U.S. patent application number 14/216586 was filed with the patent office on 2015-03-19 for bioreactor system.
The applicant listed for this patent is KIYATEC INC.. Invention is credited to Hal Crosswell, Matthew R. Gevaert, David E. Orr.
Application Number | 20150079584 14/216586 |
Document ID | / |
Family ID | 51538083 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079584 |
Kind Code |
A1 |
Gevaert; Matthew R. ; et
al. |
March 19, 2015 |
BIOREACTOR SYSTEM
Abstract
A three dimensional cell culture and bioreactor system is
provided. The system comprises one or more cell culture chamber.
Each cell culture chamber comprises an inlet port and an outlet
port in fluid communication with the cell culture chamber. The cell
culture chambers may be segregated or in fluid communication with
one another. The systems may be used to conduct drug efficacy test,
isolate certain cell types from a complex tissue sample of multiple
cell types, allow for the ex vivo culturing of patient tissue
samples to help guide the course of treatment, and conduct
co-culture experiments.
Inventors: |
Gevaert; Matthew R.;
(Greenville, SC) ; Orr; David E.; (Piedmont,
SC) ; Crosswell; Hal; (Greenville, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KIYATEC INC. |
Greenville |
SC |
US |
|
|
Family ID: |
51538083 |
Appl. No.: |
14/216586 |
Filed: |
March 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61791432 |
Mar 15, 2013 |
|
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Current U.S.
Class: |
435/6.1 ; 435/26;
435/29; 435/32; 435/373 |
Current CPC
Class: |
C12N 5/0062 20130101;
C12N 2533/80 20130101; C12N 5/0693 20130101; C12M 35/08 20130101;
C12N 2513/00 20130101; C12M 29/04 20130101; C12N 5/0068 20130101;
C12M 29/10 20130101; G01N 33/5011 20130101; G01N 2333/904 20130101;
G01N 2500/10 20130101; C12N 5/0695 20130101; G01N 33/5073 20130101;
G01N 33/5014 20130101; C12M 23/44 20130101; C12N 2533/30
20130101 |
Class at
Publication: |
435/6.1 ; 435/29;
435/32; 435/373; 435/26 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12N 5/09 20060101 C12N005/09; C12N 5/095 20060101
C12N005/095; C12N 5/00 20060101 C12N005/00 |
Claims
1. A method for testing the efficacy of pharmaceutical agents using
three-dimensional cell culturing systems comprising; culturing a
target cell population in a cell culture chamber of a
three-dimensional (3D) cell culturing system; exposing the target
cell population in the cell culture chamber to a pharmaceutical
agent for a defined period of time and under a defined set of
exposure conditions; and determining a cell viability or other
measurable parameter for the target cell population, wherein the
target cell population is responsive to the pharmaceutical agent if
the cell viability or other measurable parameter of the target cell
population is less than the cell viability or other measurable
parameter of a control cell population; wherein the cell culture
chamber of the 3D cell culturing system comprises an inlet port and
an outlet port that are in communication with an interior volume of
the cell culture chamber, the interior volume of the cell culture
chamber housing a cell-scaffold construct and the target cell
population.
2. The method of claim 1, wherein the target cell population is a
cancer cell population, and the pharmaceutical agent is an
anti-cancer agent.
3. The method of claim 2, wherein the cancer cell population is
obtained from a patient biopsy sample.
4. The method of claim 1, wherein the 3D culture system further
comprises two or more cell culture chambers.
5. The method of claim 4, wherein the control cell population is
cultured in one of the two or more cell culture chambers.
6. The method of claim 4, wherein the two or more cell culture
chambers are connected, and wherein the two or more cell culture
chambers are separated by an impermeable membrane portion.
7. The method of claim 4, wherein the two or more cell culture
chambers are connected, and wherein the two or more cell culture
chambers are separated by a permeable membrane portion that allows
fluid communication between the cell chambers.
8. The method of claim 1, wherein the cell-culture chamber further
comprises an access port through which an analytical probe is
inserted to access either the interior or exterior of the cell
chamber.
9. A method for isolating residual cell populations from tissue
samples after exposure to pharmaceutical agents comprising;
culturing a primary cell population derived from at least a portion
of a subject biopsy sample in a cell culture chamber of a 3D cell
culturing system, wherein the primary cell population comprises one
or more cell types; treating the primary cell population with a
first dose of one or more pharmaceutical agents, wherein exposure
to the one or more pharmaceutical agent results in an increased
ratio of a first residual cell type relative to other cell types in
the primary cell population; and culturing the residual cell type;
wherein the cell culture chamber of the 3D cell culturing system
comprises an inlet port and an outlet port that are in
communication with an interior volume of the cell culture chamber,
the interior volume of the cell culture chamber housing a
cell-scaffold construct and the target cell population.
10. The method of claim 9, further comprising treating the residual
cell type with a second dose of the one or more pharmaceutical
agents to obtain an enriched residual cell type.
11. The method of claim 9, further comprising; treating the
cultured residual cell type with a second dose of the one or more
pharmaceutical agents, wherein the second dose of the one or more
pharmaceutical agents is a higher does than the first dose;
determining an efficacy of the second dose based at least in part
on a cell viability of the residual cell type after treatment with
the second dose.
12. The method of claim 9, further comprising; treating the
cultured residual cell type with a second dose of one or more
different pharmaceutical agents.
13. The method of claim 9, wherein the residual cell type comprises
tumor stem cells, and the one or more pharmaceutical agents are
anti-cancer agents.
14. The method of claim 9, wherein the cell-culture chamber further
comprises an access port through which an analytical probe is
inserted to access either the interior of the cell chamber.
15. A method for determining appropriate dosage regimens for one or
more pharmaceutical agents, comprising culturing, in a first cell
culture chamber of a 3D cell culture system, a residual cell
population obtained from a tissue sample of a subject in need of
treatment using the method of claim; culturing, in a second cell
culture chamber of a 3D cell culture system, a primary cell
population derived from a tissue sample from the subject;
sequentially treating the enriched residual cell population and the
primary cell population with increasing doses of one or more
pharmaceutical agents; determining a cell viability of the enriched
residual cell population and the primary cell population at each
dose; and selecting a final dose of the one or more pharmaceutical
agents for administration to the subject, wherein the final dose of
the one or more pharmaceutical agents is the dose that demonstrate
the greatest decrease in cell viability of both the primary cell
population and the enriched residual cell population; wherein each
cell culture chamber of the 3D cell culturing system comprises an
inlet port and an outlet port in communication with an interior
volume of the cell culture chamber, the interior volume of the
culture chamber housing a cell-scaffold construct and the target
cell population.
16. The method of claim 15, wherein the tissue sample is a cancer
tissue sample, and the one or more pharmaceutical agents are
anti-cancer agents.
17. The method of claim 15, wherein the first cell culture chamber
and the second cell culture chamber are connected, and wherein the
first cell culture chamber and the second cell culture chamber are
separated by an impermeable membrane portion.
18. The method of claim 15, wherein the two or more cell culture
chambers are connected, and wherein the two or more cell culture
chambers are separated by a permeable membrane portion that allows
fluid communication between the cell culture chambers.
19. The method of claim 15, wherein each cell-culture chamber
further comprises an access port through which an analytical probe
is inserted to access either the interior or exterior of the cell
culture chamber.
20. A method for monitoring and adjusting patient treatment
regimens over a course of treatment comprising; culturing a patient
cell population obtained from a diseased tissue of a patient in
need of treatment in a cell culture chamber of a 3D cell culture
system; treating, prior to initiating or changing the treatment
regimen, the patient cell population with one or more candidate
pharmaceutical agents at one or more doses; determining a cell
viability or other measured parameter of the patient cell
population for each candidate pharmaceutical agent at each dose;
and selecting a pharmaceutical agent and a dose of the
pharmaceutical agent for the treatment regimen, wherein the
pharmaceutical agent and the dose that demonstrates the greatest
decrease in the cell viability or other measured parameter of the
patient cell population; wherein each cell culture chamber of the
3D cell culturing system comprises an inlet port and an outlet port
in communication with an interior volume of the cell culture
chamber, the interior volume of the cell culture chamber housing a
cell-scaffold construct and the target cell population.
21. The method of claim 20, the patient cell population comprises
cancer cells.
22. The method of claim 20, wherein the cell-culture chamber
further comprises an access port through which an analytical probe
is inserted to access either the interior or exterior of the cell
culture chamber.
23. A method for co-culturing cells comprising; culturing a first
cell population in a first culture chamber of a 3D culture system,
the first culture chamber comprising an inlet port and an outlet
port through which a cell culture medium can flow, the inlet port
connected to an opening in a first side of a cell culture chamber,
and the outlet port connected to a second side on an opposing side
of the cell culture chamber, and comprising an additional opening
on a bottom side of the cell culture chamber; culturing a second
cell population in a second culture chamber of the 3D culture
system, the second culture chamber comprising an inlet port and an
outlet port through which a cell culture medium can flow, the inlet
port connected to an opening in a first side of the cell culture
chamber, and the outlet port connected to a second side on an
opposing side of the cell culture chamber, and comprising an
additional opening on a top side of the cell culture chamber;
wherein the bottom side of the first culture chamber and the top
side of the second culture chamber are connected to one another,
and wherein the first culture chamber and the second culture
chamber are separated by a membrane portion.
24. The method of claim 23, further comprising treating the first
and second cell population with one or more pharmaceutical agents;
and determining an efficacy, a toxicity, or both of the one or more
pharmaceutical agents on the first and second cell populations by
determining a cell viability for the first and second cell
populations.
25. The method of claim 23, wherein the first cell population is a
diseased cell population isolated from a biological organism, and
the second cell population is a disease free cell population
isolated from the biological organism.
26. The method of claim 25, wherein the diseased cell population is
a cancer cell population.
27. The method of claim 23, wherein the first cell culture chamber
and the second cell culture chamber are connected, and wherein the
first cell culture chamber and the second cell culture chamber are
separated by an impermeable membrane portion.
28. The method of claim 23, wherein the first cell culture chamber
and the second cell culture chamber are connected and stacked
together, and wherein the first and second cell culture chambers
are separated by a permeable membrane portion that allows fluid
communication between the two or more cell culture chambers.
29. The method of claim 23, wherein each cell-culture chamber
further comprises an access port through which an analytical probe
is inserted to access either the interior or exterior of the cell
culture chamber.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/791,432 filed Mar. 15, 2013 and entitled
"Bioreactor System." The entire contents of the above-identified
application are hereby fully incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure related to three-dimensional cell
culture systems and uses thereof.
BACKGROUND
[0003] The ability to culture in vitro viable three-dimensional
cellular constructs that mimic natural tissue has proven very
challenging. One of the most difficult of the many problems faced
by researchers is that there are multiple dynamic biochemical and
mechanical interactions that take place between and among cells in
vivo, many of which have yet to be fully understood, and yet the
complicated in vivo system must be accurately modeled if successful
development of engineered tissues in vitro is to be accomplished.
The ideal in vitro system should accurately model the physical
environment as well as the essential cellular interactions found in
vivo so as to enable utilization of the product, for instance as an
in vivo model or as transplantable tissue.
[0004] Many existing culture systems are simple well plate designs
that are static in nature and do not allow for manipulation of the
local environment beyond the gross chemical inputs to the system.
As such, the development of more dynamic culture systems has become
of interest, because it introduces the possibility of
advantageously changing the local environment over the course of a
cell culture experiment. However, known dynamic systems have not
been widely implemented in the field of cell culture, as they are
labor intensive, cost prohibitive, have configurations which limit
their experimental flexibility and lack inter- and intra-lab
comparability because there is no universal standard procedure.
[0005] In another aspect, there are many advantages to culturing
cells in 3D (as opposed to historic 2D cell culture) that are being
increasingly appreciated with a societal focus on higher and higher
fidelity in vitro models of in vivo human physiology. One of these
many advantages relates to the cultured cells' phenotype. It is
known that conventional 2D culture of cells is often associated
with a loss of phenotype and cell damage while 3D culture has been
shown to retain cell phenotype. See Mayne, R. et al (1976) PNAS,
73, 5; Brodkin, K. R. et al (2004) Biomaterials, 25, 28; Elowsson,
L. (2009) PhD Thesis (University of Sheffield, UK); Benya, P. D.
and Schaffer, J. D. (1982) Cell, 30, 1; Bonaventure, J. et al
(1994) Experimental Cell Research, 212, 1; and Osiecka, I. et al
(2008) Molecular Medicine Reports, 1, 6. However, current
technology does not allow for the exploitation of 3D culture
advantages and requires innovation to address the practical
difficulties of 3D culture when compared to its simpler, 2D cell
culture, predecessor.
[0006] One such exemplary technology lag area has been the process
of cell passaging, in which a relatively small number of cells are
repeatedly doubled for the sole purpose of creating a large number
of cells (e.g., to achieve the number of cells necessary for a
particular experiment). As one skilled in the art will appreciate,
passaging cells in 2D is convenient and ubiquitously standardized.
