U.S. patent application number 17/013100 was filed with the patent office on 2021-02-04 for advanced tissue engineering system.
The applicant listed for this patent is OCTANE BIOTECH INC.. Invention is credited to Rupert HAGG, Yves LARCHER, Lowell MISENER, Guy ORAM, Sydney M. PUGH, Timothy J.N. SMITH, Roberto TOMMASINI.
Application Number | 20210032583 17/013100 |
Document ID | / |
Family ID | 1000005153969 |
Filed Date | 2021-02-04 |
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United States Patent
Application |
20210032583 |
Kind Code |
A1 |
SMITH; Timothy J.N. ; et
al. |
February 4, 2021 |
ADVANCED TISSUE ENGINEERING SYSTEM
Abstract
The invention is an automated advanced tissue engineering system
that comprises a housing in which one or more tissue engineering
modules are accommodated together with a central microprocessor
that controls functioning of the tissue engineering modules. In one
embodiment, the tissue engineering module comprises a housing
supporting one or more bioreactor chamber assemblies and a fluid
reservoir operationally engageable with the housing. The bioreactor
chamber assemblies may be selected depending on the end product
option desired and may include, for example, a cell therapy
bioreactor chamber, a single implant bioreactor chamber and a
multiple (mosaic) implant bioreactor chamber.
Inventors: |
SMITH; Timothy J.N.;
(Kingston, CA) ; PUGH; Sydney M.; (Glenburnie,
CA) ; MISENER; Lowell; (Kingston, CA) ; ORAM;
Guy; (Kingston, CA) ; HAGG; Rupert; (Kingston,
CA) ; TOMMASINI; Roberto; (Kingston, CA) ;
LARCHER; Yves; (Kingston, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OCTANE BIOTECH INC. |
Kingston |
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CA |
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Family ID: |
1000005153969 |
Appl. No.: |
17/013100 |
Filed: |
September 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15293611 |
Oct 14, 2016 |
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17013100 |
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11597550 |
Oct 11, 2007 |
9499780 |
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PCT/CA05/00783 |
May 26, 2005 |
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15293611 |
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60574223 |
May 26, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/04 20130101;
C12M 41/12 20130101; C12M 23/44 20130101; C12M 27/16 20130101; C12M
23/24 20130101; C12M 25/14 20130101; C12M 45/09 20130101; C12M
21/08 20130101; C12M 23/34 20130101 |
International
Class: |
C12M 3/00 20060101
C12M003/00; C12M 1/12 20060101 C12M001/12; C12M 1/00 20060101
C12M001/00; C12M 1/04 20060101 C12M001/04; C12M 3/06 20060101
C12M003/06; C12M 1/34 20060101 C12M001/34 |
Claims
1. A product chamber assembly, comprising: a bioreactor; a set of
flexible bags that are used to contain processing fluids and for
automated flow to and from the bioreactor, wherein at least one
flexible bag operates at reduced temperature from the bioreactor,
and wherein at least one flexible bag is operationally engaged with
a collection reservoir; and at least one biosensor for the
monitoring of parameters of the flexible bags and cells within the
bioreactor, the at least one biosensor relaying variable parameter
information to a microprocessor to process the parameters and
dynamically adjust and adapt to specific needs of cells during cell
proliferation.
2. The product chamber assembly of claim 1, wherein the set of
flexible bags and the bioreactor are connected via at least one of
a passageway, tubing, connector, valve, pump, filter, fluid access
port, in-line gas exchange membrane, and in-line sensor.
3. The product chamber assembly of claim 1, wherein the product
chamber assembly provides an environment for at least one of the
following: cell sorting, cell washing, cell concentrating, cell
seeding, cell proliferation, cell storage, and cell transport.
4. The product chamber assembly of claim 1, wherein the bioreactor
is a cell therapy bioreactor.
5. The product chamber assembly of claim 4 wherein the cell therapy
bioreactor collects and holds proliferated cells for cell therapy
applications.
6. The product chamber assembly of claim 1, wherein the at least
one biosensor has the ability to monitor the specific performance
of cells in the bioreactor and thereby, allow the system to
accommodate for the requirements of cells of an individual in the
product chamber assembly.
7. The product chamber assembly of claim 1, wherein the product
chamber assembly is portable.
8. The product chamber assembly of claim 1, wherein the bioreactor
is removable from the product chamber assembly.
9. The product chamber assembly of claim 1, wherein the bioreactor
further comprises at least one of a gas permeable membrane, flow
interrupters, and vibratory elements.
10. A product chamber assembly, comprising: a bioreactor, the
bioreactor having a bioreactor lid connected to a vertical chamber
and a bioreactor base, the bioreactor containing a conical cell
collector; a set of flexible bags that are used to contain
processing fluids and for automated flow to and from the
bioreactor, wherein at least one flexible bag is in fluid
communication with the bioreactor; and at least one biosensor for
monitoring parameters of the flexible bags and cells within the
bioreactor, the at least one biosensor relaying variable parameter
information to a microprocessor to process the parameters and
dynamically adjust and adapt to specific needs of the cells.
11. The product chamber assembly of claim 10, further comprising a
temperature controller to allow for reduced temperature
operation.
12. The product chamber assembly of claim 10, further comprising a
collection reservoir.
13. The product chamber assembly of claim 10, further comprising a
fluid pump.
14. The product chamber assembly of claim 10, further comprising
one or more fluid control valves.
15. A product chamber assembly comprising: a bioreactor; a set of
flexible bags that are used to contain processing fluids and for
automated flow to and from the bioreactor, wherein at least one
flexible bag is in fluid communication with the bioreactor; a flow
control valve housing comprising one or more control valves; and at
least one biosensor for monitoring parameters of the flexible bags
and cells within the bioreactor, the at least one biosensor
relaying variable parameter information to a microprocessor to
process the parameters and dynamically adjust and adapt to specific
needs of the cells.
16. The product chamber assembly of claim 15, wherein the
bioreactor is a cell therapy bioreactor.
17. The product chamber assembly of claim 15, further comprising a
collection reservoir.
18. The product chamber assembly of claim 15, further comprising a
fluid pump.
19. The product chamber assembly of claim 15, wherein the at least
one flexible bag is fluidly connected to the bioreactor via a
needless connection.
20. The product chamber assembly of claim 15, wherein the flow
control valve housing is a fluid pathway tube interconnect and
fluid direction interface between the bioreactor and the at least
one flexible bag.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 15/293,611, filed Oct. 14, 2016, which is a
divisional of U.S. application Ser. No. 11/597,550, filed Oct. 11,
2007, now U.S. Pat. No. 9,499,780, which is a U.S. National Phase
under 35 U.S.C. .sctn. 371 of PCT/CA05/00783, filed May 26, 2005,
which claims benefit of U.S. Provisional Patent Application No.
60/574,223, filed May 26, 2004, the disclosures of each of which
are incorporated by reference herein in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to a tissue engineering system. More
specifically, the invention relates to an autologous advanced
tissue engineering system for automated cell therapy and tissue
engineering for clinical hospital settings.
BACKGROUND OF THE INVENTION
[0003] Different types of cell culture and tissue engineering
devices have been developed as are described for example in U.S.
Pat. Nos. 5,688,687, 5,792,603, 5,846,828, 5,994,129, 6,060,306,
6,048,721, 6,121,042, 6,228,635 and 6,238,908. The major drawbacks
of these devices are the fact that they have limited functional
capabilities and are useful only for the culture and expansion of
cells. The devices are not designed for the production of
autologous tissue implants. Furthermore, these devices are complex
to use, bulky and thus not very portable, and still require user
intervention in many aspects of the cell culturing process.
