primary human dermal fibroblasts (HDF) were
maintained and expanded in Medium 106 supplemented with 2% FBS, 1
.mu.g/ml of hydrocortisone, 10 ng/ml of human epidermal growth
factor, 3 ng/ml of basic fibroblast growth factor, 10 .mu.g/ml of
heparin, 100 U/ml Penicillin, and 0.1 mg/ml streptomycin (all from
Invitrogen Corp., Carlsbad, Calif.) at 37.degree. C. and 5%
CO.sub.2. Confluent cultures of HDF between passage 4 and 8 were
used in all studies.
NovoGel.TM. Solutions and Mold
[0299] Preparation of 2% and 4% (w/v) NovoGel.TM. solution: 1 g or
2 g (for 2% or 4% respectively) of low melting point NovoGel.TM.
(Ultrapure LMP) was dissolved in 50 ml of Dulbecco's phosphate
buffered saline (DPBS). Briefly, the DPBS and NovoGel.TM. were
heated to 85.degree. C. on a hot plate with constant stirring until
the NovoGel.TM. dissolves completely. NovoGel.TM. solution was
sterilized by steam sterilization at 125.degree. C. for 25 minutes.
The NovoGel.TM. solution remains in liquid phase as long as the
temperature is maintained above 66.5.degree. C. Below this
temperature a phase transition occurs, the viscosity of the
NovoGel.TM. solution increases and the NovoGel.TM. forms a solid
gel.
[0300] Preparation of NovoGel.TM. mold: A NovoGel.TM. mold was
fabricated for the incubation of cellular cylinders using a
Teflon.RTM. mold that fit a 10 cm Petri dish. Briefly, the
Teflon.RTM. mold was pre-sterilized using 70% ethanol solution and
subjecting the mold to UV light for 45 minutes. The sterilized mold
was placed on top of the 10 cm Petri dish and securely attached.
This assembly (Teflon.RTM. mold+Petri dish) was maintained
vertically and 45 ml of pre-warmed, sterile 2% NovoGel.TM. solution
was poured in the space between the Teflon.RTM. mold and the Petri
dish. The assembly was then placed horizontally at room temperature
for 1 hour to allow complete gelation of the NovoGel.TM.. After
gelation, the Teflon.RTM. print was removed and the NovoGel.TM.
mold was washed twice using DPBS. Then, either 17.5 ml of HASMC
culture medium was added to the NovoGel.TM. mold for incubating
HASMC-HAEC mixed cell cylinders or 17.5 ml of HDF culture medium is
added to the NovoGel.TM. mold for incubating HDF cell
cylinders.
Cellular Cylinders
[0301] Fabrication of HASMC-HAEC mixed cellular cylinders: To
prepare mixed cellular cylinders HASMC and HAEC were individually
collected and then mixed at pre-determined ratios. Briefly, the
culture medium was removed from confluent culture flasks and the
cells were washed with DPBS (1 ml/5 cm.sup.2 of growth area). Cells
were detached from the surface of the culture flasks, by incubation
in the presence of trypsin (1 ml/15 cm.sup.2 of growth area) for 10
minutes. HASMC were detached using 0.15% trypsin while HAEC were
detached using 0.1% trypsin. Following the incubation appropriate
culture medium was added to the flasks (2.times. volume with
respect to trypsin volume). The cell suspension was centrifuged at
200 g for 6 minutes followed by complete removal of supernatant
solution. Cell pellets were resuspended in respective culture
medium and counted using a hemacytometer. Appropriate volumes of
HASMC and HAEC were combined to yield a mixed cell suspension
containing 15% HAEC and remainder 85% HASMC (as a percentage of
total cell population). The mixed cell suspension was centrifuged
at 200 g for 5 minutes followed by complete removal of supernatant
solution. Mixed cell pellets were resuspended in 6 ml of HASMC
culture medium and transferred to 20 ml glass vials, followed by
incubation on an orbital shaker at 150 rpm for 60 minutes, and at
37.degree. C. and 5% CO.sub.2. This allows the cells to aggregate
with one another and initiate cell-cell adhesions. Post-incubation,
the cell suspension was transferred to a 15 ml centrifuge tube and
centrifuged at 200 g for 5 mins. After removal of the supernatant
medium, the cell pellet was resuspended in 400 .mu.l of HASMC
culture medium and pipetted up and down several times to ensure all
cell clusters were broken. The cell suspension was transferred into
a 0.5 ml microfuge tube placed inside a 15 ml centrifuge tube
followed by centrifugation at 2000 g for 4 minutes to form a highly
dense and compact cell pellet. The supernatant medium was aspirated
and the cells were transferred into capillary tubes (OD 1.0 mm, ID
0.5 mm, L 75 mm) by aspiration so as to yield cell cylinders 50 mm
in length. The cell paste inside the capillaries was incubated in
HASMC medium for 20 minutes at 37.degree. C. and 5% CO.sub.2. The
cellular cylinders were then deposited from the capillary tubes
into the grooves of the NovoGel.TM. mold (covered with HASMC
medium) using the plunger supplied with the capillaries. The
cellular cylinders were incubated for 24 hours at 37.degree. C. and
5% CO.sub.2.