Additionally, during conventional passaging cells in 2D procedures,
most cells enter into a state of rapid proliferation which
decreases the time necessary to achieve the desired large number of
cells. As one skilled in the art will appreciate, in conventional
2D passaging, cells respond to the stiffness of the material on
which they attach. See, Attachment A Micro- and Nanoengineering of
the Cell Microenvironment, Technologies and Applications
(Engineering in Medicine & Biology), Ali Khademhosseini
(Editor)).
[0007] Relative to tissues found in the body formed from organic
materials, tissue culture lab ware is typically formed from stiff
materials. For example and without limitation, the moduli of soft
mammalian tissues ranges from about 100 Pa to about 950 kPa.
[0008] Exemplary tissue culture lab ware formed from polystyrene
has an elastic modulus of 3-3.5 GPa, which is higher than the
modulus of tissues formed from organic materials but not as high as
the elastic modulus of bone (9 GPa). In this aspect, bone is a
composite made up of inorganic minerals with high bulk moduli and
organic materials which are much softer, and the contribution of
the inorganic materials increases the modulus correspondingly. See,
Journal of Biomedical Materials Research Part A Volume 67A, Issue
3, Pages 886-899 Published Online: 20 Oct. 2003, (bulk hydroxyl
apatite modulus of 34-117 GPa).
[0009] Currently there are no commercially available products
designed for 3D cell passaging. Published research on this topic to
date has explored aspects of the potential use of 3D hydrogels (of
hyaluronic acid and poly(NIPAM) respectively) for 3D cell passaging
and has shown benefits of phenotype retention. See, TERMIS-EU 2010
Oral Presentation "Thermally-responsive Polymers for 3D Chondrocyte
Culture;" and U.S. patent application Ser. No. 11/473,870 to Singh,
which is incorporated herein by reference in its entirety. However,
hydrogel matrices are not in the stiffness range of tissue culture
polystyrene or bone. What is needed in the art is a method for
culturing cells in a dynamic environment in which the physical and
biochemical conditions can be advantageously changed over the
course of time. Moreover, what is needed is a system in which cells
can be developed to form a three-dimensional construct, while
maintaining the isolation and purity of the developing product
cells. In another aspect, what is needed is a material and method
for 3D cell passaging that the use of a stiff culture material in a
3D cell culture environment while maintaining a desired level of
phenotype retention.
SUMMARY
[0010] In one aspect, the present invention is directed to a
bioreactor system. The disclosed bioreactor system can comprise a
single or a multiple chamber culture system. In one aspect, a
bioreactor system of the invention can comprise at least one
culture chamber defining an inlet, an outlet, and a port that are
in communication with an interior volume of the at least one
culture chamber. In one non-limiting example, the at least one
culture chamber comprises a first culture chamber and a second
culture chamber. In this aspect, the first culture chamber defines
a first inlet and a first outlet that is configured to allow fluid
to selectively flow through the interior of the first culture
chamber. In a further aspect, the first culture chamber defines a
first port that is in communication with the interior of the first
culture chamber. The second culture chamber defines a second inlet
and a second outlet that is configured to allow a second fluid to
selectively flow through the interior of the second culture
chamber. In a further aspect, the second culture chamber defines a
second port that is in communication with the interior of the
second culture chamber.
[0011] In another aspect, the system can also comprise a membrane,
which can be positioned, for example and without limitation,
between the respective ports of adjoining first and second culture
chambers. The membrane can be semi-permeable and can have a
porosity that is configured to allow passage of cellular expression
products through the membrane, but prevent passage of the cells,
which are disposed therein either chamber, through the membrane. In
one embodiment, the membrane can be formed of a material, for
example and without limitation a polycarbonate, which can
discourage cellular attachment to the membrane.
[0012] In a further aspect, the bioreactor systems of the invention
can comprise a cellular anchorage in one or both of the respective
culture chambers. Suitable cellular anchorage can be formed of
multiple discrete scaffolds or single continuous scaffolds.
Multiple discrete scaffolds can be maintained within a culture
chamber through utilization of a retaining mesh that can hold the
scaffolding materials within the chamber and prevent the loss of
the scaffolding materials through the outlet of the culture
chamber.
[0013] In one aspect, a cellular anchorage can be maintained at a
predetermined distance from the membrane that separates the
chambers. In one aspect, this predetermined distance can be
selected to effect prevention or minimization of attachment of
cells to the membrane and can act to maintain the physical
isolation of different cell types within their respective culture
chambers.
[0014] The bioreactor system can also be capable of incorporating
additional culture chambers that can be in biochemical
communication with one or both of the other two culture chambers.
For instance, the at least one chamber can further comprise a third
chamber that can be configured to selectively house cells that can
be selectively positioned in biochemical communication with the one
or more of the system culture chambers, optionally with a membrane
separating the first and third chambers, though this aspect is not
a requirement of the system.
[0015] It is contemplated that, in operation, the bioreactors and
the cells disposed therein can optionally be subjected to at least
one mechanical stimuli. For example and without limitation,
pressurized fluid perfusion through a culture chamber can subject
developing cells to shear stress; an adjacent pressure module can
be utilized to subject the interior of a culture chamber to
hydrostatic loading, and the like.
[0016] It is also contemplated that the bioreactors of the system
can be used for growth and development of isolated cells in various
different applications. For instance, three-dimensional cellular
constructs can be formed including only the cells that are isolated
in one of the culture chambers of the reactor system. In one
exemplary aspect, a culture chamber can be seeded with
undifferentiated cells, and the method can comprise triggering
differentiation of the cells via the biochemical triggers provided
from the cells of the second culture chamber.
[0017] In a further aspect, it is contemplated that for tissue
passaging, a material composition comprising two or more materials
can be used. In one example, the material composition can comprise
a stiff culture material having substantially large porosity into
which a soft material has been introduced. In one example, and
without limitation, the stiff culture material can be formed from
metal, synthetic polymer, ceramic and the like. In one example, and
without limitation, the soft material can be formed from a hydrogel
or uncrosslinked oligomers of polymers either synthetic or of
natural origin, and the like. In one aspect, the soft material can
be configured or otherwise have a means for releasing the soft
material from the stiff material. In one exemplary aspect, the
releasing means can comprise chemical degradation or other change
initiated by light, temperature, pH, chemical catalyst, and the
like.
[0018] In yet another aspect, a method of 3D cell passaging is
provided that comprises providing a population of cells to be
passaged and introducing the population of cells into the material
composition to which they attach to at least portions of the soft
material. At a desirable and or predetermined time after the cells
attachment, the method can further comprise causing the soft
material to disassociate from the stiff material, thereby releasing
the soft material and the cells from the stiff material of the
material composition. In another aspect, the method can further
comprise dividing the recovered cells, with or without remnants of
the soft material, into multiple populations and repeating the
method using the subdivided populations. It is of course
contemplated that this process can be done recursively.
[0019] These and other aspects, objects, features, and advantages
of the example embodiments will become apparent to those having
ordinary skill in the art upon consideration of the following
detailed description of illustrated example embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A and 1B are views of one embodiment of the cell
modules of the bioreactor system;
[0021] FIG. 2 is a schematic diagram of the embodiment of FIG. 1
following assembly such that the two cell modules are adjacent and
allow biochemical communication between cells held in the two
adjacent modules;
[0022] FIG. 3 is one embodiment of a bioreactor system of the
present invention including two adjacent cell modules having
independently controlled flow characteristics there through.
[0023] FIG. 4 is a schematic of a bioreactor system as herein
disclosed including multiple cell culture chambers in biochemical
communication with one another; and
[0024] FIG. 5 illustrates another embodiment of the bioreactor
system in which at least one of the cell modules of the bioreactor
system can be subjected to periodic variation in hydrostatic
pressure.
[0025] FIG. 6 shows a set of schematic drawings (top and bottom)
providing cross sections that show flow characteristics, and an
actual image (middle) of example 3D culture system assemblies.
[0026] FIG. 7 shows one embodiment of a fluid circuit system
assembly.
[0027] FIG. 8 is a set of schematic drawings depicting an example
assembly for mono-culture (top) and co-culture (bottom).
[0028] FIG. 9 is a panel of images of cultured product cells
produced via the EV3D.TM. study using different mesh filters with
pore sizes ranging from 200-500 .mu.m.
[0029] FIG. 10 a is a panel of images of cultured product cells
produced via the EV3D.TM. study using mesh filters with pore sizes
ranging from 100-200 .mu.m.
[0030] FIG. 11 is a panel of images of cultured product cells
produced via the EV3D.TM. using mesh filters with a pore size
ranging from 40-100 .mu.m.
[0031] FIG. 12 shows an exemplary mono-culture.
[0032] FIG. 13 shows an exemplary co-culture.
[0033] FIG. 14 shows a graph illustrating fibroblast expansion in a
segregated co-culture.
[0034] FIG. 15 shows a graph illustrating EV3D relative response to
control and to other introduced drugs.
[0035] FIG. 16 is a set of graphs demonstrating the results of a
PrestoBlue analysis (top) and LDH release analysis (bottom) in a 2D
culture system and an example 3D culture system with and without
perfusion.
[0036] FIG. 17 is a graph demonstrating perfusion and fibroblast
co-culture support viability of HepG2 cells over 7 days in an
example 3D culture system.
[0037] FIG. 18 is a panel of micrographs showing HepG2 cells
cultured in an example 3D culture system with and without Dil(C)12
stained fibroblast. The top panels demonstrate preformed HepG2
spheroids in 3D culture system without (left) and with fibroblast
(right), and the bottom panel provides an inverted fluorescent
image of DiL(C)12 stained fibroblast cultured in a an example 3D
culture system and demonstrating stellate morphology.
[0038] FIG. 19 is a set of graphs showing vemurafenib activity in
2D and example 3D culture systems, with and without perfusion.
[0039] FIG. 20 is a graph showing vemurafenib sensitivity of cells
grown in an example 3D culture system.
DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS
Overview
[0040] The present invention can be understood more readily by
reference to the following detailed description, examples,
drawings, and claims, and their previous and following description.
However, before the present devices, systems, and/or methods are
disclosed and described, it is to be understood that this invention
is not limited to the specific devices, systems, and/or methods
disclosed unless otherwise specified, as such can, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular aspects only and is not
intended to be limiting.
[0041] The following description of the invention is provided as an
enabling teaching of the invention in its best, currently known
embodiment. To this end, those skilled in the relevant art will
recognize and appreciate that many changes can be made to the
various aspects of the invention described herein, while still
obtaining the beneficial results of the present invention. It will
also be apparent that some of the desired benefits of the present
invention can be obtained by selecting some of the features of the
present invention without utilizing other features. Accordingly,
those who work in the art will recognize that many modifications
and adaptations to the present invention are possible and can even
be desirable in certain circumstances and are a part of the present
invention. Thus, the following description is provided as
illustrative of the principles of the present invention and not in
limitation thereof.
[0042] As used herein, the singular forms "a," "an," and "the"
comprise plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to a "chamber" comprises
aspects having two or more such chamber unless the context clearly
indicates otherwise.
[0043] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect comprises from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0044] As used herein, the terms "optional" or "optionally" mean
that the subsequently described event or circumstance may or may
not occur, and that the description comprises instances where said
event or circumstance occurs and instances where it does not.
[0045] In simplest terms, disclosed herein are bioreactor systems.
In one aspect, the bioreactor systems disclosed herein comprise at
least one cell module defining a culture chamber, an inlet, an
outlet, and at least one port opening. The cell modules of the
bioreactor system can be engaged to form multi-chambered bioreactor
systems. Thus, in one aspect, disclosed herein are bioreactor
systems comprising at least one first cell module defining a first
cell culture chamber, an inlet, an outlet, and a port opening,
wherein the port opening is on one end of the cell culture chamber
and at least one second cell module defining a second cell culture
chamber, an inlet, an outlet, and a port opening, wherein the port
opening is on one end of the cell culture chamber. It is understood
that the first and second culture chambers respectively defining
the first and second cell modules can be separated by a barrier
such as a membrane. Thus, in another aspect, disclosed herein are
bioreactor systems comprising at least one first cell module
defining a first cell culture chamber, an inlet, an outlet, and a
port opening, wherein the port opening is on one end of the cell
culture chamber and at least one second cell module defining a
second cell culture chamber, an inlet, an outlet, and a port
opening, wherein the port opening is on one end of the cell culture
chamber; a membrane positioned between the open port of said first
cell module and the open port of said second cell module.
[0046] It is further understood that the first and second cell
module can be physically engaged. Thus, in still another aspect,
disclosed herein are bioreactor systems comprising at least one
first cell module defining a first cell culture chamber, an inlet,
an outlet, and a port opening, wherein the port opening is on one
end of the cell culture chamber and at least one second cell module
defining a second cell culture chamber, an inlet, an outlet, and a
port opening, wherein the port opening is on one end of the cell
culture chamber; a membrane positioned between the open port of
said first cell module and the open port of said second cell
module; and wherein the first cell module and second cell module
are sealingly engaged securing the membrane between the first and
second module.