[0004] The Applicant has developed a fully automated tissue
engineering system described in International Patent Application
No. WO 03/0872292 (the disclosure of which is incorporated herein
in its entirety). The system is a user-friendly and fully automated
system for facilitating different physiological cellular functions
and/or the generation of one or more tissue constructs from cell
and/or tissue sources.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention is an automated
advanced tissue engineering system designed for further simplicity
of use while maintaining aseptic conditions. The advanced tissue
engineering system comprises a single housing operated by a central
microprocessor unit that holds one or more tissue engineering
modules that can be independently operated. The tissue engineering
modules comprise a housing having one or more chamber assemblies
and, in aspects, comprise a tissue digestion chamber assembly, a
proliferation chamber assembly and a product chamber assembly. The
product chamber assembly is selected based on the desired end use
such as for cell collection for cell therapy or for various implant
formation. The housing is operationally engageable to a separate
fluid reservoir that, in one aspect, snaps onto the bottom of the
housing. The tissue engineering module may also have a variety of
biosensors to provide feedback with respect to the conditions
within any of the chamber assemblies provided on the module as well
as any fluids provided by the fluid reservoir and associated fluid
tubing. Integrity sensors for monitoring that all parts are present
and connected together correctly on the module may also be
incorporated.
[0006] The advanced tissue engineering system can bring turn-key
production capability for autologous cell therapy and tissue
engineering to a hospital clinic. The system can be designed for
ease-of-use while maintaining aseptic conditions. The system can
avoid the inherent hazards and cost related to the transportation
and centralized processing of human cells for various types of
tissue repair and the advanced system can also provide for
autologous, rather than allogenic or xenogenic sources of cells,
tissue or serum.
[0007] According to an aspect of the present invention is an
advanced automated tissue engineering system, the system
comprising:
[0008] a housing;
[0009] one or more tissue engineering modules supported within the
housing; and
[0010] a central microprocessor that controls functioning of the
one or more tissue engineering modules.
[0011] According to an aspect of the present invention is an
advanced automated tissue engineering system, the system
comprising:
[0012] a housing;
[0013] one or more tissue engineering modules supported within the
housing;
[0014] at least one biosensor associated with the housing and/or
the one or more tissue engineering modules; and
[0015] a central microprocessor that controls functioning of the
one or more tissue engineering modules.
[0016] According to another aspect of the present invention is a
network of automated tissue engineering systems, wherein at least
one of the automated tissue engineering systems is the automated
tissue engineering system of present invention.
[0017] In aspects of the invention, the central microprocessor unit
(CPU) of the system is used to programme and control the
functioning and operation of the entire system and the tissue
engineering module(s) contained therein. For example, the CPU is
used to release the tissue engineering module using an automatic
sequence triggered by a user command on the touch screen
display.
[0018] According to another aspect of the present invention is a
tissue engineering module comprising:
[0019] a housing supporting at least one chamber assembly, the at
least one chamber assembly selected for at least one of tissue
digestion, cell proliferation, cell differentiation and implant
formation;
[0020] a fluid reservoir operationally engageable with the housing;
and
[0021] at least one biosensor for the monitoring of parameters
within at least one of the fluid reservoir and the at least one
chamber assembly.
[0022] According to another aspect of the present invention is a
tissue engineering module, the module comprising:
[0023] a housing supporting a number of chamber assemblies selected
for tissue digestion, cell proliferation, cell differentiation
and/or implant formation;
[0024] a fluid reservoir operationally engageable with the housing;
and
[0025] at least one biosensor for the monitoring of parameters
within the chamber assemblies and/or within the fluid
reservoir.
[0026] According to another aspect of the present invention is a
tissue engineering module, the module comprising;
[0027] a housing supporting a tissue digestion chamber assembly, a
proliferation chamber assembly and a product chamber assembly;
[0028] a fluid reservoir connected to the housing and in fluid
communication with the tissue digestion chamber assembly, the
proliferation chamber assembly and the product chamber assembly;
and
[0029] at least one biosensor associated with one or more of the
fluid reservoir, the tissue digestion chamber assembly, the
proliferation chamber assembly and the product chamber assembly,
the at least one biosensor being in communication with a remote
central processor.
[0030] According to yet another aspect of the present invention is
a tissue engineering module, the module comprising;
[0031] a housing, the housing supporting a removable tissue
digestion chamber assembly, a fixed proliferation bioreactor and a
removable product chamber assembly;
[0032] a fluid reservoir connected to the housing and in fluid
communication with the tissue digestion chamber assembly, the
proliferation bioreactor and the product chamber assembly; and
[0033] at least one biosensor associated with one or more of the
fluid reservoir, the tissue digestion chamber assembly, the
proliferation bioreactor and the product chamber assembly, the at
least one biosensor being in communication with a remote central
processor.
[0034] In aspects, the tissue engineering module is capable of
conducting at least one of tissue digestion, cell proliferation,
cell differentiation and implant formation; individually,
sequentially, predetermined sequences or partial sequences.
[0035] In aspects, the product chamber assembly is configured
depending on the end product option desired and may be selected to
include for example a cell therapy bioreactor that collects and
holds proliferated cells for cell therapy applications; and a
differentiation bioreactor for the differentiation of cells into
either a single implant, multiple (mosaic) implant or a cell matrix
implant.
[0036] According to yet another aspect of the present invention is
a tissue digestion chamber assembly, the assembly comprising:
[0037] a protective containment unit comprising a unit lid and a
unit base; and
[0038] a tissue digestion bioreactor within the protective
containment unit.
[0039] According to still another aspect of the present invention
is a tissue digestion chamber assembly, the assembly
comprising:
[0040] a protective containment unit comprising a unit lid and unit
base; and
[0041] a tissue digestion bioreactor supported within the
protective containment unit.
[0042] In aspects, the tissue digestion chamber assembly is
portable and can be mounted within a tissue engineering module. The
tissue digestion chamber assembly is primarily for the digestion of
patient biopsy material to retrieve cells for further
proliferation, differentiation and/or implant formation. However,
the tissue digestion chamber can also be used in aspects to
directly receive patient cells without the need for any
digestion.
[0043] According to yet another aspect of the present invention is
a proliferation chamber assembly, the assembly comprising:
[0044] a proliferation bioreactor comprising a proliferation
chamber having a base, a lid for containment of fluid, and a
channel system therein.
[0045] According to still another aspect of the present invention
is a proliferation chamber assembly, the assembly comprising:
[0046] a proliferation bioreactor comprising a substantially flat
base with a large surface area, the base having a channel system
therein for flow of medium and cells; at least one biosensor to
detect and provide feedback on the condition of cell culture and
proliferative activity; and a lid to provide a chamber for
containment of fluid.
[0047] In aspects of the invention, the proliferation bioreactor is
mounted within a housing of a tissue engineering module. This
mounting may in aspects be fixed. The proliferation bioreactor may
also in aspects comprise a gas permeable membrane, flow
interrupters, and vibratory elements. In other aspects, the
proliferation bioreactor may be provided having one or more bases
stacked on top of one another to provide additional surface area
for the proliferation of cells. The base(s) may also be mounted
within the bioreactor at an angle to provide an elevational change
from inlet to outlet.
[0048] According to another aspect of the present invention is a
product chamber assembly, the assembly comprising:
[0049] a protective containment unit comprising a unit lid and a
unit base; and
[0050] a product bioreactor within the protective containment
unit.
[0051] According to yet another aspect of the present invention is
a product chamber assembly, the assembly comprising:
[0052] a protective containment unit comprising a unit lid and a
unit base; and
[0053] a product bioreactor supported within the protective
containment unit.
[0054] According to still another aspect of the present invention
is a product chamber assembly, the assembly comprising:
[0055] a protective containment unit comprising a unit lid and unit
base; and
[0056] a differentiation bioreactor supported within the protective
containment unit.
[0057] In aspects, the differentiation bioreactor is configured for
the collection of cells, for the generation of one or more
implants, or for the generation of cell matrix implant. In aspects
of the invention, the product chamber assembly can be reversibly
mounted to a tissue engineering module.
[0058] In other aspects of the invention, any one of the chamber
assemblies can be engageable with a tissue engineering module. This
can be done in a reversible manner or in an irreversible manner
with any of the assemblies as desired. For example, it may be
desirable in aspects to have the tissue digestion chamber assembly
non-removable after initial installation to ensure that the digest
bioreactor is not re-used. By default, this ensures that the
remainder of the tissue engineering module cannot be re-used.
[0059] It is noted that although the tissue engineering module is
shown to have a tissue digestion chamber assembly, a proliferation
chamber assembly and a product chamber assembly, not all of these
assemblies need to be used to create a final end product for
clinical use. As a non-limiting example, cells provided by
enzymatic digestion of a surgical biopsy in the tissue digestion
chamber assembly can be moved either to the proliferation chamber
assembly or directly to the product chamber assembly.