[0302] Fabrication of HDF cell cylinders: HDF cylinders were
prepared using a method similar to preparing HASMC-HAEC mixed
cellular cylinders. Briefly, the culture medium was removed from
confluent HDF culture flasks and the cells were washed with DPBS (1
ml/5 cm.sup.2 of growth area). Cells were detached from the surface
of the culture flasks by incubation in the presence of trypsin
(0.1%; 1 ml/15 cm.sup.2 of growth area) for 10 minutes. Following
the incubation HDF culture medium was added to the flasks (2.times.
volume with respect to trypsin volume). The cell suspension was
centrifuged at 200 g for 6 minutes followed by complete removal of
supernatant solution. Cell pellets were resuspended in 6 ml of HDF
culture medium and transferred to 20 ml glass vials, followed by
incubation on an orbital shaker at 150 rpm for 75 minutes, and at
37.degree. C. and 5% CO.sub.2. Post-incubation, the cell suspension
was transferred to a 15 ml centrifuge tube and centrifuged at 200 g
for 5 minutes. After removal of the supernatant medium, the cell
pellet was resuspended in 400 .mu.l of HDF culture medium and
pipetted up and down several times to ensure all cell clusters were
broken. The cell suspension was transferred into a 0.5 ml microfuge
tube placed inside a 15 ml centrifuge tube followed by
centrifugation at 2000 g for 4 minutes to form a highly dense and
compact cell pellet. The supernatant medium was aspirated and the
cells were transferred into capillary tubes (OD 1.0 mm, ID 0.5 mm,
L 75 mm) by aspiration so as to yield cell cylinders 50 mm in
length. The cell paste inside the capillaries were incubated in HDF
culture medium for 20 minutes at 37.degree. C. and 5% CO.sub.2. The
cellular cylinders were then deposited from the capillary tubes
into the grooves of the NovoGel.TM. mold (covered with HDF medium).
The cellular cylinders were incubated for 24 hours at 37.degree. C.
and 5% CO.sub.2.
Fabrication of Multi-Layered Vascular Tubes
[0303] Preparation of NovoGel.TM. base plate: A NovoGel.TM. base
plate was fabricated by dispensing 10 ml of pre-warmed
(>40.degree. C.) NovoGel.TM. (2% w/v) into a 10 cm Petri dish.
Immediately after dispensing, the NovoGel.TM. was evenly spread so
as to cover the entire base of the dish and form a uniform layer.
The Petri dish was incubated at room temperature for 20 minutes to
allow the NovoGel.TM. to gel completely.