[0047] The disclosed bioreactor systems can be assembled to allow
for single or multiple cultures of tissues or cells. Thus, in one
aspect, the bioreactor system is directed to multi-chambered
systems, such as a co-culture bioreactor system, and can, for
example, be utilized for the growth and development of isolated
cells of one or more cell types in a dynamic in vitro environment
more closely resembling that found in vivo. For instance, the
multi-chambered bioreactor system can allow biochemical
communication between cells of different types while maintaining
the different cell types in a physically separated state, and
moreover, can do so while allowing the cell types held in any one
chamber to grow and develop with a three-dimensional aspect. In
addition, the presently disclosed bioreactor system can allow for
variation and independent control of environmental factors within
the individual chambers. For instance, it is contemplated that the
chemical make-up of a nutrient medium that can flow through a
chamber as well as the mechanical force environment within the
chamber including the perfusion flow, hydrostatic pressure, and the
like, can be independently controlled and maintained for each
separate culture chamber of the disclosed systems
[0048] In yet another embodiment, undifferentiated stem cells can
be located in a first chamber of the bioreactor system, and one or
more types of feeder cells can be located in adjacent chamber(s),
which, as one skilled in the art will appreciate, can selective be
in biological communication with the first chamber. Such a
bioreactor system can be utilized to, for example and without
limitation, retain the differentiation state of cells in the first
chamber and/or direct the course of their differentiation, as
desired.
[0049] Cells and tissues used in the disclosed bioreactor systems
and methods can be obtained by any method known to those of skill
in the art. Examples of sources of cells and tissues include
without limitation purchase from a reliable vendor, blood
(including peripheral blood and peripheral blood mononuclear
cells), tissue biopsy samples (e.g., spleen, liver, bone marrow,
thymus, lung, kidney, brain, salivary glands, skin, lymph nodes,
and intestinal tract), and specimens acquired by pulmonary lavage
(e.g., bronchoalveolar lavage (BAL)). The source of cells and
tissues obtained from blood, biopsy, or other direct ex vivo means
can be any subject having tissue or cells with the desired
characteristics including subject with abnormal cells or tissues
which are characteristic of a disease or condition such as, for
example, a cancer patient. Thus, it is contemplated herein that the
subject can be a patient. It is also understood that there may be
times where one of skill in the art desires normal tissues or
cells. Thus, also disclosed herein are tissues and cells obtained
from a normal subject or from normal tissue wherein a "normal"
subject or tissue refers to any subject or tissue not suffering
from a disease or condition that affects the tissues or cells being
obtained. It is further understood that the subject can comprise an
organism such as a mouse, rat, pig, guinea pig, cat, dog, cow,
horse, monkey, chimpanzee or other nonhuman primate, and human.
[0050] Therefore, it is contemplated that exemplary cell types
comprise, at least partially and without limitation, cells having
the following exemplary morphologies: Acinar cells, Adipocytes,
Alveolar cells, Ameloblasts, Annulus Fibrosus Cells, Arachnoidal
cells, Astrocytes, Blastoderms, Calvarial Cells, Cancerous cells
(Adenocarcinomas, Fibrosarcomas, Glioblastomas, Hepatomas,
Melanomas, Myeloid Leukemias, Neuroblastomas, Osteosarcomas,
Sarcomas) Cardiomyocytes, Chondrocytes, Chordoma Cells, Chromaffin
Cells, Cumulus Cells, Endothelial cells, Endothelial-like cells,
Ensheathing cells, Epithelial cells, Fibroblasts, Fibroblast-like
cells, Germ cells, Hepatocytes, Hybridomas, Insulin producing
cells, Intersticial Cells, Islets, Keratinocytes, Lymphocytic
cells, Macrophages, Mast cells, Melanocytes, Meniscus Cells,
Mesangial cells, Mesenchymal Precursor Cells, Monocytes,
Mononuclear Cells, Myeloblasts, Myoblasts, Myofibroblasts, Neuronal
cells, Nucleus cells, Odontoblasts, Oocytes, Osteoblasts,
Osteoblast-like cells, Osteoclasts, Osteoclast precursor cells,
Oval Cells, Papilla cells, Parenchymal cells, Pericytess,
Peridontal Ligament Cells, Periosteal cells, Platelets,
Pneumocytes, Preadipocytes, Proepicardium cells, Renal cells,
Salisphere cells, Schwann cells, Secretory cells, Smooth Muscle
cells, Sperm cells, Stellate Cells, Stem Cells, Stem Cell-like
cells, Stertoli Cells, Stromal cells, Synovial cells, Synoviocytes,
T Cells, Tenocytes, T-lymphoblasts, Trophoblasts, Urothelial cells,
Vitreous cells, and the like; said cells originating from, for
example and without limitation, any of the following tissues:
Adipose Tissue, Adrenal gland, Amniotic fluid, Amniotic sac, Aorta,
Artery (Carotid, Coronary, Pulmonary), Bile Duct, Bladder, Blood,
Bone, Bone Marrow, Brain (including Cerebral Cortex), Breast,
Bronchi, Cartilage, Cervix, Chorionic Villi, Colon, Conjunctiva,
Connective Tissue, Cornea, Dental Pulp, Duodenum, Dura Mater, Ear,
Endometriotic cyst, Endometrium, Esophagus, Eye, Foreskin,
Gallbladder, Ganglia, Gingiva, Head/Neck, Heart, Heart Valve,
Hippocampus, Iliac, Intervertebral Disc, Joint, Jugular vein,
Kidney, Knee, Lacrimal Gland, Ligament, Liver, Lung, Lymph node,
Mammary gland, Mandible, Meninges, Mesoderm, Microvasculature,
Mucosa, Muscle-derived (MD), Myeloid Leukemia, Myeloma, Nasal,
Nasopharyngeal, Nerve, Nucleus Pulposus, Oral Mucosa, Ovary,
Pancreas, Parotid Gland, Penis, Placenta, Prostate, Renal,
Respiratory Tract, Retina, Salivary Gland, Saphenous Vein, Sciatic
Nerve, Skeletal Muscle, Skin, Small Intestine, Sphincter, Spine,
Spleen, Stomach, Synovium, Teeth, Tendon, Testes, Thyroid, Tonsil,
Trachea, Umbilical Artery, Umbilical Cord, Umbilical Cord Blood,
Umbilical Cord Vein, Umbilical Cord (Wartons Jelly), Urinary tract,
Uterus, Vasculature, Ventricle, Vocal folds and cells, and the
like; said tissues which originate, for example and without
limitation, in any of the following species: Baboon, Buffalo, Cat,
Chicken, Cow, Dog, Goat, Guinea Pig, Hamster, Horse, Human, Monkey,
Mouse, Pig, Quail, Rabbit, and the like.
[0051] Referring to FIGS. 1A and 1B, a view of one embodiment of
the bioreactor system is illustrated. In one aspect, the bioreactor
system 2 comprises at least one individual culture chamber 10,
which is defined therein a cell module 12. The dimensions and
overall size of a cell module 12, and culture chamber 10, are not
critical to the invention. In general, a cell module 12 can be of a
size so as to be handled and manipulated as desired, and so as to
provide access to the culture chambers either through disassembly
of the device, through a suitably located access port, or according
to any other suitable method. As one skilled in the art will
appreciate, the culture chamber 10 defined by the module 12 can
generally be of any size as long as adequate for the assigned task.
In one aspect, nutrient flow can be maintained throughout a
three-dimensional cellular construct growing in the culture chamber
10, so as to prevent cell death at the construct center due to lack
of nutrient supply.
[0052] Thus, in one aspect, one embodiment is a cell module 12.
Though each cell module 12 of the embodiment illustrated in FIG. 1
can comprise a single culture chamber 10, or, optionally, a single
cell module 12 can comprise multiple culture chambers. In the
latter aspect, each culture chamber of the module can comprise
individual access ports (described further below), so as to provide
individualized flow through each culture chamber and independent
control of the local environmental conditions in each culture
chamber. While the materials from which the module 12 can be formed
can generally be any moldable or otherwise formable material, the
surface of the culture chamber 10, as well as any other surfaces of
the module that may come into contact with the cells, nutrients,
growth factors, or any other fluids or biochemicals that may
contact the cells, should be of a suitable sterilizable,
biocompatible material. In one particular embodiment, components of
the system can also be formed so as to discourage cell anchorage at
the surfaces.
[0053] It is also contemplated herein that the cell module 12 and
the components that make up the cell module 12 can be constructed
from a single mold rather than attaching individual pieces. That
is, disclosed herein are bioreactor systems wherein each cell
module comprises a monolithic construction. The advantage of such
construction provides increased sterility and removes possibilities
of leaks forming. Thus, in one aspect, the cell module 12 can be
constructed of any material suitable to being formed in a mold.
[0054] In one embodiment two cell modules 12 can be selectively
coupled via a compression fitting so form two culture chambers 10
that are adjoined and are in selective biological communication
with each other. Thus, in one aspect, the cell modules 12 can
comprise a means for sealingly engaging the top surface of one
module with the top surface of another module. It is understood
that once fully engaged, the two cell modules can selectively, and
optionally releasably, lock into place. In one aspect, it is
contemplated that the means for sealingly engaging the respective
cell modules will cause a compressive force to be effected on the
adjoined surfaces of the respective modules. It is understood that
there are many means for sealingly engaging two cell modules 12.
One such method is shown in FIG. 1A. In this aspect, a male
compression fitting 35 can be configured to sealingly engage the
fitting 36 to form a compression fitting. In one aspect, the
fitting 36 can have a raised portion and the male compression
fitting 35 an indentation that when aligned form a lock. It is
understood that such an engagement means could be engaged using a
press and twisting motion. It is further understood that said
engagement means could be disengaged by twisting in the opposite
direction. It is further understood that the cell module 12
comprises both the male compression fitting 35 and female fittings
36 on the same or opposing faces of the cell module 12. For
example, the cell module 12 can comprise male compression fittings
on one face and female fittings 36 on the opposite face.
Alternatively, the cell module 12 can comprise male compression
fittings 35 and female fittings 36 on the same face. It is
understood that the placement of the male fittings 35 and female
fittings 36 is such that compression and stability are maximized,
for example, with male compression fittings 35 being at opposite
corners or sides from each other but adjacent to female fittings 36
which are on opposite sides or corners from each other.
[0055] Alternatively, the two cell module system can comprise a
first cell module 12 and a second cell module 12, wherein the first
and second module comprise identical cell chambers 10, inlets, and
outlets, but wherein the first cell module 12 comprises one or more
male compression fittings and the second cell module 12 comprises
one or more female fittings. For example, the first cell module 12
can comprise only male compression fittings 35 and the second cell
module 12 can comprise only female fittings 36. In an alternative
example, the top surface of the first cell module 12 can comprise a
raised perimeter with a convex bevel located at the mid point to
three fourths point on the interior wall of the raised perimeter.
The top surface of the second cell module 12 can have a perimeter
relief that is of a depth to receive the male fitting on the first
cell module. Additionally, the relief on the second cell module 12
can have a concave indentation which can form a lock when the
convex bevel of the first cell module 12 is engaged. Similarly, the
first and second cell modules 12 can be threaded in such a manner
to allow the first module to be screwed down on the second
module.
[0056] Thus, in one aspect a cell culture system can comprise first
and second cell modules 12 capable of engaging wherein the first
and second cell module are identical and interchangeable. In
another aspect, the cell culture system can comprise a first and
second cell module 12, wherein the first and second cell modules
are not identical or interchangeable but capable of being
engaged.
[0057] It is further contemplated that the cell culture systems
disclosed herein can comprise one or more first and second cell
modules. The cell culture systems can have cell modules 12
independently controlled or serially linked through the outlet of
one first and second cell module to the inlet of a second first and
second cell module. The connections of inlets and outlets to media
source, reagents, or flow source can be regulated by valves or
linked directly to said source. Alternatively when serial linking
is used, the outlet of one cell module 12 can be directly linked to
the inlet of a second cell module 12 or have a controlled
connection such as with a valve.
[0058] The culture chamber 10 can generally be of a shape and size
so as to cultivate living cells within the chamber. In one
preferred embodiment, culture chamber 10 can be designed to
accommodate a biomaterial scaffold within the culture chamber 10,
while ensuring adequate nutrient flow throughout a cellular
construct held in the culture chamber 10. For instance, a culture
chamber 10 can be between about 3 mm and about 10 mm in any cross
sectional direction. In another embodiment, the culture chamber can
be greater than about 5 mm in any cross sectional direction. For
instance, the chamber can be cylindrical in shape and about 6.5 mm
in both cross sectional diameter and height. The shape of culture
chamber 10 is not critical to the invention, as long as flow can be
maintained throughout a cellular construct held in the chamber.
[0059] It is understood that the formation of the culture chamber
creates a volumetric reservoir or a size determined by the cross
sectional direction and depth of the chamber. Accordingly, it is
understood that the disclosed culture chambers 10 can be between 1
.mu.L and 50 mL, 50 .mu.L and 1 mL, 100 .mu.L and 500 .mu.L, or 250
.mu.L or any volume therebetween. Typically the culture chamber is
circular or oval in cross sectional shape. However, it is further
understood that the cross sectional shape of the culture chamber 10
can also be hexagonal, heptagonal, octagonal, nonagonal, decagonal,
hendecagonal, dodecagonal, or larger polygon in shape.
Additionally, it is contemplated herein that the closed end of a
cell culture chamber 10 can be flat or convex. It is understood
that fewer angles and abrupt changes in plane encourages cells to
avoid adhering to the walls of the culture chamber and reduce
turbulence of fluids passing through the chamber. Thus, it is
contemplated herein that the shape of the culture chamber can be
selected based on the particular characteristics one of skill in
the art desires to replicate.
