U.S. patent application number 15/002292 was filed with the patent office on 2016-05-19 for compositions and methods of functionally enhanced in vitro cell culture system.
This patent application is currently assigned to HUREL CORPORATION. The applicant listed for this patent is Robert Freedman, Yaakov Nahmias, Eric Novik, Martin Yarmush. Invention is credited to Robert Freedman, Yaakov Nahmias, Eric Novik, Martin Yarmush.
Application Number | 20160137984 15/002292 |
Document ID | / |
Family ID | 42226369 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160137984 |
Kind Code |
A1 |
Yarmush; Martin ; et
al. |
May 19, 2016 |
COMPOSITIONS AND METHODS OF FUNCTIONALLY ENHANCED IN VITRO CELL
CULTURE SYSTEM
Abstract
Compositions and methods described herein provide a cell culture
system in which cells are in high metabolic states from the onset
of the culture. Combinations of various cell culture components
disclosed and employed herein allow cells to be in high metabolic
states useful for drug testing immediately after the start of cell
culture.
Inventors: |
Yarmush; Martin; (Newton,
MA) ; Freedman; Robert; (Beverly Hills, CA) ;
Nahmias; Yaakov; (Plymouth, MN) ; Novik; Eric;
(Edison, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yarmush; Martin
Freedman; Robert
Nahmias; Yaakov
Novik; Eric |
Newton
Beverly Hills
Plymouth
Edison |
MA
CA
MN
NJ |
US
US
US
US |
|
|
Assignee: |
HUREL CORPORATION
Beverly Hills
CA
GENERAL HOSPITAL CORPORATION
Boston
MA
|
Family ID: |
42226369 |
Appl. No.: |
15/002292 |
Filed: |
January 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13131041 |
Feb 2, 2012 |
9243221 |
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PCT/US2009/065781 |
Nov 24, 2009 |
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15002292 |
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61118362 |
Nov 26, 2008 |
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Current U.S.
Class: |
435/29 ;
435/289.1; 435/373; 435/395; 435/397; 435/402 |
Current CPC
Class: |
C12M 23/16 20130101;
C12M 35/08 20130101; C12N 5/0671 20130101; C12N 2500/02 20130101;
C12M 23/12 20130101; C12N 2500/90 20130101; C12N 2513/00 20130101;
C12M 25/14 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1. A cell culture system comprising at least one compartment for
culturing cells, a culture of a first population of metabolically
active cells and at least two cell culture components selected
from: (i) a cell culture environment with an oxygen concentration
higher than the atmospheric concentration; (ii) a second cell
population for co-culture with said first population of cells
and/or a structure configured for three-dimensional culture of said
first population of cells with or without a second cell population
for co-culture with said first population of cells; (iii) a
serum-free culture medium or culture medium with a low
concentration of serum; and (iv) a cell culture medium that is
configured to contact or come into proximity with said first
population of cells under at least one condition of actuated
perfusion or flow.
2. The cell culture system of claim 1, wherein said first
population of cells comprise hepatocytes.
3. The cell culture system of claim 1, wherein said structure for
three-dimensional culturing comprises a gel sandwich culture, a gel
overlay culture, a micropatterned overlay culture, a scaffold, a
tissue slice, a tissue segment culture, or an artificial tissue
construct.
4. The cell culture system of claim 1, wherein said second cell
population comprises fibroblasts, glial cells, endothelial cells,
non-parenchymal cells, or stromal cells.
5. The cell culture system of claim 1, wherein said cell culture
environment comprises about 95% oxygen and about 5% CO.sub.2.
6. The cell culture system of claim 1 comprising a cell culture
environment comprising about 95% oxygen and about 5% CO.sub.2, a
three-dimensional culture comprising hepatocytes and fibroblasts
and a serum-free culture media.
7. The cell culture system of claim 1 further comprising a cell
binding material on said compartment for attachment of said
cells.
8. The cell culture system of claim 1 further comprising at least
one subcellular component contained in the at least one compartment
or another separate compartment.
9. The cell culture system of claim 1 wherein said culture medium
is circulated to said at least one compartment under actuated flow
or perfusion through at least one microfluidic channel.
10. A method of culturing a first population of metabolically
active cells comprising culturing said cells in the presence of at
least two cell culture components selected from: (i) a cell culture
environment with an oxygen concentration higher than the
atmospheric concentration; (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
11. The method of claim 10, wherein said first population of cells
comprises hepatocytes.
12. The method of claim 10 wherein said first population comprises
kidney cells or keratinocytes.
13. The method of claim 10, wherein said oxygen concentration is
higher than atmospheric for the seeding of said first population of
cells.
14. The method of claim 10, wherein said structure for
three-dimensional culturing comprises a gel sandwich culture, a gel
overlay culture, a micropatterned overlay culture, a scaffold, a
tissue slice, a tissue segment culture, or an artificial tissue
construct.
15. The method of claim 10, wherein said second cell population
comprises stromal cells, non-parenchymal cells or immune cells.
16. The method of claim 10, wherein said cell culture environment
comprises about 95% oxygen and about 5% CO.sub.2.
17. The method of claim 10, wherein primary cell hepatocytes are
seeded onto a three-dimensional cell culture structure together
with secondary cells comprising fibroblasts in an environment
comprising about 95% oxygen and about 5% CO.sub.2 and cultured in a
serum-free medium.
18. The method of claim 10, wherein said compartment further
comprises a coating of a binding material for attachment of said
cells.
19. A method of screening a material for its pharmacologic,
metabolic, pharmacokinetic, or toxicological properties comprising
culturing a first population of metabolically active cells in the
presence of at least two cell culture components selected from: (i)
a cell culture environment with an oxygen concentration higher than
the atmospheric concentration; (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
20. The method of claim 19, further comprising measuring an
activity of said first population of cells.
21. The method of claim 19, wherein said primary cells comprise
hepatocytes.
22. The method of claim 19, wherein said primary cells comprise
kidney cells or keratinocytes.
23. The method of claim 20, wherein said activity of said first
cells comprises a metabolite, a biomarker, gene expression, or
protein activity.
24. The method of claim 19, wherein said oxygen concentration is
higher than atmospheric for the seeding of said primary cells.
25. The method of claim 19, wherein said structure for
three-dimensional culturing comprises a gel sandwich culture, a gel
overlay culture, a micropatterned overlay culture, a scaffold, a
tissue slice, a tissue segment culture, or an artificial tissue
construct.
26. The method of claim 19 further comprising contacting said first
cells or the cell culture media from said first cells with a
subcellular component.
27. The method of claim 26, further comprising measuring an
activity of said subcellular component.
28. A kit for culturing cells comprising: (i) a device for
culturing cells containing at least one compartment, each
compartment filled with serum-free cell culture medium; (ii) a vial
of a first population of cryopreserved metabolically active cells;
(iii) a vial of a second population of cryopreserved cells; and
(iv) a canister of gases comprising 95% oxygen and 5% CO.sub.2.
29. The kit of claim 28, wherein said first population of cells
comprise hepatocytes.
30. The kit of claim 28, further comprising a scaffold for
three-dimensional cell growth at least one compartment.
31. The kit of claim 28, wherein said device comprises a microtiter
plate.
32. The kit of claim 28, wherein said device comprises a chip with
at least one microfluidic channel adapted to flow culture media
through said at least one compartment.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/118,362, filed Nov. 26, 2008, which application
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The liver constitutes a central site in the absorption,
binding, distribution, metabolism, excretion, and toxicogenicity
(absorption, distribution, metabolism, excretion and toxicity,
"ADME-T") of xenogenous materials (i.e. materials foreign to the
body in their origination). When a xenobiotic entity, such as a
drug, pharmaceutical, or nutriceutical, enters a human body, it is
frequently cleared (i.e., metabolically disposed of) in the liver
by oxidation, reduction, hydroloysis, and/or hydration steps of
biochemical reaction. Of the over a dozen different cell types that
comprise the liver, the hepatocyte is the type primarily
responsible for playing the role of "clearing house" or
"biotransformation driver," metabolically disposing of xenogenous
material. In the liver, the hepatocyte is the cell type wherein a
family of enzymes named cytochrome P-450 or CYP450 are chiefly
expressed, along with other enzymes that also mediate the Phase I
as well as the Phase II metabolic disposition of drugs, and other
xenogenous materials. The various CYP450 isozymes collectively
comprise the most important group of metabolizing enzymes that
perform the role of clearing house. The field of study of how the
body disposes of xenobiotic entities is frequently called Drug
Metabolism and Pharmacokinetics, or DMPK. The term
"pharmacokinetics" is often used in contradistinction to the term
"pharmacodynamics." Pharmacodynamics signifies the impacts and
effects that a drug may biochemically exert upon a cell, an organ
or an entire animal; whereas pharmacokinetics signifies the
impacts, effects and ultimate disposition that a cell, organ or
entire animal may biochemically exert upon a xenogenous chemical
entity. In everyday language, pharmacodynamics comprises what the
drug does to the body, while pharmacokinetics comprises what the
body does to the drug. Toxicity, including hepatotoxicity, is a
major category of pharmacodynamic effect (drug efficacy being
another such major category); while drug absorption, metabolism,
distribution, and excretion comprise the major categories of
pharmacokinetic effects.