[0304] Multi-layered vascular tube: Vascular tubes consisting of an
outer layer of HDF and an inner layer of HASMC-HAEC were fabricated
utilizing HDF cylinders, and HASMC-HAEC mixed cell cylinders. A
geometrical arrangement as shown in FIG. 5 was utilized. Briefly,
at the end of the 24-hour incubation period mature HDF and
HASMC-HAEC cylinders were aspirated back into the capillary tubes
and placed in appropriate culture medium until further use. The
support structure consisting of NovoGel.TM. rods was prepared as
follows: Pre-warmed 2% NovoGel.TM. was aspirated into the capillary
tubes (L=50 mm) and rapidly cooled in cold PBS solution (4.degree.
C.). The 5 cm long gelled NovoGel.TM. cylinder was deposited from
the capillary (using the plunger) and laid down straight on the
NovoGel.TM. base plate. A second NovoGel.TM. cylinder was adjoined
to the first one and the process was repeated until 10 NovoGel.TM.
cylinders were deposited to form the first layer. At this point 20
.mu.l of PBS was dispensed above the NovoGel.TM. cylinders to keep
them wet. Further six NovoGel.TM. cylinders were deposited on top
of layer 1 at positions as shown in FIG. 5 (layer 2). Three HDF
cylinders were then deposited at positions 4, 5, and 6 to complete
layer 2. After dispensing each HDF cylinder 40 .mu.l of HDF culture
medium was dispensed on top of the deposited cylinder to assist the
deposition of the subsequent cylinder as well as to prevent
dehydration of the cellular cylinders. Next NovoGel.TM. cylinders
for layer 3 were deposited followed by HDF cylinders at positions 3
and 6. Following rewetting of the structure with HDF culture
medium, HASMC-HAEC mixed cylinders were laid down in positions 4
and 5. Subsequently, 40 .mu.l of HASMC medium and 40 .mu.l of HDF
medium were dispensed on top of the cell cylinders. Layer 4 was
completed by depositing NovoGel.TM. cylinders at positions 1 and 7,
HDF cylinders at positions 2 and 6, HASMC-HAEC mixed cylinders at
positions 3 and 5, and finally a 4% NovoGel.TM. cylinder at
position 4. Layers 5, 6, and 7 were completed similarly by laying
down NovoGel.TM. cylinders followed by HDF cylinders and finally
HASMC-HAEC cylinders at positions shown in FIG. 5. Once the entire
construct was completed 0.5 ml of warm NovoGel.TM. was dispensed
over each end of the construct and allowed to gel at room
temperature for 5 minutes. Following gelation of that NovoGel.TM.,
30 ml of HASMC medium was added to the Petri dish (to ensure the
entire construct was completely submerged). The construct was
incubated for 24 hours at 37.degree. C. and 5% CO.sub.2 to allow
for fusion between the cellular cylinders.
[0305] At the end of 24 hours, the surrounding NovoGel.TM. support
structure was removed from the fused multi-layered vascular
tube.
Example 3
Bioprinter
[0306] A bioprinter was assembled. The bioprinter contained a
printer head having a collet chuck grip for holding a cartridge,
and a piston for dispensing the contents of the cartridge. The
cartridges used were glass microcapillary tubes having a length of
75-85 mm. A new capillary tube was loaded each time bio-ink or
support material was required.
[0307] In order to print structures, a dispense position
repeatability of .+-.20 .mu.m was required for the duration of the
printing process, i.e., when new capillaries were loaded into the
printer head. In order to maintain repeatability of all loaded
capillary tubes relative to the same point in the x-, y-, and
z-directions, the bioprinter contained a laser calibration system
for calibrating the position of the microcapillary tube. The laser
calibration system calibrated the position of all capillary tips to
a common reference location. All printing moves were made relative
to this reference position.
[0308] All three axes (x-, y-, and z-axes) were calibrated through
usage of a single laser distance measurement sensor. The system
consisted of a laser sensor and a laser beam. The sensor threshold
was the maximum sensing distance of the laser sensor. The sensor
was configured to ignore all signals further away than a
pre-defined threshold. The sensor used triangulation to determine
distance to the object (the capillary tip). The laser sensor was
orientated with the beam aimed vertically up (+z-axis).