[0060] In one aspect, the culture chamber 10 is defined by an open
end port on the top surface of the cell module 12 and a closed end
on the bottom surface of cell module 12. The open end allows for
the addition for cell anchorage and cells and can be sealed by a
membrane 23 (see FIG. 2). It is also contemplated that the culture
chamber can be open at both ends and, in this aspect, the open ends
of the culture chamber 10 are defined in the respective top and
bottom surfaces of the cell module 12. In another aspect the
culture chamber 10 is defined by an opened end port on both the top
and bottom surface. As one skilled in the art will appreciate, when
the culture chamber 10 is defined by two opened ports, the open
ended ports can be closed by mating the cell module 12 with a
second cell module 12 and placing a membrane between the respective
cell culture chambers 10. Thus, in another aspect, disclosed herein
are bioreactor systems further comprising at least one third cell
module, wherein the third cell module comprises a cell chamber open
at both ends, wherein the cell chamber of the third cell module is
closed by sealingly engaging the first and second cell modules on
opposite faces of the third cell module 12.
[0061] In another aspect, the system can comprise a material
composition. For example, it is contemplated that the material
composition can be configured to serve as a cell anchorage that can
be contained in the culture chamber 10. The term "cell anchorage"
as utilized herein refers to one or more articles upon which cells
can attach and develop. For instance, the term "cell anchorage" can
refer to a single continuous scaffold, multiple discrete scaffolds,
or a combination thereof. The terms "cell anchorage," "cellular
anchorage," and "anchorage" are intended to be synonymous. It is
contemplated that any suitable cell anchorage as is generally known
in the art can be located in the culture chamber 10 to provide
anchorage sites for cells and to encourage the development of a
three-dimensional cellular construct within the culture chamber
10.
[0062] For purposes of the present disclosure, the term continuous
scaffold is herein defined to refer to a construct suitable for use
as a cellular anchorage that can be utilized alone as a single,
three-dimensional entity. A continuous scaffold is usually porous
in nature and has a semi-fixed shape. Continuous scaffolds are well
known in the art and can be formed of many materials, e.g., coral,
collagen, calcium phosphates, synthetic polymers, and the like, and
are usually pre-formed to a specific shape designed for the
location in which they will be placed. Continuous scaffolds are
usually seeded with the desired cells through absorption and
cellular migration, often coupled with application of pressure
through simple stirring, pulsatile perfusion methods or application
of centrifugal force.
[0063] Discrete scaffolds are smaller entities, such as beads,
rods, tubes, fragments, or the like, for example tubes for the
formation of vascular tubes. When utilized as a cellular anchorage,
a plurality of identical or a mixture of different discrete
scaffolds can be loaded with cells and/or other agents and located
within a void where the plurality of entities can function as a
single cellular anchorage device. Exemplary discrete scaffolds
suitable for use in the present invention that have been found
particularly suitable for use in vivo are described in U.S. Pat.
No. 6,991,652, which is incorporated herein in it's entirety by
reference. A cellular anchorage formed of a plurality of discrete
scaffolds can be preferred in certain embodiments of the bioreactor
system as discrete scaffolds can facilitate uniform cell
distribution throughout the anchorage and can also allow good flow
characteristics throughout the anchorage as well as encouraging the
development of a three-dimensional cellular construct.
[0064] In one embodiment, for instance when considering a cellular
anchorage including multiple discrete scaffolds, the anchorage can
be seeded with cells following assembly and sterilization of the
system. For example, an anchorage including multiple discrete
scaffolds can be seeded in one operation or several sequential
operations. Optionally, the anchorage can be pre-seeded, prior to
assembly of the system. In one aspect, the anchorage can comprise a
combination of both pre-seeded discrete scaffolds and discrete
scaffolds that have not been seeded with cells prior to assembly of
the bioreactor system.
[0065] The good flow characteristics possible throughout a
plurality of discrete scaffolds can also provide for good transport
of nutrients to and waste from the developing cells, and thus can
encourage not only healthy growth and development of the individual
cells throughout the anchorage, but can also encourage development
of a unified three-dimensional cellular construct within the
culture chamber. Thus, it is understood the scaffolds and matrices
utilized herein can comprise shapes akin to real tissues with
meaningful volumes.
[0066] The materials that are used in forming an anchorage can
generally be any suitable biocompatible material. In one
embodiment, the materials forming a cellular anchorage can be
biodegradable. For instance, a cellular anchorage can comprise
biodegradable synthetic polymeric scaffold materials such as, for
example and without limitation, polylactide, chondroitin sulfate (a
proteoglycan component), polyesters, polyethylene glycols,
polycarbonates, polyvinyl alcohols, polyacrylamides, polyamides,
polyacrylates, polyesters, polyetheresters, polymethacrylates,
polyurethanes, polycaprolactone, polyphophazenes, polyorthoesters,
polyglycolide, copolymers of lysine and lactic acid, copolymers of
lysine-RGD and lactic acid, and the like, and copolymers of the
same. Optionally, an anchorage can comprise naturally derived
biodegradable materials including, but not limited to, chitosan,
agarose, alginate, collagen, hyaluronic acid, and carrageenan (a
carboxylated seaweed polysaccharide), demineralized bone matrix,
and the like, and copolymers of the same.
[0067] It is contemplated that exemplary scaffold materials can
comprise, at least partially and without limitation: Collagen;
PLA/poly(lactide); PLGA/poly(lactic-co-glycolic acid;) Chitosan;
PCL/poly(.epsilon.-caprolactone); Alginate/sodium alginate;
PGA/poly(glycolide); Hydroxyapatite; Gelatin; Matrigel.TM.; Fibrin;
Acellular/Allogenic Tissue (all forms); Hyaluronic Acid;
PEG/poly(ethylene glycol); Peptide; Silk Fibroin; Agarose/Agar;
Calcium phosphate; PU/polyurethane; TCP/tri calcium phosphate;
Fibronectin; PET/poly(ethylene terephthalate); Bioglass;
PVA/Polyvinyl alcohol; Laminin; GAG/glycosaminoglycan; Cellulose;
Titanium; DBP/demineralized bone powder; Silicone;
PEGDA/PEG-diacrylate; Fibrinogen; Acellular/Allogenic Tissue-SIS;
PDMS/polydimethylsiloxane; Acellular/Allogenic Tissue-Bone; ECM (in
situ derived); Polyester; Elastin; PS/polystyrene; Glass;
PBT/polybutylene terephthalate; Dextran; PEG/poly(ethylene
glycol)-other modified forms; PES/polyethersulfone;
PLL/poly-l-lysine; MWCNT/multiwalled carbon nanotube;
PHBV/poly(hydroxybutyrate-co-hydroxyvalerate); Coral; Starch;
PPF/poly(propylene fumarate);
PLCL/poly(lactide-co-.epsilon.-caprolactone); Chondroitin Sulfate;
PAM/polyacrylamide; PC/polycarbonate; PEUU/poly(ester
urethane)urea; Calcium carbonate; Atelocollagen;
PHB/poly(hydroxybutyrate); Polyglactin; Gelfoam.RTM.;
Acellular/Allogenic Tissue-Vasculature; PuraMatrix.TM.;
PAA/poly(acrylic acid); PA/polyamide (Nylon); Clot;
PDO/polydioxanone; PMMA/poly(methyl methacrylate) (acrylic);
Acellular/Allogenic Tissue-Heart Valve; PHEMA/poly(hydroxyethyl
methacrylate); PVF/polyvinyl formal; PGS/poly(glycerol sebacate);
PEO/poly(ethylene oxide); Acellular/Allogenic Tissue-Cartilage;
Pluronic.RTM. F-127; PHBHHx/PHB-co-hydroxyhexanoate; PHP/polyHIPE
polymer; Polyphosphazene; Silicate; Poly-D-lysine; Poly
peptide/MAXI; Aluminum oxide; PTFE/polytetrafluoroethylene;
Silica/silicon dioxide; SWCNT/single-walled carbon nanotube;
Cytomatrix.RTM. (Tantalum); PLG/poly(L-lactide-glycolide);
ORMOCER.RTM.; POSS/polyhedral oligomeric silsesquioxanes;
Acellular/Allogenic Tissue-Tendon; HEWL/Hen egg white lysozyme;
Polyelectrolyte; Polyamidoamine; POC/poly(octanediol citrate);
PEI/polyethyleneimine; Hyaff-11.RTM.; PTMC/poly(trimethylene
carbonate); PAAm/Poly(allylamine); Polyester utethane; Lactose;
PNiPAAm/poly(N-isopropylacrylamide); Polyurethane-urea; Keratin;
Cyclic Acetal; NiPAAm; Poly HEMA-co-AEMA; PE/polyethylene (all
forms); PLDLA/poly(L/D)lactide; Vitronectin; PDL/poly-D-lysine;
Corn starch; TMP/trimethylolpropane; Poloxamine;
Acellular/Allogenic Tissue-Skin; Gellan gum; PEMA/poly(ethyl
methacrylate); Tantalum; DegraPol.RTM.; Silastic; Akermanite;
Polyhydroxyalkanoate; AlloDerm.RTM.; Polyanhydrides; Zirconium
Oxide; Polyether; TMC/trimethylene carbonate; Sucrose;
PEVA/poly(ethylene-vinyl alcohol); PMAA/poly(methacrylic acid);
Hydrazides; Poly(diol citrate); PVDF/polyvinylidene fluoride;
COBB/Ceramic Bovine Bone; PVLA/polyvinylbenzyl-D-lactoamide;
PCU/poly(carbonate-urea)urethane; MBV; Chitin; Synthetic elastin;
PBSu-DCH/diisocyanatohexane-extended poly(butyl); PANI/polyaniline;
Polyprenol; Zein; Egg Shell Protein; EVA/Ethylene Vinyl Acetate;
Gliadin; HPMC/hydroxypropyl methylcellulose; PE/phthalate ester;
Thrombin; PP/Polypropylene; OptiCell.TM.; PEEP/poly(ethyl ethylene
phosphate); OCP/Octacalcium Phosphate; PEA/poly(ester amide);
Aggrecan; Graphite; NovoSorb.TM.; PLO/poly-L-ornithine;
DOPE/dioleoyl phosphatidylethanolamine; ELP/Elastin-like
polypeptide; LDI/lysine diisocyanate; PPC/poly (propylene
carbonate); Plasma; Fe(CO)(5)/Iron pentacarbonyl; Asbestos;
PPE/polyphosphoester; Azoamide; Triacrylate; PRP/platelet-rich
plasma; Dextran (modified forms);
PGSA/poly(glycerol-co-sebacate)-acrylate; Polyorthoester;
SPLE/sodium polyoxyethylene lauryl ether sulfate; Methacryloyloxy;
TGA/thioglycolic acid; PCTC/poly(caprolactone-co-trimethylene
carbonate; SU-8; SLG/sodium N-lauroyl-L-glutaminate; Polysulfone;
Phosphosphoryn; HEA/hydroxyethyl acrylate; PSSNa/poly(sodium
styrene sulfonate); Carbon Foam; PFOB/perfluorooctyl bromide;
Lecithin; Mebiol.RTM.; BHA/butylated hydrorxyanisole;
Surgisis.RTM.; OsSatura.TM.; Skelite.TM.; Cytodex.TM.;
COLLOSS.RTM.; E; Magnesium; PAN/polyacrylonitrile;
HPMA/hydroxypropyl-methacrylamide; Lutrol.RTM. F127;
PDTEc/poly(desaminotyrosyl-tyrosineethyl esterc; Rayon (commercial
product); Organo Clay; Portland Cement; Xyloglucan; Vaterite
Composites (SPV); PRx/polyrotaxane; AW-AC/anti-washout apatite
cement; Starch acetate; Nicotinamide; POR/poly-L-ornithine
hydrobromide; AM-co-VPA/acrylamide-co-vinyl phosphonic acid;
Calcium Silicate; Carbylan GSX; Colchicine;
GPTMS/glycidoxypropyltrimethoxysilane; Phosphorylcholine;
PLE/polyoxyethylene lauryl ether; Tartaric acid;
HPA/hydroxyphenylpropionic acid;
PLVA/poly-N-p-vinylbenzyl-D-lactonamide;
PEOT/polyethyle-neoxide-terephtalate; Adipose Tissue Powder;
SLS/sodium lauryl sulfate; KLD-12 peptide;
PDTOc/poly(desaminotyrosyl-tyrosine octylesterc;
Si-TCP/silicate-substituted tricalcium phosphate;
PCLF/polycaprolactone fumarate;
PAMPS/poly(acrylamidomethylpropanesulfonicsodiu; Bio-Oss.RTM.;
MGL/mono glyceryl laurate; DMA/fullerene C-60 dimalonic acid;
THF/tetrahydrofuran; Polyphosphoester; Paper; Calcium-silicon;
PPD/poly-p-dioxanone; BME/Basement Membrane Extract (generic); and
OPF/oligo[poly(ethylene glycol) fumarate].