[0003] In addition to metabolic function, signaling interactions
constitute another important category of cellular function in the
liver and in other organs. Classes of proteins called chemokines or
cytokines, among others, frequently effectuate signaling
interactions. Modem biotechnology has led to the delineation of a
variety of molecular signaling pathways in the cell. These
signaling pathways not only have provided insights into the
mechanisms of a cell, but also have opened opportunities to
intervene with cellular processes or abnormalities. Antibodies,
vaccines and other forms of chemical entities have been utilized to
specifically promote, inhibit, induce, or reduce one or more
signaling pathways.
[0004] Many attributes of a molecular entity must be investigated
and in some cases chemically modified or improved in the course of
developing that molecular entity into a therapeutically efficacious
and safe compound that regulatory agencies approve for marketing
and clinical use. One challenge is investigating and if necessary
overcoming any toxicity that the drug candidate may directly or
indirectly induce. Another set of challenges is to understand and
in some circumstances to improve the pharmacokinetic properties of
the molecular entity. Studying and if possible improving the
efficacy of the molecular entity to achieve an intended biochemical
result comprises a third set of challenges to be addressed along
the path of discovering, developing, testing, and ultimately
receiving marketing approval for a new drug.
[0005] To address the kinds of challenges enumerated above, in
vitro cell-based assay systems are frequently utilized to simulate,
measure and/or predict various functional attributes (including
without limitation those elaborated in the paragraphs above) of
liver cells as well as of cells from other organs comprising a
mammalian organism, and of the various organs themselves. These
assays may be variously utilized for analytic, therapeutic,
diagnostic, or industrial purposes. However, at the current state
of the art, such in vitro cell-based assay systems possess
limitations that impose high costs, or that limit the simulative or
predictive capacities of the assay (which in turn imposes high
costs when the simulative or predictive results of the assay are
found to be of no or limited use). One form of limitation that
currently, frequently occurs in in vitro cell-based assay systems
is that the configuration of the system causes the degree of
metabolic or other functional competency of the cultured cells to
remain at too low a level to yield accurate, or measurable, results
that afford a useful prediction of how a chemical entity being
tested on the system will interact with a cell, an organ, an organ
system or an entire organism in vivo. Another challenge of
cell-based systems is that the system is configured such that a
level of cellular functionality, once achieved, cannot be
maintained over a desirable duration of time. Another such form of
limitation is that the amount of time that must be devoted to the
initial incubation and/or culture of the cellular materials, prior
to the time when the cells assume the higher or highest degrees of
functionality of which they become capable, is a time of long
duration. Improvements to the state of the relevant art, which may
serve to reduce any of these or other limitations or their impacts,
will provide more accurate and cost-effective means of using
cellular cultures.
SUMMARY OF THE INVENTION
[0006] Present herein is a system in which cells are maintained in
a metabolically highly active state from close to the onset of the
culture. The system may be utilized to test the potential
toxicological impacts of constituent components of consumer and
industrial products, to study the environmental impacts of
pollutants and other chemicals, to detect the presence of chemical
and bioweapons, to analyze the pharmacologic, pharmacokinetic and
toxicological properties of molecular entities, and to study other
interactions between cellular materials and chemical or molecular
entities.
[0007] From a commercial standpoint, certain cell types are not
useful if they exist in a low metabolic state. For example, testing
drugs for hepatotoxicity frequently cannot be adequately
accomplished if the hepatocytes are in a low metabolic state.
Therefore, the period during which the cells are in a low metabolic
state constitutes a period of low, no, or wasted productivity in
economic terms.
[0008] Despite the absence or loss of productivity in the time
during which the low metabolic state obtains which may be as long
as a week or longer, such initial phase has been viewed and
accepted by industrial and academic scientists as an unavoidable
prerequisite that must be endured in order to reach at a later
point of time a culture characterized by a higher metabolic or
other functional state.
[0009] As presented herein there are embodiments in which the
initial phase of low metabolic state is reduced significantly to an
extent that useful applications of cultured cells are provided. In
other aspects, other embodiments are described in which other
limitations associated with traditional cell culture are reduced
significantly to an extent that useful applications of cultured
cells are provided.
[0010] Compositions and methods described herein utilize multiple
cell culture components. Each of the components of the system plays
a role distinct from the other components. Combinations of various
cell culture components give rise to various embodiments of
compositions and methods described herein. Also, each cell culture
component may have one or more embodiments. These cell culture
components combine together to provide unexpectedly rapidly arising
and high level of metabolic or other cellular function of the
cultured cells.
[0011] In one aspect, a cell culture component is a high oxygen
environment during cell seeding.
[0012] In one aspect, a cell culture component is a high oxygen
environment during cell culture.
[0013] In one aspect, a cell culture component is the absence of
serum in the culture media. Alternatively, said cell culture
component is a low level of serum in the culture media.
[0014] In one aspect, a cell culture component is the use of
material that enables the cells to assume a three-dimensional
("3D") relationship to each other. In one embodiment, the cell
culture component is a three-dimensional scaffold. Alternatively,
said cell culture component is a gel sandwich, a gel overlay, a
micropatterned array of cells, cells configured in a spheroid, a
tissue segment, a tissue slice, or an artificial tissue
construct.
[0015] In one aspect, a cell culture component is a cell cultured
in the presence of at least one additional cell type.
[0016] In one aspect, a cell culture component is liquid or gaseous
cell culture medium that is configured to contact cultured cellular
materials including cells cultured in a mono-layer, cells cultured
in a 3D relationship to each other, cells cultured in a co-culture,
subcellular materials, subcellular components, and cellular
products--under at least one condition of actuated perfusion or
flow.
[0017] Compositions and methods described herein comprise a cell
culture system comprising at least one compartment for culturing
cells, a culture of a first population of metabolically active
cells and at least two cell culture components selected from: (i) a
cell culture environment with an oxygen concentration higher than
the atmospheric concentration, (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
[0018] In one aspect of said cell culture system, said first
population of cells comprises hepatocytes.
[0019] In one aspect of said cell culture system, said structure
for three-dimensional culturing comprises a gel sandwich culture, a
gel overlay culture, a micropatterned overlay culture, a scaffold,
a tissue slice, a tissue segment culture, or an artificial tissue
construct.
[0020] In one aspect of said cell culture system, said second cell
population comprises non-parenchymal cells, stromal cells or immune
cells.
[0021] In one aspect of said cell culture system, said cell culture
environment comprises about 95% oxygen and about 5% CO.sub.2.
[0022] In one aspect of said cell culture system, said culture
medium is circulated to said at least one compartment under
actuated flow or perfusion through at least one microfluidic
channel.
[0023] In one aspect, said cell culture system comprise a cell
culture environment comprising about 95% oxygen and about 5%
CO.sub.2, a three-dimensional culture comprising hepatocytes and
fibroblasts and a serum-free culture media. In another aspect, said
cell culture system further comprises a cell binding material on
said compartment for attachment of said cells. In another aspect,
said cell culture system further comprises at least one subcellular
component contained in the at least one compartment or another
separate compartment.
[0024] Compositions and methods described herein comprise a method
of culturing a first population of metabolically active cells
comprising culturing said cells in the presence of at least two
cell culture components selected from: (i) a cell culture
environment with an oxygen concentration higher than the
atmospheric concentration, (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
[0025] In one aspect of said method of culturing, said first
population of cells comprises hepatocytes. Alternatively, said
first population comprises kidney cells or keratinocytes.
[0026] In one aspect of said method of culturing, said oxygen
concentration is higher than atmospheric for the seeding of said
first population of cells.
[0027] In one aspect of said method of culturing, said structure
for three-dimensional culturing comprises a gel sandwich culture, a
gel overlay culture, a micropatterned overlay culture, a scaffold,
a tissue slice, a tissue segment culture, or an artificial tissue
construct.
[0028] In one aspect of said method of culturing, said second cell
population comprises fibroblasts, glial cells, endothelial cells,
stromal cells or non-parenchymal cells.
[0029] In one aspect of said method of culturing, said cell culture
environment comprises about 95% oxygen and about 5% CO.sub.2.
[0030] In one aspect of said method of culturing, primary cell
hepatocytes are seeded onto a three-dimensional cell culture
structure together with secondary cells comprising fibroblasts in
an environment comprising about 95% oxygen and about 5% CO.sub.2
and cultured in a serum-free medium.
[0031] In one aspect of said method of culturing, said compartment
further comprises a coating of a binding material for attachment of
said cells.
[0032] Compositions and methods described herein comprise a method
of screening a material for its pharmacologic, metabolic,
pharmacokinetic, or toxicological properties comprising culturing a
first population of metabolically active cells in the presence of
at least two cell culture components selected from: (i) a cell
culture environment with an oxygen concentration higher than the
atmospheric concentration; (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
[0033] In one aspect of said method of screening, said method
further comprises measuring an activity of said first population of
cells.