Vertical Laser Calibration
[0309] For calibration in the x-axis: The capillary tip was moved
in the range of the laser sensor, with the tip to the left (-x) of
the laser beam. The capillary was moved to in the +x direction
until the sensor detected the capillary edge, and this position was
recorded. The above steps were repeated from the opposite side
(i.e., the tip was positioned at the right (+x) of the laser beam
and moved in the -x direction until the sensor detected the
capillary edge). The positions from both steps were averaged to
calculate the mid-point of the capillary. Optionally, the above
process was repeated for different y-positions and the calculated
mid-points were averaged.
[0310] For calibration in the y-axis: The above procedure (for the
x-axis) was repeated for the y-axis.
[0311] For calibration in the z-axis: The capillary tip was moved
to above the sensor beam so that the bean hit the bottom surface of
the capillary, and the tip was just outside of the sensor range
threshold. The capillary was lowered until the sensor threshold was
reached, and that position was recorded as the z-position.
Optionally, the above steps were repeated at multiple points on the
capillary tip surface and measured heights were averaged.
Horizontal Laser Calibration
[0312] For calibration in the y-axis: The capillary was moved so
that the tip was just below the laser beam height, and the
capillary was off to one side (in the y-direction). The capillary
was moved in the y-direction towards the laser. The capillary was
stopped when the laser sensor detected the beam reflected off the
capillary, and this position was recorded. The above steps were
repeated with the capillary off to the other side of the laser, and
moved in the -y direction). The mid-point from the above steps was
recorded as the y-position.
[0313] For calibration in the x-axis: Using the results of the
calibration in the y-axis, the y-axis was moved so that the laser
was centered on the capillary. The capillary was moved past the
sensor threshold and moved towards the sensor. The capillary was
stopped as soon as the capillary crossed the sensor threshold and
the sensor output changed. This position, plus Vi the capillary
width (from the y-calibration) was recorded as the x-position.
[0314] For calibration in the z-axis: The capillary was moved up
from the x-position until it was clear of the laser beam. The
capillary tip was moved down towards the laser beam, and stopped as
soon as the laser beam was interrupted (using the same process as
for the y-axis). This position was recorded as the z-position.
Capillary Priming
[0315] Before printing from a capillary, the bio-ink or support
material inside the capillary was primed so that the bio-ink or
support material would begin printing at the very tip of the
capillary. The calibration laser was used to prime the capillary.
The capillary tip was moved just above the laser beam, with the
beam centered in the y-axis. The tip was between 20-100 .mu.m above
the laser beam. The dispensing piston in the printer head was
driven down until the bio-ink or support material started to
dispense out of the capillary tip and interrupted the laser beam.
The dispensed bio-ink or support material was aspirated back into
the capillary tube by driving the piston in the reverse direction
(20-100 .mu.m). The capillary was then primed and ready to
dispense.
NovoGel.RTM. Capillary Cleaning
[0316] NovoGel.RTM. was used as a support material. In order to
remove excess NovoGel.TM. sticking to the outside surface of the
capillary tube and to avoid the excess NovoGel.TM. from affecting
print quality, the excess NovoGel.RTM. was removed. A wiping
feature was integrated into a bulk NovoGel.RTM. vessel. A bulk
NovoGel.RTM. vessel was fitted with a standard medical vial with an
open cap for a septum to be attached. A septum was configured with
a cross cut in the center of 1-2 mm thick silicone. By dipping the
capillary into the bulk NovoGel.RTM. vessel through the septum and
aspirating NovoGel.RTM. excess NovoGel.RTM. was wiped from the
capillary as it exited the vessel, and remained in the bulk
vessel.
Printing of a Vascular Structure
[0317] The bioprinter and cartridge was assembled as above. The
bioprinter had a stage having a Petri dish for receiving structures
generated by the bioprinter. The Petri dish was coated with
NovoGel.TM..