[0068] A biodegradable anchorage can comprise factors that can be
released as the scaffold(s) degrade. For example, an anchorage can
comprise within or on a scaffold one or more factors that can
trigger cellular events. According to this aspect, as the
scaffold(s) forming the cellular anchorage degrades, the factors
can be released to interact with the cells. Referring again to
FIGS. 1A and 1B, in those embodiments including a cellular
anchorage formed with a plurality of discrete scaffolds, a
retaining mesh 14 can also be located within the culture chamber
10. The retaining mesh 14 can be formed of any suitable
biocompatible material, such as polypropylene, for example, and can
line at least a portion of a culture chamber 10, so as to prevent
material loss during media perfusion of the culture chamber 10.
Alternatively, the retaining mesh can be a located at the opening
of the inlet and outlet of the culture chamber 10. The retaining
mesh 14 can be an integral part of the inlet and outlet so as to be
made of the same material and in the same form as the cell module
12 such that the retaining mesh 14 is not removable for the cell
module 12. A porous retaining mesh 14 can generally have a porosity
of a size so as to prevent the loss of individual discrete
scaffolds within the culture chamber 10. For example, a retaining
mesh 14 can have an average pore size of between about 10 .mu.m and
about 1 mm, between about 50 .mu.m and about 700 .mu.m, or between
about 150 .mu.m and about 500 .mu.m.
[0069] Upon assembly of the bioreactor system, two (or more)
culture chambers 10 can be aligned so as to be immediately adjacent
to one another. In one aspect, to help create a fluid-proof seal of
the system, a gasket 16 and a permeable membrane portion 23 can be
positioned between the adjoining surfaces of the cell modules to
selectively prevent fluid leakage from between the respective open
ends (the respective ports of the culture chambers). In one aspect,
the gasket 16 and the membrane portion 23 can be formed as a single
integrated structure. It is contemplated that the membrane portion
23 of gasket 16 can be positioned between the respective ports
adjoined culture chambers 10 and can have a porosity that can allow
biochemical materials, for instance growth factors produced by a
cell in one chamber, to pass through the membrane and into the
adjoining chamber, where interaction can occur between the
biochemical material produced in the first chamber and the cells
contained in the second chamber.
[0070] Optionally, the two or more culture chambers 10 can be
aligned with only the membrane portion 23 positioned between the
adjoining surfaces of the cell modules and in over/underlying
relationship to the respective ports of the adjoining chambers. In
operation, by interlocking two cell modules 12, the membrane
portion 23 can be compressed therebetween the adjoining surface to
effect a fluid-proof seal around the ports of the culture chamber
10. Thus, in this exemplary aspect, the membrane acts as a gasket.
In a further alternative embodiment, at least one of the cell
modules can comprise a raised convex concentric ring which
encircles the open end, the port, of the culture chamber 10 on the
top surface of the cell module 12. In this aspect, when the two
cell modules are interlocked the added pressure placed on the
raised area effects a seal on the membrane that is interposed
therebetween. In a further aspect, the cell modules can comprise a
male and female cell module where the male module comprises a
raised convex concentric ring which encircles the open end, the
port, of the culture chamber 10 on the top surface of the cell
module 12 and the female cell module comprises a concave concentric
ring which encircles the culture chamber 10 on the top surface of
the female cell module 12. When the male and female cell modules
are engaged, the male and female rings form a bight in the membrane
creating a seal and aid in alignment of the culture chambers
12.
[0071] In bioreactor systems where a membrane is used without a
gasket, the membrane becomes a gasket by compressing the membrane
under the compression formed by the interlocking of two or more
cell modules 12. Therefore, it is understood and herein
contemplated that the membrane can comprise a compressible material
that is conducive to the formation of a gasket. Such materials are
well known to those of skill in the art.
[0072] In various aspects, it is contemplated that the membrane 23
can be a solid, non-porous, or semi-permeable (i.e., porous)
membrane. The porosity can be small enough to prevent passage of
the cells or cell extensions from one chamber to another. In
particular, the membrane porosity can be predetermined so as to
discourage physical contact between the cells held in adjacent
chambers, and thus maintain isolation of the cell types. Suitable
porosity for a membrane can be determined based upon specific
characteristics of the system, for instance the nature of the cells
to be cultured within the chamber(s). Such determination is well
within the ability of one of ordinary skill in the art and thus is
not discussed at length herein.
[0073] Additionally, the membrane 23 can comprise not only material
that affects the transmission of physical parameters, but optical
transmission as well. Thus, contemplated herein are membranes 23
wherein the membrane only allows the transmission of certain
wavelengths of light to pass from one side of the membrane to the
other or excludes specific wavelengths of light.
[0074] Alternatively, the membrane 23 can comprise a composite
structure of both porous and non-porous or solid membranes, which
allow the removal of one non-porous membrane while the other porous
membrane remains in place between the culture chambers 10. In one
aspect, the non-porous or solid membrane can be affixed to the
porous membranes and separated from the porous membrane without
needing to remove the semi-permeable membrane. Thus, the solid
membrane allows for separate culturing conditions and media usage;
whereas a porous membrane allows for the passage of biochemical
materials. In another aspect, the membrane 23 comprising a porous
and solid or nonporous membrane can be placed between adjoined
culture chambers to allow for separate culture conditions and after
a period of time the solid or non-porous membrane can be removed to
allow for passage of biochemical materials.
[0075] In another alternative, the membrane 23 can comprise a
biodegradable material. Through the use of a biodegradable material
for the membrane 23, porosity can be electively increased over the
course of the usage of the membrane. For example, a non-porous
membrane 23 made of biodegradable material can be used which
prevents the exchange of culture conditions. In operation, as the
material is used, the membrane degrades allowing for the exchange
of biochemical materials. In a further alternative, the membrane 23
can comprise biodegradable and non-biodegradable material such as a
porous non-biodegradable membrane where the pores are sealed with a
biodegradable material or coating. As the biodegradable material or
coating is dissolved, the non-degradable porous membrane is
revealed.
[0076] In another embodiment the cells contained in a culture
chamber 10 can be maintained at a distance from the membrane 23 to
discourage physical contact between cells held in adjacent culture
chambers. For instance, in this example, retaining mesh 14 can be
located between a cell anchorage held in a culture chamber and the
membrane located between two adjacent chambers. The width of the
retaining mesh 14 can prevent contact of the cells with the
membrane 23. Optionally, the retaining mesh 14 can be at a distance
from the membrane 23, providing additional separation between the
membrane 23 and cells held in the culture chamber 10. In another
embodiment, a continuous scaffold can be located in a culture
chamber 10 at a distance from the membrane 23 so as to discourage
physical contact between the cells held in the culture chamber and
the membrane 23. While a preferred distance between the membrane 23
and cells held in the chamber will vary depending upon the specific
characteristics of the system as well as the cells to be cultured
in the system, in general, the distance between the two can be at
least about 100 microns.
[0077] Each culture chamber 10 of the system can comprise the
capability for independent flow control through the chamber. For
example, and referring again to FIGS. 1A and 1B, each individual
culture chamber 10 can comprise an inlet 8 and an outlet 9 through
which medium can flow. In this exemplary aspect, the inlet 8 and
outlet 9 can be connected to medium perfusion tubing via
quick-disconnect luers 18 and stopcock valves, but this particular
arrangement is not a requirement of the invention, and any suitable
connection and perfusion system as is generally known in the art
can be utilized. In another embodiment, the connection can be an
integral portion of a single formed module 12. For example, the
luers 18 can be formed at the outward ends of the inlet 8 and
outlet 9 as shown in FIG. 1. It is understood and herein
contemplated that other means for attaching tubing and stopcock
valves are well known in the art and can be used in the present
invention as an alternative to a luer lock. Such attachment
mechanisms comprise but are not limited to compression fittings,
threaded fittings, and friction.
[0078] It is contemplated that at least portions of the respective
inlet 8 and outlet 9 can be straight or can comprise one or more
bends. It is understood that the inlet 8 and outlet 9 do not have
to line up within the culture chamber 10, but can be situated at
opposing ends (i.e., one at the top and another the bottom as
reflected in the middle module in FIG. 4). It is contemplated that
the respective shapes of the inlet and outlet can be configured to
affect the desired flow characteristics within the chamber.
[0079] Referring to FIG. 2, one aspect of a pair of adjoined
modules 12 following assembly is shown. As can be seen, the
embodiment comprises two modules 12, each of which comprises a
single culture chamber 10. Upon assembly, the two culture chambers
10 are aligned with the permeable membrane portion 23 of gasket 16
positioned therebetween the ports of the culture chambers. In this
particular embodiment, a plurality of discrete scaffolds 15 has
been located within each of the two culture chambers 10 as a
cellular anchorage. In addition, each culture chamber 10 can be
lined with a retaining mesh 14, as shown. Upon assembly, desired
media can be independently perfused through each culture chamber 10
via the separate inlets 8 and outlets 9.
[0080] FIG. 3 illustrates one embodiment of a bioreactor system
according to the present invention. This aspect comprises two
assembled modules 12, such as those illustrated in FIG. 2, each in
line in a flow circuit that is completely independent of the other
that includes a pump 17, for instance a peristaltic pump and a
media container 19. In this aspect, gas exchange can be facilitated
by two methods, including a first method utilizing a coiled length
of a gas permeable tubing 18 such as, for example, a platinum-cured
silicone tubing, as well as a second method including an air filter
22 located, in this aspect, at the media container 19. Any gas
exchange method as is known can alternatively be utilized,
however.
[0081] One skilled in the art will appreciate that one of the many
benefits of the disclosed invention is the versatility of the
system and cell modules. For example, in the bioreactor system
illustrated in FIG. 3, the design attributes allow convenient and
flexible reversal of the perfusion flow for a particular
experimental protocol.
[0082] The bioreactor systems are not limited to single culture
bioreactor systems or co-culture bioreactor systems in which only
two independently controlled culture chambers are located adjacent
to one another. In other aspects, additional cell modules can be
selectively added to the bioreactor system such that a single
culture chamber can be in selective biochemical communication with
the contents of two or more other culture chambers. For example, a
third chamber can house cells that can be in biochemical
communication with the first culture chamber, optionally with a
membrane separating the first and third chambers, though this
aspect is not a requirement of the system such as for example in
the instance stacked arrangement as illustrated in FIG. 4.
[0083] In one aspect, it is contemplated that the number of
additional third chambers, which can be interior cell modules 12,
which can be employed is not limited to a single interior cell
module (i.e., three total cell modules 12 (one interior cell module
and two end cell modules)), but can comprise 2, 3, 4, 5, 6, 7, 8,
9, 10 or more interior cell modules (i.e., 4, 5, 6, 7, 8, 9, 10,
11, 12, or more total cell modules 12, respectively). Thus, as a
further embodiment disclosed herein are cell modules 12 that can be
utilized as interior cell modules in a stacked configuration. Such
interior cell modules 12 can comprise two top surfaces. Because the
interior cell modules 12 comprise two top surfaces, the culture
chamber 10 of these modules is open at both ends to allow for
biochemical passage between the interior module and each of the
exterior modules. As with the exterior cell modules, the top
surface of the interior cell modules 12 can comprise means of
sealingly engaging the top surface of other cell modules 12. Thus,
it is contemplated herein that both of the top surfaces of the
interior module 12 can comprise female fittings, male compression
fittings, or a combination of both on each surface. Moreover, it is
understood that the top surfaces of the interior cell module 12 can
be identical or comprise an orientation with a male and a female
end.
[0084] In another embodiment, one or more of the culture chambers
of the system can be designed so as to provide the capability of
subjecting the interior of the culture chamber to variable dynamic
mechanical stimuli such as mechanical loading or variation in fluid
flow through the culture chamber in order to vary the associated
stress on the developing cells. Additionally one or more culture
chambers of the system can be designed as to provide the capability
of subjecting the interior of the culture chamber to electric
current or a light source. Such an embodiment can be utilized to,
for instance, trigger differentiation and development of stem cells
contained in a culture chamber. In addition, cyclical hydrostatic
loading patterns can be established, if desired, by simply cycling
the pressurized fluid through the pressure chamber 24 through use
of a solenoid valve and a time-delay relay, computer automation, or
any other method that is generally known to one of ordinary skill
in the art. Also, electrical currents can be provided through the
use of an electrical probe in culture chamber of an adjacent cell
module 12.
[0085] For example, according to one aspect, as illustrated in FIG.
5, a cell module 12 can be located immediately adjacent to a second
cell module (not shown in FIG. 5), as described above. In addition,
the cell module 12 can, on a second side of the module 12, be
aligned with a pressure module 32 that can be utilized to vary the
hydrostatic pressure on the contents of the culture chamber 10.
According to this embodiment, the culture chamber 10 can be aligned
with a pressure chamber 24 defined by pressure module 32, and the
two adjacent chambers 10, 24 can be separated by an impermeable
diaphragm 26. The introduction of pressurized fluid, e.g., air,
into the pressure chamber 24, can deflect the diaphragm 26, as
shown in FIG. 5B, and transfer the pressure to the volume of fluid
in the culture chamber 10. In one embodiment, fluid flow through
the culture chamber 10, as well as through other adjacent culture
chambers, can be stopped prior to pressurizing the system, so as to
develop a fixed volume of fluid within the affected portion of the
system.