[0034] In one aspect of said method of screening, said primary
cells comprise hepatocytes. Alternatively, said primary cells
comprise kidney cells or keratinocytes.
[0035] In one aspect of said method of screening, said activity of
said first cells comprises a metabolite, a biomarker, gene
expression, or protein activity.
[0036] In one aspect of said method of screening, said oxygen
concentration is higher than atmospheric for the seeding of said
primary cells.
[0037] In one aspect of said method of screening, said structure
for three-dimensional culturing comprises a gel sandwich culture, a
gel overlay culture, a micropatterned overlay culture, a scaffold,
a tissue slice, a tissue segment culture, or an artificial tissue
construct.
[0038] In one aspect of said method of screening, said method
further comprises contacting said first cells or the cell culture
media from said first cells with a subcellular component. In
another aspect of said method of screening, said method further
comprises measuring an activity of said subcellular component.
[0039] Compositions and methods described herein comprise a kit for
culturing cells comprising: (i) a device for culturing cells
containing at least one compartment, each compartment filled with
serum-free cell culture medium; (ii) a vial of a first population
of cryopreserved metabolically active cells; (iii) a vial of a
second population of cryopreserved cells; and (iv) a canister of
gases comprising about 95% oxygen and about 5% CO.sub.2.
[0040] In one aspect of said kit, said first population of cells
comprise hepatocytes.
[0041] In one aspect of said kit, said kit further comprises a
scaffold for three-dimensional cell growth at least one
compartment.
[0042] In one aspect of said kit, said device comprises a
microtiter plate.
[0043] In one aspect of said kit, said device comprises a chip with
at least one microfluidic channel adapted to flow culture media
through said at least one compartment.
[0044] In one embodiment of compositions and methods described
herein, the system comprises multiple cell culture components.
[0045] In one embodiment of compositions and methods described
herein, the system employs three cell culture components.
[0046] In one embodiment of compositions and methods described
herein, the system employs two cell culture components.
INCORPORATION BY REFERENCE
[0047] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The novel features of compositions and methods described
herein are set forth with particularity in the appended claims. A
better understanding of the features and advantages of compositions
and methods described herein will be obtained by reference to the
following detailed description that sets forth illustrative
embodiments, in which the principles of compositions and methods
described herein are utilized, and the accompanying drawings of
which:
[0049] FIGS. 1 and 1A illustrates an embodiment of compositions and
methods described herein comprising multiple cell culture
components.
[0050] FIGS. 2 and 2A is illustrates an embodiment of compositions
and methods described herein comprising multiple cell culture
components.
[0051] FIGS. 3A and 3B illustrates effects of various cell culture
components on the metabolic state of cultured cells.
[0052] FIGS. 4A, 4B, and 4C illustrates effects of various cell
culture components on the metabolic state of cultured cells.
[0053] FIGS. 5A and 5B illustrates clearing of drugs by hepatocytes
cultured according to one embodiment of present invention.
[0054] FIG. 6 is a pro forma, prophetic graphical illustration of
the unexpected benefit which would be produced by an embodiment of
compositions and methods described herein comprising multiple cell
culture components.
DETAILED DESCRIPTION OF THE INVENTION
[0055] While preferred embodiments of compositions and methods
described herein have been shown and described herein, it will be
obvious to those skilled in the art that such embodiments are
provided by way of example only. Numerous variations, changes, and
substitutions will now occur to those skilled in the art without
departing from compositions and methods described herein. It should
be understood that various alternatives to the embodiments of
compositions and methods described herein may be employed in
practicing compositions and methods described herein. It is
intended that the following claims define the scope of compositions
and methods described herein and that methods and structures within
the scope of these claims and that their equivalents be covered
thereby.
[0056] Described herein are configurations of elements of a cell
culture system for the purpose of producing, in an in vitro
environment, the enhanced cellular functionality of the cells
cultured therein, their subcellular components, cellular products,
or cellular materials, to an extent determined either in terms of
at least one of either (a) improved degree of superior
functionality achieved or (b) the improved rapidity with which any
particular degree of functionality is achieved than may be
achievable by any alternative cell culture system that does not
comprise the component elements that are configured in compositions
and methods described herein. Enhanced cellular functionality may
comprise the cell's enhanced ability to synthesize proteins or
other cellular products; to maintain the functionality of
organelles or other subcellular components, such as mitochondria;
to produce cytokines; to express genes; or to transport or
metabolize xenogenous materials with which the cell comes in
contact; or to perform any other cellular function Enhanced
cellular functionality may comprise the degree, or extent, to which
the cell manifests any of the foregoing functions.
[0057] Presented herein is a cell culture system in which cells
rapidly achieve and maintain a metabolically highly active state
from close to the onset of the culture. Various combinations of the
cell culture components described provide highly functional cells
while diminishing the time required to achieve such high
functionality, thus reducing time lost for productive use of the
cultures.
[0058] From a commercial standpoint, certain cell types are not
useful if they exist in a low metabolic state, i.e., at a low level
of metabolic competency. For example, with respect to cultured
hepatocytes, testing for the hepatotoxicity or clearance of
chemical entities, or the generation of metabolites derived from
such entities, is not effective if the hepatocytes exist in a low
metabolic state. The period in which cells are in said low
metabolic state thus constitutes a period of low, or no, or wasted
productivity in economic terms. This limitation may diminish the
utility of culturing cells for a wide range of potentially
desirable uses including without limitation not only the study of
drug toxicity or drug metabolism, but also therapeutic uses (such
as a device comprising cultured cells that is used to treat
patients), diagnostic uses (such as, for example, a device
comprising cultured cells that is configured to measure some marker
in the blood of a patient), or industrial uses (such as, to cite
several examples, a device comprising cultured cells that is
configured to test for the potential toxicity of molecules that are
constituent elements of industrial or consumer products, or a
device comprising cultured cells that is configured to test for
levels of environmental pollutants or for the presence of chemical
or biological warfare agents). With the cell culture systems
described herein, this limitation is ameliorated and such uses as
those elaborated above become more effective, cost-effective, and
achievable. The embodiments presented herein contains various
combinations of components and conditions in which the initial
phase of low metabolic state is reduced providing quick achievement
of highly functional and metabolically active cell cultures useful
for a variety of purposes.
[0059] Described are multiple cell culture components. Each
component of the system is distinct from the other components and
each contributes to the unexpected benefits shown. Combinations of
various cell culture components give rise to different embodiments.
Also, each cell culture component may have one or more
embodiments.
[0060] In one aspect, a cell culture component employed in
compositions and methods described herein is a higher than normal
atmospheric oxygen environment. This may include high oxygen
conditions for seeding the cells and/or for the growth and
culturing of the cells. High oxygen conditions or environment
comprise a concentration of oxygen that is higher than a normally
occurring, atmospheric concentration of oxygen. In one embodiment,
the concentration of oxygen is about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 100% of the gaseous composition present in
the system. In another embodiment, the concentration of oxygen is
about 52%, 57%, 62%, 67%, 72%, 77%, 82%, 87%, 92%, or 97% of the
total gaseous composition present in the system. In some instances
the concentration of oxygen may be only slightly higher than the
normal atmospheric concentration. The oxygen concentration may be
held constant or relatively constant throughout the seeding or
culturing, or it may be changed over the course of the seeding or
culturing period.
[0061] There may be one or more non-essential gases present in the
total atmospheric composition utilized for high oxygen conditions.
The non-essential gas can be air, CO.sub.2, N.sub.2, or any type of
inert gas.
[0062] High oxygen seeding is accomplished by a number of
approaches. For example, high oxygen seeding is accomplished by
seeding the cells in an air-tight culture chamber. In an exemplary
embodiment, cells comprising a single cell type are seeded, i.e.,
plated, onto a suitable physical substrate and then placed in an
air-tight chamber and the atmosphere is exchanged to high oxygen
immediately following the seeding. Alternatively, high oxygen
seeding can be accomplished by the use of a preconditioned chamber
in which the chamber already maintains a high level or
concentration of oxygen.
[0063] For high oxygen seeding, the duration in which oxygen level
is maintained higher than atmospheric level may vary from as little
as the time required for the seeding of the cell to as long as
about 24 hours. In one embodiment, as soon as the seed culture is
placed in a cell culture chamber, oxygen level is returned to
normal atmospheric level. In another embodiment, the oxygen level
is maintained higher than atmospheric level for 24 hours.
[0064] Alternatively or in addition, high oxygen conditions may be
utilized after seeding. One could seed under normal oxygen
concentration and then increase the gaseous level of oxygen for the
period of time the cells are cultured. It is possible to utilize
high oxygen conditions for cell culture, for about 1, 2, 3, 4, 5,
6, 7, 8 or more days, or for any greater or lesser length of time.
In one embodiment, the high oxygen is used for seeding. In another
embodiment the high oxygen is utilized for the entire culturing
period. In another embodiment the high oxygen is utilized for a
portion of the culturing period. In another embodiment the high
oxygen is used intermittently.