[0318] A two dimensional representation (see e.g., FIG. 5) of a
vascular structure was inputted by a user into a software program
into a computer which was connected to the bioprinter. The two
dimensional representation of the vascular structure consisted of
rods of HASMC-HAEC mixed cellular cylinders, HDF cylinders, and
NovoGel.RTM. rods defining the voids of the vascular structure and
surrounding the vascular structure. HASMC-HAEC mixed cellular
cylinders and HDF cellular cylinders were prepared as in Example 1,
and aspirated into capillary tubes for insertion into the collet
chuck of the printer head. Alternatively, capillary tubes were
loaded into the printer head and dipped into the bulk NovoGel.RTM.
vessel and NovoGel.RTM. was aspirated into the capillary tube. The
capillary tubes were calibrated using the vertical laser
calibration system.
[0319] When the commands from the software program were provided to
the bioprinter, the bioprinter would print the three-dimensional
structure, alternating between HASMC-HAEC rods, HDF rods and
NovoGel.RTM. rods, onto the Petri dish, in predetermined locations.
See Example 2. After each rod was laid down on the Petri dish, the
rod was wetted with a small amount of culture medium. Once the
entire construct was completed warm NovoGel.RTM. was dispensed over
each end of the construct and allowed to gel at room temperature,
and cell culture medium was added to the Petri dish to submerge the
entire construct. The construct was then incubated at 37.degree. C.
and 5% CO.sub.2 to allow for fusion between the cellular cylinders.
At the end of the incubation time, the surrounding NovoGel.RTM.
support structure was removed from the fused multi-layered vascular
tube.
Example 4
Bioprinting of UV Cross-Linked PEG-DA
[0320] A solution of 10% (w/v) PEG-DA (Glycosan BioSystems, Inc.;
Salt Lake City, Utah) in water is mixed with a curing agent (0.1%
w/v PEGcure Photoinitiator) (Glycosan BioSystems, Inc.; Salt Lake
City, Utah). The resultant solution is kept away from light in a
vial chamber in a bioprinter. This solution is then aspirated using
a 300 .mu.m or 500 .mu.m sized glass capillary via extrusion method
and exposed to UV light (wavelength 365 nm, intensity 15,000-20,000
.mu.W/cm.sup.2) for at least 3 minutes to allow polymerization via
cross-linking to occur. The cross-linking converts the liquid
hydrogel into semi-solid structure. The semi-solid hydrogel is
bioprintable with or without cells and be used to create
geometrically complex structures such as sheets, sacs, tubes,
conduits, cylinders, tissues, organs, etc.
Cross-Linked PEG-DA with Cells
[0321] In various applications, PEG-DA is used to encapsulate
cells, proteins, or other biological material prior to being
extruded from the bioprinter. For applications where a UV
cross-linkable material (e.g., PEG-DA, etc.) including cells is
bioprinted, an attenuation filter is added to the UV module to
achieve reduced UV light intensities. In one application, the
PEG-DA is exposed to UV light at an intensity and/or exposure time
not optimal for full cross-linking. In a particular case, PEG-DA
including mammalian cells was exposed to UV light with an intensity
of 800 mW/cm.sup.2 and the UV source at a distance of 2 inches away
from the PEG-DA material. The degree of cross-linking is optionally
controlled by altering the composition of UV cross-linkable
material and photoinitiator. See Table 2.
TABLE-US-00002 TABLE 2 Composition Ratios UV Exposure Result 5%
PEGDA + 5% 1:100/1:50 6 minutes <50% cross-linked Irgacure 2959
5% PEGDA + 15% 1:100/1:50 6 minutes <75% but >50% Irgacure
2959 cross-linked 5% PEGDA + 5%/ 1:25 6 minutes 100% cross-linked
15% Irgacure
[0322] The bioprinter then prints successive layers of PEG-DA and
cells in a structure where the cells are partially or completely
encapsulated by the PEG-DA. Lastly, the entire structure is further
exposed to UV to promote full cross-linking of the PEG-DA
surrounding the cells. This procedure is optionally used to
encapsulate other types of biological materials either in liquid or
semi-solid state.