[0060] According to yet another aspect of the present invention is
a tissue engineering module comprising at least one of the tissue
digestion chamber assembly, the proliferation chamber assembly, and
the product chamber assembly of described herein.
[0061] According to another aspect of the present invention is a
tissue engineering module comprising:
[0062] a housing supporting a fluid reservoir and at least one of
the tissue digestion chamber assembly, the proliferation chamber
assembly, and the product chamber assembly of described herein;
and
[0063] at least one biosensor for the monitoring of parameters
within at least one of the fluid reservoir and said at least one
chamber assembly.
[0064] Other features and advantages of the present invention will
become apparent from the following detailed description and
drawings. It should be understood, however, that the detailed
description and drawing while indicating embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from the
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] Figure I shows a perspective view of an advanced tissue
engineering system of one embodiment of the present invention;
[0066] FIG. 2 shows the advanced tissue engineering system of
Figure I with one of the bay doors in the open position;
[0067] FIG. 3 shows a perspective view of one side of an embodiment
of a tissue engineering module of the present invention removed
from the bay door of FIG. 2;
[0068] FIG. 4 shows a perspective view of the other side of the
tissue engineering module of FIG. 3;
[0069] FIG. 5A shows a perspective view of one embodiment of a
tissue digestion chamber assembly of the present invention;
[0070] FIG. 5B shows a partial exploded view of the tissue
digestion bioreactor assembly of FIG. 5A showing a protective unit
lid, a tissue digestion bioreactor, and a unit base;
[0071] FIG. 5C shows a partial exploded view of the tissue
digestion bioreactor of FIG. 5B;
[0072] FIG. 6 shows a perspective view of an embodiment of a
proliferation chamber assembly of the present invention;
[0073] FIG. 7A shows a perspective view of one embodiment of a
product chamber assembly of the present invention;
[0074] FIG. 7B shows a partial exploded view of the product chamber
assembly of FIG. 7A showing a protective unit lid, a
differentiation bioreactor, and a unit base;
[0075] FIG. 7C shows a partial exploded view of the differentiation
bioreactor of FIG. 7B;
[0076] FIG. 8A a perspective view of another embodiment of a
product chamber assembly of the present invention;
[0077] FIG. 8B shows a partial exploded view of the product chamber
assembly of FIG. 8A showing a protective unit lid, a cell therapy
bioreactor, and a unit base;
[0078] FIG. 8C shows a partial exploded view of the cell therapy
bioreactor of FIG. 8B;
[0079] FIG. 9 shows a perspective view of an embodiment of a fluid
reservoir of the present invention;
[0080] FIG. 10 shows a perspective view of an embodiment of a flow
control valve housing of the tissue engineering module of FIG.
3;
[0081] FIG. 11 shows an exploded view of the tissue engineering
module of FIG. 4 with the fluid reservoir of FIG. 9;
[0082] FIG. 12 shows a scheme of a general methodology for clinical
tissue engineering using the tissue engineering module of FIG. 3;
and
[0083] FIG. 13 shows an embodiment of a fluid flow schematic.
DETAILED DESCRIPTION OF THE INVENTION
[0084] The present invention is an improvement to the Applicant's
automated tissue engineering system described in International
Patent Application No. WO 03/0872292 (the disclosure of which is
incorporated herein in its entirety). The advanced automated tissue
engineering system of the invention has a variety of improvements
incorporated therein in order to increase the ease-of-use, maintain
the aseptic conditions of the system and simplify the manufacture
of the system, yet basically operates in the same manner as the
system of Applicant's International Patent Application No.
WO03/0872292.
[0085] The present invention will now be described in more detail
with reference to the Figures. FIG. 1 shows the advanced tissue
engineering system 100. The system comprises a housing 102,
multiple bays 106 each accommodating a tissue engineering module
(not shown) and a user interface touch screen 110. FIG. 2 shows the
tissue engineering system 100 with one of the bays 106 and the
associated door in the open position. The automated insertion and
retraction system is supported by a guide rail system 120. Each of
the bays can contain one tissue engineering module 118 which is
secured to the bay by automated latches residing in the bay (not
shown) and can be programmed to be removed through the entry of a
specific password via the touch screen to prevent unauthorized or
unscheduled access or tampering.
[0086] FIGS. 3 and 4 show two side perspective views of the
assembled tissue engineering module 118 removed from the bay. The
module 118 has two main components; the upper housing 200 and the
lower fluid reservoir 300. The upper housing 200 has a tissue
digestion chamber assembly 500, a proliferation chamber assembly
600, and a product chamber assembly 700. There are fluid access
ports 210 located on one side of the module 118 and internally
connected to the fluid pathway (not shown) within the module 118,
which can be used for quality control sample removal and component
addition to the internal fluid system if required. There are flow
control valves 212 located on the other side of the module 118.
[0087] The chamber assemblies 500, 600 and 700, the lower fluid
reservoir 300, and the remainder of the module 118 are each
described below, respectively.
[0088] Figure SA shows the tissue digestion chamber assembly 500.
Figures SB and SC illustrate the components of the tissue digestion
chamber assembly 500. The chamber assembly 500 comprises a tissue
digestion bioreactor 510, described more fully with respect to
Figure SC, and an outer protective containment unit 520. The outer
protective containment unit 520 comprises a protective unit lid 522
and a unit base 524 to enhance the protection and isolation of the
contents of the bioreactor 510 therein. The tissue digestion
bioreactor 510 is connected to the protective unit lid 522 via
sterile needleless connectors 526. Each of the connectors 526 is in
direct fluid communion with a corresponding mating connector 528 of
the protective unit lid 522. Once the tissue digestion bioreactor
510 is connected to the protective unit lid 522, the protective
unit lid 522 is then connected to the unit base 524.
[0089] The tissue digestion bioreactor 510 shown in FIG. 5C has
four primary components: a bioreactor base 530 that substantially
forms a tissue digestion chamber 532 of an appropriate size to
accommodate one or more tissue samples such as a tissue biopsy (not
shown); a removable bioreactor lid 534; a port filter 536, and an
optional port filter (not shown).
[0090] The bioreactor lid 534 provides for sterile needleless
connectors 526 located at the ends of internal ports 538 and 540.
The term "needleless connector" is understood to be a connector
with no sharp needles (e.g. a blunt cannula). When the bioreactor
lid 534 is assembled to the bioreactor base 530, internal port 538
is in fluid communion with the central tissue digestion chamber
532. Fluid may be transferred from port 538 to and from chamber 532
across an optional port filter (not shown). Similarly, internal
port 540 within the bioreactor lid 534 is in fluid communion with
the bioreactor base 530, which in turn is in fluid communion with
the tissue digestion chamber 532. Fluid may be transferred from
port 540 to the tissue digestion chamber across the port filter
536. The role of the port filters 536 is to retain tissue
aggregates and biopsy debris within the tissue digestion chamber
532 while allowing the passage of disassociated cells out of the
tissue digestion chamber 532, via port 540.
[0091] Loading of a tissue biopsy into the tissue digestion chamber
532 is performed with the bioreactor lid 534 removed from the
bioreactor base 530. Following loading, the lid 534 and base 530
are assembled together and the tissue digestion chamber 532 is
operationally engaged with the module 118 and then filled under
automated control with an enzyme solution through port 540. The
addition of enzyme solution to the tissue digestion chamber 532 is
balanced by air escaping through air vent 542. Biopsy digestion
takes place under continuous or intermittent recirculation of the
enzyme solution, thereby keeping the released cells in suspension
and maximizing the exposure of the biopsy to the enzyme reagents.