[0065] For high oxygen conditions, the media can be treated by
oxygen bubbling. The high oxygen conditions in the closed chamber
may be accomplished just by utilizing oxygen bubbling through the
medium. Alternatively, the media can be used without the bubbling.
In one embodiment, the high oxygen content is achieved by
increasing the partial pressure of oxygen in the gaseous
environment in the cell culture device. It can be useful to begin
with the high oxygen content or to close the system including the
cells and then alter the oxygen content in the system. One may also
enhance the oxygen concentration of the media by the addition of an
oxygen carrier, such as hemoglobin or perfluorocarbon.
[0066] The level of oxygen can be monitored by various methods
including blood gas analyzer, monitoring atmospheric tension,
oxygen monitor, or other measuring devices known in the art.
Monitoring of the oxygen will permit one to maintain the conditions
at the desired oxygen level and for the period of time desired.
[0067] The oxygen level of the cell culture can be maintained by an
automated feed-back regulator type device. For example, a
computerized process can be employed to monitor the oxygen level
and adjust the input oxygen level accordingly. Such devices are
well known and available for this regulation.
[0068] In one aspect, a cell culture component is the absence of
serum in the culture media. Alternatively, said cell culture
component is low level of serum in the culture media. In one
embodiment, the cells are cultured in serum-free or substantially
serum-free media. A substantially serum-free medium might contain
trace level of serum. Serum-free media can be prepared or purchased
from material provided by serum-free media manufacturers. One
example of such media is Dulbecco's Modified Eagles Medium.
Serum-free media may include additives such as non-essential amino
acids, antibiotics, L-glutamine, or tryptophan. In another
embodiment, the cells are cultured in low-serum containing media.
Examples of low serum concentration level include about 0.1%, 0.2%,
0.5%, 0.7%, 1%, 1.2%, 1.5%, 1.7%, 2.0%, 2.2%, 2.5%, 2.7%, 3.0%,
3.2%, 3.5%, 3.7%, 4.0%, 4.2%, 4.5%, 4.7%, 5.0%, 5.2%, 5.5%, 5.7%,
6.0%, 6.2%, 6.5%, 6.7%, 7.0%, 7.2%, 7.5%, 7.7%, 8.0%, 8.2%, 8.5%,
8.7%, 9.0%, 9.2%, 9.5%, 9.7%, 10%, 10.2%, 10.5%, 10.7%, or 11%, or
any intermediate value between any of the forgoing. Alternatively,
examples of low serum level include about 11%, 11.5%, 12%, 13.5%,
14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%,
19.5% or 20% or any intermediate value between any of the
forgoing.
[0069] Exemplary sources of serum include, but not limited to,
horse, chicken, donkey, rabbit, cow, or rat.
[0070] In one aspect, a cell culture component is the use of
material that allows the cells to assume three-dimensional ("3D")
relationships to each other.
[0071] One approach for a 3D configuration comprises hepatocytes
cultured in a "gel sandwich" configuration, wherein matrices of
collagen fibers are configured both below and above the
hepatocytes, and the hepatocytes are cultured in between the
layers. In a variant 3D approach a monolayer of hepatocytes which
is adhered to a rigid coated or uncoated surface is overlaid with a
layer of collagen gel. A third 3D approach relies on the
aggregation of hepatocytes into spheroids following the seeding of
the cells on a soft gel such as Matrigel.TM. or a weakly adhering
surface.
[0072] In an embodiment, the material that allows the cells to
assume 3D relationships is a three-dimensional scaffold. The
three-dimensional scaffold can be made with a biologically
non-toxic, inert material. The scaffold can be made with material
that allows cellular attachment. One example of such material is a
biocompatible gel formula made with calcium alginate which forms a
material for culturing the cells. Other examples of biological
coating materials include, but are not limited to, methylcellulose,
MATRIGEL.TM., BIOCOAT.TM., collagen, fibrinogen, fibronectin,
gelatin, laminin hyaluronin, hyluronic acid, or any of the family
of polyamines such as polylysines. Such materials encourage binding
and enhance culturing and also may provide a micro-scaffold in
which cells can multiply while maintaining three-dimensional
relationship to each other. In one embodiment, the scaffold is made
of a gel matrix. In another embodiment, the scaffold is
self-assembling peptide hydrogel. In one embodiment there is no
binding material on the scaffold. The cross-sectional dimensions of
the structural filaments or fibers of the scaffold may range from
20 microns to 1000 microns, although any size that accommodates the
cells, in the interstitial spaces of the scaffold, known as the
pores, will generally function. Pore sizes may range in size from 1
micron to 200 microns, or larger.
[0073] In an aspect, the culture device includes one or more
chambers at least one of which includes a physical structure
containing a material for the three-dimensional cell culture. Such
structure may be a microscale scaffold for the stable lodging of
cellular material.
[0074] In a variant approach, hepatocytes can be entrapped in
microscale scaffolds such as calcium alginate, inside which the
hepatocytes may assume a three-dimensional configuration with
respect to each other, such as a spheroid configuration. Yet other
forms of 3D hepatocyte culture are comprised of culturing a slice
or a segment of actual tissue drawn directly from the liver of a
recently deceased organism; or an artificial tissue construct. 3D
cell cultures may also comprise tissue slices or tissue segments
drawn from other organs, such as slices or segments of brain
tissue, or slices or segments of kidney tissue.
[0075] In one aspect, a cell culture component is a co-culture
system. In one embodiment, a co-culture system comprises a first
cell population and a second cell population wherein the second
cell population plays a supportive role in the metabolic or other
functionality of the first cell population. In an alternative
aspect, each cell population's presence in the co-culture system
serves to enhance the functionality of the other cell population
and of the co-culture as a whole. Examples of such a supportive
role include, but are not limited to, providing cytokines,
providing cell-cell contact, providing anchorage, excreting or
secreting extracellular material, and providing an environment
mimicking the in vivo environment of the first cell population. The
second cell population can be sub-lethally irradiated to prevent
the population from growing. The second cell population can be a
layer of cells attached to the planar surface of, or to a scaffold
configured inside, the chamber. The second cell population can be
configured to be cultured interspersed in or contiguous to the
first cell population. Alternatively, the second population can be
in suspension in the culture media. Exemplary sources of the second
cell population include stromal cells, non-parenchymal cells,
fibroblasts, glial cells, or immune cells. One can utilize various
immune cells, such as lymphocytes, dendritic or Kupffer cells. In
one embodiment, the second cell population is 3T3-J2 fibroblasts.
In one embodiment, the first and second cell populations are from
the same species of animal, such as human. In other cases, the cell
populations are from different species. It may be useful to obtain
the cells from any species of animal, including mammalian species
such as human, monkey, mouse, rat, pig, cow, horse, dog or sheep.
The cells may be freshly isolated cells, primary cells, engineered
cells, preserved cells, a cell line, a tissue slice, a tissue
segment, an artificial tissue construct, cryopreserved cells, or
stem cells.
[0076] In one embodiment, the co-culture system is comprised of two
different cell populations representing two different parenchymal
cell populations or representing a parenchymal cell population and
non-parenchymal cell population. For example, the first cell
population can be a kidney glomerular parietal cell population and
the second population can be a glomerular podocyte population. In
another example, the first cell population can be hepatocytes, and
the second population can be fibroblasts, endothelial cells, or
stellate cells. The proportion between the first cell population
and the second cell population can vary depending on the type of
cells. The ratio can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,
1:8, 1:9, 1:10, or vice versa, or any intermediate ratio between
any two of the foregoing, or any other ratio. In one embodiment,
hepatocytes are cultured with 3T3-J2 fibroblasts in a 10:1 ratio.
In another embodiment, hepatocytes are cultured with 3T3-J2
fibroblasts in a 2:1 ratio. In yet another embodiment, hepatocytes
are cultured with 3T3-J2 fibroblasts in a 1:1 ratio. Another
example is a gut epithelial population and a goblet cell
population. Another example is a Type I pneumocyte population and
an endothelial cell population. In another example the first
population is a keratinocyte population and the second population
is a Langerhan cell or dendritic cell population. In one
embodiment, the co-culture system is comprised of three or more
different cell populations representing parenchymal cell
populations, non-parenchymal cell populations, and immune cell
populations; for example, the first cell population can be
hepatocytes; the second population can be fibroblasts, endothelial
cells, or stellate cells; and the third population can be Kupffer
cells or T cells. Another example of a co-culture comprising three
or more different cell populations is a kidney glomerular parietal
cell population, a glomerular podocyte population, and an
endothelial cell population. Another such example is a co-culture
comprising a keratinocyte population, a fibroblast population, and
a Langerhan cell or dendritic cell population. Another such example
is a gut epithelial population, a goblet cell population, and a T
cell population. Another example is a Type I pneumocyte population,
a Type II pneumocyte population, and an endothelial cell
population. In one embodiment, members of the respective cell
populations are configured to be cultured in a particular geometric
arrangement and/or in particular proportions to one another by
means of a micropatterning technique.
[0077] A variety of configurations of the physical substrate for
adhering, holding or containing the cell culture system are
possible, including a flat substrate; or, in an embodiment, the
physical substrate may be configured to comprise at least one
chamber or compartment. The chamber comprises a planar surface and
walls surrounding and partially or completely enclosing the planar
surface. The planar surface is coated or treated to allow cellular
attachment. In an aspect, the chamber is open in one dimension,
thereby assuming the configuration of an open compartment or well.
In an alternative aspect, the chamber further comprises an
additional element configured to be continuously contiguous with
the walls of the chamber, so as to comprise a closed, air-tight
chamber suitable for the containment, without leakage, of gaseous
or liquid cell culture medium. The cell culture chamber is
structured to contain media as well as to withstand atmospheric
pressure accompanied with oxygenation. The chamber may or may not
comprise at least one opening through which sampling of the media
or atmosphere can be performed, or through which cell culture
medium can enter and/or exit the chamber, thereby perfusing any
cellular materials configured therein. In an embodiment, the cell
culture chamber is microscale, wherein microscale means being
configured to possess at least one physical feature that is
characterized by having at least one linear dimension (length,
width, height or depth) measuring less than one millimeter. Often
microscale is considered to be having dimensions of 10 nm to 1 mm.
In an aspect, for ease and efficiency of use, the cell culture
chamber is repetitively configured with multiple embodiments
integrated proximally one to another into a single piece of
laboratory ware, such as a multi-well microtiter plate.
[0078] In one embodiment, a cell culture chamber can be used to
accommodate a monolayer cell culture. In an alternative embodiment,
the cell culture chamber is used to accommodate a cellular
co-culture, a culture of cells in a 3D configuration, subcellular
materials, cellular products, or subcellular components, any of
which may be primary, naturally occurring, man-made, artificial, or
engineered. The subcellular material in the culture device may be a
cellular product. Exemplary cellular products may include without
limitation an enzyme, a nucleic acid, a protein, a lipid, and a
carbohydrate. The cellular product may be man-made. The cellular
product may comprise a naturally occurring or man-made cellular
product in conjunction with some other biochemical entity. The
subcellular material may comprise a subcellular component.
Exemplary subcellular component may include without limitation a
microsome, mitochondrion, nucleus, ribosome, plasma membrane, and
the like. The subcellular component may be man-made. The
subcellular component may comprise a naturally occurring or
man-made subcellular component in conjunction with some other
biochemical entity. For example, the subcellular component may be
an engineered enzyme, protein or artificial cellular structure.
Such subcellular component may be included in any of the
compartments during the culturing step. In one embodiment, the
subcellular component is involved in the metabolic or toxicological
process of the system. For example, a material to be analyzed may
be cultured with the first population of metabolically active cells
to produce a product from the culturing step, which may interact
with the subcellular component. The result of that interaction may
be measured or analyzed to provide an evaluation of the
material.
[0079] In another embodiment the physical substrate for cell
culture is a biochip. The biochip comprises a microscale channel or
channels fluidically connected to or otherwise fluidically
integrated with at least one chamber or compartment for culture of
cells or subcellular materials. The at least one channel and the at
least one chamber are configured to facilitate the actuated,
microfluidic circulation or recirculation of cell culture medium in
contact with or in proximity to the cell culture system, under a
condition of perfusion or flow. In an aspect the configuration of
the at least one compartment and the at least one chamber comprises
a linear flow path for the circulation or recirculation of the cell
culture medium. In an alternative embodiment the flow path may
bifurcate and become multilinear. The biochip may be microscale.
The at least one compartment of the biochip may be configured to be
either open in one dimension, thereby assuming the configuration of
an open compartment or well; or closed so as to comprise a closed,
air-tight chamber suitable for the containment, without leakage, of
gaseous or liquid cell culture medium, as well as to withstand
atmospheric pressure accompanied with oxygenation. In an aspect,
the chamber also comprises an inlet and an outlet for flow of
culture medium.
[0080] An embodiment of the biochip may contain a single
compartment (e.g., a chamber); or alternatively, another embodiment
of the biochip may contain two compartments, where one compartment
contains cells, subcellular materials, subcellular components, or
cellular products and the other compartment is an open reservoir
for the addition or withdrawal of culture medium. In another
aspect, more than one chamber of the biochip may each contain cells
cultured in a monolayer, cells cultured in a co-culture,
subcellular materials, subcellular components, or cellular
products, wherein the cell culture system contained in one chamber
is different from the cell culture system contained in the at least
one other chamber. In an alternative embodiment, the cell culture
system in one compartment of the biochip is identical to the cell
culture system in the at least one other compartment of the
biochip. In another embodiment the biochip may contain at least one
compartment and in some instances three or more compartments.
[0081] Another embodiment of the biochip may further comprise a
pumping mechanism, wherein the pumping mechanism may either be
integrated in the biochip or separate from and external to the
biochip. In one such embodiment the pumping mechanism may be
hydraulic, such as a syringe pump or a peristaltic pump. In one
such embodiment, the pumping mechanism may be electro-kinetic or,
alternatively, an alternative embodiment may comprise a diaphragm
pump that is mechanically actuated or pneumatically actuated. In
another embodiment, the biochip may further comprise a de-bubbler
located within the biochip or external to the biochip. In another
embodiment the biochip may comprise at least one sensor for
obtaining signals from the cultured cells, subcellular materials,
subcellular components, or cellular products, wherein at least one
sensor may be a biosensor and the biosensor may comprise a
waveguide.
[0082] The biochip may be microfabricated. The biochip may be
manufactured from a microfabricated master. The biochip may be
manufactured by mass production that causes the geometry of the
device (including the provision for the rate of fluid flow in and
through the device) to be substantially the same from one such
manufactured copy, specimen or iteration of the device to the next.
The process of mass production may include that the biochip is
manufactured from a microfabricated master.
[0083] The well, chamber, microtiter plate, biochip, or other
physical substrate may be comprised of glass, silicon, a plastic
such as polycarbonate, cyclic oxide copolymer (COC), polystyrene,
or other plastic formulations, or any other material that may
comprise a suitable physical substrate for in vitro cell
culture.
[0084] In one aspect, a cell culture component employed in
compositions and methods described herein is cell culture medium
that is configured to come into contact or proximity with a cell
culture system under a condition of perfusion or flow. The biochip,
the cell culture system and the pump may be configured together to
achieve desired or optimal levels of metabolic or other cellular
functions under the at least one condition of perfusion or flow.
The condition of perfusion or flow may be configured to maintain
desired values or ranges of certain parameters, such as the amount
of shear stress brought to bear upon the cells by the perfusate
culture medium, the flow volume or flow velocity of the perfusate
culture medium as it contacts or comes into proximity to the cell
culture system, the residence time during which a single molecule
of a chemical entity dissolved or suspended in the perfusate
culture medium remains within a compartment of the biochip or
remains in contact with the at least one cell culture system
contained in the biochip, and the like. Maintaining desires values
or ranges of these and other parameters may cause the cell culture
system to manifest desired or optimal levels of cellular function.
In one aspect, a desired value or range of at least one such
parameter simulates a value found in a living organism.
[0085] In an embodiment, the condition of perfusion or flow is
maintained during cell seeding. In an alternative embodiment, the
condition of perfusion or flow is maintained during cell culture.
Parameters that characterize the flow may be controlled so as to
achieve desired or optimal levels of cellular or subcellular
functionality. For example, in an embodiment the shear stress
exerted by the flowing cell culture medium upon the cell culture
system is less than 14 dynes per square centimeter (dyn/cm.sup.2).
In an alternative embodiment the shear stress exerted by the
flowing cell culture medium upon the cell culture system is less
than 2 dyn/cm.sup.2. In other embodiments, the shear stress exerted
by the flowing cell culture medium upon the cell culture system and
cells is less than 1 dyn/cm.sup.2, less than 0.5 dyn/cm.sup.2, less
than 0.2 dyn/cm.sup.2, less 0.1 dyn/cm.sup.2, less than one order
of magnitude less than 0.1 (0.01) dyn/cm.sup.2, less than two
orders of magnitude less than 0.1 (0.001) dyn/cm.sup.2, less than
three orders of magnitude less than 0.1 (0.0001), less than
0.00001, less than 0.000001 dyn/cm.sup.2, less than 0.0000001
dyn/cm.sup.2, less than 0.00000001 dyn/cm.sup.2, less than
0.000000001 dyn/cm.sup.2, less than 0.0000000001 dyn/cm.sup.2. In
another embodiment, the cell culture system is configured in a
biochip and the at least one compartment of the biochip is in turn
configured with a series of ridges and depressions in its planar
surface, such that the cell co-culture is seeded down in the
depressions of the compartment while the ridges, which extend
higher up into the flow path than the cell culture system does,
serve to mechanically shield the cell culture system from the most
forceful contacts with the perfusate, such that the shear stress
exerted upon the cell culture system is minimal. In an alternative
aspect, the compartment is configured to comprise a permeable
membrane that segregates the cell culture system from the flowing
perfusate and thereby shields it from shear stress while permitting
chemical entities dissolved or suspended in the perfusate culture
medium to come into contact and interact with the cell culture
system by diffusing across the membrane. In another aspect, the
flow rate of the perfusate cell culture medium through the biochip
is two nanoliters per minute (2 nL/min). In an alternative aspect,
the flow rate of the perfusate is five microliters per minute (5
.mu.L/min). In a third exemplary aspect, the flow rate is one
milliliter per minute (1 mL/min). In another embodiment, the flow
rate is any intermediate value between any of the foregoing rates.
The flow rate may be held constant or, in an alternative
embodiment, it may vary or be intermittent during the period of
cell culture or during the period of cell seeding. In another
embodiment, the geometry of the biochip design and the speed of the
pumping mechanism are together configured to produce a residence
time in the at least one compartment of the biochip of about 0.5
seconds (sec.), 0.75 sec., 1.0 sec., 5.0 sec., 20 sec., 30 sec., 60
sec., 2 minutes (min), 5 min, 30 min, 60 min., 2 hours (hr.), 3
hr., 4 hr., 6 hr., 12 hr., 24 hr., 48 hr., 96 hr., 1 week (wk.), 2
wk., or any time value between any of the foregoing. In one aspect,
the residence time is configured to simulate at least one residence
time found in vivo in the human liver.
[0086] An example of an embodiment that comprises the multiple cell
culture components is high oxygen seeding in the presence of 95%
oxygen; cell culture media comprising a low concentration of serum;
non-parenchymal cells as the second cell population; and cell
culture medium that is configured to be pumped so as to
re-circulate through the compartment, where it comes into contact
with cells from at least one of the cell populations under at least
one condition of perfusion or flow. In another embodiment, an
example of the multiple cell culture components is high oxygen
seeding in the presence of 87.5% oxygen, cell culture medium
comprising no serum, non-parenchymal cells as the second cell
population, and cell culture medium that is configured to perfuse
the cell culture system in a biochip at a rate of 5 mL/min.
Alternatively, the cell culture system comprises multiple cell
culture components comprising high oxygen seeding in the presence
of 95% oxygen, cell culture media with 0.5% serum concentration,
non-irradiated fibroblasts as the second cell population, and a
three dimensional cell culture scaffold --including a layer of a
binding material, such as MATRIGEL.TM.. Another alternative example
of the multiple cell culture components comprises high oxygen
seeding in the presence of 100% oxygen, cell culture media with 1%
serum concentration, a co-culture comprising cryopreserved primary
human hepatocytes as the first cell population and 3T3-J2
fibroblasts as the second cell population, and flowing cell culture
medium that perfuses the co-culture (configured in a compartment of
a biochip) while maintaining a residence time in the compartment
similar to at least one value for residence time obtained in vivo
in the liver of an adult human, and while exerting in the
compartment a shear stress of less than 2 dyn./cm.sup.2. Another
embodiment includes high oxygen culturing in 95% oxygen, cell
culture media in serum free media, non-irradiated fibroblasts as
the second cell population, and a three dimensional cell culture
scaffold including a layer of a binding material, such as
MATRIGEL.TM..
[0087] In one embodiment, the cell culture system comprises
multiple cell culture components. One example of the multiple cell
culture components includes high oxygen seeding in the presence of
95% oxygen, serum-free cell culture media, non-irradiated
fibroblasts as the second cell population, and a cell culture
scaffold provided by MATRIGEL.TM.. Another example of the multiple
cell culture components is high oxygen seeding in the presence of
100% oxygen, serum-free cell culture media, 3T3-J2 fibroblasts as
the second cell population, and a cell culture scaffold provided by
MATRIGEL.TM.. Yet another example of the multiple cell culture
components is high oxygen seeding in the presence of 95% oxygen and
5% CO.sub.2, serum-free media, 3T3-J2 fibroblasts as the second
cell population, a cell culture scaffold provided by collagen
coating.
[0088] In another embodiment, the cell culture the system comprises
three cell culture components. One example of the three cell
culture components are high oxygen seeding in the presence of about
95% oxygen, serum-free cell culture media, and non-irradiated
fibroblasts as the second cell population. Another example of the
three cell culture components are high oxygen seeding in the
presence of 100% oxygen, serum-free cell culture media, 3T3-J2
fibroblasts as the second cell population. Yet another example of
the three cell culture components are high oxygen culturing in the
presence of about 95% oxygen and about 5% CO.sub.2, serum-free cell
culture media, 3T3-J2 fibroblasts as the second cell
population.
[0089] In one embodiment, the cell culture the system comprises two
cell culture components. One example of the two cell culture
components is high oxygen seeding in the presence of 95% oxygen and
cell culture media with serum-free media. Another example of the
two cell culture components is high oxygen seeding in the presence
of 100% oxygen and cell culture media with 0.1% serum. Yet another
example of the two cell culture components is high oxygen culturing
in the presence of about 95% oxygen and about 5% CO.sub.2 and
serum-free cell culture media.
[0090] It is possible to use any mammalian cell type, whether
comprising a primary cell or a cell line, for the cultures,
including without limitation hepatocytes, enterocytes,
keratinocytes, neural cells, cardiac muscle cells, pancreatic
cells, renal cells, and stem cells. Various embodiments of the
culture system disclosed herein can be customized as to cell type
so as to obtain a high metabolic or other functional state of the
cells from close to the onset of the culture; or to obtain at least
one other unexpected benefit from the culture. Compositions and
methods described herein can be useful for maintaining for an
extended period of time primary cell culture, i.e., cells freshly
isolated from tissue or an organ; or alternatively for maintaining
for an extended period of time cell lines, i.e., engineered cells
adapted to grow and/or be maintained and to function under in vitro
conditions.
[0091] Primary cells can be obtained by any techniques known in the
art. Examples of such techniques include but are not limited to
surgical separation, isolation, fluorescence activated cell
sorting, magnetic activated cell sorting, use of a cell sieving
device, centrifugation, volume cell sorting, and chemotactic cell
sorting methods. In practice one may utilize multiple-parallel
formats wherein the format allows parallel culturing of cells under
the disclosed different combinations of cell culture components. In
one embodiment, the format is implemented in a micro-titer plate.
Examples of micro-titer plates include 6-well plate, 12-well plate,
24-well plate, 48-well plate, 96-well plate, 256-well plate,
384-well plate, and 1536-well plate.
[0092] Compositions and methods described herein can be
manufactured as a kit. In one embodiment a kit comprises a
microtiter plate, oxygen tank containing a mix of gases with
pre-determined ratio of high oxygen content, frozen vials
containing one or more cell population(s), and serum-free or low
serum culture media. In one embodiment a kit comprises a microtiter
plate coated with a binding agent and lyophilized cells and
serum-free or low serum media. In another embodiment the kit
comprises a microfluidic chip including channels for circulation of
media to permit contacting the cells under perfusion or flow, one
or more compartments for culturing cells, which compartments may
include a variety of designs and materials, such as biocompatible
coatings of binding agents, scaffolds for three-dimensional cell
growth, reservoirs for fluids, and ports for the injection of
materials, gases, and monitoring of oxygen or metabolic activity
and products. Such chip may include cells and media. Alternatively,
the kits may include the chip, vials of the primary and secondary
cells and media for cell growth. The kit may also include
educational or training materials such as an instruction manual or
CD.
[0093] A variety of methods may be used to measure the metabolic,
toxicological, or other functional state of the cells. Methods of
measurement include any method to measure gene expression (e.g.
reverse transcription polymerized chain reaction ("RT-PCR")
analysis), any method to measure the expression level of enzymes or
other proteins produced in or by the cell (e.g. western blot or
ELISA), any method to measure the activity of enzymes or other
proteins produced by the cell (e.g. EROD
(ethoxyresorufin-O-deethylase), MROD
(methoxyresorufin-O-deethylase), PROD
(pentoxyresorufin-O-deethylase), BROD
(benzyloxyresorufin-O-deethylase) assays, or metabolite formation
by liquid chromatographic/mass spectroscopic analysis), any method
to measure active molecular transport into or out of the cell, any
method to measure the metabolic activity of organelles such as
mitochondria, any method to measure the production or secretion of
cytokines or chemokines, any method to measure the presence or
quantity of a biomarker indicative of a toxicological, signaling,
or other cellular process, or any other method used to measure an
aspect of cellular functionality. In one embodiment, hepatocytes
are cultured in a culture system comprising multiple cell culture
components and their metabolic states are measured by the level of
albumin. In another embodiment, hepatocytes are cultured in a
culture system having two, three or multiple components for three
days and their collective metabolic state is measured by reverse
transcription polymerized chain reaction ("RT-PCR") analysis. In
one embodiment, hepatocytes are cultured for a period of one, two,
three, or four days, or longer, in a culture system having two,
three or multiple components, in the presence of a potential
toxicant dissolved or suspended in the culture medium, and the
cells and the culture medium are subsequently assayed to determine
the presence of a biomarker indicative of the onset of an apoptotic
or otherwise cytotoxic process having incepted in the cell. In some
embodiments, the system may include five cell components--serum
free media, higher than atmospheric oxygenation, co-culture in a 3D
structural relationship and perfusion of the culture media. In one
embodiment, primary enterocytes or CACO-2 cells are cultured for a
period of one, two, or three days, or longer, in a culture system
having two, three or multiple components, in the presence of a
molecular entity dissolved and suspended in the culture medium,
wherein the primary enterocytes or CACO-2 cells are cultured on a
physical substrate that is configured with the enterocytes or
CACO-2 cells as a permeable membrane; and the cells and the culture
medium are subsequently assayed to determine the metabolic action
of CYP3A enzymes expressed in said cells upon said molecular
entity; and also to determine whether the cells have afforded the
absorption of the molecular entity through the permeable membrane.
In an embodiment, primary glomerular cells isolated from a human
kidney are cultured for a period of one, two, or three days, or
longer, in a culture system having two, three or multiple
components, in the presence of a molecular entity dissolved and
suspended in the culture medium, and the cells and the culture
medium are subsequently assayed to determine the presence of a
biomarker indicative of the onset of an apoptotic or otherwise
nephrotoxic process having incepted in the cell.
[0094] The devices and methods disclosed herein can be useful for
drug discovery and development or for consumer and industrial
product testing, or for environmental testing or biodefense
applications. Such testing includes, but is not limited to in vitro
drug toxicity testing and in vivo-surrogate testing. In one
embodiment, the culture systems described herein is used for the
study of drug clearance. In one embodiment it is used for the study
of metabolite generation. In one embodiment it is used for the
study of mechanisms of active or passive molecular transport. In
one embodiment it is used for the testing of the toxicity of
substances constituent to consumer products such as toothpaste,
shampoo, hair dye, or makeup. In one embodiment it is used for the
testing of the toxicity of substances constituent to industrial
products such as paint or insulation materials. In one embodiment
it is used for measuring the level of environmental pollutants such
as dioxin. In one embodiment, an environmental sample is extracted
from water or atmosphere and introduced into a culture medium,
where it may subsequently incite in the cell culture a response
indicative of toxicity, inflammation or hypersensitivity. In one
embodiment it is used for detecting the presence of a chemical or
biological warfare agent, such as aflatoxins. With the disclosed
cell culture systems, enhanced cellular function is manifested with
respect to at least one type of cellular function to produce a
higher percentage of the functionality that the cells would
evidence in vivo, compared to what those cells could produce under
in vitro cell culture conditions that were not so enhanced.
[0095] Alternatively, enhanced cellular functionality may comprise
the greater speed, or rapidity--i.e., the reduced time required--in
which cells may recover an equivalent degree of any one such type
of cellular functionality compared to the time in which those cells
could recover that same functionality under in vitro cell culture
conditions that were not so enhanced. The benefit of such
time-based enhanced cellular functionality is that it may increase
the aggregate amount of time during which the cell manifests the
full level of functionality it achieves in in vitro culture, or
diminish the time that elapses during the cell or tissue culture
process, before that high level of full in vitro functionality
incepts, thereby increasing the total time available, and
therefore, the total potential benefit derivable, from the cell
culture while in subsequent experimental use. In an alternative
aspect the benefit of such time-based, enhanced cellular
functionality is that it minimizes the time that is lost, and the
attendant labor and cost that is expended, compared to cell culture
methods that do not benefit from the enhanced functionality yielded
by compositions and methods described herein, which require longer
periods of time in culture.
[0096] The systems may be utilized to evaluate the toxicity,
efficacy, and pharmacokinetic disposition of new drugs more
efficiently than is currently possible because of the quicker
achievement of a high metabolic state by the cells. One aspect of
the culturing systems disclosed is the ability to detect the
formation, accumulation and/or further metabolic clearance of
secondary metabolites as well as the potential to elucidate the
role of drug transporters in drug clearance. For example, a parent
molecular entity can be exposed to the system and the culture
medium can subsequently be repeatedly sampled at predetermined time
points; the samples can then by analyzed by mass spectroscopy to
investigate the prospective formation and clearance of primary
metabolites of the parent, secondary metabolites of the parent,
tertiary metabolites of the parent, and subsequent generations of
metabolites of the original parent compound.
Examples
Exemplary Cell Culture System Comprising Multiple Cell Culture
Components
[0097] The system depicted in FIG. 1 comprises a compartment 102
with a layer of binding material 103 configured to facilitate the
physical adherence of cellular materials to the physical substrate
101. The binding material 103 is comprised of at least one material
drawn from the group consisting of MATRIGEL.TM., any of the cell
culture systems provided under the trade name BIOCOAT.TM.,
collagen, fibrinogen, fibronectin, gelatin, laminin hyaluronin or
hyaluronic acid, or any of the family of polyamines such as
polylysines. The system 100 depicted in FIG. 1 also comprises a
co-culture of at least one type of metabolically active cell 104
located in proximity with at least one type of non-parenchymal,
stromal cell 105. The system 100 depicted in FIG. 1 also comprises
a liquid cell culture medium containing no serum 106 and a cell
culture environment 107 having an oxygen content higher than
atmospheric concentration.
[0098] The system depicted in FIG. 1A illustrates an alternative
embodiment of the system. Hepatocytes 104 and fibroblasts or other
types of stromal cells 105 are affixed in a particular geometry and
proximity to each other within a compartment 102 on the physical
substrate 101 in predetermined quantities and patterns using a
micropatterning technique. Binding material 103 is selectively
placed so as to cause the cells 104 and 105 to assume their desired
positions and geometry in the micropattern. The system 100 depicted
in FIG. 1A also shows a liquid cell culture medium containing no
serum 106 and a cell culture environment 107 having an oxygen
content higher than atmospheric concentration.
[0099] FIG. 2 illustrates an alternative embodiment of the system.
The system 100 depicted in FIG. 2 comprises protein layers 203A and
203B that are configured above and below the metabolically active
cells 204 that are configured in a monolayer between them. The
protein layers 203A and 203B and the metabolically active cells 204
collectively comprise a type of three-dimensional cell culture
configuration. Alternatively, the lower protein layer 203A may not
be not present while the upper protein layer 203B is configured to
be present in the system, comprising along with the cells 204 a
type of three-dimensional cell culture configuration. In yet
another embodiment of the system 100, the lower protein layer 203A
is not present but in its place is configured a layer of binding
material 103 within the compartment 102 of the substrate 101. Also
shown are the system 100, a liquid cell culture medium 106 and a
cell culture environment 107 having an oxygen content higher than
atmospheric concentration.
[0100] FIG. 2A illustrates an alternative embodiment of the system.
The at least one compartment 102 of the physical substrate 101
contains a material that is configured to comprise a type of
three-dimensional cell culture microscale scaffold 301 for the
stable lodging and/or adherence of cellular material. The scaffold
301 may be comprised of calcium alginate. The cross-sectional
dimensions of the scaffold 301 may vary in size. A protein binding
material 303 is configured to affix and stabilize points of the
scaffold 301 to the compartment 102. Alternatively, protein binding
material 303 may be absent. Metabolically active cells 304 are
configured to be lodged and stabilized in the pores of the
scaffold, agglomerating into non-linear configurations of multiple
cells such as (but not limited to) spheroids. Also shown are the
system 100, a liquid cell culture medium 106 and a cell culture
environment 107 having an oxygen content higher than atmospheric
concentration.
[0101] Effect of Various Cell Culture Components on the Metabolic
State of Cultured Cells
[0102] In FIGS. 3A and B, freshly isolated hepatocytes are
co-cultured with 3T3-J2 fibroblasts, in the presence of
serum-containing media and atmospheric oxygen. Albumin production
slowly increases over time stabilizing at 80 .mu.g/1.times.10.sup.6
cells/24 hrs following 11 days of co-culture. Gene expression
analysis of Phase I and Phase II enzymes such as CYP450 enzymes in
the case of Phase I enzymes, or glucuronidation enzymes or
sulfation enzymes in the case of Phase II enzymes], as well as
transporter proteins is carried out by quantitative reverse
transcription polymerase chain reaction (qRT-PCR). Gene expression
at the onset of culture is minimal and is significantly lower than
in vivo levels of transcription.
[0103] In FIG. 4A, the gene expression of hepatocytes co-cultured
with non-parenchymal cells in serum free media, and under high
oxygen tensions (95% O.sub.2, 5% CO.sub.2) at the first day of
culture (Day 1), is compared to that of fresh hepatocytes. Gene
expression analysis of phase I and phase II enzymes, as well as
transporter proteins is carried out by quantitative reverse
transcription polymerase chain reaction (qRT-PCR). Gene expression
at the onset of culture is comparable to in vivo levels of gene
transcription.
[0104] In FIG. 4B, long-term synthetic function of oxygenated
cultures is tested. In FIG. 4C, long-term cyp1A1/2 activity is
measured. During the first day of culture, albumin secretion is
4-fold higher in oxygenated, serum-free, co-cultures compared to
hepatocytes co-culture with 3T3-J2 fibroblasts under standard
conditions (P=0.011 N=3) demonstrating the more rapid achievement
of metabolic function. No significant difference is detected at day
seven of culture. To compare long-term cyp1A1/2 activity to
hepatocytes in suspension, freshly isolated hepatocytes as
described above is compared to isolated hepatocytes following 2
hours of incubation at 37.degree. C. to control for the rapid loss
of function in suspension. FIG. 4C shows that cyp1A1/2 activity in
oxygenated cultures is comparable to that of hepatocytes in
suspension. During the first day of culture, the activity is 135%
higher than that of hepatocytes co-cultured with 3T3-J2 under
standard conditions (P=0.001 N=3). However, at day 7 of culture,
there is no significant difference between the cultures,
demonstrating a benefit of the combinations.
[0105] Clearing of Drugs in Hepatocytes Cultured According to One
Embodiment--
[0106] Cryopreserved human hepatocytes are cultured with 3T3-J2
fibroblasts under serum free conditions. Following overnight
incubation in high oxygen tension (95% O.sub.2, 5% CO.sub.2) the
cells are exposed to one of the following drugs: antipyrine,
buspirone, metoprolol, sildenafil, and timolol. FIG. 5A shows the
clearance of all drugs including slow clearing drugs such as
antipyrine, sildenofil, and timolol which could not be assayed
using suspension cultures. FIG. 5B shows the long term accumulation
of 4-OH midazolam in our cultures in the presence and absence of
rifampicin an inhibitor of the drug transporter Oatp-2. The
accumulation of 4-OH midazolam in the culture media can be detected
over 30 hours of culture. This allows the completion of long term
experiments and evaluations during the first few days of
culture.
[0107] High Metabolic State of Hepatocyte at the Onset of Cell
Culture
[0108] High metabolic state may comprise the cell's enhanced
ability to synthesize proteins or other cellular products; to
maintain the functionality of organelles or other subcellular
components, such as mitochondria; to produce cytokines; to express
genes; or to transport or metabolize xenogenous materials with
which the cell comes in contact; or to perform any other cellular
function High metabolic state may comprise the degree, or extent,
to which the cell manifests any of the foregoing functions. High
metabolic state may therefore be manifested by cell culture systems
the unique configurations of which endow their constituent cells
with the ability with respect to at least any one type of cellular
function to produce a higher percentage of the functionality that
they would evidence in vivo, compared to what those cells could
produce under in vitro cell culture conditions that were not so
enhanced.
[0109] Table 1 illustrates the advantages of the transition to the
components of compositions and methods described herein comprised
by serum-free (or alternatively, low serum concentration) media
formulation, higher than atmospheric oxygen concentration, or both.
In this experimental configuration hepatocytes are cultured alone
or co-cultured with non-parenchymal cells, in the presence or
absence of serum. Two seeding conditions were evaluated: one was
normal atmospheric condition and the second was 95% oxygen, both
under 5% CO.sub.2 as is common practice. Following overnight
seeding, the cultures were washed and the level of demonstrated
cellular functionality at first day of culture, as exemplified by
CYP1A1/2 activity, was measured by the ethoxyresorufin-O-deethylase
(EROD) assay under normal oxygen condition. The result shows that,
compared to the baseline condition of monocultured hepatocytes,
each respective embodiment of compositions and methods described
herein confers an improved cellular functionality at first day of
culture. It should be emphasized that the data in Table 1 is not
simply the attainment of a higher level of cellular function, but
the attainment of that higher level very rapidly, at the first day
of culture, thereby enabling the culture to be productively
utilized on as many as six, nine, or twelve or more additional,
earlier days than would be afforded under traditional cell culture
or co-culture methods.
TABLE-US-00001 TABLE 1 CYP1A1/2 activity at first day of culture
demonstrated by various embodiments of compositions and methods
described herein Cell culture components comprised in various
embodiments: none serum- (hepatocyte co- high free CYP1A1/2
activity monoculture) culture oxygen media (nM/min/1 .times.
10.sup.6 cells) X 0.89 .+-. 0.20 X X 1.45 .+-. 0.35 X X 2.44 .+-.
0.20 X X 2.63 .+-. 0.13 X X X 3.45 .+-. 0.21
[0110] Table 2 compares the metabolic function of hepatocytes
co-cultured with non-parenchymal cells in serum free media, and
under high oxygen tensions (95% O.sub.2, 5% CO.sub.2) at the first
day of culture (Day 1), to that of freshly isolated hepatocytes
cultured in suspension (Day 0). The activities of CYP1A1/2 and
CYP2B1/2 are measured using the EROD, MROD, PROD and BROD assays
respectively. As can be seen, the metabolic activity of hepatocytes
in oxygenated co-cultures is equivalent or higher than that of
hepatocytes in suspension.
TABLE-US-00002 TABLE 2 Cyp1a1/2 and Cyp2b1/2 activity (nM/min/1
.times. 10.sup.6 cells) EROD MROD PROD BROD Suspension 2.73 .+-.
0.07 0.74 .+-. 0.10 0.24 .+-. 0.03 0.04 .+-. 0.08 (Day 0)
Oxygenated co- 3.10 .+-. 0.40 0.83 .+-. 0.10 0.85 .+-. 0.35 0.23
.+-. 0.07 culture (Day 1)
[0111] FIG. 6 illustrates, on a pro forma, prophetic, conceptual
basis, the time-based benefit derived from an embodiment of a cell
culture system described herein, wherein the benefit area is
depicted in the Figure as the cross-hatched area. In FIG. 6, the
low bell curve that shows cellular functionality enduring at a low
level from 0-10 days represents the performance of plated
cryopreserved primary hepatocytes cultured in a mono-culture under
traditional culture methods and conditions. The second bell curve
that shows cellular functionality rising to a relatively higher
level by Day 14 represents the performance of a traditional culture
of a co-culture of plated cryopreserved primary hepatocytes with
3T3 fibroblasts. This represents co-culture without any at least a
second cell culture component. The large bell curve that
encompasses both the cross-hatched area and the area bounded by the
second bell curve reflects the level of cellular function
envisioned by at least one embodiment, including materially more
rapid attainment of high functionality very close to the time of
onset of the cell culture. The high cellular function beginning on
the first day after culture permits use of the cell cultures more
quickly than in the past; reduces tissue culture, labor, and
inventory carrying costs; creates additional days of high cellular
function during which the cell culture can be utilized for
economically productive purposes, which days would otherwise have
to be devoted to the economically unproductive, prerequisite task
of additional tissue incubation and culture; and provides a more
efficient system.
[0112] A Multi-Well Format Embodiment of Compositions and Methods
Described Herein
[0113] A 96-well plate is coated with hydrogel. Each well is filled
by serum-free media. 3T3-J2 fibroblasts are placed to each well and
the plate is cultured overnight. On the following day, human
hepatocytes are thawed from a cryopreserved vial and placed in 95%
oxygen chamber with serum-free media. The cells are counted. To
each well, discrete, pre-determined numbers of cells are seeded
while maintaining high oxygen level. The 96-well plate is then
culture overnight in the presence of 95% oxygen. The plate is
subsequently moved to a cell culture incubator with normal oxygen
level. 12 different drugs are diluted in serum free media and then
added to the first row of 96-well plate. The addition is repeated
in subsequent rows of 96-well plate. 5 days later, MROD assays are
conducted with cell extracts obtained from each well.
[0114] In an alternative multi-well embodiment, the cell culture
system is comprised in one compartment of a multi-well assay
platform that is configured to comprise a culture of artificially
or naturally occurring cellular material in at least one additional
compartment of the platform. The several compartments of the assay
platform are microfluidically interconnected by at least one
microscale channel configured to conduct liquid or gaseous cell
culture medium in a circulating or re-circulating pathway between
and among the compartments.
[0115] In an alternative embodiment, the cell culture system is
configured to comprise a cell culture compartment that is
microfluidically connected to at least one microscale input or
output channel, wherein the channel and the compartment are
configured to conduct liquid or gaseous cell culture medium in a
circulating or re-circulating pathway, and wherein the cell culture
medium being configured to come into contact with the cell culture
comprising compositions and methods described herein under at least
one condition of perfusion or flow comprises an additional,
separate and distinct component of compositions and methods
described herein.
[0116] Kits--
[0117] A kit is manufactured including the following components:
microtiter plate, oxygen tank containing mix of gases with
pre-determined ratio, an instruction manual, an instruction compact
disc, frozen vials containing second cell population, and culture
media, which may be low serum or serum free media. Alternatively, a
kit is prepared containing a microtiter plate coated with a binding
agent layer or a material to form a microscaffold and containing
serum-free or low serum media. Included are vials of cryopreserved
primary and secondary cells. In another embodiment, the kit
comprises a chip including at least one compartment connected with
microfluidic channels to permit perfusion of the culture media,
oxygen containers with a mixture of gases to provide a higher than
atmospheric concentration of oxygen, and a serum free culture
media.
* * * * *