Cross-Linked PEG-DA without cells
[0323] In another application, PEG-DA is used as the basis of a
scaffold onto which cells and other material is bioprinted. A
pre-formed PEG-DA structure is created via mechanical molding with
exposure to UV light. For example, PEG-DA is dissolved in a 50 mM
Tris HCL buffer and cross-linked using 5% Irgacure 2959 at a 1:250
dilution ratio. Long-wave UV light (i.e. 365 nm) with an intensity
of 800 mW/cm.sup.2 is utilized to achieve complete cross-linking in
5 minutes of exposure. The preformed PEG-DA structure is placed in
the bioprinter and cells and other materials are printed onto the
structure. Once the cells are printed, they are allowed to fuse and
the PEG-DA scaffold is subsequently removed. This technique is
capable of producing cellular structures in the shape of organs
such as a bladder and other complex shapes.
Example 5
Bioprinting of UV Cross-Linked Methacrylated Hydrogel
[0324] Cross-linked methacrylated hydrogel is bioprinted from a
NovoGen MMX.TM. Bioprinter including a UV module using the
following parameters: [0325] Hydrogel Concentration: 5% [0326]
Irgacure 2959 Concentration: 0.5% [0327] UV Intensity: 1.50
W/cm.sup.2 [0328] UV Exposure Time: 15 sec [0329] Dispense Height:
0.5 mm (1.times.D using a 500 .mu.m capillary)
[0330] Optionally, a longer exposure time is used in combination
with a lower UV intensity. Because some methacrylated hydrogels
expand to a bioprintable length approximately 40% longer than the
aspiration amount, a variable pump speed dispense command is
configured to dispense a shorter length of material from the
dispense pump at a slower speed than the actual robot axes
movements. To print a 50 mm long piece of methacrylated hydrogel in
the +Y axis at 2 mm/sec. The following calculations are made to
determine the correct amount of material to aspirate and the
corresponding pump speed to result in a 50 mm line: [0331]
Calculate the amount of methacrylated hydrogel to aspirate: 50
mm/1.4=35.71 mm [0332] Calculate pump speed: 2 mm/sec/1.4=1.43
mm/sec [0333] The bioprinter software script commands are
configured as follows: [0334] Aspirates 35.71 mm of fluid and then
moves to the UV exposure chamber for 15 seconds. [0335] Moves the
gel capillary to the zero point at a dispense height of 0.5 mm.
[0336] Moves the robot 50 mm in the Y-axis at a speed of 2 mm/sec
and moves the gel dispense pump 35.71 mm at a speed of 1.43 mm/sec.
The gel pump and Y-axis should stop at the same time, resulting in
a 50 mm line.
Example 6
Coaxial Nozzle--Varying Flow Rates
[0337] Nozzle modification allows for spatial portioning of cell
types, materials, and/or mixtures of cells. This experiment
investigates the use of model compounds of a given viscosity
extruded through the nozzle at a variety of flow rates in order to
assess optimal condition for minimizing mantle-core mixing due to
volumetric outflow mismatch. Specifications for the nozzle were as
follows: inner diameter of nozzle for bio-ink flow of the core
layer: 150 .mu.m; inner diameter of nozzle for bio-ink flow of the
mantle layer: 500 .mu.m; outer diameter of the nozzle: 800 .mu.m
(see FIG. 29). The materials were initially a blue-tinted 30%
P-F127 and clear 30% P-F127. Later, because of rapid diffusional
normalization of the blue tint, an opaque viscous proteinaceous
material with viscosity similar to P-F127 was employed. Assessment
was qualitatively determined by the naked eye for proper
mantle/core formation. Results are shown below. Higher flow rates
induced flow instability and therefore should be considered the
upper limit of sustainable flow for the nozzle and bio-inks of this
experiment.
TABLE-US-00003 Core Flow Mantle Flow (mL/h) (mL/h) Qualitative
result 1 4 4 Core and mantle side by side, asymmetrical positioning
2 6 4 Coaxial structure, but mantle is too thin 3 8 4 Good
formation of mantle thickness, symmetrical 4 10 4 Instability
forms, oscillation in flows
[0338] A follow-up experiment was conducted with 1% alginate as the
mantle material, with model cells (NIH3T3) comprising the core
layer. Flow rates were determined from the initial experiment.