[0086] In another embodiment, each cell module can be designed to
allow for the direct sampling and observation of the culture
chamber such as optical and spectrophotometric analysis. Such
designs can comprise but are not limited to optically transmissive
culture chamber 10 such that the bottom of the well of the culture
chamber comprises optical glass or plastic (i.e., a cell module
comprising optically transmissible material). Thus, a microscope
can directly visualize the culture chamber 10 by focusing through
the optically transmissive culture chamber on the bottom side of
the cell module 12. Additionally, high resolution and
three-dimensional imaging modalities including, but not limited to,
laser confocal microscopy, multiphoton microscopy, optical
coherence tomography, and nuclear magnetic resonance can be used to
visualize the cell culture. The cell module 12 can be made from
opaque material, for example and without limitation, the cell
module can be made from opaque white material for luminescent
detection or opaque black material for fluorescent detection to
effectively limit endogenous background signal. Additionally, the
cell module 12 can comprise transluscuent, photoreactive, or
optically filtering glass or polymers. For example, the cell module
12 can comprise a polymer that allows the passage of certain
wavelengths of light or filters out ultraviolet light. Similarly,
the culture chambers can comprise an inlet through which an
analytical probe may be inserted.
[0087] It is understood that when sampling an observation of
culture chamber is undertaken, it can be useful to provide a
mechanism for securing the cell module 12 on any device used for
observation such as a microscope or plate reader such as a
spectrometer. Thus, disclosed herein are cell modules mounted in a
microscope stage adaptor. Also disclosed are cell modules 12
mounted to well plate adaptor for use in instrumentation, i.e.,
spectrometer plate reader.
[0088] In yet another embodiment, an electrical current can be
provided to the interior of a culture chamber 10 through the use of
a piezoelectric membrane 23. The piezoelectric membrane upon
compression generates an electric current which is supplied to the
culture chamber. In an alternative aspect, the electric current can
be supplied through the use of cell anchorage constructed with a
piezoelectric material. For example, as pressure is applied through
the introduction of a pressurized fluid, an electrical current is
emitted from the cell anchorage. Alternatively, the bioreactor
systems disclosed herein can comprise an electrically charging or
piezoelectric scaffold.
[0089] In various aspects, multiple independent bioreactor systems
can be provided that can incorporate various combinations of
experimental stimuli, which can provide real time comparisons of
the differing stimuli on the developing cellular constructs.
[0090] In a further aspect, a bank of multiple and identical
systems can be established that can provide replication of a single
experimental procedure and/or to provide larger cumulative amounts
of the product cells that are grown, developed or otherwise
produced within each of the individual culture chambers.
[0091] It is contemplated that the disclosed culture systems can be
incorporated into a singular instrument to allow for the control of
temperature, gas exchange, media contents and flow rate, external
and mechanical stresses, and endpoint analysis. The instrumentation
can comprise multiple modular components each designed to
accomplish a specific task. Thus, for example, the disclosed
instrumentation can comprise one or more of a means for seeding
cells onto anchorages, a means for controlling the flow of media, a
means for adding or changing media, a means for subjecting the
culture to mechanical stress or pressure, an analytical probe, and
a device for manipulating the parameters of the various modules as
well as collecting and analyzing data (for example, a computer and
a computer program designed to accomplish these tasks).
[0092] The culture systems disclosed herein have many uses known to
those of skill in the art. For example, the disclosed culture
systems and cell modules can be used in tissue engineering where a
3D bioreactor is useful to properly model tissue.
[0093] In a further aspect, it is contemplated that for tissue
passaging, a material composition comprising two or more materials
can be used. In one example, the material composition can comprise
a stiff culture material having substantially large porosity, such
as, for example and without limitation, having pores of average
size between 50 .mu.m and 2 mm, into which a soft culture material
has been introduced. "Stiff culture material" is defined herein as
tissue culture material having a tensile elastic modulus, or
Young's modulus, of about 1 GPa or greater and "soft culture
material is defined as tissue culture material having a tensile
elastic modulus, or Young's modulus, of about 500 MPa or less. In
one example, and without limitation, the stiff culture material can
be formed from metal, synthetic polymer, ceramic and the like. In
one example, and without limitation, the soft culture material can
be formed from a polymer of biological origin, a synthetic polymer,
or a combination of biological and synthetic polymers. The soft
culture material may then be formed as a hydrogel or an
uncrosslinked oligomers of polymers either synthetic or of natural
origin, and the like.
[0094] It is understood that as the bioreactor systems disclosed
herein are utilized for passaging cells in culture, that in one
aspect, the disclosed bioreactor systems comprise cells. It is
further understood that the cells attach to the culture material.
Thus, in one aspect, disclosed herein are bioreactor systems
further comprising cells attached to the culture material. In one
aspect, the cells are attached to the soft culture material. The
cells can be introduced to the soft culture material before or
after introduction of the soft culture material to the stiff
culture material. Thus, in one aspect, the cells are introduced to
the soft culture material and with the soft culture material
introduced to the stiff culture material. For example, the cells
can be encapsulated in a hydrogel soft culture material and then
introduced to the stiff culture material.
[0095] In one aspect, the soft culture material can be configured
or otherwise have a means for releasing the soft material from the
stiff culture material. In one exemplary aspect, the releasing
means can comprise chemical degradation or other change initiated
by light, temperature, pH, chemical catalyst, and the like.
[0096] In another aspect, the material composition may further
comprise a biocompatible aqueous solvent such as, for example and
without limitation, Minimum Essential Medium (MEM), developed by
Harry Eagle, and its many altered forms. In this aspect, it is
contemplated that the biocompatible aqueous solvent can provide the
basic benefits of cell culture media, which include, without
limitation, provision of nutrients and removal of cell waste.
[0097] Further, in addition to the usual or conventional soluble
factors known to be generally beneficial for the sustained culture
of cells, such as, for example and without limitation, Inorganic
Salts: CaCl2 (anhydrous), Fe(NO3)3.9H2O, MgSO4 (anhydrous), KCl,
NaHCO3, NaCl, NaH2PO4H2O; Amino Acids: L-Alanine, L-Arginine.HCl,
L-Asparagine.H2O, L-Aspartic Acid, L-Cysteine.HCl, L-Cystine.2HCl,
L-Glutamic Acid, L-Glutamine, Glycine, L-Histidine.HCl.H2O,
L-Isoleucine, L-Leucine, L-Lysine.HCl, L-Methionine,
L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,
L-Tyrosine.2Na.2H2O, L-Valine; Vitamins: L-Ascorbic Acid.Na,
D-Biotin, Choline Chloride, Folic Acid, myo-Inositol, Lipoic Acid,
Nicotinamide, D-Pantothenic Acid, (hemicalcium), Pyridoxine.HCl,
Riboflavin, Thiamine.HCl; Other: Adenosine, Cytidine,
2'-Deoxyadenosine, 2'-Deoxycytidine.HCl, 2'-Deoxyguanosine,
D-Glucose, Glutathione (reduced), HEPES, Phenol Red (Sodium Salt),
Pyruvic Acid, Sodium Pyruvate, Thioctic Acid, Thymidine, Uridine,
and the like, the aqueous solvent can provide specific soluble
factors and/or stimulants which are known to affect, e.g., either
increase, decrease or stabilize, cell proliferation within the
exemplary stiff/soft material composition. In another aspect, the
aqueous solvent may comprise oligomers or fragments of the soft
material that are known to effect cell attachment to the soft
material originally contained within the stiff culture material,
such as, for example and without limitation, natural materials
common to mammalian tissue such as collagen, chondroitin sulfate,
fibrin, fibrinogen, glycosaminoglycan, hyaluronic acid, keratin,
laminin, or thrombin; natural materials common to non-mammalian
tissue or derived from non-mammalian organisms such as chitin,
chitosan, dextran, starches or other polysaccharides, gelatin,
silks, or synthetic materials such as HEMA/hydroxyethyl
methacrylate, PCL/poly(.epsilon.-caprolactone), PEG/poly(ethylene
glycol), PEMA/poly(ethyl methacrylate), PEO/poly(ethylene oxide),
PEVA/poly(ethylene-vinyl alcohol), PGA/poly(glycolide),
PHEMA/poly(hydroxyethyl methacrylate), PLA/poly(lactide),
PLG/poly(L-lactide-glycolide), PLGA/poly(lactic-co-glycolic acid),
PLL/poly-l-lysine, PLLA/poly(L-lactic acid), PVA/Polyvinyl alcohol
and the like.
[0098] Optionally, the material composition can comprise a stiff
tissue culture material in the physical form of discrete beads or
microparticles to or within which a soft culture material has been
introduced. In one example, and without limitation, the stiff
culture material can be formed from metal, synthetic polymer,
ceramic and the like. In one example, and without limitation, the
soft culture material can be in the physical form of a hydrogel or
uncrosslinked oligomers of polymers either synthetic or of natural
origin, and the like. In one example, and without limitation, the
soft culture material can be formed from polymers of biological
origin, synthetic polymers, or combinations of synthetic and
biological polymers. As one skilled in the art will appreciate,
aside from this physical change in the initial physical form of the
stiff culture material, the material composition of this exemplary
aspect has the same properties and behavior as the aspect
previously disclosed.
[0099] In yet another aspect, a method of 3D cell passaging is
provided that comprises providing a population of cells to be
passaged and introducing the population of cells into the material
composition, that is comprised of the combination of stiff and soft
tissue cultural materials. In this method, it is contemplated that
at least a portion of the population of cells will attach to at
least portions of the soft culture material. It is further
contemplated that the cells can attach prior to or after
introduction of the soft culture material to the stiff culture
material. For example, the cells can be encapsulated into a soft
culture material such as a hydrogel and thereafter introduced to
the stiff culture material. In one aspect, it is further
contemplated that the "attached" population of cells can be
cultured under conditions that are typical for the growth of the
respective cells, i.e., using appropriate temperature, humidity,
and/or gas exchange. At a desirable and or predetermined time after
the cells attachment, the method can further comprise causing the
soft culture material to disassociate from the stiff culture
material, thereby releasing the soft culture material and the cells
from the stiff culture material of the material composition. In
another aspect, the method can further comprise dividing the
recovered cells, with or without remnants of the soft culture
material, into multiple populations and repeating the method using
the subdivided populations. It is of course contemplated that this
exemplary process can be done recursively.
[0100] In one exemplary aspect of the method of 3D cell passaging,
the at least one cell module can be preloaded with a predetermined
quantity of material composition, such as, the exemplary stiff/soft
culture material composition. It is contemplated that the material
composition can fill between about 5 and 100% of the available
space in the cell culture chamber. For example and without
limitation, in a configuration having a cell culture chamber with a
250 .mu.L volume, a material composition of between 12.5 .mu.L and
250 .mu.L can be utilized. It is also contemplated that the
exemplary stiff/soft culture material composition can have multiple
configurations to include, without limitation, providing a material
composition that allows for a 3D environment within the at least
one cell module of appropriate desired density and surface area to
support growth of the particular cells being cultured. Therefore,
it is contemplated that the matrix has the surface area to allow
for attachment of the cells as well as the surface area to allow
for proliferation of the cells and flow through of any growth
factors, nutrients, media, environmental factors, chemokines,
chemicals, cytokines, and the like to which it is desired the cells
be exposed. Optionally it is contemplated that the exemplary
stiff/soft culture material composition can provide a material
composition that allows for a 3D environment within the at least
one cell module of appropriate desired density, in which the matrix
upon which the population of cells attaches has less free space if
it is desirable to maintain a stable cell population rather than
proliferating the cell population.
[0101] Subsequently, an operator can couple the at least one cell
module into the bioreactor flow circuit and introduce the
population of cells into the at least one cell module. After
introduction of the cells and the attachment to the soft culture
material of the material composition, the cells can then be
cultured in the at least one cell module for a desired and or
predetermined period of time to effect either doubling or
maintenance of the introduced population of cells. It is
contemplated that, during the culture process, the operator can
affect cell behavior (e.g. differentiation, growth, metabolite,
vector production, and the like) by using media containing specific
soluble factors, such as, for example and without limitation,
inorganic salts, amino acids, vitamins, ribonucleosides,
deoxyribonucleosides, and the like, that can encourage a desired
outcome.
[0102] Upon the desired and or predetermined period of time
expiring, the operator can introduce a trigger or stimulus designed
to disassociate the soft culture material from the stiff culture
material of the material composition. In this aspect, the soft
culture material can be configured or otherwise have a means for
releasing the soft culture material from the stiff culture
material. In one exemplary aspect, the releasing means can comprise
chemical degradation or other change initiated by light,
temperature, pH, chemical catalyst, and the like. Therefore, it is
contemplated that the operator triggered stimulus can comprise,
without limitation, chemical stimuli, i.e., introduced into media
supplied to the at least one cell module, pH stimuli, thermal
stimuli, i.e., for soft culture material of the material
composition configured to fall apart or otherwise degrade upon
increase or decrease in temperature, light stimuli, and like
stimuli. Next, the operator can collect cells and soft culture
material fragments downstream of the at least one cell module.
Optionally, the operator can separate the cells from the soft
material fragments or can allow the cells and the soft culture
materials to remain unseparated.