During recirculation, the enzyme solution enters the bottom of the
tissue digestion chamber 532 through the port filter 536 via port
540 and leaves the top of chamber 532 via port 538. This creates a
fluid flow path in a direction opposite to the gravity vector such
that the biopsy is suspended and tumbled to maximize the
effectiveness of the enzyme reagents. Digestion may be enhanced by
gentle agitation of the digestion medium within the digestion
chamber via a mixing diaphragm (not shown). The air vent 542 may be
closed during any recirculation steps, as any residual air bubbles
present in the fluid flow system are trapped and retained in the
upper half of the bioreactor, above the inlet of port 538. Upon
completion of the digestion sequence, the application of reverse
flow of either air or medium via port 538 into the top of the
tissue digestion chamber 532 results in the dispensing of the
disassociated cells past the port filter 536 and out of the
bioreactor via port 540 to either a proliferation chamber assembly
600 or a cell collection vessel. It is understood by one of skill
in the art that the tissue digestion bioreactor 500 can be
optionally loaded with cells instead of a tissue, in this case
digestion of the cells is not required.
[0092] Once the tissue digestion bioreactor 510 is assembled and
placed within the outer protective containment unit 520, as shown
in FIG. 5A, twin layers of containment and protection are present
for transport from the operating room, where the biopsy is
harvested, to the clinical area containing the system 100. Peel
tape seals are present over the sterile needleless connectors 528
such that the sterility of these connectors is maintained until it
is time to install this assembly 500 into the tissue engineering
module 118.
[0093] FIG. 6 shows the proliferation chamber assembly 600. The
proliferation chamber assembly 600 comprises a proliferation
bioreactor 602 that has a proliferation chamber 604. The
proliferation chamber 604 has a base 606 having a proliferation
surface 610 suitable for cell attachment and growth and a lid 611
for containment of fluid. To adjust or maintain the levels of
dissolved gases in the medium, a gas permeable membrane (not shown)
may be incorporated to the top surface of the proliferation chamber
604 that allows the transport of gases such as oxygen and CO.sub.2.
Separation walls 612 divide the internal space of the proliferation
chamber 604 into a channel system that forces medium to follow a
predefined pathway from an inlet port 616 to an outlet port
618.
[0094] The design of the proliferation chamber assembly 600 has
several important operational features. Relatively uniform cell
seeding can be obtained by the infusion of a cell suspension
through the channel system. Furthermore, the channel configuration
ensures that media flow is well distributed over the whole
proliferation surface 610, thereby reducing potential low-flow
regions that may compromise local cell vitality due to reduced
nutritional supply or waste product removal. Confluence sensors 620
may be distributed through the chamber 604 to automate the
detection of final cell confluence. These sensors 620 provide
feedback on the progress of the cell culture activity to facilitate
automatic control over the entire process. In addition, information
generated from the sensor data enables the operator to obtain
advanced notification of processing status such that related
clinical activities may be scheduled as appropriate.
[0095] At the conclusion of the proliferation sequence, continuous
or intermittent recirculation of an appropriate enzyme solution
through the channel system induces cell detachment due to the
effect of the enzyme reaction and the low-level sheer stresses
generated by the fluid flow. Accordingly, cell harvest is achieved
without the need for mechanical shaking or rotation of the
proliferation chamber assembly 600.
[0096] The channel system used herein can provide for a uniform
distribution of cells to enable homogeneous cell feeding of the
proliferation surface.
[0097] The inlet and outlet ports 616 and 618 connect with the
proliferation chamber 604 via ducts (not shown) that increases in
width as the base 606 of the proliferation chamber 604 is
approached. This reduces the streamlining of the flow and allows a
more uniform fluid distribution into and out of the proliferation
chamber 604.
[0098] The interior height of the proliferation chamber 604 within
the proliferation chamber assembly 600 has been optimized to obtain
an intermediate height between a low height that allows air bubbles
to bridge between the lid 611 and the proliferation surface 610
(causing cell necrosis), and a high height where the fill volume is
excessive and air removal is problematic.
[0099] The proliferation bioreactor 602 optionally includes flow
interrupters (not shown) that deliberately cause controlled
turbulence along the length of the proliferation chamber 604. These
interrupters are placed perpendicular to the flow as irregularities
in the top surface of the proliferation chamber 604. The flow
interrupters cause controlled mixing along the length of the
chamber 604 so that free cells (particularly post release after
confluence detection) remain in suspension and can be moved
efficiently toward the outlet 618.
[0100] The proliferation bioreactor 602 optionally includes a
progressive change in elevation along the length of the fluid
pathway from inlet 616 to outlet 618 to enable the more complete
exhaust of all contents. This elevation change is accomplished with
both the proliferation surface 610 of the base 606 and the top
surface of the chamber 604 changing in elevation at the same rate,
thereby maintaining a consistent separation between the top surface
and proliferation surface 610.
[0101] The proliferation bioreactor 602 optionally includes a
vibratory element (not shown) that facilitates cell release from
the proliferation surface 610. This element is optionally mounted
directly onto the chamber 604.
[0102] The proliferation chamber 604 optionally includes multiple
bases 606 (e.g. proliferation surfaces 610) in a stacked geometry
whereby each level is either in series or in parallel in terms of
fluid flow.
[0103] FIG. 7A shows the product chamber assembly 700. FIGS. 7B and
7C illustrate the components of the product chamber assembly 700.
The chamber assembly 700 comprises a differentiation bioreactor
710, described more fully with respect to FIG. 7C, and an outer
protective containment unit 720. The outer protective containment
unit 720 comprises a protective unit lid 722 and a unit base 724 to
enhance the protection and isolation of the contents of the
differentiation bioreactor 710 therein. The differentiation
bioreactor 710 is connected to the protective unit lid 722 via
sterile needleless connectors 726. Each of the connectors 726 is in
direct fluid communion with a corresponding mating connector (not
shown) of the protective unit lid 722. Once the differentiation
bioreactor 710 is connected to the protective unit lid 722, the
protective unit lid 722 is then connected to the unit base 724.
This forms twin layers of containment and protection. Additionally,
peel tape seals are present over the sterile needleless connectors
such that the sterility of these connectors is maintained until it
is time to install this assembly 700 into the tissue engineering
module 118.
[0104] The differentiation bioreactor 710, designed to promote cell
differentiation and subsequent tissue construct formation, is shown
in FIG. 7C. The differentiation bioreactor 710 has four primary
components: a bioreactor base 730 that substantially forms a
differentiation/tissue formation chamber 732; a removable
bioreactor lid 734; a permeable membrane tube 736; and a
scaffold/membrane/matrix 738. The permeable membrane tube 736
tightly encircles the scaffold/membrane/matrix 738 to form a cell
and tissue growth compartment 740 above the
scaffold/membrane/matrix 738. The tissue growth compartment 740 may
extend within the scaffold/membrane/matrix 738 according to the
pore size of the scaffold/membrane/matrix 738 and the placement of
the scaffold/membrane/matrix 738 within the membrane tube 736. The
membrane tube 736 is also affixed to the inlet port 742, such that
the membrane is physically located within the
differentiation/tissue formation chamber 732. This divides the
bioreactor into two independent compartments, a cell and tissue
growth compartment 740 and an outer cell-free medium compartment
744, all within chamber 732. The pore size of the membrane tube 736
is selected on the basis of being impermeable for cells but
permeable for nutrients, waste products, growth factors, etc.,
within the culture medium. If desired, membrane pore size can be
chosen in a manner to exclude molecules of a certain molecular
weight from passing through the membrane.
[0105] The inlet port 742 is required for loading a cell suspension
into the tissue growth compartment 740 and for the perfusion of the
emerging tissue construct with culture medium. During the delivery
of the cell suspension into the empty tissue growth compartment,
entrapped air within the tissue growth compartment 740 is allowed
to exit through air vent 746. In a similar fashion, the outer cell
free compartment 744 of chamber 732 is loaded with media via port
748 and entrapped air may escape via air vent 750.
[0106] The design of the differentiation bioreactor 710 allows
direct perfusion of the tissue construct through media delivery to
port 742 or indirect media supply to the surrounding cell free
compartment 744 of chamber 732 via port 748. The indirect media
supply is located away from that region of the implantable
scaffold/membrane/matrix 738 that is seeded with cells so as to
minimize the potential for damaging sheer stresses that could
compromise the formation of cell aggregates. Typically, ports 746
and 750 are closed during perfusion and port 752 serves as a media
outlet; however, various alternate media supply scenarios are
possible based on specific tissue engineering requirements or
advanced cell culture requirements. An important aspect of the
media perfusion strategy is that the permeable membrane 736, which
forms part of the tissue growth compartment 740, allows fresh
culture medium to permeate into the tissue growth compartment 740
without any loss of cells away from the scaffold. Furthermore,
nutrition is provided to the cells from essentially all directions
without restrictions from any impermeable bioreactor walls.
[0107] The differentiation bioreactor 710 complete with the
protective containment unit installed, as shown in FIG. 7A,
represents the pre-assembled and sterilized format for the assembly
700. In use, this assembly 700 is removed from the tissue
engineering module 118 upon completion of the biological processing
and is transferred to the operating room. By virtue of the
progressive layers of containment, the assembly is ideally suited
for operating room aseptic procedures.
[0108] FIGS. 8A to 8C show the configuration of another embodiment
of a product chamber assembly of the present invention. FIG. 8A
shows a product chamber assembly 800. FIGS. 8B and 8C illustrate
the components of the product chamber assembly 800. The chamber
assembly 800 comprises a cell therapy bioreactor 810, described
more fully with respect to FIG. 8C, and an outer protective
containment unit 820. The outer protective containment unit 820
comprises a protective unit lid 822 and a unit base 824 to enhance
the protection and isolation of the contents of the cell therapy
bioreactor 810 therein. The cell therapy bioreactor 810 is
connected to the protective unit lid 822 via sterile needleless
connectors 826. Each of the connectors 826 is in direct fluid
communion with a corresponding mating connector (not shown) of the
protective unit lid 822. Once the cell therapy bioreactor 810 is
connected to the protective unit lid 822, the protective unit lid
822 is then connected to the unit base 824. This forms twin layers
of containment and protection. Additionally, peel tape seals are
present over the sterile needleless connectors such that the
sterility of these connectors is maintained until it is time to
install this assembly 800 into the tissue engineering module
118.
[0109] The cell therapy bioreactor 810 receives cells following
proliferation and cell washing. The bioreactor 810 is shown in FIG.
8C. The cell therapy bioreactor 810 has a bioreactor lid 828
connected to a vertical chamber 830 and a bioreactor base 832 that
contains a conical cell collector 834. When the lid 828 is
connected to the bioreactor base 832 for operation in the module
118, a cell suspension may be introduced into the bioreactor 810
and under quiescent conditions the cells settle by gravity into the
conical cell collector 834. The combination of a sealed bioreactor
chamber shelf 836 and the fluid return tube 838 allows the
remaining fluid above the conical cell collector 834 to be removed
from the bioreactor 810 thus leaving concentrated cells within the
conical cell collector 834 ready for implantation.
[0110] The cell therapy bioreactor 810 complete with the protective
containment unit installed, as shown in FIG. 8A, represents the
pre-assembled and sterilized format for the assembly 800. In use,
this assembly 800 is removed from the tissue engineering module 118
upon completion of the biological processing and is transferred to
the operating room. By virtue of the progressive layers of
containment, the assembly is ideally suited for operating room
aseptic procedures.
[0111] FIG. 9 shows the fluid reservoir 300. The internal elements
of the fluid reservoir 300 are a set of flexible bags (not shown)
used to contain all of the processing fluids and waste fluids.
These bags are connected to the fluid pathway (not shown) via the
needleless connections 910. Prior to connection of the fluid
reservoir 300 to the upper housing 200 of the tissue engineering
module 118, connections 910 are also used to install fluids into
each bag, as well as provide access for adding final components
(such as autologous serum). In order to ensure that the fluids
contained within the bags in the fluid reservoir 300 remain viable
for the extended periods required for cell culture and tissue
engineering, the fluid reservoir 300 has been designed to allow for
reduced temperature operation as compared with the remainder of the
tissue engineering module 118, which typically operates at
37.degree. C. The reduced temperature within the fluid reservoir
300 is attained via a temperature controller, such as Peltier1M
cooling elements on the base of the system bay (not shown), which
protrude upwardly into the fluid reservoir 300 through holes 912 to
provide local cooling to the bags. The bag temperature sensor (not
shown) resides on the base of the system bay (not shown) and
protrudes through a hole 914 in the fluid reservoir 300 to provide
the control feedback necessary for temperature control. Local
cooling within the overall module 118 for the tissue engineering
system 100 has the advantage of minimizing the power required for
cooling and also minimizing problems associated with condensation.
To ensure that there is adequate insulation for the inner fluid
reservoir bags, the fluid reservoir 300 is constructed to be a
double walled reservoir 916. An air space is present between the
two walls thereby improving the insulation properties of the
reservoir 300 while, preferably, maintaining optical clarity for
inspection. Through active cooling of the fluid reservoir 300, the
contents remain in a refrigerated state thereby minimizing or
eliminating operator intervention to replace fluids that would
otherwise expire if maintained continuously at 37.degree. C.
Latches 918 on the comers of the fluid reservoir 300 provide the
connection points that assemble the fluid reservoir 300 to the
upper housing 200 of the tissue engineering module 118.
[0112] FIG. 10 shows a flow control valve housing 1000 of the upper
housing 200, which is used as the main fluid pathway tube
interconnect and fluid direction interface between the chamber
assemblies 500, 600 and 700 and the fluid reservoir 300. The flow
control valve housing 1000 is constructed as a molded plastic
component that makes connections to the fluid reservoir 300 through
the series of cannula connections 1010. Fluid control is obtained
via flow control valves 212 that are installed into the flow
control valve housing 1000. The flow control valve headers 1012 and
1014 provide a common fluid connection between the flow control
valves 212 and enables the internal fluid pathway connections
between the chamber assemblies 500, 600 and 700 and the fluid
reservoir 300. This housing 1000 and related vertical plates (not
shown) dramatically simplifies the internal tubing complexity for
the fluid management system used within the tissue engineering
module 118.
[0113] FIG. 11 shows an exploded view of the tissue engineering
module 118 of FIG. 4 with the fluid reservoir 300 of FIG. 9. FIG.
11 shows the two main components of the tissue engineering module
118 in the final stage of installation; the upper housing 200 and
the lower fluid reservoir 300. To summarize, the upper housing 200
contains the installed tissue digest bioreactor 510 within a
protective containment unit 520; the proliferation chamber assembly
600; the installed differentiation bioreactor 710 within the
protective containment unit 720 or the cell therapy bioreactor 810
within the protective containment unit 820; and the flow control
valve housing 1000. The tissue engineering module 118 contains
internal crossflow cell concentrator (not shown) and interconnect
tubing and valves to complete the fluid handling system (not
shown). Connection of the upper housing 200 to the fluid reservoir
300 automatically engages a series of fluid connectors that enable
fluid communication between the two components 200 and 300. Both
components 200 and 300 are held together by molded latches (not
shown) on the upper housing 200 and molded latches 918 on the fluid
reservoir 300.
[0114] FIG. 12 shows a scheme of a general methodology for clinical
cell therapy and tissue engineering using the tissue engineering
module 118 of FIG. 3, operating in the tissue engineering system
100 of FIGS. 1 and 2, and autologous cartilage tissue engineering
as a representative example. In such an example, cells (i. e.
chondrocytes) are obtained from a surgical biopsy of a patient and
placed in the tissue digestion bioreactor 510 of the tissue
digestion chamber assembly 500. The tissue digestion chamber
assembly 500 is engaged with the tissue engineering module 118
containing the proliferation chamber assembly 600 and product
chamber assembly 700. A central microprocessor is present within
the tissue engineering system and controls and customizes the
internal environment of the bioreactor/chambers, and hence
facilitates tissue growth therein, resulting in the stimulation of
cell growth and subsequent matrix expression to generate an
implant. Sensors within the bioreactor provide feedback to the
microprocessor to ensure that the cells are seeded, expanded and
differentiated in a desired and controlled manner to provide an
autologous tissue implant. Once the implant is generated, the
product chamber assembly 700 is removed from the module 118 and
transported to the operating room for surgical implantation into
the patient. The present system provides an advantageous way to
provide autologous tissue engineered implants in a sterile, safe,
convenient and efficacious manner. Furthermore, the ability to
prepare tissue engineered implants in a clinical setting allows
considerable flexibility in the locations for undertaking the
tissue engineering process. While the system can be used in a
centralized location, the design and automated operation of the
system enables clinical use at regional centers. Such widespread
availability precludes the transportation of biological material to
and from centralized cell/tissue processing facilities, thereby
improving the cost effectiveness and efficiency of the tissue
engineering process while avoiding shipment, tracking and
regulatory complications.
[0115] FIG. 13 illustrates an embodiment of a fluid flow schematic
in which the bioreactors/chambers of FIGS. 5 and 6, and either 7 or
8 may be employed. A tissue digestion bioreactor 510 is present
that accommodates a tissue biopsy. A proliferation chamber assembly
600 is present that is configured to accept cells from the tissue
digestion bioreactor 510 and allows seeding of the proliferation
surface 610. Bubble traps within the tissue digestion bioreactor
510 remove air bubbles from the input line to the proliferation
chamber assembly 600 and therefore prevents these bubbles from
entering the proliferation chamber assembly 600 and potentially
compromising localized cell populations. A cell washing reservoir
220 is present to accept the expanded cell numbers from the
proliferation chamber assembly 600 and to serve as a temporary
holding container during a cell washing and cell concentration
procedure performed with the aid of a cross flow filtration module
222. One of several alternative product bioreactors (e.g. cell
therapy bioreactor 810, differentiation/tissue formation bioreactor
710, a multi-implant bioreactor 1310, or a cell matrix implant
bioreactor 1312) is also present and is configured to accept the
cells from reservoir 220 after the washing and concentration
step.
[0116] Tissue engineering reagents (i.e. media, enzyme solutions,
washing solutions, etc.) and waste fluids are stored in a fluid
reservoir 300 such as that shown in FIG. 9. Fluid flow through the
system is directed by the operation of a fluid pump 224, flow
control valves 212a-212g and 212r-212v according to control inputs
from a central microprocessor. Air filters allow the transfer of
air into or out of the system as required during operation without
compromising system sterility. Furthermore, in-line gas exchange
membranes (not shown) may be deployed at various locations within
the fluid flow paths to facilitate the control of dissolved gases
in the culture medium.
[0117] In one non-limiting example of the system operation, a
tissue biopsy is inserted into the tissue digestion bioreactor 510
of the tissue digestion chamber assembly 500. A digestion medium
containing enzymes is pumped into the tissue digestion bioreactor
510 from the fluid reservoir 300 to initiate the digestion process.
The digestion medium may be continuously or periodically
re-circulated via pump 224, thereby keeping the released cells in
suspension and maximizing reagent exposure to the biopsy.
Introduction of a proliferation culture medium from the fluid
reservoir into the top of the tissue digestion bioreactor 510
transfers the cell suspension to the proliferation chamber assembly
600 and simultaneously dilutes the enzyme solution to a
concentration that is tolerable for cell proliferation. The
transfer of partially digested tissue out of the digestion
bioreactor 510 is precluded by port filter that is sized to allow
passage of disassociated cells and retention of cell aggregates.
Cells generated from the biopsy digestion process are homogeneously
distributed throughout the proliferation chamber assembly 600
either by the recirculation of the cell suspension via the
activation of valves 212 and the pump 124, or by the automated
application of gentle shaking of the proliferation chamber assembly
600.
[0118] Following a quiescent period to allow attachment of the
cells to the proliferation surface 610, the proliferation medium is
periodically or continuously replaced with fresh proliferation
medium from the fluid reservoir 300. During a medium replacement
step, the supply of fresh medium from the fluid reservoir 300 is
balanced by the discharge of waste fluid to a waste container in
the fluid reservoir 300 via valve 212g.
[0119] Once the cell culture approaches confluence, the media
within the proliferation chamber assembly 600 is evacuated into the
waste container within the fluid reservoir. In this process, the
removal of fluid from the proliferation chamber assembly 600 is
balanced by incoming sterile air delivered via an air filter or by
incoming PBS wash solution from the fluid reservoir 300.
[0120] The cells are subsequently released from the proliferation
surface 610 through an automated sequence, such as the delivery of
enzymes (for example trypsin) and the timed recirculation of the
cell suspension or the timed application of impact or agitation to
the bioreactor via an impact drive. In order to remove the enzymes
and to collect the cells in a relatively small volume of medium for
subsequent transfer to a selected product bioreactor (710, 810,
1310, or 1312) of the product chamber assembly, the cell suspension
is transferred from the proliferation chamber assembly 600 to the
cell washing reservoir 220. The cell suspension is then
continuously recirculated via valves 212 and pump 224 through the
cross-flow filtration module 222. The membrane in the cross-flow
filtration module 222 prevents the loss of cells but allows a
certain percentage of media (permeate) to be removed via valve 212g
to the waste container in the fluid reservoir 300. The result is a
reduction of the suspension volume and/or dilution of any enzymes
present, provided the removal of permeate is compensated by the
supply of fresh medium from the fluid reservoir 300. The continuous
flow reduces the potential for cells to become entrapped within the
membrane of the cross-flow filtration module 222.
[0121] Cell delivery to the product bioreactor is achieved by
transferring the washed cells from the reservoir 220 via the valves
212 and pump 224. Following cell transfer to the product
bioreactor, fresh media may be introduced into the product
bioreactor through the operation of pump 224. During biological
processing, the medium is periodically or continuously replaced
with fresh medium from the fluid reservoir 300. During a medium
replacement step, the supply of fresh medium from the fluid
reservoir 300 is balanced by the discharge of waste fluid to a
waste container in the fluid reservoir via valve 212g. In between
the medium replacement steps, the fluid within the product
bioreactor is continuously or periodically recirculated under the
control of pump 224 and valves 212. In order to ensure that
environmental conditions within the different bioreactors promote
normal cellular activity, conditions are monitored and controlled
for the period necessary for the successful collection of expanded
cells in the case of cell therapy or formation of one or more
tissue constructs in the case of tissue engineering. Once the
collection of cells or formation of tissue implants is complete,
the product bioreactor is removed and transported to the operating
room for subsequent clinical use
[0122] It should be noted that the system/module of the invention
is not limited to a particular type of cell or tissue. For example,
a skeletal implant may be prepared for use in the reconstruction of
bone defects. In this application, bone marrow could be used as the
source of the primary and/or precursor cells required for the
tissue engineering process. Accordingly, there is no requirement to
perform tissue digestion; hence, the bioreactor chamber assembly
may be of the type that only supports proliferation and
differentiation. Depending on the available cell population and the
required size of the implant, even proliferation may not be
required. In this case, the configuration of the bioreactor chamber
assembly may be directed to the single stage of cell
differentiation and ongoing tissue formation. The final tissue
construct could be comprised of an implantable scaffold, which may
be composed of a bone biomaterial such as Skelite.TM., with active
bone cells lining the open pores of the scaffold and actively
laying down new mineralized matrix (osteoid). Such an implant would
be quickly integrated at the implant site thereby accelerating the
recovery process.
[0123] When two or more chamber assemblies are used in the module,
the chamber assemblies may be independently operable or
co-operatively operable. For example, the chamber assemblies may be
operatively connected such that there is an exchange of fluids,
cells and/or tissues from chamber to chamber or the chamber
assemblies may operate independent of one another. The chamber
assemblies may be connected via at least one of a passageway,
tubing, connector, valve, pump, filter, fluid access port, in-line
gas exchange membrane, and in-line sensor.
[0124] The tissue engineering system of the present invention is
designed to perform activities under aseptic operating conditions.
The system is fully automated, portable, multifunctional in
operation and performs/provides without being limited thereto, one
or more of the following: [0125] sterile reception/storage of
tissue biopsy; [0126] automated monitoring of digestion process
[0127] digestion of biopsy tissue to yield disassociated cells;
[0128] cell sorting and selection, including safe waste collection;
[0129] cell seeding on or within a proliferation substrate or
scaffold [0130] proliferation of cells to expand cell populations;
[0131] cell washing and cell collection; [0132] cell seeding on or
within a tissue engineering scaffold, membrane and/or matrix;
[0133] cell differentiation to allow specialization of cellular
activity; [0134] tissue formation; [0135] mechanical and/or
biochemical stimulation to promote tissue maturity; [0136]
harvesting the tissue engineered constructs/implants for
reconstructive surgery; and [0137] storage and transportation of
implantable tissue.
[0138] The tissue engineering system of the present invention may
be pre-programmed to perform each of the above noted steps and/or
other steps, individually, sequentially or in certain predetermined
sequences or partial sequences as desired and required.
Furthermore, each of these steps, or any combination thereof, are
accomplished within one or more chamber assemblies on a tissue
engineering module. In operation, the tissue engineering system is
pre-programmed and automatically controlled thus requiring minimal
user intervention and, as a result, enhances the efficiency and
reproducibility of the cell culture and/or tissue engineering
process while minimizing the risks of contamination. Therefore, in
one example, the automated tissue engineering system of the present
invention is capable of multi-functionally carrying out all of the
steps of a biopsy tissue digestion to yield disassociated cells,
subsequent cell seeding on a proliferation substrate, cell number
expansion, controlled differentiation, tissue formation and/or
production of a tissue implant within a single system.
[0139] The tissue engineering system and tissue engineering module
is not to be limited to tissue engineering per se. The system and
module can be utilized, for example, for cell therapy. Therefore,
the applicability of the system and module of the present invention
ranges from tissue engineering; to the formation of cells and/or
tissues on and/or within at least one scaffold, membrane and
matrix; to, simply, the expansion of cells for cell therapy
applications. It is noted that the scaffolds/membranes/matrices can
be any suitable shape, such as contoured, circular, have an
irregular perimeter. The term "cell matrix implant" used herein is
understood to encompass cells within and/or on a scaffold, matrix,
and/or membrane, such as, and without being limited thereto, a
pre-tissue.
[0140] Cells and tissues may be selected from, and without being
limited thereto, non-cartilage tissue, such as cardiac tissue,
vascular implants, and skin grafts, and skeletal tissues such as
bone, cartilage, tendon, disc, related bone and cartilage precursor
cells, and combinations thereof. More specifically, cells suitable
for use in chamber assemblies, module and system of the invention
are selected from but not limited to the group consisting of
embryonic stem cells, adult stem cells, osteoblastic cells,
pre-osteoblastic cells, chondrocytes, nucleus pulpous cells,
pre-chondrocytes, skeletal progenitor cells derived from bone, bone
marrow or blood, including stem cells, and combinations thereof.
The cells or tissues may be of an autologous, allogenic, or
xenogenic origin relative to the recipient of an implant formed by
the cell culture and tissue engineering functions of the invention.
It is also understood that the term tissues, as used herein, is not
to be limited only to connective tissues but can include a variety
of tissues such as, and without being limited thereto, cardiac
tissue, vascular implants, and skin grafts.
[0141] The chamber assemblies of the present invention may provide
an environment for at least one of the following selected from the
group consisting of storage of tissue biopsy, digestion of tissue
biopsy, cell sorting, cell washing, cell concentrating, cell
seeding, cell proliferation, cell differentiation, cell storage,
cell transport, tissue formation, implant formation, storage of
implantable tissue and transport of implantable tissue.
[0142] The sensors used herein, such as, for example, confluence
sensors, may have ability to monitor the specific performance of
cell populations/tissue in said at least one chamber assembly from
various donors and thereby, allow the system to accommodate for the
requirements of cells/tissue of individual donors in said at least
one chamber assembly. For example, as a result of these sensors,
the system has the ability to adapt to the needs of specific
cells/tissues from different donors. For instance, cells from an
older donor may grow at a slower rate compared to a younger donor,
therefore, the sensors would permit the system to adjust
accordingly to permit longer growth times.
[0143] In addition, the tissue engineering module of the present
invention can have at least one additional chamber assembly that
can share a common process with an existing chamber assembly such
that the additional chamber assembly can be removed in order to
provide analysis and/or evaluation of the contents of the chamber
that parallels the contents of the existing chamber. The contents
may be media, tissue and/or cells.
[0144] The advanced tissue engineering system of the present
invention has several advantages compared to methods and systems of
the prior art. In particular, the turn-key operation of the device
enables complex tissue engineering procedures to be performed under
automated control in the clinic, thereby precluding the need to
transport cells to centralized facilities for biological
processing. The system is simple to use and obviates the existing
time consuming and expensive manual human tissue culture procedures
which can lead to implant contamination and failure. The tissue
engineering modules and associated subsystem assemblies may be
customized for the type of cell or tissue to be cultured and may be
fabricated from any suitable biocompatible and sterilization
tolerant material. The entire tissue engineering module or specific
components thereof are replaceable and may be considered
disposable. The tissue engineering module may be provided in a
single-use sterile package that simplifies system set-up and
operation in clinical settings. In other embodiments, any
components such as the tissue digestion chamber assembly and
product chamber assembly as well as the housing and the fluid
reservoir can be provided separately packaged for use as a kit. In
embodiments of the invention, the tissue digestion chamber assembly
and the product chamber assembly with a selected bioreactor
therein, may be provided separately packaged and as such can be
provided as a kit to be used with a tissue engineering module. The
proliferation chamber assembly in aspects is fabricated already
attached to the housing of the tissue engineering module. All
detachable aspects of the tissue engineering module are designed to
ensure that assembly can only be done with the correct orientation
and once assembled is essentially tamperproof.
[0145] It is understood by those skilled in the art that the tissue
engineering module and device of the present invention can be
fabricated in various sizes, shapes and orientation. The device can
be fabricated to incorporate a single tissue engineering module or
multiple modules in vertical or horizontal formats. Accordingly,
the subassemblies can be made to correspond to the spatial format
selected for the tissue engineering device. As such, different
types of tissue engineering can be simultaneously conducted in a
single device with each tissue engineering sequence being
automatically monitored and controlled on an individual basis. It
is also within the scope of the invention to have a plurality of
automated tissue engineering systems operating and networked under
the control of a remote computer.
[0146] The present invention is an improvement to the Applicant's
automated tissue engineering system described in International
Patent Application No. WO 03/0872292. The improvements to the
advanced tissue engineering system of the present invention are
generally discussed below.
[0147] In one aspect of the invention, the tissue engineering
module still contains multiple bioreactors provided within chamber
assemblies to allow multistage processing
(digest/proliferation/differentiation); however, there are new
aspects to the tissue engineering module as follows: [0148] The
flow pathway of the advanced system is comprehensively revised
reducing the number of valves. This can be achieved through an
innovative use of check valves with specific cracking pressures;
[0149] Since the advanced system can be comprised of a series of
disposable components assembled in the clinic at the time of use
(disposable tissue engineering module, fluid reservoir, chamber
assemblies (tissue digestion, proliferation and one of four or more
product chambers)), the tissue engineering module can include
assembly integrity sensors that monitor that all parts are present
and are connected together correctly; [0150] Predictive software
can be included in combination with biosensor feedback to enhance
control over the bioprocessing of the advanced system enabling the
implantation surgery to be forecast in advance; [0151] The advanced
system can accommodate the use of autologous (patient) serum as
well as autologous cells, thereby minimizing risk; [0152] The
advanced system can allow for multiple sample ports for the removal
of media and/or cell and media samples; [0153] In addition, in the
advanced system, ports can be available to allow mid process
loading of additional media and/or additives, in the event this is
necessary for certain clinical activities; and [0154] In addition
to the assessment of cell vitality and cell number as part of a
quality control kit, the advanced tissue engineering system can
support the innovative use of assays in the form of microarrays and
protein expression arrays. This is facilitated by the fact that a
user may have access to the various components of the module such
as for example the proliferation chamber assembly. In this manner,
cells may be tested for expression of certain genes and proteins at
various steps during the processing and operation of the
system.
The Reservoir
[0154] [0155] The fluid reservoir is installed as a separate unit
(as shown in FIG. 11) and, in one embodiment, fluid connections are
provided via connectors, such as an array of needleless ports, on
the top surface that engages with mating connectors present in the
upper housing of the tissue engineering module. The connectors and
mating connectors are in fluid communication with one another;
[0156] The fluid reservoir is optionally pre-filled; [0157] The
fluid reservoir is optionally pre-sterilized; [0158] The fluid
reservoir may be structurally rigid for ease of handling; [0159]
The attachment of the fluid reservoir to the tissue engineering
module may be via a one-way snap-on connection. Once attached, the
reservoir cannot be detached; thereby, precluding potentially
hazardous (and contra-indicated) re-use; [0160] The fluid reservoir
may be clear for inspection of contents and to allow visible
confirmation of additive loading prior to connection to the
cassette; [0161] The fluid reservoir may have open "windows" in the
base to enable thermal union with Peltier (or similar) cooling
members that emerge from the base of the bay present on the
instrument; [0162] The fluid reservoir may be designed with twin
walls to maximize the insulation properties when operated at
4.degree. C. and the remainder of the instrument at 37.degree.
C.
The Bioreactors
[0163] The bioreactor design is significantly different than the
Applicant's earlier PCT application. While the basic internal
working of the tissue digestion bioreactor, the proliferation
bioreactor, and the differentiation bioreactor for implant
formation are per the Applicant's earlier International Patent
Application No. WO 03/0872292, the design has been improved. In one
particular embodiment, a novel design for the provision of a double
containment for selected bioreactors has been implemented to
improve and maintain aseptic conditions during transport of these
bioreactors to or from the clinic or operating room. The chamber
assembly for the tissue digestion bioreactor and/or the product
bioreactor (differentiation bioreactor) comprises an outer
protective unit that houses the selected bioreactor therein. The
outer protective unit comprises a unit lid and unit base. Engaged
within the outer protective unit is a desired bioreactor that
comprises a bioreactor lid and bioreactor base. The bioreactor lid
may be supported and engageable with a portion of the unit lid by
needleless injectors.
Chamber Assembly--with Tissue Digestion Bioreactor [0164] The
chamber assembly having a tissue digestion bioreactor therein is
designed to accept a tissue biopsy (for example but not limited to
a cartilage biopsy, in other aspects may be loaded with cells) and
facilitates directed flow as outlined in the Applicant's
International Patent Application No. WO 03/0872292. In one
embodiment, the tissue digestion bioreactor within the assembly is
formed with the bioreactor base chamber containing an integral
lower tubing connection such that all the flow port connections
occur at the top. This enables connection to a top manifold through
sterile needleless connections (FIGS. 5B and 5C). [0165] In
addition the tissue digestion bioreactor chamber assembly may be
produced with two "containment layers" whereby the tissue digestion
bioreactor and connection ports are loaded into an outer protective
containment unit with a further set of connection ports (Figures SB
and SC). The ports are designed to allow aseptic docking once the
protective port covers (tabs or adhesive labels) are removed. This
twin level of protection provides important added security to
prevent inadvertent contamination as the tissue digestion
bioreactor within the chamber assembly is being transported from
the site of biopsy collection (e.g. operating room) to the location
of the advanced tissue engineering system (e.g. clinical lab).
Chamber Assembly--with a Proliferation Bioreactor
[0166] The proliferation bioreactor is similar to the "s-channel"
bioreactor shown in Applicant's International Patent Application
No. WO 03/0872292. However, there are several changes made thereto
that provide important improvements: [0167] One embodiment of the
layout of the proliferation bioreactor is in a race-track
configuration (FIG. 6) that is similar but not limited to the
letter "C". This allows the inlet and outlet to be placed at the
center of the tissue engineering module, thereby facilitating
tubing connections. [0168] The race-track has inlet and outlet
ports that enter the proliferation chamber with a duct that
increases in width as the chamber is approached. This reduces the
streamlining of the flow and allows a more uniform fluid
distribution into and out of the chamber. [0169] The volume of the
proliferation bioreactor was considered in terms of the resulting
dilution that occurs when the incoming cell suspension released
from the digest bioreactor is mixed with incoming media to fill the
proliferation chamber. It was found that residual enzymes initially
used in the digestion process do not need to be physically removed
or deactivated (to preclude cell complications during
proliferation) when dilutions of for example about 10:1 occurs
during the loading of the proliferation chamber. [0170] The height
of the proliferation bioreactor can be optimized to obtain an
intermediate height between a low height that allows air bubbles to
bridge between the top surface and the active cell surface (causing
cell necrosis), and a higher height where the fill volume is
excessive and air removal is problematic. [0171] The proliferation
bioreactor can optionally include flow interrupters that
deliberately cause controlled turbulence along the length of the
proliferation surface. These interrupters would be placed
perpendicular to the flow as irregularities in the ceiling of the
proliferation chamber. The objective is to cause controlled mixing
along the length of the proliferation surface so that free cells
(particularly post release after confluence detection) remain in
suspension and can be moved efficiently toward the outlet. [0172]
The proliferation bioreactor may optionally include a slight
elevation change from inlet to outlet (cork-screw style or similar
to a spiral ramp) to enable the more complete exhaust of all
contents. This elevation change would be accomplished with both the
floor and ceiling of the cavity decreasing in elevation at the same
rate, thereby maintaining a consistent interior height. [0173] The
proliferation bioreactor includes sensors to monitor the onset of
cell confluence. In one aspect, sensor electrodes reside on the
proliferation surface and are exposed to the media to monitor the
changes in impedance that occurs with increasing cell growth.
[0174] The proliferation bioreactor optionally includes a vibratory
element that facilitates cell release from the proliferative
surface of the chamber. This element is mounted directly in the
chamber. [0175] In addition, multiple proliferation bioreactors may
be incorporated. Chamber Assembly--with Product (i.e. Implant)
Bioreactor [0176] Four different product bioreactors may be
selected for use in the product chamber assembly (identified
generically as differentiation bioreactors in the Applicant's
International Patent Application No. WO 03/0872292). The product
formats are: [0177] Cell therapy bioreactor--In one representation,
there is a vial contained within the overall bioreactor (FIG. 8C)
where the vial enables cell sedimentation. After cell sedimentation
the design of the cell therapy bioreactor provides for the removal
of the media supernatant leaving the concentrated cells in a small
cone at the base of the vial. This approach provides a vial with
concentrated cells as is now provided by centralized cell therapy
providers. With this automated technique, additional use of a
centrifuge to concentrate the cells at the end of the process as
per conventional manual techniques is not required. [0178] Single
(TE) tissue engineered bioreactor--The bioreactor facilitating all
ports at the top for easy connection with the system via needleless
connectors (FIGS. 7B and 8B). The approach to the formation of the
tissue engineered implant is similar to that described in
Applicant's International Patent Application No. WO 03/0872292)
[0179] Multiple (TE) tissue engineered implants--This is similar to
that defined in the Applicant's International Patent Application
No. WO 03/0872292. [0180] Cell matrix implant bioreactor--This is a
hybrid of cell therapy and tissue engineering where cells are
cultured for a short period on a matrix to allow attachment but
minimal tissue formation. The cell matrix may be flexible. This
technique has been references as MACI approach (matrix induced
autologous chondrocyte implantation). [0181] The product bioreactor
(in any of the aforementioned formats) also benefits from the twin
"containment layers" (as shown in FIGS. 7A, 7B, 8A, and 8B) as
substantially employed for the tissue digestion bioreactor. In the
case of a product bioreactor, the value of the twin layers (i.e.
the outer containment unit) is to allow the bioreactor contained
therein to be disassembled in a sequence consistent with operating
room practice. That is to say that the exterior can be removed and
the interior parts handled while maintaining aseptic practices.
Other Components
[0181] [0182] Beyond the different bioreactor chamber assemblies
that incorporate the double containment system, a high efficiency
cross-flow filter can be implemented to enable cell concentration
post proliferation collection. This component eliminates
centrifugation. This component is shown in the flow diagram (FIG.
13). [0183] In aspects, a space and cost efficient valve manifold
has been designed that provides the fluid management described in
the Applicant's International Patent Application No. WO 03/0872292
while also providing the structural support for the valve array,
the critical fluid interconnects between the valves the bioreactors
and the fluid reservoir, and the attachment points for the tissue
engineering module latches.
[0184] A cell collection reservoir has been included in the design
as a staging area and to allow for warming of fluid from the
reservoir prior to infusion into the different chamber
assemblies.
[0185] Although preferred embodiments have been described herein,
it is understood by one of skill in the art that variations may be
made thereto without departing from the spirit of the
invention.
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