Expected bulk morphological results (H & E staining) are shown
in FIG. 30, indicating that the compact cell core remains intact
after extrusion.
[0339] Both sets of experiments demonstrate the feasibility of the
production of complex bilayer structures using a coaxial nozzle.
Variations in volumetric flow rates to the mantle and core impacted
the morphological outcome of the extruded components, but utilizing
a core flow that was approximately twice that of the mantle flow
generated a stable structure.
Example 7
Coaxial Nozzle--Printing of Vascular Vessels
[0340] The functionality of a coaxial nozzle (specifications for
the nozzle: inner diameter of nozzle for bio-ink flow of the core
layer: 514 .mu.m; inner diameter of nozzle for bio-ink flow of the
mantle layer: 819 .mu.m; outer diameter of the nozzle: 3200 .mu.m)
was tested on a non-cellular material-only system. An alginate
gelatin mixture was extruded through the outer nozzle around
Novogel-containing calcium chloride extruded through the center.
The presence of the calcium chloride in the center was sufficient
to crosslink the outer shell. The Novogel was flushed out with PBS
by manual use of a syringe and needle. Once verified in the
material-only system, a cell/alginate/gelatin mixture was extruded
to create a hollow tube (inner diameter 820 .mu.m, outer diameter
2300 .mu.m). The extruded cell/material tube was flushed to
establish patency, and then segmented into six 50 mm tubes. Tubes
were cultured both under static and flow conditions.
[0341] In a subsequent experiment, 50:50 normal human lung
fibroblasts (NHLFs): human pulmonary endothelial cells (HPAECs)
bio-ink was utilized in the printing of vascular vessels. It was
hypothesized that creation of a strong oxygen gradient (high
outside the vessel/low inside the vessel) would reverse the
undesirable effect seen previously where the HPAECs automatically
migrated to the abluminal surface of the vessel under standard
conditioning protocols. FC40, an oxygen earner, was utilized in
conjunction with aerated media in order to generate a high oxygen
abluminal compartment in the bioreactor. Two (6.times.1) vessels of
aforementioned composition were printed and conditioned under the
high abluminal oxygen conditions for 5 days. At day 5 the vessels
were fixed and given for histological analysis.
[0342] Resulting compartmentalization of HAPECs in response to the
high abluminal oxygen gradient is seen in FIG. 31. CD31 staining
clearly showed mass migration of HPAECs into the lumen of the
vessel, compared with previous results (not shown here) indicating
that in the absence of such a gradient, HPAEC preference is to
migrate to and to colonize the abluminal surface of the vessel.
[0343] These results suggest that the oxygen gradient method can be
utilized for this particular cell type (HPAECs) to force migration
of cells to the inner compartment, or (in using a reverse gradient)
to the outer compartment. The technique of driving cells to a
specific compartment can be utilized not only in tubular elements
or spherical elements, but in other geometric constructions.
Example 8
Coaxial Nozzle--Bioprinting with I-bio-ink
Experiment 1:
[0344] With a cellular admixture consisting of 70% normal human
lung fibroblasts (NHLFs): 20% bronchial smooth muscle cells
(BSMCs): 10% human adipose derived mesenchymal stem cells (ADSCs),
500-.mu.m diameter bio-printed vessels were generated and evaluated
qualitatively at 12 hours for cohesion, surface smoothness (a sign
of proper intra-bio-ink fusion), shortening (contraction), and
ability to form tubular structures through standard bio-printing
protocols (a sign of inter-bio-ink fusion). After assessment,
vessels were embedded in a bioactive hydrogel and perfused with
pulsatile flow. Following time on the bioreactor, the hydrogel with
embedded vessels was sent for histological examination which
demonstrated expected cellularity and morphology.
