U.S. patent application number 16/580468 was filed with the patent office on 2020-03-19 for co-culture bioreactor system.
This patent application is currently assigned to KIYATEC INC.. The applicant listed for this patent is KIYATEC INC.. Invention is credited to Matthew R. Gevaert, David E. Orr.
Application Number | 20200088719 16/580468 |
Document ID | / |
Family ID | 43357062 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200088719 |
Kind Code |
A1 |
Gevaert; Matthew R. ; et
al. |
March 19, 2020 |
CO-CULTURE BIOREACTOR SYSTEM
Abstract
Disclosed herein are bioreactor systems and methods of utilizing
said systems.
Inventors: |
Gevaert; Matthew R.;
(Greenville, SC) ; Orr; David E.; (Piedmont,
SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KIYATEC INC. |
Greenville |
SC |
US |
|
|
Assignee: |
KIYATEC INC.
|
Family ID: |
43357062 |
Appl. No.: |
16/580468 |
Filed: |
September 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15406396 |
Jan 13, 2017 |
10466232 |
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16580468 |
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13379152 |
Mar 21, 2012 |
9575055 |
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PCT/US2010/039119 |
Jun 18, 2010 |
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15406396 |
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61218097 |
Jun 18, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2503/00 20130101;
C12M 29/04 20130101; C12M 35/08 20130101; C12M 23/44 20130101; G01N
33/5029 20130101; G01N 33/5091 20130101; C12N 2502/00 20130101;
C12N 2500/00 20130101; C12N 5/0693 20130101; C12M 23/34 20130101;
C12N 2503/02 20130101; G01N 33/5011 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12M 1/00 20060101 C12M001/00; C12M 3/00 20060101
C12M003/00; C12M 1/42 20060101 C12M001/42; C12N 5/09 20060101
C12N005/09 |
Claims
1-23. (canceled)
24. A bioreactor system, comprising: a. 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; b. 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; and c. a membrane positioned between the open port of said
first cell module and the open port of said second cell module,
wherein the membrane is formed of a material which discourages
cellular attachment; wherein the first cell module and second cell
module are sealingly engaged securing the membrane between the
first and second module.
25. The bioreactor system of claim 24, wherein the bioreactor
system is a co-culture bioreactor system.
26. The bioreactor system of claim 24, further comprising a
retaining mesh, wherein the retaining mesh forms an integral part
of the inlet and outlet.
27. The bioreactor system of claim 24, wherein the cell module
comprises an optically transmissible material.
28. The bioreactor system of claim 24, wherein the first cell
module and the second cell module are identical.
29. The bioreactor system of claim 24, wherein the first cell
module and second cell module comprise identical cell chambers,
inlets, and outlets, but the first cell module comprises male
fittings and the second cell module comprises complementary female
fittings.
30. The bioreactor system of claim 24, 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.
31. The bioreactor system of claim 24, wherein each cell module
comprises a monolithic construction.
32. The bioreactor system of claim 24, wherein the coupling of the
cell modules forms a fluid-proof seal with the membrane.
33. The bioreactor system of claim 24, wherein the first and/or
second cell culture chamber comprises a biomaterial scaffold.
34. A method of maturing three-dimensional one or more tissues for
in vivo implantation comprising culturing said tissue or cells in
the bioreactor system of claim 24.
35. The method of claim 34, wherein the one or more tissues is
preferentially stimulated by another cell population through
soluble factor exchange across a membrane.
36. The method of claim 34, wherein the one or more tissues is at
least two tissues.
37. The method of claim 36, wherein the two or more tissues are
connected through soluble factor exchange across a membrane.
38. The method of claim 36, wherein the two or more tissues are
independently matured with no soluble factor exchange across a
membrane.
39. A method of pharmacokinetic screening, comprising: a. culturing
one or more cells in the cell culture chamber of a first cell
module in the bioreactor system of claim 24; b. passing an agent
through the inlet and outlet of the first cell module; c. detecting
the presence of an increase, decrease, or no change in the rate or
amount of a pharmacokinetic effect on the one or more cells in the
first cell culture chamber, wherein an increase, decrease, or no
change in the pharmacokinetic effect relative to a control provides
information on the pharmaceutical properties of the agent.
40. The method of claim 39, wherein the one or more cells are
obtained from the biopsy of a subject.
41. A method of screening for an agent that modulates cell
migration/invasion, comprising: a. culturing one or more cells in
the cell culture chamber of a first cell module; b. passing an
agent through the inlet and outlet of a second cell module; wherein
the cell culture chamber of the first cell module and the cell
culture chamber of the second cell module are separated by a
membrane that is formed of a material which discourages cellular
attachment; and c. detecting the presence of an increase, decrease,
or no change in the rate or amount of cellular migration across the
membrane, wherein an increase or decrease in cellular migration in
the presence of the agent relative to a control indicates an agent
that modulates cell migration/invasion.
42. The method of claim 41, wherein the one or more cells are
obtained from a biopsy of a subject.
43. The method of claim 41, wherein the membrane has a pore size
between 0.2 .mu.m and 10 .mu.m.
44. The method of claim 41, wherein the one or more cells of step a
are cancer cells; wherein the cancer is selected from the group
consisting of lymphoma; B cell lymphoma; T cell lymphoma; mycosis
fungoides; Hodgkin's Disease; myeloid leukemia; bladder cancer;
brain cancer; nervous system cancer; head and neck cancer; squamous
cell carcinoma of head and neck; kidney cancer; lung cancers such a
small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma; ovarian cancer; pancreatic cancer;
prostate cancer; skin cancer; liver cancer; melanoma; squamous cell
carcinomas of the mouth, throat, larynx, and lung; colon cancer;
cervical cancer; cervical carcinoma; breast cancer; epithelial
cancer; renal cancer; genitourinary cancer; pulmonary cancer;
esophageal carcinoma; head and neck carcinoma; large bowel cancer;
hematopoietic cancers; testicular cancer; colon and rectal cancers;
prostatic cancer; and pancreatic cancer.
45. The method of claim 41, wherein the first and/or second cell
culture chamber comprises a biomaterial scaffold.
46. A method of screening for an agent that inhibits a cancer
comprising performing the method of claim 41.
47. A method of pharmacokinetic screening, comprising: a. culturing
one or more cells of a first cell type in the cell culture chamber
of a first cell module; b. culturing one or more cells of a second
cell type in the cell culture chamber of a second cell module; c.
passing an agent through an inlet and outlet of the first cell
module; wherein the cell culture chamber of the first cell module
and the cell culture chamber of the second cell module are
separated by a membrane that is formed of a material which
discourages cellular attachment; d. detecting the presence of an
increase, decrease, or no change in the rate or amount of a
pharmacokinetic effect on the one or more cells in the second cell
culture chamber; and wherein an increase, decrease, or no change in
the pharmacokinetic effect relative to a control provides
information on that agent's pharmaceutical properties.
48. The method of claim 47, wherein the one or more cells of the
first or second cell type are obtained from a patient biopsy.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/406,396, filed Jan. 13, 2017, which is a
continuation of U.S. patent application Ser. No. 13/379,152, filed
Mar. 21, 2012, which is the National Stage of International
Application No. PCT/US2010/039119, filed Jun. 18, 2010, which
claims the benefit of U.S. Provisional Application No. 61/218,097,
filed Jun. 18, 2009. The entire contents of the above-identified
priority applications are hereby fully incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 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
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 mechanical environment as
well as the essential cellular interactions found during in vivo
development while providing purity of the desired product construct
so as to enable utilization of the product, for instance as
transplantable tissue. For example, it is commonly desired that the
product cells be isolated and free from extraneous cells of other
phenotypes, and in particular those previously shown to exhibit
unfavorable attributes following implant (e.g., tumor generation or
immune system reaction). However, biochemical interaction between
those less than desirable cell types with the product cells may be
necessary for the healthy growth and development of the product
cells, for example due to their introduction of growth stimulation
factors into the culture environment.
[0003] Many existing co-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 co-culture systems
has become of interest. However, known dynamic systems, similar to
the static systems, often provide only a single source of
nutrients/growth stimulants/etc. to all of the cell types held in
the system.
[0004] Moreover, the different cell types that are co-cultured in
both static and dynamic systems are usually maintained in actual
physical contact with one another, preventing the development of an
isolated cell population, and also limiting means for better
understanding the biochemical communications between the cell types
during growth and development.
[0005] There are some systems in which an attempt has been made to
physically separate cell types in dynamic systems, for instance
through location of a porous substrate between the two cell types.
However, in these systems, all cell-types cultured in the system
are still subjected to the same culture media, similar to the
above-described static systems. Additionally, the porous substrate
usually also serves as the support scaffold to which cells are
intended to attach and grow. Attachment of cells to the porous
substrate will alter the flow characteristics of biochemicals
across and through the substrate, which in turn affects
communication between the cells.
[0006] What is needed in the art is a method for co-culturing
multiple cell types in a dynamic environment in which the different
cell types can communicate biochemically, and yet can be separated
physically. 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 and at the same time allowing for biochemical communication
between cells of different types.
SUMMARY
[0007] In one aspect, the present invention is directed to a
bioreactor system. The disclosed bioreactor system can comprise a
single or a multiple culture system, such as a co-culture system.
Thus, in another aspect, the present invention is directed to a
bioreactor system that can maintain different cell types in
physically isolated environments without soluble factor exchange.
In yet another aspect, the present invention is directed to a
bioreactor system wherein the bioreactor system is a co-culture
bioreactor system that can maintain different cell types in
physically isolated environments but can allow biochemical
communication between the different cell types. 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate certain aspects
of the instant invention and together with the description, serve
to explain, without limitation, the principles of the invention.
Like reference characters used therein indicate like parts
throughout the several drawings.
[0015] FIGS. 1A and 1B are views of one embodiment of the cell
modules of the bioreactor system;
[0016] 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;
[0017] 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;
[0018] FIG. 4 is a schematic of a bioreactor system as herein
disclosed including multiple cell culture chambers in biochemical
communication with one another;
[0019] FIGS. 5A and 5B illustrate 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, with FIG. 5A showing media flow with no air
pressure and FIG. 5B showing deflection of the diaphragm due to air
pressure in the absence of media flow;
[0020] FIG. 6 illustrates cellular metabolic activity over a
seven-day duration for bioreactor cell study 1 as described in
Example 1;
[0021] FIG. 7 illustrates average cumulative metabolic data over
21-day duration for bioreactor cell studies 2 and 3 as described in
Example 1;
[0022] FIG. 8 illustrates cell viability on day 28 for the
experimental and control setups for bioreactor cell study 4 as
described in Example 1;
[0023] FIG. 9 illustrates cumulative glucose consumed over 28-day
duration for bioreactor cell study 4 as described in Example 1;
[0024] FIG. 10 illustrates cumulative lactic acid produced over
28-day duration for bioreactor cell study 4 as described in Example
1;
[0025] FIG. 11 illustrates cumulative glucose consumed in the
bioreactor study described in Example 2;
[0026] FIG. 12 illustrates cumulative lactic acid produced in the
bioreactor study described in Example 2;
[0027] FIG. 13 illustrates AlamarBlue.TM. cell viability assay
results in the bioreactor study described in Example 2;
[0028] FIG. 14 illustrates total protein content assay results in
the bioreactor study described in Example 2;
[0029] FIG. 15 illustrates alkaline phosphatase activity in the
bioreactor study described in Example 2;
[0030] FIG. 16 illustrates calcium content assayed in the
bioreactor study described in Example 2; and
[0031] FIG. 17 illustrates phosphorous content assayed in the
bioreactor study described in Example 2.
[0032] FIG. 18 shows cross section and actual image of 3D culture
system assemblies.
[0033] FIG. 19 shows fluid circuit system assembly.
[0034] FIGS. 20A, 20B show assembly for (20A) mono-culture and
(20B) co-culture.
[0035] FIG. 21 shows the difference between 2D and 3D culture
systems utilizing the same culture conditions.
[0036] FIG. 22 shows cellular metabolism assays results following
3D culture.
[0037] FIG. 23 shows a first and second cell module 12 engaged
through the use of male fittings and female fittings. FIG. 23
further shows said cell modules mounted in a microscope stage
adapter.
[0038] FIG. 24 shows six cell modules 12 mounted to well plate
adapter for use in instrumentation, i.e., spectrometer plate
reader.
DETAILED DESCRIPTION OF THE INVENTION
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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 (see, for
example, FIGS. 18, 20A, and 20B).
[0047] In still another aspect, disclosed herein are methods of
using the bioreactor systems disclosed herein for the growth and
maturation of three-dimensional tissue for in vivo implantation.
The disclosed methods can comprise culturing of one or more tissues
using the disclosed bioreactor systems. The disclosed methods can
further comprise single or co-culture applications. Thus, disclosed
herein are methods of growing three-dimensional tissue comprising
the maturation of a single tissue preferentially stimulated by
another cell population through soluble factor exchange across a
membrane. Also disclosed herein are methods of growing
three-dimensional tissue for use in in vivo implantation comprising
the simultaneous maturation of two tissues connected through
soluble factor exchange across a membrane 23. In another aspect,
the disclosed methods can comprise the simultaneous and independent
maturation of two tissues with no soluble factor exchange. It is
understood that such a method could be accomplished through the use
of a non-permeable membrane.
[0048] In one aspect, it is contemplated that the bioreactor
systems can be utilized for culturing product cells for medical
use, for example and without limitation, for use as a
drug-discovery test system for pharmacokinetics (for example,
toxicology and Absorption Distribution Metabolism and Elimination
(ADME)), for culturing a biopsy for use as a tissue-based
diagnostic, for transplant to a patient; or for manufacture of a
protein product; such as a biopharmaceutical. Thus, for example
disclosed herein are methods of pharmacokinetic screening,
comprising a) culturing one or more cells in the cell culture
chamber of a first cell module in the bioreactor system disclosed
herein; b) passing an agent through the inlet and outlet of the
first cell module; and c) detecting the presence of an increase,
decrease, or no change in the rate or amount of a pharmacokinetic
effect on the one or more cells in the second cell culture chamber;
wherein an increase, decrease, or no change in the pharmacokinetic
effect relative to a control provides information on the
pharmaceutical properties of the agent. Also disclosed are methods
of pharmacokinetic screening, comprising a) culturing one or more
cells of a first cell type in the cell culture chamber of a first
cell module; b) culturing one or more cells of a second cell type
in the cell culture chamber of a second cell module; c) passing an
agent through the inlet and outlet of the first cell module;
wherein the cell culture chamber of the first cell module and the
cell culture chamber of the second cell module are separated by a
membrane; and d) detecting the presence of an increase, decrease,
or no change in the rate or amount of a pharmacokinetic effect on
the one or more cells in the second cell culture chamber; wherein
an increase, decrease, or no change in the pharmacokinetic effect
relative to a control provides information on that agent's
pharmaceutical properties.
[0049] According to the pharmacokinetic aspects disclosed herein,
cells can be grown in an environment that comprises the biochemical
products of different cell types, at least some of which may be
necessary for the growth and development of the desired cells.
However, it is contemplated that cell types can be maintained in a
physically isolated state during their growth and development. As
such, possible negative consequences due to the presence of
aberrant or undesired cell types in the desired product cells can
be avoided.
[0050] In another aspect, it is contemplated that the bioreactor
systems disclosed herein can be utilized for the replication of
biological conditions such as cell migration/invasion such as, for
example and without limitation, wound healing, metastasis,
vasculogenesis, immune responses, angiogenesis, tumor formation,
and chemotaxis. Additionally the bioreactor systems disclosed
herein can be utilized for the replication of biological conditions
involved in cellular proliferation, cell survival, and attachment.
Used in such a manner the bioreactor system provides the
extracellular contact and milieu to more closely replicate the
intact biological system. In a like manner, the bioreactor systems
disclosed herein can be utilized as the framework for cell
migration assays. It is contemplated that cell migration/invasion
assays can look at the movement of cells across a membrane through
the use of a Boyden chamber or in a manner similar to a Boyden
chamber utilizing a permeable membrane 23 with a pore size similar
or identical to that used in a Boyden chamber. For example, the
membrane 23 can have a pore size between 0.2 .mu.m and 10 .mu.m. As
contemplated herein, cells are deposited into a first chamber with
a permeable membrane separating the cells from a second chamber.
Media is placed in a second chamber that encourages migration (such
as the presence of or a higher concentration of serum, cytokine, or
chemokine). Alternatively, factors such as electric current,
pressure, and media flow rate can be utilized to encourage or
discourage migration. Migratory cells move across the membrane
while non-migratory cells remain in the first chamber. In one
example, after the cells have been allowed to migrate, the
migratory cells can be disassociated from the membrane utilizing
conventional means, such as, without limitation, a suitable
detachment buffer. In another example, cell migration does not stop
on the opposing side of the membrane 23, but continues to a
scaffold in the second chamber.
[0051] In another aspect, the bioreactor system disclosed herein
can be used for the separation and isolation of cells. That is, the
disclosed bioreactor system can be used to separate and isolate
cells of one type from a mixed population of cells or tissue. For
example, the disclosed bioreactor system can be used for the
separation and isolation of stem cells from a mixed population of
cells such as bone marrow, or umbilical cord blood.
[0052] The use of bioreactor systems as devices for performing cell
separation and isolation, cell migration/invasion assays, or
replicating bioreactor systems is of significant importance to the
study of many diseases and conditions. For example, diseases where
excessive angiogenesis has been implicated include, but are not
limited to, rheumatoid arthritis, cancer, psoriasis, and diabetic
retinopathy. Diseases where insufficient angiogenesis has been
implicated include but are not limited to stroke, heart disease,
ulcers, infertility, and scleroderma. Accordingly, the bioreactor
systems disclosed herein and their use in cell migration/invasion
assays have considerable use and commercial significance to the
identification of regulatory pathways or migration/invasion and
modulators of migration/invasion. Thus, in one aspect, disclosed
herein are methods of screening for an agent that modulates cell
migration/invasion, comprising culturing one or more cells in the
cell culture chamber of a first cell module; passing an agent
through the inlet and outlet of a second cell module; wherein the
cell culture chamber of the first cell module and the cell culture
chamber of the second cell module are separated by a membrane
detecting the presence of an increase, decrease, or no change in
the rate or amount of cellular migration across the membrane; and
wherein an increase or decrease in cellular migration in the
presence of the agent relative to a control indicates an agent that
modulates cell migration/invasion.
[0053] In another application, the bioreactor system can be used to
more closely study the biochemical communication between different
cell types and the influence of this biochemical communication on
the growth and development of cells. As the local environment
within each culture chamber of the bioreactor system can be
substantially independently controlled while biochemical
communication between chambers can be maintained, information
regarding the growth and development of cells and the influence of
the local environment on that growth and development can be
examined through use of the bioreactor system.
[0054] 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, study the triggering mechanisms involved in stem cell
differentiation or to provide isolated, differentiated cells for
implantation.
[0055] 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 wherein a "normal" subject refers to any
subject 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.
[0056] Therefore, it is contemplated that exemplary cell types
comprise, at least partially and without limitation: Primary-hBM
SC; Primary-hSkin FB; Primary-cow CC; primary-rat BMSC; Primary-h
CC; MC3T3-E1; Primary-hUVEC; Primary-rabbit CC; NIH 3T3;
Primary-CC; Primary-rat Liver Hep; Primary-hSkin Keratinocyte;
MG63; HEP-G2; L929; Primary-BM SC; Primary-rabbit BM SC;
Primary-pig CC; Primary-hBone OB; MCF-7; Primary-rat Heart CM;
Primary-h Foreskin FB; Primary-hAdipose SC; Primary-hFB; # NIA;
Primary-hAdipose SC; Primary-PB; Primary-ratAortaSMC; Primary-Bone;
Primary-dog CC; 3T3 (nonspecific); C2C12; MDA-MB-231; SaOS-2;
Primary-mouse BM SC; Primary-rat CC; Primary-h Mesoderm Mes Pre C;
Primary-rat Brain Neuronal; PC12; Primary-Cancerous; Primary-h Skin
EC; Primary-rat BM OB; Primary-mouse Embryo SC; MCF-10A; Primary-h
Bone OB-like; Primary-goat BMSC; Primary-h Aorta SMC; MDCK
(Madin-Darby Canine Kidney); Primary-hl DAnnulus C; Primary-ratBone
OB; Primary-h Adipose Preadipocyte; Primary-SC; Primary-rat
Skeletal Muscle Myoblast; Primary-Heart CM; Primary-cow AortaEC;
Primary-dog BM SC; Primary-sheep BM SC; Primary-sheep CC;
Primary-pig BMSC; Primary-cow BMSC; Primary-h BladderSMC;
Primary-pig Aorta EC; Primary-h Cornea Epi C; Primary-h Aorta EC;
Primary-h Cornea FB; Primary-pig Aorta SMC; Primary-mouse Liver
Hep; A549; Primary-Bone OB; Primary-h Bladder Uro; Primary-h UV
SMC; Swiss 3T3; Primary-Liver Hep; Primary-h Lig FB; Primary-h
Coronary Artery SMC; Primary-OB-like; Primary-h Teeth Mes Pre C;
HT1080; Primary-rat Heart FB; Primary-pig HV Intersticial C; C3A;
Primary-h Breast Cancerous; Primary-h Foreskin Keratinocyte;
Primary-h Oral Mucosa Keratinocyte; Primary-mouse Ovary Oocytes;
Primary-h Vase SMC; 3T3-L1; Primary-h Lung FB; Primary-chicken
Ganglia Neuronal; Primary-h U CStC; Primary-cow Aorta SMC;
Primary-mouse Embryo FB; Primary-h Bronchi EpiC; CHO-K1; Primary-h
Liver Hep; Primary-hSaphVEC; Primary-hTeethPDL; Primary-rat Skin
FB; Primary-pig Liver Hep; PC-3; Primary-SMC; Primary-hMVEC;
Primary-mouseFB; Primary-h Nasal Chondrocyte;
Primary-hCorneaKeratinocyte; Primary-hOvaryCancerous; Primary-h U
CBSC; Primary-rat Heart EC; Primary-Vase; Primary-mouse Skin FB;
Primary-h Tendon TC; Primary-rat Brain Astrocyte; Primary-rat Nerve
SC; Ha CaT; Primary-h Gingiva FB; Primary-Neural; Primary-cow Bone
OB; Primary-rat Adipose SC; Primary-mouse Bone OB; Primary-h Teeth
PC; Primary-h Blood Mononuclear; Primary-rat Hippocampus Neuronal;
D3; HeLa; HEK293; Cl 7.2; Primary-h Skin Melanocyte; Primary-h
Blood EC-like; HOSTE85; Primary-h UC SC-like; Primary-h Cornea SC;
Primary-rat Aorta EC; Primary-h Saph VSMC; Primary h UCBEC;
Primary-mouse Heart CM; D1 ORL UVA; Primary-h Coronary Artery EC;
Primary-h Aorta Myo FB; HT-29; Primary-h Tendon FB; RAW 264;
Primary-rat Dental Pulp SC; 3T3-J2; Hl; Primary-pig Teeth;
Primary-rat Sciatic Schwann; Primary-rabbit Bone OB-like;
Primary-sheep Aorta EC; Primary-rabbit Cornea Epi C; Primary-h
Ovary Epi C; Primary-rabbit Ear Chondrocyte; SH-SY5Y; Primary-h
Teeth FB; Primary-h Oral Mucosa FB; Primary-rabbit FB; C6;
Primary-rat Testes Stertoli; Primary-cow Arterial EC;
Primary-pigHVEC; Primary-cow Nucleus Pulposus Cells; Primary-rat
Ganglia Neuronal; Primary-dog Bladder SMC; Primary-Vase SMC;
129/SV; Primary-pig Ear Chondrocyte; ED27; Primary-rabbit Bone B;
Primary-h Brain Glioblast; Primary-rat Adipose Preadipocyte;
Primary-h Cartilage Synov; Primary-rat Pancreas Insulin;
Primary-hEC; Primary-sheep Aorta SMC; Primary-h Endometrium EpiC;
U251; Primary-h Endometrium StC; Primary-pig Bladder SMC; Primary-h
HVIintersticial C; Primary-pig Esoph SMC; Primary-h NP Neuronal;
Primary-rabbit Aorta SMC; Primary-h NSC; Primary-rabbit CorneaFB;
Primary-h ral Cancerous; Primary-rabbit Lig FB; Primary-h SC;
Primary-rat BMOB-like; Primary-h Skeletal Muscle Myoblast; COS-7;
C-28/12; HK-2; Primary-h Uterus Cancerous; Primary-rat Ventricle
CM; Primary-h Vasc EC; Primary-sheep Carotid Artery SMC; HCT-116;
ROS 17/2.8; Primary-h Vocal FB; UMR-106; Primary-mouse Aorta SMC;
H9; RI; Primary-rat Fetal Neuronal; Primary-chicken Ear EpiC; Huh7;
Primary-rat Vase SMC; Primary-h NP SC; ES-D3; IMR-90; Primary-rat
Bladder SMC; 293T; Primary-h Foreskin VascularEC; Primary-h
Placenta EC; Primary-h Lung EpiC; Primary-h Prostate EpiC; U-87 MG;
Primary-dog Carotid Artery SMC; Primary-rabbit Cornea StC;
Primary-dog ID Annulus Fibrosus; Primary-chicken Embryo
Chondrocyte; Primary-EC; HFF; Vero; HFL-1; Primary-h Adipose FB;
Primary-cow FB; Primary-h UTSMC; Primary-rat Ventricle FB; AH 927;
Primary-sheep Vase FB; DU-145; ST2; B16.F10; Primary-h Nasal EpiC;
Primary-ID Annulus C; Primary-h Dental Pulp SC; 3HIOTI/2;
Primary-Heart Valve; Primary-h Bone Alveolar; Primary-rabbit Tendon
FB; Primary-mouse Kidney Insulin; HEPM; Primary-baboon Aorta SMC;
HTK; Primary-mouse MDSC; Primary-rat Esoph EpiC; Primary-mouse
Nerve SC; Primary-h Fetus OB-like; Primary-mouse Skeletal Muscle
SC; hFOB 1.19; Primary-Nerve Schwann; Primary-h Ganglia Neuronal;
Caco-2; Primary-h Kidney Renal; Primary-h Breast EpiC; Primary-h
Liver SC; Primary-pig Bladder Uro; Primary-h Lung EC; Primary-h
Breast FB; Primary-sheep Jugular Vein EC; Primary-pig Esoph EpiC;
Primary-h Lymph EC; Primary-chicken CC; Primary-h Lymph TCell;
Primary-h Colon Adenocarcinoma; Primary-h Mammary EC; Primary-pig
Vocal FB; Primary-h Mammary EpiC; Primary-rabbit Adipose SC;
Primary-h Cornea EC; H9c2; Primary-h UT StC; Primary-cat Heart CM;
Primary-mouse Pancreas EpiC; HS-5; Primary-sheep Skeletal Muscle
Fetus Myoblast; Primary-cow ID; Primary-mouse BM OCpre; Primary-cow
Knee Meniscus C; Hep-3B; Primary-cow Lig FB; HL-1; HuS-E/2; RWPE1;
Primary-cow Retina EpiC; Primary-hVascMyoFB; IEC-6; Primary-mouse
Fetal Hep; HS68; OVCAR-3; Primary-dog Knee MeniscusC;
Primary-rabbit Mesoderm Mes PreC; Primary-dog Lig FB; Primary-rat
Lung Alveolar; Primary-dog Skin Keratinocyte; CRL-11372;
Primary-dog Vasc SMC; HMEC-1; Primary-Embryo SC; T-47D1;
Primary-goatCC; Primary-h UVSC-like; Primary-guineapig Ear EpiC;
Primary-Ligament; Primary-guineapig Skin FB; Primary-mouse Cortical
Neuronal; Primary-hAdipose Adipocyte; Primary-mouse Liver SC;
Primary-h Adipose FB-like; CAL72; J774; P19; Primary-h Amniotic
fluid; Primary-rabbit Cornea EC; Primary-h Amniotic FSC;
Primary-rat BMFB-like; ARPE-19; Primary-rat Kidney Mesangial;
K-562; Primary-rat Nasal Ensheathing; Primary-h Bladder StC;
Primary-chicken Embryo Proepicardium; ATDC5; Primary-sheep FB;
Kasumi-1; Primary-Skeletal Muscle; Primary-h Bone Mes PreC;
HMT-3522; Primary-h Bone Periosteal; A431; Primary-h Brain EC;
Primary-h UTFB; KLE; 143b OST; BALB/3T3; Primary-h Vase FB;
LLC-PKI; Primary-h Vase Pericyte; BHK21-C13; Primary-MammaryEpiC;
M.DUNNI; C4-2B; ZR-75; HEC-IB; Primary-h Gingiva Keratinocyte; Ul
78; Primary-h HN Cancerous; Primary-mouse Mammary EpiC; Primary-h
Keratinocyte; Primary-mouse Sciatic N Schwann; OVCA429; Primary-h
Kidney EpiC; Primary-pig Esoph FB; MBA-15; Primary-pig Mandible
FB-like; Primary-h Liver Cancerous; Primary-rabbit Bladder Uro;
GD25beta1A; Primary-rabbit ID AnnulusC; HSC-T6; Primary-rabbit NP
Neuronal; DOV13; HEY; Primary-h Mammary FB; HTB-94; BZR-T33;
Primary-chicken CorneaFB; MiaPaCa2; Primary-rat Mucosa Ensheathing;
Primary-hOvaryFB; Primary-rat Salivary Acinar; Primary-h Ovary
Oocyte; Primary-rat Testes Germ; Primary-h Pancreas Cancerous;
Primary-chicken Embryo StC; Primary-h Pancreas Stellate Cells;
Primary-sheep Carotid Artery FB; MLO-Y4; Primary-chicken Retina
SC-like; Primary-h Prostate Cancerous; Primary-chicken Ten TC;
Primary-h Saph V Myo FB; Primary-Synoviocyte; MTLn3; Primary-Vase
EC; Primary-h Skeletal Muscle Pre; RT4-D6P2T; C2; SCA-9; HOC-7;
T31; Primary-h UC EpiC; TR146; HCS-2/8; EA.hy926; Primary-rat
Ebryo; SW480; Primary-sheep Fetus CC; Primary-dog Pancreas Insulin;
KS-IMM; BPH-1; Primary-rat Pancreas SC; M2139; RIN-5F;
Primary-hGallbladderCancerous; E14/TG2a; M4E; HES3; GS;
Primary-hConjunctivaFB; Primary-dogSaphVEC; LN CaP; Primary-dog
Saph V SMC; M4T; Primary-h Fetus CC; BR-5; Primary-pig UT Uro;
Primary-Hippocampus Neuronal; PE- 0041; Primary-dog Skin FB;
Primary-rabbit Skeletal Muscle MyoBlast; Primary-cowDenta 1pulp;
CGR8; Primary-dog Teeth PDL; Primary-rat Fetus Hep; Primary-dog
Tendon FB; Primary-rat Mammary; Primary-h Knee C; Primary-rat SMC;
BRC6; Primary-sheep Artery FB; Primary-dog Vase EC; Primary-cow
Mammary Alveolar; pZIP; 293 cell line; BMC9; Primary-h Lung
Cancerous; SKOV-3; IOSE; TEC3; MCF-12A; Primary-rabbitBladderEpiC;
Gli36DeltaEGFR; Primary-rabbit Conjunctiva EpiC; Primary-h Lung
Neuronal; Primary-rabbit Endometrium EpiC; 1205Lu; Primary-rabbit
MDSC; 3T3-A31; Primary-rabbit Tendon Tenocyte; MDA-MB-435;
Primary-h Cancerous; Primary-cow EC; Primary-rat Cornea FB;
Primary-EpiC; Primary-rat Fetal Cardiac; Primary-h Meninges
Arachnoidal; COS-1; Primary-Eye; Primary-rat Liver Oval C;
GLUTag-INS; Primary-rat Oral Mucosa Keratinocyte; GM3348; CRFK;
21NT; Primary-rat Testes EC; Primary-h Nasal FB; Primary-h Dura
MaterSC; Primary-h Nasal OB; Primary-dog NP Neuronal; Primary-h
Nasal Secretory; Primary-sheep Lung FB; AC-1M59; BHPrEl; MING;
Primary-UT; MKN28; RAT-2; MLO-A5; RT1 12; CRL-2266; S91; GM5387;
SK-ChA-1; Primary-horse CC; SPL201; Primary-horse Tendon FB;
Primary-h Fetus Mes PreC; D283; Primary-pig Thyroid EpiC; H1299;
Par-C10; AE-6; Primary-rabbit Blood Platelet; Primary-goat Carotid
EC; Primary-rabbit Bone OC; Primary-goat Carotid FB; Primary-cow
Cornea FB-like; Primary-h Pancreas SC; Primary-rabbit CT Pericyte;
Primary-goat Carotid SMC; Primary-rabbit Esophagus SMC; Primary-h
Parotid Acinar; Primary-baboon Blood EC; A498; Primary-h Bronchi
SMC; Primary-h Placenta SC; Primary-rabbit Sphincter SMC;
Primary-cow Retina SC; 7F2; MM-Sv/HP; A10; Primary-h Prostate StC;
Primary-buffalo Embryo SC-like; Primary-h Salivary Cancerous;
CH0-4; Primary-h Salivary Salisphere; Primary-rat Cortical
Neuronal; H13; Primary-rat Embryo Neuronal; Primary-guineapig
Pancreas EpiC; Primary-rat Fetal OB; H144; CNE-2; MPC-11; 21PT;
Primary-cow Synovium; Primary-rat Liver EC; Primary-cow Fetus CC;
BEAS-2B; H2122; LM2-4; Detroit 551; C18-4; FLC4; Ishikawa;
Primary-rat Skin Keratinocyte; H35; Primary-rat Tendon; Primary-h
SMC; HTR8; Primary-h Synovial CC; E8.5; H460M; HL-60; MUM-2B;
CRL-1213; MUM-2C; CRL-12424; W20-17; Lovo; Primary-dog Blood EC;
Primary-sheep Nasal CC; HAK-2; Primary-sheep Skin FB; Primary-h
Testes Sertoli; Primary-h Thyroid Cancerous; Primary-Trachea;
Primary-h Trachea; LRM55; Primary-h UASC-like; Primary-Colon FB;
Primary-hUASMC; r-CHO; HAT-7; RN22; HC-11; Primary-h Eye Vitreous;
AEC2; S2-020; HCC1937; CRL-2020; AG1522; SCC-71; N18-RE-105;
SK-N-AS; Primary-h Uterus SMC; SLMT-1; IMR-32; STO; NB4; Swan 71;
Primary-h Alveolar Perosteum; Primary-dog Oral Mucosa EpiC;
Primary-h Amnion EP; Primary-h Fetus Schwann; Primary-dog Bone OB;
Primary-pig UTSMC; 184A1; Pane 1; NCTC 2544; 46C; Primary-cow
Cornea EC; B6-RPE07; Primary-hamster EC; cBAL111; Primary-hamster
Retina Neuronal; HEPA-1C1c7; NEB1; CCE; NHPrEl; Primary-rabbit
ConjunctivaFB; 410; Hepa RG; Primary-Keratinocyte; PMC42-LA;
Primary-dog Cartilage Synov; 21MT; NOR-Pl; Primary-rabbit
Endometrium StC; Primary-Lymphnode Lymphocyte; DLD-1;
Primary-Lymphnode TCell; Primary-rabbit Lacrimal Gland Acinar;
AB2.1; primary-rabbit Lung Pneumocyte; Primary-monkey Embryo; ES-2;
Primary-monkey Kidney FB-like; Primary-rabbit Penis SMC;
Primary-mouse Adipose StC; Primary-rabbit Skin FB; NR6;
Primary-Blood SC; Primary-mouse BM Macrophage; 786-0; AT2;
Primary-rat Adrenal Chromaffin; AT3; CCF-STTGI; Primary-mouse Bone
Calvarial; Primary-rat Bladder Uro; HCT-8/E11; CE3; Primary-mouse
Brain Neuronal; CFK2; Primary-mouse Breast Cancerous; L6;
Primary-mouse Chondrocytes; HeyA8; Primary-mouse Colon EpiC;
Primary-rat Cortical Astrocyte; Primary-dog CFB; Primary-buffalo
Ovary EpiC; Primary-dog Cornea Chondrocyte; Primary-rat Embryo CM;
Primary-mouse Embryo Neuronal; A2780; C5.1 8; Primary-dog MV EpiC;
Primary-mouse Esophagus SC; Primary-rat Fetal Renal; HEKOO1; A357;
EFO-27; Primary-chicken Bone OB; Primary-mouse Fetal Lung;
Primary-rat Heart SC-like; Primary-mouse Germ; Primary-rat Kidney;
EN Stem-ATM; Primary-rat Lacrimal Acinar; U-251 MG; Primary-dog
Myofibroblasts; A4-4; Primary-rat Liver SC-like; Primary-cow Brain
EC; Primary-rat Lung FB; Primary-mouse Kidney Renal; BEL-7402; NT2;
HIAE-101; Primary-h BM Mononuclear; Primary-rat Ovary;
Primary-mouse Lymph FB-like; Primary-rat Pancreas Islets;
Primary-dog Esophageal EpiC; Primary-rat Renal EpiC; Primary-mouse
Mast; Primary-chicken Embryo Blastoderm; NTera2/c1.D1; G-415; Null;
Primary-rat Small Intestine; Primary-mouse Ovary Cumulus C;
Primary-rat Teeth SC-like; HEL-299; Primary-rat Tendon Tenocyte;
KB; b-End-2; Primary-mouse Pancreas Insulin; Primary-rat Vase EC;
Primary-mouse Salivary Salisphere; Primary-h Duodenum EpiC;
Primary-h Bone Fetus OB; Primary-Respiratory EpiC; Primary-mouse
Skeletal Muscle Myoblast; Primary-sheep Amniotic fluid; 0C2;
Primary-chicken Heart CM; Daudi; Primary-sheepArtery MyoFB;
Primary-mouse SkinKeratinocyte; Primary-sheep Bone OB-like;
Primary-mouse Small Intestine; Primary-chicken Heart ECM;
Primary-mouse Spleen Tcell; LNZ308; Primary-mouse Teeth
Odontoblast; Primary-sheep ID Annulus Fibrosus; Primary-mouse
Testes SC; Primary-sheep Jugular Vein SMC; Primary-mouse Testes
Sperm; Primary-sheep Lung SC; Primary-mouse UT Uro; Primary-sheep
Saph VEC; Primary-mouse Uterus EpiC; Primary-sheep Skin EC; OCT-1;
Primary-sheep Vase EC; HELF; Primary-sheep Vase SMC; CAC2; HL-7720;
OPC1; Primary-Teeth PDL; Primary-dog Heart SC;
Primary-UCB/Mononuclear; Primary-pig Artery Carotid EC; Primary-h
Endometriotic CystStC; Primary-pig Artery Carotid SMC;
Primary-Colon Cancerous; Primary-pig Artery Coronary SMC; QCE-6;
Primary-pig Bladder FB; R221A; OSCORT; LS180; B35; RIF-1; Calu-1;
RL-65; Calu-3; Primary-cow Adrenal ChrC; B5/EGFP; RT-112;
Primary-pigEC; RW.4; Primary-pig ESC; S2-013; OVCAR-5; S5Y5;
Primary-h Bone OC-like; SA87; INT-407; SAV-I; Primary-pig Fetus
Hep; SCC-68; P69; HNPSV-1; CaSki; SK-0015; Primary-pig Iliac EC;
SK-N-DZ; Hep2; SKOV3ip.1; Primary-pig Mandible Ameloblast; SNB19;
Primary-cow Joint Synovial; Primary-h Fetus FB; Primary-pig
Mandible Odontoblast; SWI 353; Primary-pig NP Neuronal; SW948;
Primary-pig Oral MucosaEpiC; CRL-2102; Primary-pig Pancreasislets;
T4-2; Primary-pig PulmonarySMC; TE-85; Primary-pig Salivary Acinar;
THP-1; Primary-pig SynoviumSC; BME-UV1; KG-1; D4T; HUES-9;
Primary-mouse Hippocampus Neuronal; ECV304; NRK; Primary-mouse
Kidney Mesangial; D407; 1OT1/2 cell line; and Primary-h Foreskin
Melanocyte.
[0057] 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.
[0058] Thus, in one aspect, one embodiment is a cell module 12.
Though each cell module 12 of the embodiment illustrated in FIGS.
1A and 1B 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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 module are
not identical or interchangeable but capable of being engaged.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] The system can also comprise 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.
[0068] 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.
[0069] 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 its 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.
[0070] 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.
[0071] 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.
[0072] 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,
polycarbonate s, 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.
[0073] 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(e-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-1-lysine; MWCNT/multiwalled carbon nanotube;
PHBV/poly(hydroxybutyrate-co-hydroxyvalerate); Coral; Starch;
PPF/poly(propylene:fumarate); PLCL/poly(lactide-co-e-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; PEI 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;
Polyhydroxyalk:anoate; 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(C0)(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;
Phosphophoryn; HEAi hydroxyethyl acrylate; PSSNa/poly(sodium
styrene sulfonate); Carbon Foam; PFOB/perfluorooctyl bromide;
Lecithin; Mebiol.RTM.; BHA/butylated hydorxyanisole; 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-omithine 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-substitutedtricalcium 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].
[0074] 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 or between about
150 .mu.m and about 500 .mu.m.
[0075] 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.
[0076] 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. fu 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] In yet another alternative, the membrane 23 can comprise a
porosity large enough to allow for the passage of cells between
culture chambers 10. For example, the membrane can comprise pores
from 0.05 .mu.m to 100 .mu.m, 0.1 .mu.m to 20 .mu.m, 1 to 10 .mu.m,
1 to 5 .mu.m, or 3 .mu.m. Thus, examples of pore sizes for membrane
23, include but are not limited to 0.2 .mu.m, 0.45 .mu.m, 1 .mu.m,
3 .mu.m, and 8 .mu.m. Through the use of such a porous membrane 23,
cell migration/invasion assays can be performed that look at the
effect of chemokines, cytokines, morphology, electricity, pressure,
or modulators on the migratory process. Thus, in one aspect, a
porous membrane 23, can be used to screen for modulators of
cellular migration/invasion.
[0083] Physical isolation of cellular contents of adjacent chambers
can also be encouraged through selection of membrane materials. In
one aspect, materials used to form the membrane 23 can be those
that discourage anchorage of cells onto the membrane 23. Attachment
of cells to the membrane 23 can be discouraged to prevent physical
contact between cells held in adjacent culture chambers as well as
to prevent interference with flow between the adjacent chambers.
Flow interference could interfere with the biochemical
communication between the adjacent culture chambers. One exemplary
material that can discourage cellular attachment is a polymer, such
as, for example and without limitation, a polycarbonate membrane.
Other suitable materials generally known to those of skill in the
art comprise but are not limited to polyvinyl, polypropylene,
polyethersulfone, Polyvinylidene Fluoride (PVDF), polycarbonate,
polyolefin, and polytetrafluoroethylene (PTFE). Examples of
suitable membranes can be purchased directly from Millipore, Pall,
Whatmann, and Sartorious.
[0084] 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.
[0085] 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 FIGS. 1A and 1B. 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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. In addition, the disclosed bioreactor
systems can be utilized to allow biochemical communication with
physical separation between two or more different cell types for a
variety of applications including, for example and without
limitation, bone development (osteocyte/preosteoblast or stem
cell/preosteoclast), breast tissue replacement
(preadipocyte/endothelial), stem cell research (stem cells/feeder
cells), and regeneration or replacement of damaged liver cells
(hepatocyte/endothelial). Due to the liver's significant role in
drug metabolism, hepatocytes are the most relevant cell type for
many drug discovery applications. Unfortunately, primary
hepatocytes cultured in monolayer often lose phenotype-specific
functionality and undergo apoptosis. Biosynthesis of
drug-metabolizing enzymes, essential for pharmaceutical toxicity
assays, is among the first functions to be lost. Hepatocyte cell
lines have similar drawbacks, ranging from significant to complete
loss of important CYP-related enzyme functionality. These and other
cell culture challenges require advanced solutions.
[0090] It is understood and herein contemplated that cell migration
and invasion are critical aspects to the development and
proliferation of cancer. Additionally, it is further understood
that the disclosed bioreactor systems are ideal for the
identification of agents that affect cell migration, proliferation,
cell invasion and adherence. Accordingly the disclosed bioreactor
systems can be used in methods to screen for agents that modulate
cancer. Thus, disclosed herein are methods of screening for an
agent that inhibits a cancer comprising culturing cancer cells in a
first cell chamber 10 of a first cell module 12, administering an
agent into a second cell chamber 10 of a second cell module 12,
wherein the first and second cell chambers are separated by a
membrane 23, and wherein an agent that inhibits proliferation,
migration, or invasion of cells from the first cell chamber 10
across the membrane 23 into the second cell chamber 10 is an agent
that inhibits cancer.
[0091] It is understood that the cell cultured in the first cell
chamber 10 can be from a cell line or obtained from a biopsy or
other tissue sample. It is further understood that the cancer can
be selected from the group of cancers consisting of lymphoma, B
cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's
Disease, myeloid leukemia, bladder cancer, brain cancer, nervous
system cancer, head and neck cancer, squamous cell carcinoma of
head and neck, kidney cancer, lung cancers such as small cell lung
cancer and non-small cell lung cancer, neuroblastoma/glioblastoma,
ovarian cancer, pancreatic cancer, prostate cancer, skin cancer,
liver cancer, melanoma, squamous cell carcinomas of the mouth,
throat, larynx, and lung, colon cancer, cervical cancer, cervical
carcinoma, breast cancer, and epithelial cancer, renal cancer,
genitourinary cancer, pulmonary cancer, esophageal carcinoma, head
and neck carcinoma, large bowel cancer, hematopoietic cancers;
testicular cancer; colon and rectal cancers, prostatic cancer, or
pancreatic cancer.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] For example, according to one aspect, as illustrated in
FIGS. 5A and 5B, a cell module 12 can be located immediately
adjacent to a second cell module (not shown in FIGS. 5A and 5B), 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.
[0096] Such an embodiment may be particular beneficial in
orthopedic related research studies. This system can provide
improved ex vivo simulation of the physiological in vivo
environment for bone that comprises both hydrostatic compression
and perfusion fluid flow as a result of normal skeletal loading.
For example, in one embodiment of the system, a hydrostatic loading
cycle of 0.5 Hz and fluid pressures exceeding 300 kPa can be
demonstrated. These values closely approximate those found in the
lacunar-canalicular porosity of the human femur during normal gait,
i.e., 0.5-2.0 Hz and 270 kPa.
[0097] 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 translucent, 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.
[0098] 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 (FIG. 23). Also disclosed are cell modules
12 mounted to well plate adaptor for use in instrumentation, i.e.,
spectrometer plate reader (FIG. 24).
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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).
[0103] 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.
Example 1
[0104] Cell Culture: A 3T3 mouse fibroblast cell line (available
from ATCC, Manassas, Va.) was used in three studies (numbered 1-3
below) to examine cell viability when subjected to perfusion fluid
flow in a system. A fourth study (Study 4) used a Dl cell line
(ATCC) of adult mouse bone marrow stromal cells and incorporated
hydrostatic compression in addition to the perfusion flow. The Dl
cell line was selected due to its demonstrated multi-potent
potential including favorable osteogenic properties.
[0105] Anchorage Fabrication: Multiple discrete poly-L-lactide
(PLL) hollow beads with an average diameter of 0.8 mm served as the
discrete tissue engineering scaffolds for all studies. Briefly,
scaffold fabrication was completed using solvent emulsion
techniques, beginning with an 8% (m/v) solution of Purasorb
polylactide pellets (Cargill, Minneapolis, Minn.) and
dichloromethane (Mallinckrodt Baker, Phillipsburg, N.J.). A
quantity of 5 ml of PLL solution was dispensed, using a 20 cc glass
syringe (BD, Franklin Lakes, N.J.) and 16-gauge needle (BD), into
500 mi of a stirred 0.1% aqueous solution of polyvinyl alcohol
(PVA) (Sigma-Aldrich, St. Louis, Mo.). PVA solution in the amount
of 300 ml was siphoned out of the beaker, after which, 200 ml of 2%
isopropyl alcohol solution (VWR, West Chester, Pa.) was added.
Following three minutes of stirring, 300 ml of solution was
siphoned out and 400 ml of PVA solution was added back to the
remaining 100 ml volume. The total volume of 500 ml was then
stirred for three minutes, resulting in PLL bead formation. The
hollow beads were strained from the solution and allowed to dry
under vacuum to remove any residual solvent.
[0106] Modular bioreactor assembly was completed with the inclusion
of a porous volume of 0.2 ml of PLL hollow beads within a culture
chamber. Four sets of clamping socket screws, washers and nuts were
used to form a tightly sealed assembly. The assembled bioreactor,
including adjacent luers, stopcock valves and enclosed PLL beads,
was sterilized with ethylene oxide gas at room temperature and
degassed for several days under 25 inches Hg vacuum. Flow circuit
tubing and quick-disconnect luers were also sterilized with
ethylene oxide gas while the medium storage bottle was
autoclaved.
[0107] The complete bioreactor flow circuit including a single cell
module located immediately adjacent to a single pressure module
such as that illustrated in FIGS. 5A and 5B but with only a single
culture chamber, was assembled in a standard laminar flow hood to
prevent contamination. A volume of 60 ml of medium was added to the
storage bottle. Initial culture medium for studies 1-3 consisted of
Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad,
Calif.) supplemented with 10% fetal bovine serum (FBS) (Mediatech,
Herndon, Va.), 5 ml antibiotic/antimycotic (Invitrogen), 1 ml
fungizone (Invitrogen), 5 ml L glutamine (Invitrogen) and 10 pg
fibroblast growth factor (Fisher): Initial culture medium used for
study 4 was Dulbecco's Modified Eagle's Medium (ATCC) supplemented
with 10% FBS. For all studies, the system was primed with medium to
prewet the PLL beads prior to cell seeding. Cell seeding was
accomplished by pipetting the appropriate cell suspension into the
medium storage bottle and then allowing the system to perfuse the
cells through the flow circuit to the discrete scaffolds within the
cell module. The entire perfusion flow circuit was contained in an
incubator that was maintained at 37.degree. C. and 5% CO.sub.2.
Each of the four preliminary studies differed either in cell type,
quantity, passage, duration or mechanical stimulus as described
below in Table 1.
TABLE-US-00001 TABLE 1 Cell Seeding (per ml l Quantity Mechanical
Study Cell Type Passage scaffold volume) Seeded Duration Stimulus 1
3T3 37 1.2E7 2.4E6 7 days Perfusion 2 3T3 29 3.0E6 6.0E5 21 days
Perfusion 3 3T3 31 3.0E6 6.0E5 21 days Perfusion 4 DI 24 3.0E6
6.0E5 28 days Perfusion & Hydrostatic indicates data missing or
illegible when filed
[0108] Study 1 provided initial observations regarding metabolism
and viability of cells cultured within the confines of the culture
chamber. A medium perfusion volumetric flow rate of 4.8 ml per
minute was continuous throughout the seven-day duration. The medium
was not changed for the seven-day duration of the study. Lactic
acid and glucose levels were measured at days 0 and 7 using a YSI
2700 SELECT Biochemistry Analyzer (YSI, Yellow Springs, Ohio).
Visual inspection of live fibroblast attachment to the PLL beads
was demonstrated at day 7 through the use of a LIVE/DEAD.RTM.
Viability/Cytotoxicity Kit (Invitrogen) and fluorescent microscopy.
A seven-day acellular control study was conducted in parallel to
confirm that rising lactic acid levels were due to cell metabolic
activity and not PLL hydrolysis.
[0109] Studies 2 and 3 extended the culture time to 21 days and
provided opportunity to refine endpoint assay techniques. As in
Study 1, the volumetric flow rate of 4.8 ml per minute was
continuous for the 21-day duration with the exception of brief
periods of medium changes and aliquot retrieval. Medium aliquots
were taken every two days, beginning on day 4, and complete medium
changes occurred on days 8 and 16. Aliquots were monitored for
metabolic solute levels, and live/dead fluorescent microscopy was
completed on day 21. Unlike previous studies, study 4 incorporated
hydrostatic compression in the experimental protocol beginning on
day 4. Daily routine involved 8 hours of cyclic hydrostatic
compression, approximately 330 kPa at 0.1 Hz (5 seconds on, 5
seconds off), followed by 16 hours of continuous perfusion flow at
4.8 ml per minute. Hydrostatic compression was interrupted after 4
hours with 5 minutes of perfusion flow to deliver fresh nutrients
to the cells and prevent the buildup of waste products. A control
system was used that did not receive hydrostatic compression
loading. The control system's stopcock valves adjacent to the cell
module were closed during the eight-hour hydrostatic segment but no
pressure was applied. The inclusion of D1 adult mouse bone marrow
stromal cells was strategic due to the demonstrated osteogenic
characteristics. These properties provided opportunities to observe
changes in phenotype differentiation in response to the applied
mechanical loading regimens. Beginning at day 3, osteogenic media
for both the experimental and control systems were supplemented
with 50 pg/ml L-ascorbic acid (Sigma) and 10 mMI3-glycerophosphate
(Sigma). Assays commonly used to evaluate osteogenic
differentiation, including alkaline phosphatase activity, calcium
content and total protein, were practiced for application in future
experiments. Metabolic activity was observed through assessment of
medium aliquots as well as relative AlamarBlue.TM. (Biosource,
Camarillo, Calif.) fluorescent emission.
[0110] The graphs of FIGS. 6-10 represent mean.+-.standard error of
the mean with n=1, where n represents the number of replications of
a given study. Nested measurements were made within each study at
the respective time points; thus, variability is due to subsampling
within the experimental assay. Therefore, no statistical
comparisons could be made for the preliminary experiments.
Microsoft Excel was used for all numerical analysis.
[0111] The acellular control setup for study 1 presented no
numerical elevation in lactic acid levels due to PLL hydrolysis
(FIG. 6). Cumulative levels of glucose consumption and lactic acid
production increased over the 21-day duration of studies 2 and 3
(FIG. 7). Live/dead imaging depicted a confluent cell layer at the
PLL bead surface (not shown). Comparative cellular metabolic
activity of the experimental and control systems of study 4
demonstrated similar numerical values for cumulative metabolic
solutes and AlamarBlue.TM. relative fluorescence units (FIGS.
8-10).
[0112] As can be seen, the modular bioreactor provided an in vitro
environment conducive for cell growth on three-dimensional
scaffolds. Metabolic data demonstrated that cells continued to
flourish over the duration of each preliminary study. Endpoint
evaluations with AlamarBlue.TM. and live/dead fluorescent
microscopy provided additional evidence as to the ongoing viability
of cells cultured in the bioreactor system.
Example 2
[0113] Dynamic bioreactor systems were utilized to examine the
influence of multiple mechanical stimuli on the differentiation
traits of adult mesenchymal stem cells in addition to a variety of
other cell types. The bioreactor was designed to model the in vivo
conditions through available system conditions including
hydrostatic loading. Perfusion of medium through the system was
also adjusted to provide physiological levels of fluid shear stress
across the cells.
[0114] Bioreactor systems such as that illustrated in FIGS. 5A and
5B were prepared. Each 0.2 mL volume culture chamber was loaded
with 45 mg PolyGraft.TM. granular material. A silicone
diaphragm/gasket and four clamping socket screws were used to
provide leak-free assembly of the culture chamber adjacent a
pressure module.
[0115] All systems utilized a DI cell line grown in quantity using
standard cell culture flasks (Corning). The initial cell culture
medium consisted of DMEM (ATCC) supplemented with 10% FBS and 1%
antibiotic/antimycotic. Each bioreactor flow circuit, including the
culture chamber loaded with the granular scaffold, was primed and
pre-wetted with medium prior to cell seeding. Each medium storage
bottle was filled with 30 mM of the initial cell culture medium.
Each bioreactor setup was seeded with a total of 6.0E5 cells by
injecting one milliliter of the cell suspension directly into the
flow circuit and culture chamber.
[0116] Five different treatment regimens were designed having
different combinations of perfusion flow rate and hydrostatic
compression characteristics. According to the regimens, perfusion
flow was set to static, low or high. Low perfusion flow was set as
0.35 mL/min, and high perfusion flow was 0.70 mL/min. For those
treatment regimens requiring only perfusion flow (i.e., no
hydrostatic compression component), continuous perfusion of the
bioreactor flow circuit occurred for the duration of the study with
the exception of medium changes and aliquot sampling.
[0117] Hydrostatic compression was either low, at 0 kPa, or high,
at 200 kPa. Treatment regimens applying both perfusion flow and
hydrostatic compression underwent a daily schedule including 22
hours of continuous perfusion flow followed by two hours of cyclic
hydrostatic compression. Within the two hour time period, cyclic
hydrostatic compression was applied for 10 minute increments
followed by a five minute session of perfusion flow to provide
cells with fresh nutrients and remove damaging waste products. The
experimental protocol of shifting from compression to perfusion and
back to compression was repeated for the duration of the two hour
period. Compression was applied at 200 kPa and cycled at 0.5 Hz
(one second on and one second off). Specific treatment regiments
were as shown below in Table 2.
TABLE-US-00002 TABLE 2 Regimen Number Perfusion (mL/min)
Compression (kPa) 1 0.35 0 l2 0.70 0 3 0.35 200 f4. 0.70 200 5
Static Static
[0118] The initial supplemented medium of DMEM, FBS and
antibiotic/antimycotic was used through day 2. At day 2, the
initial medium was modified with the following additional
supplements to formulate an osteogenic differentiation medium: 50
.mu.g/mL-ascorbic acid 2-phosphate, 3 mM 13-glycerophosphate, 10 nM
Dexamethosone. Complete change of the supplemented medium took
place on days 2, 8, 14 and 20.
[0119] Lactic acid production and glucose consumption were
monitored throughout the course of the runs by taking medium
samples every two or three days. Each treatment regimen was carried
out in six bioreactor setups. Each regimen was assayed at day 7 and
day 21 to provide a variety of quantitative, comparable data across
all treatment combinations. Upon reaching a designated endpoint of
either day 7 or 21, each of three of the setups were disassembled,
and a variety of assay methodologies were carried out.
[0120] FIGS. 11-17 graphically illustrate the assay results
obtained for determination of cumulative glucose consumed,
cumulative lactic acid produced, AlamarBlue.TM. cell viability,
total protein content, alkaline phosphatase activity, calcium
content, and phosphorous content, respectively.
[0121] Visual observation of the cell-scaffold constructs
uponremoval from the culture chamber recognized that the constructs
retained much of the bulk shape as packed in the culture chamber.
Samples undergoing high hydrostatic compression appeared to
maintain a tighter packing of the granular cluster following
transfer from the culture chamber. The inclusion of mechanical
stimuli in the experimental protocol clearly upregulated the
cumulative metabolic requirements of cells when compared to static
culture conditions. Hydrostatic compression also appeared to
further increase the' rate of glucose consumption and lactic acid
production as can be seen with reference to FIGS. 11 and 12. While
the dynamic experimental conditions appeared to possess higher
metabolic rates, the endpoint cell viability ascertained through
the AlamarBlue.TM. assay conveyed a result of consistency
regardless of whether dynamic or static conditions were applied, as
can be seen with reference to FIG. 13.
[0122] The dynamic mechanical stimulus of the culture chambers
appeared to prompt extra-cellular matrix (ECM) production as
indicated by the total protein content results (FIG. 14). The
culture chamber design was intended to mimic in vivo conditions and
in effect cause the cells to product bone-like ECM. Total protein
content was clearly improved by day 21 as compared to the static
culture protocol. As occurs in vivo, the active loading within the
culture chamber can force a cell to lay down new ECM to surround
itself with new "bone" and afford the cell's 30 long-term anchorage
to the scaffold for continued survival.
[0123] ALP activity has often been used to indicate cell
differentiation toward the osteogenic lineage. In this case, both
the static culture and high compression protocols indicated
increased levels of cell differentiation through the statistical
increase in ALP activity from day 7 to day 21 (FIG. 15).
[0124] Calcium and phosphorous contents (FIGS. 16 and 17) appeared
to uniquely vary over time as well as in respect to the static
culture regimens. Studies that did not undergo hydrostatic
compression demonstrated a statically significant increase in both
calcium and phosphorous content from day 7 to day 21. Studies
including hydrostatic compression showed no statistical change and
even slight numerical reduction in calcium and phosphorous content
over time. It is believed that the effect of compression may have
broken up the more brittle mineralized portion of the ECM, allowing
fragments to be swept out of the chamber by the ensuing perfusion
flow, thereby reducing the endpoint level of calcium and
phosphorous within the three dimensional construct.
[0125] These and other modifications and variations to the present
disclosure can be practiced by those of ordinary skill in the art,
without departing from the spirit and scope of the present
disclosure, which is more particularly set forth in the appended
claims. In addition, it should be understood that aspects of the
various embodiments can be interchanged both, in whole, or in part.
Furthermore, those of ordinary skill in the art will appreciate
that the foregoing description is by way of example only and is not
intended to limit the disclosure so further described in such
appended claims.
Example 3
Novel 3D Cell Culture System
[0126] The use of injection-molded culture chambers (FIG. 18)
allows the researcher to load the desired 3D scaffold material
(FIG. 18, component A) 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. 18, component B) molded directly into the
fluid ports.
[0127] 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. 18, component C) between the opposing
culture chambers allows for two independent samples (n=2) within
each plasticware assembly. Each chamber receives an independent
perfusion (FIG. 18, component D) 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. 18,
component E) to allow transfer of soluble factors and metabolites
between different cell populations retained within the opposing
chambers.
Peristaltic Assembly
[0128] The system assembly (FIG. 19) incorporates a two chamber
bioreactor assembly (FIG. 19, component A) into a
peristaltic-driven closed fluid circuit (FIG. 19, component B) with
medium reservoir (FIG. 19, component C) to provide continuous
perfusion into the culture chamber. Three-way valves (FIG. 19,
components D and E) allow the system to switch to syringe
pump-driven perfusion (FIG. 19, component F) for periodic delivery
of growth hormone and collection (FIG. 19, component G) 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).
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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 4
[0133] Dynamic 3D Co-Culture Vs. Static 2D
[0134] A comparison of 3D co-culture and 2D static systems was
conducted on two oncogenic cell lines, the Human Liver cell line,
HepG2 and MCF7 Human Breast Cancer Line. Both cell lines were
obtained from the ATCC. Cell passage for the HepG2 cells was
conducted 4 times in the 3D culture and 11 times in the 2D static
system. Cells were plated at a concentration of 9.0.times.10.sup.5
cells/chamber (3D) and 1.0.times.10.sup.5 cells/chamber (2D). In
the assays utilizing MCF7 cells, 3D passage was conducted 5 times
with a plating of 3.3.times.10.sup.5 cells/chamber. The 2D static
culture of MCF7 cells was passaged 6 times and seeded at a
concentration of 9.0.times.10.sup.4 cells/chamber. For both cell
types and culture systems, cells were grown in minimum essential
eagle medium supplemented with Sodium bicarbonate 1500 mg/L, Sodium
pyruvate 1 mM, FBS 10%, and Antibiotic 1%. A flow rate of 1 mL/min
was used in all experiments. A 3D Scaffold of Alginate beads (cells
encapsulated) with a scaffold diameter of .about.1 mm was used to
support the cell culture. 2D static cultures were conducted in
12-well plates with no scaffolds. Cells were assayed at 0, 2 and 6
days following culture. Albumin production was measured in the
HepG2 cells, Cathepsin D activity in the MCF7 cells and Hoechst dye
cell counts conducted with both cell types (see FIG. 21). 3D
confocal laser microscopy was performed in situ via 3D plasticware
imaging window. 3D cell populations demonstrated restrained growth
curves while 2D culture grew in an exponential fashion. Numerical
trends in cell function assays demonstrated good physiological
response by cells in 3D culture while 2D cells were limited or
trended down in their response. 3D confocal microscopy provided
good images of cells stained with calcein and ethidium homodimer-1.
Image depth was limited by the alginate bead scaffold material.
Example 5
[0135] HepG2 Human Liver Line obtained from the ATCC were passaged
14 times before being seeded in a cell chamber 10 at a
concentration of 6.3.times.10.sup.5 cells/chamber. Cells were grown
in minimum essential eagle medium supplemented with Sodium
bicarbonate 1500 mg/L, Sodium pyruvate 1 mM, FBS 10%, and
Antibiotic 1%. A flow rate of 1 mL/min was used to move media
through the inlet into the cell chamber and out through the outlet.
A 3D Scaffold of Alginate beads (cells encapsulated) with a
scaffold diameter of .about.1 mm was used to support the cell
culture. Cells were assayed at 0, 2 and 6 days following culture.
The assays conducted on the cells included Hoechst dye cell count,
AlamarBlue cell metabolism, and 3D confocal laser microscopy in
situ (see FIG. 22). Hoechst cell count and alamarBlue cell
metabolism assays demonstrated excellent correlation of the
averages (0.995) in assessing the 3D cell populations at various
timepoints. DiIC12(3) fluorescent dye also proved useful for longer
duration staining and imaging of live cells under 3D confocal
microscopy.
* * * * *