[0103] Optionally, it is contemplated that the collected cells can,
if desired, comprise cells that can be divided to form a plurality
of "new" populations of cells that can be subsequently individually
introduced into separate cell modules that are preloaded with the
material composition, such as, for example, the exemplary
soft/stiff material composition. It is contemplated that the "new"
population(s) of cells can be introduced into/onto the soft culture
material prior to introduction of the soft culture material to the
stiff culture material. For example, the "new" population(s) of
cells can be encapsulated in a soft culture material such as a
hydrogel.
[0104] It is contemplated that at least portions of the material
composition can be recycled. For example, if the stiff culture
material forming a portion of the material composition is valuable,
e.g., a metal, the spent cell module can be recycled. In one
non-limiting example, it is contemplated that the stiff culture
scaffold of the material composition could be recovered from the
spent cell module, cleansed, and then subsequently preloaded into a
cell module for future use.
[0105] In an optional aspect, it is contemplated that the
bioreactor systems can be used for culturing product cells, for
example and without limitation, for use as a drug discovery test
system for efficacy. In such tests, the effects of a pharmaceutical
agent or agents on a target cell population is measured. Thus in
one aspect, disclosed herein are methods of screening for a
pharmaceutical agent comprising culturing one or more test cells in
a cell culture chamber of a first cell module in a bioreactor
system, passing an agent through the inlet and outlet of the first
cell module, and detecting the presence of an increase, decrease,
or stasis in the growth rate or viability or other measurable
parameter of the one or more test cells, wherein an increase,
decrease, or stasis in the growth rate or viability or other
measurable parameter in the one or more test cells relative to a
control cell or cells indicates whether the agent has an effect on
the one or more test cells.
[0106] In yet another aspect, it is contemplated that the
bioreactor systems can be used for culture of both diseased cells
and normal cells from the same biological organism. In this aspect,
the different cell populations can be connected through soluble
factor exchange across a membrane but maintain physical separation.
Thus, for example, disclosed herein are methods of monitoring the
effects of a diseased cellular population on neighboring normal
cells comprising culturing a diseased cell or cell in a first cell
module in a bioreactor system, culturing one or more normal cells
in a second cell module in a bioreactor system wherein the first
and second cell modules are separated by a semi-permeable membrane
and wherein soluble factor exchange occurs across the membrane.
[0107] In another aspect, the disclosed methods can comprise the
simultaneous and independent maturation of two tissues with no
soluble factor exchange. One skilled in the art will appreciate
that, in one non-limiting example, such a method can be
accomplished through the use of a non-permeable membrane. In
another aspect, it is understood and herein contemplated that the
ability to culture two independent cell populations such as one
diseased and another normal separate cell modules in a bioreactor
system while providing for the transmission of soluble factor
exchange allows for direct and simultaneous assessment of efficacy
and/or toxic effects of therapeutic agents on both cell
populations. For example, an agent can be administered into the
bioreactor system and a determination can be made on the
specificity of the agent for the diseased cell population or if not
specific whether the effects of the agent harm the normal cell
population. In another aspect, such methods can be used to
determine if the effects on the diseased cell population cause the
release of toxic factors from the diseased cells which have a
deleterious effect on the normal cells.
[0108] The utility of such a differential efficacy-toxicity
readout, by way of example and not limitation, is illustrated by
application to the class of pharmaceutical agents known as EGFR
inhibitors, which are known to have a classic skin toxicity as a
dose limiting issue. In that particular example using EGFR
inhibitors, applying the contemplated culture strategy can create a
tumor--skin model useful for determining the optimal does of the
pharmaceutical compound prior to starting its clinical
administration to a patient. A second non-limiting example is the
case of the class of pharmaceutical agents known as PI3K
inhibitors, which are known to cause an increase in glucose as a
surrogate of pharmacodynamics effect. In that particular example
using PI3K inhibitors, applying the contemplated culture strategy
can create a tumor-adipose model useful for determining the optimal
does of the pharmaceutical compound prior to starting its clinical
administration to a patient.
[0109] In yet another aspect, it is contemplated to the use of the
disclosed bioreactors and culture techniques for the testing of the
biometabolism of an active agent. In one example, through
substantially simultaneous culture of appropriately selected and
sourced tumor cells and liver cells biometabolism of an active
agent can be tested. In this aspect, for example and without
limitation, using techniques currently known to one skilled in the
art, a bank of iPSC derived hepatocytes can be created from one,
several or many donors. The iPSC bank can be frozen or otherwise
stored via conventional methods so as to provide a generally
available source of cells by which to create, on demand, a
surrogate 3D liver construct.
[0110] At an early point in treatment, a blood sample and tumor
biopsy can be procured from a cancer patient. Using techniques
currently known to one skilled in the art, peripheral blood
mononuclear cells can be isolated from the patient's blood (e.g.,
ficoll gradiants) and used to perform a cyp-specific genotypic
analysis of the patient. Having characterized that patient's cells'
cyp metabolism, the corresponding iPSC derived hepatocytes can be
chosen from the aforementioned iPSC bank of cells. The disclosed
bioreactor systems and culture techniques can then be used for the
culture of both the patient-derived tumor cells and the matched
iPSC derived hepatocytes, in order to perform an analysis of the
metabolism of one or more pharmaceutical agents concurrently with
the calculation of an IC50 of the tumor cell population. Once
calculated, the IC50 can then be used in conjunction with
pharmacokinetic data typically obtained in that agent's Phase I
clinical trial to determine if the IC50 is achievable or is
otherwise feasible (based on AUC and cMax). Using this information
and strategy, information can be concurrently derived regarding an
agent's therapeutic efficacy (using cells obtained by readily
available biopsied tissue) and, optionally, regarding the
metabolism of the agent in the same patient. One skilled in the art
will appreciate that the disclosed methodology can avoid the need
to obtain liver cells from the patient, which is a difficult,
costly and inconvenient procedure.
[0111] In another aspect, the disclosed bioreactors and culture
strategies can be used to achieve tumor stem cell enrichment. Short
term bioreactor studies such as those generally known in the art
and/or such as those described herein can provide an analysis of
the inherent resistance or sensitivity of a portion of cells
obtained from a patient biopsy towards one or multiple
pharmaceutical agents. Using these methods, the agents
demonstrating the largest desired effect (e.g. the largest
reduction in viability of the tested cell population) can be
chosen. In this aspect, it is contemplated that the measurement of
the desired effect can be accomplished using various analytical
techniques known to one skilled in the art that preserve the
viability of the analyzed cell population. In such fashion, it will
be appreciated that the cells that survive said exposure to the one
or multiple pharmaceutical agents (i.e.; cancer stem cells) can be
selectively maintained and when expanded in number over time,
enriched. It is understood and herein contemplated that the top
pharmaceutical agents thus selected can be expanded using the
disclosed bioreactors and culture strategies to produce adequate
numbers of cells that enable further testing (for example, and
without limitation, using multiple different concentrations of the
agent or agents on multiple samples).
[0112] In one aspect, after 3-4 weeks of further culture, the
remaining cells can be tested via further administration of the
agents and the appropriate starting dose for the clinical
administration of the agent or agents to the patient can be
calculated (using the IC50 as previously discussed above). In this
aspect, it is contemplated that the cell population that remains
viable after such second administration of said pharmaceutical
agent or agents can be then further propagated in the disclosed
bioreactor systems, said population of cells having been "doubly"
enriched. Thus, the disclosed bioreactor systems can be used for
multiple enrichments of the stem cell population of a given cell
mixture obtained from a patient biopsy. This process, and the IC50
values thus generated, can be selectively used to detect the
development of resistance and determine an achievable dose to
overcome resistance or indicate a measure that can be taken to
affect resistance.
[0113] The devices and methods for cancer stem cell enrichment
disclosed can be applied to either a portion or all of the cells
obtained from a patient biopsy. In the case of applying these
devices and methods to a portion of the cells obtained from a
patient biopsy, the concurrent propagation of both the original
cell mixture and the stem cell enriched cell mixture, and
subtractive and other analysis and/or comparisons are also
disclosed.
[0114] In a further aspect, having established the use of the
bioreactor systems and culture techniques disclosed to create a
cell population from a patient's tumor that is resistant to a given
pharmaceutical agent or agents, the anticipated effects of clinical
actions can be explored. For an ex vivo rapid resistance thus
developed, a combination of the devices and methods described
herein can be used to examine the effects of one or more of a) a
dose adjustment or increase; b) the addition of another
pharmaceutical agent or agents to overcome resistance; or c)
molecular analysis or phosphoproteomic analysis to determine
pathways associated with resistance.
[0115] In further aspect, also disclosed herein is the continuous
ex vivo culture of cells obtained from a patient biopsy throughout
the course of said patient's clinical treatment using the disclosed
bioreactors and cell culture techniques. This continuous ex vivo
culture can occur in parallel to the patient's clinical treatment,
which enables predictive course adjustment of the patient's
treatment according to information derived from the ex vivo culture
of their cells using the disclosed bioreactors and culture methods.
In another aspect, for example and without limitation, it is
contemplated via the methodologies disclosed herein to obtain
information on preferred pharmaceutical agents to be administered
during the patient's second course or subsequent courses of
therapy, based on an ex vivo analysis of their cells obtained from
a biopsy procured during their first course of therapy. In another
aspect, it is contemplated that the disclosed methods can be used
to assess the susceptibility or resistance of the biopsy to
treatment with a pharmaceutical agent.
EXAMPLES
Example #1
3D Cell Passaging Experiment
[0116] Herein provides an example of a method by which cells are
cultured in vitro, expanded, released from a 3D soft-stiff
composite scaffold via digestion and captured (passaged) for
purposes of reseeding into additional 3D culture vessels. This
method removes the need to culture the cells in traditional
two-dimensional (2D) conditions such as Petri dishes and well
plates, which has been shown to result in non-physiologically
relevant cell response and function. Additionally, 3D passaging
permits the continuous culture of cells in a closed system over
longer durations while maintaining the cells' natural phenotype and
function.
Materials and Methods
[0117] Culture Chamber: 3DKUBE.TM. 3D Cell Culture
Plasticware--Independent Chambers Configuration (KIYATEC Inc.,
Pendleton, S.C.)
[0118] Scaffold: A soft-stiff composite scaffold was fabricated
consisting of "stiff" polystyrene (PS) struts with interconnected
porosity and "soft" hyaluronic acid (HA) hydrogel filling the
interconnected porosity. The PS porous scaffold consisted of a
stacked crosshatch of 300 .mu.m diameter fibers forming an average
400 .mu.m pore size. Overall dimensions of the PS porous scaffold
were 5 mm in diameter and 1.8 mm in thickness. The PS scaffold was
placed in the bottom of a well of a 96-well plate. A volume of 50
.mu.L of methacrylated HA at 2% w/v including 12959 photoinitiator
was pipetted into the porosity of the PS scaffold. The HA was
crosslinked for 6.5 minutes at approximately 10 mW/cm.sup.2. The
well plate was incubated at 37.degree. C. and 95% RH for one hour
to further crosslink the "soft" HA hydrogel within the porosity of
the "stiff" PS scaffold. The entire soft-stiff composite scaffold
was frozen for two hours followed by overnight lyophilization to
create porosity in and around the contracted HA hydrogel and PS
struts.
[0119] Cells: SHEP (human neuroblastoma) cell line transfected with
a luciferase expressing gene (SHEP-Luc)
Results and Discussion
[0120] The soft-stiff composite scaffold was stored under vacuum at
20.degree. F. to prevent rehydration of the lyophilized HA hydrogel
component. Upon commencing the study, a single soft-stiff (HA-PS)
scaffold was placed in the culture chamber of a special white
opaque 3DKUBE Independent Chambers configuration. The inlet and
outlet ports of the 3DKUBE were capped with standard male luer
plugs. Prior to assembling the 3DKUBE modules, the soft-stiff
scaffold was seeded with a 100 .mu.L cell suspension of SHEP-Luc
cells (1.0E+5) in Dulbecco's Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin. Seeding was done by pipetting the cell
suspension directly onto the soft-stiff scaffold, covering the open
3D culture chamber in a 60 mm plastic culture dish and allowing
cell attachment under static conditions for two hours. Following
the two hour static period, the remaining seeding medium was gently
removed and the complete 3DKUBE was assembled and connected to the
syringe pump flow circuit consisting of a 10 mL syringe,
platinum-cured silicone tubing, standard luer connectors and a gas
exchange reservoir bag. The complete flow circuit, including the
assembled 3DKUBE, was primed slowly by hand and connected to the
syringe pump to commence 50 .mu.L/min volumetric flow rate. A total
of 10 mL of DMEM was used for the duration of the 5 day
experiment.
[0121] Samples were prepared and evaluated via Hoechst 33258 dye at
time points of Day 2 and Day 5. Upon completion of the respective
culture period (two or five days), a 3 mL syringe was used to
manually inject 500 .mu.L of 40 units/mL hyaluronidase digestion
solution (608 units/mg hyaluronidase) in PBS into the 3D culture
chamber via the luer port. Following slow perfusion of the
digestion solution into the 3D culture chamber, the system was
incubated under periodic agitation (manual infusion and withdrawal)
for 4 hours to digest the "soft" HA portion of the soft-stiff
composite scaffold. Following digestion, the contents of the 3D
culture chamber (i.e., the dissociated cells and soft culture
material) were gently drawn off into the 3 mL syringe to model the
concept of 3D cell passaging. The stiff culture material remained
in the culture chamber. The resulting cell suspension of cells and
soft culture material was placed in a microcentrifuge tube and spun
down to collect the cell pellet. The cell pellet was stored dry at
-80.degree. C. followed by Hoechst 33258 dye evaluation to quantify
the seeding efficiency. A range of known cell quantities were
cultured, frozen and evaluated in parallel with experimental
samples to provide a correlative standard curve for conversion of
fluorescent units to cell number.