[0345] Qualitative assessment showed no discernible difference from
similar bio-ink admixtures that did not contain ADSCs. Surface
characterization of bio-ink was smooth and opaque. Bio-ink
cylinders, when handled, were resilient-indicative of good
cohesion. Contraction (shortening) was on the order of 50%, which
is consistent for bio-ink generated in cylindrical shapes. Three
bioprinted vessels (ID 500 .mu.m/OD 1500 .mu.m) were fused and
patent (i.e., open and non-occluded) at 12 hours. Two
representative vessels were conditioned in a hydrogel on a
bioreactor for 3 days. Histological assessment showed consistency
with previous non-MSC containing specimens. Example histology of
embedded tubular structure and surrounding hydrogel is shown in
FIG. 32.
Experiment 2:
[0346] With two cellular admixtures [50% normal human lung
fibroblasts (NHLFs): 40% human pulmonary artery endothelial cells
(HPAECs): 10% human adipose derived mesenchymal stem cells (ADSCs);
50% normal human lung fibroblasts (NHLFs): 45% human pulmonary
artery endothelial cells (HPAECs): 5% human adipose derived
mesenchymal stem cells (ADSCs)], 500-.mu.m diameter bio-printed
vessels were generated and evaluated qualitatively at 12 hours for
cohesion, surface smoothness (a sign of proper intra-bio-ink
fusion), shortening (contraction), and ability to form tubular
structures through standard bioprinting protocols (a sign of
inter-bio-ink fusion). Representative samples were perfused in a
bioreactor. Samples from these groups were submitted for histology
with staining for morphology (H&E), apoptosis (TUNEL),
proliferation (ki67), smooth muscle cell actin, endothelial marker
(CD31), fibroblast marker (TE7), and extracellular matrix
deposition (Trichrome).
[0347] Qualitative assessment showed no discernible difference from
similar bio-ink admixtures that did not contain ADSCs. Surface
characterization of bio-ink was smooth and opaque. Bio-ink
cylinders, when handled, were resilient--indicative of good
cohesion. Contraction (shortening) was on the order of 50% which is
consistent for bio-ink generated in cylindrical shapes. Lastly,
four bioprinted vessels (ID 500 .mu.m/OD 1500 .mu.m) were fused and
patent (i.e., open and non-occluded) at 12 hours. Two vessels that
were loaded into the bioreactor were from the 50% NHLF: 45% HPAEC:
5% ADSC group due to their exceptional quality. Conditioning with
pulsatile flow occurred for 9 days and vessels were histologically
analyzed as described above. Resulting histology showed
qualitatively no difference with control constructs generated from
bio-ink without immunomodulatory cells.
Experiment 3:
[0348] With a cellular admixture consisting of 75% human adipose
derived mesenchymal stem cells (ADSCs): 25% human artery
endothelial cells (HAECs), 500-.mu.m diameter bio-printed vessels
were generated and evaluated qualitatively at 12 hours for
cohesion, surface smoothness (a sign of proper intra-bio-ink
fusion) and shortening (contraction). This same cellular admixture
was also evaluated for ability to form patch structures (5
mm.times.5 mm) through standard bioprinting protocols (a sign of
inter-bio-ink fusion). Samples from these groups were submitted for
histology with staining for morphology (H&E) & apoptosis
(TUNEL).
[0349] Qualitative assessment showed no discernible difference from
similar bio-ink admixtures that did not contain ADSCs. Surface
characterization of bio-ink was smooth and opaque. Bio-ink
cylinders, when handled, were resilient--indicative of good
cohesion. Contraction (shortening) was on the order of 50% which is
consistent for bio-ink generated in cylindrical shapes. Lastly,
four bioprinted patches (5 mm.times.5 mm) were fused and patent
(i.e., open and non-occluded) at 12 hours. The patches were
submitted for histology after 2 days. An example patch is shown in
FIG. 33.
[0350] While the invention has been described in connection with
specific embodiments thereof, it will be understood that the
inventive methodology is capable of further modifications. This
patent application is intended to cover any variations, uses, or
adaptations of the invention following, in general, the principles
of the invention and including such departures from the present
disclosure as come within known or customary practice within the
art to which the invention pertains and as may be applied to the
essential features herein before set forth and as follows in scope
of the appended claims.
* * * * *