[0122] Samples were resuspended in 400 .mu.L ddH2O and incubated
for one hour at 37.degree. C. followed by storage again at
-80.degree. C. and then thawed to room temperature. A volume of 100
.mu.L of each sample were transferred (in duplicate) to a black,
clear bottom 96-well plate. 100 .mu.l of Hoechst Reagent
(Invitrogen FluoReporter Blue Fluorometric dsDNA Quantification
Kit) was then added to each well before reading the fluorescence
using a Wallac Victor.sup.2 1420 fluorescent plate reader with a
355 nm excitation filter and a 460 nm emission filter.
[0123] The 3D culture demonstrated an increasing numerical trend
from Day 2 to Day 5 of the study. Cells were successfully recovered
from the 3D soft-stiff composite scaffold via digestion and
perfusion via the luer ports. Additional cells were found to remain
within the "soft" HA scaffold following the 4 hour digestion
procedure suggesting a recommended increase in the digestion period
to recover more cells. Ultimately, the study provides a relevant
example of 3D cell passaging using the 3DKUBE.TM. 3D Cell Culture
Plasticware in combination with a soft-stiff composite scaffold
useful to the art of cell and tissue culture.
Novel 3D Cell Culture System
[0124] The use of injection-molded culture chambers (FIGS. 6 and 8)
allows the researcher to load the desired 3D scaffold material
(FIG. 6A) of interest into two opposing culture chambers. The
culture chambers can accommodate a variety of scaffold
configurations including discrete beads, continuous porous
constructs (e.g., sponge-like), and hydrogels, all retained by an
integrated screen (FIG. 6B) molded directly into the fluid
ports.
[0125] Cells can be loaded in the scaffold material prior to
plasticware assembly or seeded post-assembly via manual syringe
perfusion through the culture chamber and scaffold. The placement
of a solid gasket (FIG. 6C) between the opposing culture chambers
allows for two independent samples (n=2) within each plasticware
assembly. Each chamber receives an independent perfusion (FIG. 6D)
of culture medium that can accommodate unique chemical or
mechanical stimulus for multiple experimental treatments.
Integrated inlet and outlet ports are standard luer connectors that
facilitate leak-free assembly within the perfusion fluid circuit.
In one aspect, the 3D cell culture plasticware can facilitate
advanced co-culture models by changing the solid gasket with a
gasket-membrane assembly (FIG. 6E) to allow transfer of soluble
factors and metabolites between different cell populations retained
within the opposing chambers.
Peristaltic Assembly
[0126] The system assembly (FIG. 7) incorporates a two chamber
bioreactor assembly (FIG. 7A) into a peristaltic-driven closed
fluid circuit (FIG. 7B) with medium reservoir (FIG. 7C) to provide
continuous perfusion into the culture chamber. Three-way valves
(FIGS. 7D,E) allow the system to switch to syringe pump-driven
perfusion (FIG. 7F) for periodic delivery of growth hormone and
collection (FIG. 7G) of soluble factors and metabolite products. A
mirror image of the system setup is used to perfuse the independent
sample in the second culture chamber (n=2).
[0127] In this example, the two chamber bioreactor assembly and the
fluid circuit system assembly are used as described. 3D
cell-scaffold constructs are maintained at 37.degree. C. in a
humidified incubator with 95%/5% air/CO.sub.2. A base culture
medium consisting of minimum essential Eagle medium supplemented
with sodium bicarbonate (1500 mg/L), sodium pyruvate (1 mM),
insulin (0.01 mg/mL) and 1% penicillin-streptomycin is used. Base
culture medium is contained in the medium reservoir and cells are
seeded in the desired scaffold material and packed within the
culture chambers. An initial volume of base medium is held in the
reservoir to maintain the 3D culture throughout the study duration.
The 3D cell-scaffold constructs experience a perfusion flow rate
throughout the study, with the exception of the syringe pump
treatment regimens.
[0128] The 3D cell-scaffold constructs undergo periodic treatment
with (+) or without (-) growth stimulant supplement (i.e., growth
factor or growth hormone) to the culture medium. 2D culture would
require manual supplementation of the base culture medium (+) or
(-) growth stimulant followed by manual aspiration and
replenishment of fresh base medium. In this 3D example, the
cell-scaffold constructs undergo a more automated transition to the
syringe pump perfusion of the treatment medium (+) or (-). The
frequency of (+) or (-) treatment application can be established to
occur multiple times daily for a variable length of time as
prescribed by the experimental protocol.
[0129] An additional experimental factor can involve supplementing
the base culture medium with pharmaceutical compounds and repeating
the 3D culture protocols performed previously in combination with
periodic treatment of (+) and (-) growth stimulant. Daily aliquots
of the culture medium are collected to assess the metabolism of the
pharmaceutical supplements. Analytical assessment of aliquots can
include metabolite and protein screening.
[0130] The endpoint for a given study can involve rinsing the 3D
cell culture with phosphate buffered saline. 3D cell-scaffold
constructs are removed from the plasticware and placed in sterile
tubes for cell isolation. Following centrifugation, supernatant is
aspirated resulting in an isolated cell pellet. The cell pellet
derived from the 3D culture can be snap frozen and stored at
-80.degree. C. Analytical assessment can include protein content,
enzyme activity, qPCR, sequencing, etc.
Example 2
Assay for Assessing Drug Efficacy and Treatment Selection
[0131] The EV3D (Ex Vivo 3D) pilot study was a prospective biology
study designed to compare EV3D.TM. results to radiographic response
and to clinical response. 20 Patients were enrolled in the course
of the study over an enrolment period of less than a year. Periodic
follow ups with the enrolled patients were conducted. The primary
aim of the study was to verify clinical correlation and a secondary
aim was to determine feasibility.
[0132] Initially desired specimens were recovered from the enrolled
patients and were initially biopsied within 30-60 minutes post
incision. The biopsied specimen was washed in HBSS and necrotic
tissues were subsequently removed. The remaining specimen was
conventionally minced into small pieces and then intubated in a
solution for 1-2 hours at 37.degree. C. to digest the tissue. The
digested tissue is then centrifuged to recover cells and the
recovered cell suspension is sequentially passed through a series
of sterile sieves.
[0133] Next, the cells are placed in an ultra low attachment plate
for a culture period that typically ran for about 24 hours. After
culturing, the cells were introduced into the 3DKUBE.TM. 3D Cell
Culture Plasticware for exemplary 3D culture/drug treatment.
Results of the EV3D study are shown in FIGS. 9-16.
Example 3
Drug Efficacy Measurement in Cell Culture System
[0134] This experiment shows that cells can be cultured under media
flow through the contemplated culture chambers, treated with
pharmaceutical agents, and the efficacy assessed using non-lytic
analytical means.
[0135] The experiment was performed in a single chamber culture
system (one chamber of a 3DKUBE in an "independent chamber"
configuration) or using wells in a 12-well plate. The 3DKUBE
chamber was cylindrical in shape and had a 6.0 mm diameter, 8.8 mm
depth, and 250 .mu.L volume. The chamber had an inlet port and an
outlet port enabling cell culture medium to flow through the
chamber if configured within a flow circuit. The inlet port was
connected to an opening in a first side of the 3DKUBE cell module
and the outlet port connected to an opening in a second side of the
cell module. The chamber featured an imaging widow enabling
non-lytic analysis inside the chamber through various
spectrophotometric and other techniques.
[0136] Human mammary epithelial cells (hMEC) were utilized. For 2D
experiments hMEC were cultured on the bottom of 12-well plates as
per typical cell culture practices (referred to as "2D"
experiments). For 3D static and perfusion experiments hMEC were
suspended in a Matrigel.TM.:Collagen (rat tail collagen Type I)
mixture which was added to silk fibroin scaffolds (H 2.5 mm.times.W
5 mm) contained in 12-well plates (referred to as "3D static") or
3DKUBEs (referred to as "3D perfusion"). Crosslinking due to
temperature increase caused the Matrigel:Collagen mixture to remain
in the silk scaffolds. Cell culture media was added to each
experiments. For the experiments in 12-well plates (2D and 3D
static), no media flow occurred. The experiments in the 3DKUBES
were configured such that the 3DKUBE was a part of a flow circuit
with a syringe at one end and featured media flow through the
chamber driven through the inlet and outlet ports. The media
perfusion was caused by the infusion and withdrawal action of a
syringe pump to which the syringe was connected.
[0137] The secretion of lactate dehydrogenase (LDH) is a non-lytic
assay of cytotoxicity. PrestoBlue.RTM. reagent is a resazurin-based
solution that functions as a cell viability indicator. In order to
calculate cytotoxicity percentages, both an untreated negative
control and a fully killed positive control is required at each
time point. The cells were cultured in these 2D, 3D static, or 3D
perfusion configurations over 7 days in media alone or in media
with increasing concentrations of cisplatin (a DNA alkylating agent
used in chemotherapy) and analyzed via PrestoBlue or LDH release
(examples of non-lytic means of assessing cell viability or
cytotoxicity). Negative controls are untreated and positive
controls were treated 2 hours prior to analysis with 2% triton to
induce maximal lysis and LDH release.
[0138] As shown in FIG. 16 PrestoBlue analysis demonstrates a
correlation between increasing cisplatin and a decrease in cell
viability that is only statistically significant in 3D static and
3D perfusion. LDH release demonstrates that reduced maximal cell
lysis by 2% triton is evident in 2D and 3D static (suggesting
background cytotoxicity that reduces the effect of the positive
control). However, in 3D perfusion, 2% triton increases LDH release
by approximately 2.5 fold which suggests increased cell viability
in the 3D perfusion system.
[0139] This data demonstrates the use of the culture system to
assess the efficacy of a pharmaceutical agent. Furthermore the data
suggests that the lack of an effect of the positive control
(triton) in 2D and 3D static is due to the increased background
cytotoxicity, whereas the positive control worked successfully in
3D perfusion, demonstrating an advantage of the system over systems
that do not feature media flow enabled by inlet and outlet
ports.
Example 4
Mixed Co-Cultures Under Static Conditions and With Perfusion
[0140] In this experiment, a mixed co-culture was established in
the 3DKUBE in order to assess the benefits of perfusion and stromal
components in mixed co-culture. HepG2 cells (ATCC) were cultured in
4 different 3D conditions over 7 days, and viability was assessed
by dsDNA staining by Hoechst 33258 and fluorometric measurement.
The four conditions included 50,000 HepG2 cells as preformed
spheroids (10,000 cells/spheroid) alone in 3D Matrigel.TM. in
static conditions (Static -FB), HepG2 cells mixed with fibroblasts
in 3D Matrigel.TM. in static conditions (Static +FB) at 2:1 ratio,
HepG2 cells alone in 3D Matrigel.TM. in perfusion at a rate of 20
uL/min (Perfusion -FB), and HepG2 cells mixed with fibroblasts in
3D Matrigel.TM. in perfusion at a rate of 20 uL/min (Perfusion
+FB). All ratios were 2:1 and other variables were exactly the
same. The data represents means of triplicates, and standard
deviation and demonstrates that HepG2 cell growth is greatest in
both perfusion and when mixed with fibroblasts over 7 days. FIGS.
17 and 18. Perfusion alone supports short term viability, whereas
both fibroblasts and perfusion are necessary to support longer term
culture (7 days).
Example 5
Ex Vivo 3D Culture of Primary Cancer Cells
[0141] This experiment demonstrates that primary cancer cells can
be cultured with or without a feeder cell population of human
foreskin fibroblasts (hFFb) in the bioreactor to assess
chemosensitivity of the cancer cell population against known drug
therapies.
[0142] The bioreactor had a first chamber loaded with tumor
spheroids encapsulated in a naturally-derived protein matrix. Tumor
spheroids derived from the heterogenic cancer cell population
ranged in size from 100-500 microns in diameter. A second chamber
was loaded with a porous scaffold pre-seeded with human foreskin
fibroblasts. The two 3D culture chambers were separated by a 0.45
micron pore size membrane to allow biochemical communication
between the cell populations to model in vivo cytokine and
secretome transfer.
[0143] The bioreactor assembly and the 3D cell culture contents
were perfused continuously for 28 days with DMEM based media,
supplemented with 10% fetal bovine serum (FBS), non-essential amino
acids and ascorbic acid at a volumetric flow rate of 20 microliters
per minute. Culture medium was changed at day 7, 14, and 21.
[0144] PrestoBlue.RTM. metabolic analysis of the hFFb cell
populations showed a numerical increase in cell metabolism from day
21 to day 28 as possible indication of an increase in cell number.
Hoechst dye analysis of double-stranded DNA quantity of the cancer
cell populations showed stable cell numbers from day 14 to 21 to
28.
[0145] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
aspects of the invention will be apparent to those skilled in the
art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *