U.S. patent application number 11/668155 was filed with the patent office on 2007-08-02 for method of drug interaction.
Invention is credited to Albert P. Li.
Application Number | 20070178441 11/668155 |
Document ID | / |
Family ID | 34556431 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070178441 |
Kind Code |
A1 |
Li; Albert P. |
August 2, 2007 |
Method of Drug Interaction
Abstract
A method of interacting a drug with an in vivo system having
multiple cell types to model a drug interaction with a living
organism in a cell culture tool having a flat plate segregated into
multiple chamber units, each of the chamber units having a chamber
wall surrounding multiple wells with the height of the chamber wall
being taller than that of the height of the surrounded multiple
wells, including depositing a different cell type in the set of
wells in each chamber, adding a fluid medium to a height above the
flat surface to fluidly interconnect the set of wells in each
chamber and adding the drug to the fluid medium in each chamber,
the dose and type of the drug being varied in each chamber.
Inventors: |
Li; Albert P.; (Columbia,
MD) |
Correspondence
Address: |
KEVIN J. MCNEELY, ESQ.
5335 WISCONSON AVENUE, NW
SUITE 440
WASHINGTON
DC
20015
US
|
Family ID: |
34556431 |
Appl. No.: |
11/668155 |
Filed: |
January 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10751983 |
Jan 7, 2004 |
7186548 |
|
|
11668155 |
Jan 29, 2007 |
|
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|
60518331 |
Nov 10, 2003 |
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Current U.S.
Class: |
435/4 |
Current CPC
Class: |
C12M 23/12 20130101;
C12M 25/04 20130101; C12M 35/08 20130101 |
Class at
Publication: |
435/004 |
International
Class: |
C12Q 1/00 20060101
C12Q001/00 |
Claims
1. A method of interacting a drug with more than one type of cell
material in a culture dish having a flat surface with a plurality
of depressions defining wells, and a containing wall surrounding
the flat surface, the method comprising: depositing different cell
types in separate wells of the culture dish; adding a fluid medium
to a height above the flat surface to fluidly interconnect the
wells; and adding the drug to the fluid medium.
2. The method of claim 1, further comprising: adding one or more
culture medium to each of the separate wells; and culturing more
than the different cell types in the separate wells.
3. The method of claim 2, wherein one of the different cell types
comprises liver cells.
4. The method of claim 2, wherein one of the different cell types
comprises kidney cells.
5. The method of claim 2, wherein one of the different cell types
comprises lung cells.
6. The method of claim 2, wherein one of the different cell types
comprises spleen cells.
7. The method of claim 2, wherein depositing different cell types
comprises depositing tissue fragments or fractions that include the
different cell types.
8. The method of claim 2, wherein depositing different cell types
comprises depositing kidney cells in a first well of the wells,
liver cells in a second well of the wells, heart cells in a third
well of the wells, lung cells in a fourth well of the wells, spleen
cells in a fifth well of the wells, and brain cells in a sixth well
of the six wells.
9. The method of claim 2, further comprising: metabolizing the drug
by the different cell types.
10. A method of metabolizing a drug in a cell culture tray having
six wells surrounded by a containing wall, the method comprising:
depositing a first cell type in a first well, a second cell type in
a second well, a third cell type in a third well, a fourth cell
type in a fourth well, a fifth cell type in a fifth well, and a
sixth cell type in a sixth well; overfilling the first, second,
third, fourth, fifth, and sixth well to cause the first, second,
third, fourth, fifth, and sixth cell type to interact via a common
fluid medium; and introducing the drug into the common fluid
medium.
11. The method of claim 10, wherein the first cell type comprises
liver cells, the second cell type comprises kidney cells, the third
cell type comprises heart cells, the fourth cell type comprises
spleen cells, the fifth cell type comprises brain cells, and the
sixth cell type comprises lung cells.
12. The method of claim 10, wherein the first, second and third
cell type comprise liver cells and the fourth, fifth, and sixth
cell type comprise heart cells.
13. A method of interacting a drug with an in vivo system having
multiple cell types to model a drug interaction with a living
organism in a cell culture tool having a flat plate segregated into
multiple chamber units, each of the chamber units having a chamber
wall surrounding multiple wells with the height of the chamber wall
being taller than that of the height of the surrounded multiple
wells, the method comprising: depositing a different cell type in
the set of wells in each chamber; adding a fluid medium to a height
above the flat surface to fluidly interconnect the set of wells in
each chamber; and adding the drug to the fluid medium in each
chamber, the dose and type of the drug being varied in each
chamber.
14. The method of claim 13, further comprising: providing the tool
having a plate segregated into 12 chamber units each having a
chamber wall surrounding 8 of the wells.
15. The method of claim 13, further comprising: providing the tool
having a plate segregated into 64 chamber units each having a
chamber wall surrounding 6 of the wells.
16. The method of claim 13, further comprising: providing the tool
having a plate segregated into 256 chamber units each having a
chamber wall surrounding 6 of the wells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/751,983 filed on Jan. 7, 2004, and claims
priority to U.S. Provisional Application No. 60/754,327 filed on
Dec. 29, 2005, and U.S. Provisional application No. 60/518,331
filed Nov. 10, 2003, which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to tissue culture vessels and, more
particularly, comprises a new and improved tissue culture vessel
for co-culturing of multiple types of cells and tissues and the
application of the cell culture device in biological studies,
including application in drug discovery and development.
BACKGROUND OF THE INVENTION
[0003] Culture tools, such as, for example, dishes, plates, flasks,
and other types of vessels, are widely used in the laboratory for a
variety of purposes. In normal use, cell and tissue culturing
involves the use of agar or medium, which covers the bottom of a
well. Cells are routinely cultured in research laboratories for
basic biochemical and cell biology research to further understand
natural biological processes. More recent uses of cell culture
systems have been in drug discovery and development in order to
test for pharmacological and toxicological effects of drug
candidates. This work generally is a monoculture test process, for
example, cells of one type are grown in a suitable medium in a
tissue culture vessel such as a well, a plate or a flask.
[0004] A normal cell culture plate consists of a chamber with a
flat bottom surrounded by vertical walls to allow filling of the
chamber with liquid and a removable cover to retain humidity and to
protect against contamination. As shown in FIGS. 1A and 1B, a
commonly used multi-well plate 100 has six identical wells 110.
Although the wells 110 can be formed integrally, as by injection or
blow molding for example, a preferred method of manufacture is to
form the plate 100 with an upper tray 120 which defines the volume
contained in each well and a lower or bottom tray 130 which defines
the bottom surface of each well. The well 110 depth, together with
the diameter of the well 110, determines the fluid capacity of each
well 110. Typically for example, each well 110 in a six well plate
is about 0.35 cm. in diameter and 2.0 cm. deep and the wells are
preferably arranged in a 2 by 3 regular rectangular array.
[0005] As shown in FIG. 1B, cells 140 are deposited in the bottom
of each of the wells 110. A fluid 150 is then added to cover the
cells 140.
[0006] Cell culture systems, commonly known as in vitro systems,
are used extensively in drug discovery and development for the
evaluation of drug properties. For example, cell culture systems
are used to evaluate drug efficacy, drug metabolism, or drug
toxicity. However, it is also recognized that an in vitro system
may not accurately predict in vivo effects due to the lack of the
complexity and interplay of biological processes for in vitro
systems. For example, by using primary liver cells (hepatocytes) in
culture, the effect of a substance on liver cells can be evaluated.
However, in vivo, the substance may be metabolized by other organs
such as the kidneys, and the resulting metabolite may have a
different effect on liver cells which would not be detected by
using liver cells alone. For this reason, interest has developed in
the co-culture of cells. Co-culturing involves growing one
population of cells in the presence of another population of cells.
Cell co-culturing has been applied in a myriad of biological
studies. In pharmacology and toxicology, co-culturing of a target
cell, such as, for example, cancer cells, with cells from a
critical organ, such as, for example, the liver, allows the
evaluation of the effect of a chemical on the target cells after
its modification by cells from the critical organ, such as, for
example, liver metabolism of a specific drug or drug candidate.
Using the normal cell culture plate, co-culture methods are
achieved by mixing the different cell types or by the use of
membranes to allow two cell types to be cultured on both sides of
the membrane. Evaluation of the individual cell types after being
physically mixed or by using membranes can be highly difficult and
tedious. Thus, a need exists for a new cell culture tool that
facilitates cell co-culturing.
SUMMARY OF THE INVENTION
[0007] In one general aspect, a cell culture tool includes a body,
an outer wall extending from the body, and more than one vessel
defined by the configuration of the body. Each vessel has a top
edge below a rim of the outer wall.
[0008] Implementation may include one or more of the following
features. For example, the body may have a flat surface with each
vessel comprising a depression in the flat surface of the body, the
depression configured to contain a volume of fluid. The vessel may
have a cylindrical wall and a circular bottom and the outer surface
of the body may be in the shape of a rectangular plate. The height
of the outer wall may be about 20 millimeters.
[0009] In one implementation, each vessel comprises a cup connected
to the body, each cup having a top edge below the rim of the outer
wall. In another implementation, the vessel includes a container
having a container wall with a top edge, the height of the
container wall being about 4 millimeters. In a further
implementation, each vessel comprises a partition wall dividing the
space defined within the perimeter of the outer wall, the partition
wall having a top edge.
[0010] In another general aspect, a multi-well culture dish
includes a base having a flat surface with a plurality of wells and
an outer wall surrounding the base. Each of the wells includes a
containing wall with a height lower than the height of the outer
wall. Implementation may include one or more of the features
described above and the dish may also include six wells.
[0011] In another general aspect, multiple culture vessels can be
connected using tubing with or without a device, such as, for
example, a pump, to circulate the fluid.
[0012] In another general aspect, a method of interacting a
substance with more than one type of cell material in a culture
dish having a plurality of wells includes depositing a different
type of the cell material in separate wells of the culture dish,
interconnecting the wells with a fluid medium, and adding the
substance to the fluid medium. In various implementations, the
substance may include a chemical or a drug.
[0013] In another general aspect, a method of metabolizing a drug
in a multi-well culture dish includes depositing different types of
cell material in separate wells of the multi-well culture dish,
connecting the separate wells with a fluid media, and introducing
the drug into the fluid media.
[0014] Implementation may include one or more of the following
features or any of the features described above. For example, the
cell material may include liver, kidney, spleen or lung cells, any
cells that can be cultured, and/or tissue fragments or
fractions.
[0015] In another general aspect, a method of metabolizing a drug
in a cell culture dish having a body with six wells and a wall
surrounding the six wells includes depositing kidney cells in a
first of the six wells, liver cells in a second of the six wells,
heart cells in a third of the six wells, lung cells in a fourth of
the six wells, spleen cells in a fifth of the six wells, and brain
cells in a sixth of the six wells, filling the dish with a fluid
medium to fluidly interconnect the six wells, and introducing the
drug into the fluid medium.
[0016] In another general aspect, a method of co-culturing
different cells in individual wells includes overfilling each well
to fluidly interconnect the wells so the different cells in the
individual wells communicate through a common fluid medium.
[0017] The method may include various implementations. For example,
the different cells in the individual wells comprise liver cells in
a first well, kidney cells in a second well, heart cells in a third
well, spleen cells in a fourth well, brain cells in a fifth well,
and lung cells in a sixth well. In another implementation, the
different cells in the individual wells comprise liver cells in a
first, second and third well and heart cells in a fourth, fifth,
and sixth well. In a further implementation, the method includes
introducing a substance into the common fluid medium so that the
different cells in the individual wells are in contact with the
same substance.
[0018] In another general aspect, a method of testing the safety
and efficacy of a drug in a culture dish having separate wells
includes depositing different cells of an organism in the separate
wells of the culture dish, depositing a harmful agent in another of
the separate wells, interconnecting the separate wells with a fluid
medium, and introducing a dose of the drug into the fluid
medium.
[0019] The method may include one or more of the following features
or any of the features described above. For example, the method may
include determining whether the different cells of the organism are
harmed by the dose of the drug, determining whether the harmful
agent is diminished by the dose of the drug, and/or increasing the
dose of the drug if the different cells of the organism are not
harmed and the harmful agent is not diminished.
[0020] The harmful agent may include tumor cells and the drug may
include an antitumor medication. The different cells of the
organism may include liver, kidney, heart, lung, spleen, and/or
brain cells of the human body.
[0021] The method may further include increasing the dose of the
drug until the drug harms the different cells of the organism and
designating the dose of the drug at which the different cells of
the organism are harmed as a toxic dose level. The method also may
include increasing the dose of the drug until the effect of the
harmful agent is reduced and designating the dose of the drug at
which the effect of the harmful agent is reduced as an effective
dose level.
[0022] The harmful agent may be cholesterol, the drug may be an
anti-cholesterol drug, and the different cells may include liver
cells. In another implementation, the harmful agent includes cancer
cells and the drug is an anti-cancer medication that has an
undesirable toxicity above a certain dose.
[0023] In another general aspect, a method of co-culturing cells in
a multi-well dish includes culturing a first cell type in a first
well of the multi-well dish and culturing a second cell type in a
second well of the multi-well dish. The cells cultured in the
second well may provide metabolites that benefit the growth of the
first cell type.
[0024] In another general aspect, a method of evaluating whether a
first cell type can enhance the growth of a second cell type
includes culturing the first cell type in a first well, culturing
the second cell type in a second well, fluidly interconnecting the
first well and the second well, and examining the impact of the
cultured first cell type on the growth of the second cell type.
[0025] The cell culture tool provides a convenient way for multiple
cell types to be co-cultured but yet physically separate so that
the individual cell types can be evaluated separately after
co-culturing in the absence of the co-cultured cells.
[0026] The tool allows the culturing of cells in individual wells
under different conditions, such as, for example, different
attachment substrate, different media, or different cell types,
followed by allowing the different wells to intercommunicate via a
common medium. After culturing as an integrated culture with a
common medium, the medium can be removed, and each well can be
subjected to independent, specific manipulations, such as, for
example, lysis with detergent for the measurement of specific
biochemicals or fixation and staining for morphological
evaluation.
[0027] As described above in the method, an application is the
culturing of multiple primary cells from different organs (e.g.
liver, heart, kidney, spleen, neurons, blood vessel lining cells,
thyroidal cells, adrenal cells, iris cells, cancer cells) so the
plate, after the establishment of individual cell types and
flooding, represents an in vitro experimental model of a whole
animal. Another application of the culture tool is to evaluate the
effect of a substance on multiple cell types. In drug discovery and
development, this culture system can be used to evaluate metabolism
of a new drug or drug candidate by cells from multiple organs or
the effect of a drug or drug candidate on the function and
viability of cells from multiple organs. An example of this
application is to culture cells from multiple organs along with
tumor cells, followed by treatment of the co-culture with an
anticancer agent to evaluate toxicity of the agent to the cells of
the different organs in comparison with its toxicity towards the
cancer cells to evaluate the therapeutic index of the agent. In
other words, each plate simulates the treatment of a whole animal
with the anticancer agent followed by examination of each organ.
Multiple tumor cell types can also be used to evaluate the efficacy
of the tested drug or drug candidate on different types of
tumors.
[0028] The tool can be utilized for the culturing of cells which
require exogenous factors from other cell types without physically
mixing the cell types, as the different cell types are placed in
different wells, with the overlaying medium allowing the exchange
of metabolites and/or secreted biomolecules.
[0029] In one general aspect, a method of culturing different cell
types from a single organ in a cell culture tool having more than
one well includes depositing the different cell types from the
single organ in separate wells, culturing the different cell types
in the separate wells and filling the cell culture tool to fluidly
interconnect the wells.
[0030] Embodiments may include one or more of the following
features. For example, the method may include introducing a
substance, such as, a drug candidate, an environmental pollutant or
an industrial chemical, into the fluidly interconnected wells and
evaluating a pharmacological effect of the substance on the
different cell types.
[0031] As another embodiment, the method may include introducing a
substance into the fluidly interconnected wells and evaluating a
toxic effect of the substance on the different cell types.
[0032] The method of separately culturing the cells may be
performed by adding a fluid medium to the individual wells.
[0033] As another embodiment, the method may include introducing a
harmful agent into the fluidly interconnected wells and adding a
therapeutic drug to the fluidly interconnected wells to determine
whether any of the cell types is harmed by the dose of the drug. It
may also include determining whether an effect of the harmful agent
is diminished by the dose of the drug and/or increasing the dose of
the drug if it does not may the cell types and the effect of the
harmful agent is not diminished.
[0034] Another feature may include increasing the dose of the drug
until it causes harm to at least one of the cell types and
designating the dose of the drug at which at least one of the cell
types is harmed as a toxic dose level. In addition, the method may
include increasing the dose of the drug until the effect of the
harmful agent is reduced and designating the dose of the drug at
which the effect of the harmful agent is reduced as an effective
dose level.
[0035] The cell culture tool may have at least three wells. The
method can be employed using the three cell culture tool for
various experiments. For example, if the single organ is a lung the
different cell types placed in separate wells may include capillary
cells lining the pulmonary blood vessels or microvascular
endothelial cells in a first well, epithelial cells lining small
airways in a second well and epithelial cells lining the large
airways in a third well.
[0036] In another experiment the organ is an eye and the different
cell types are retinal cells, corneal cells and conjunctiva cells.
In still another experiment, the organ comprises a kidney and the
different cell types comprise renal proximal tubule cells, distal
proximal tubule cells and glomeruli cells.
[0037] A four well cell culture tool may be employed for other
experiments, such as, for example, when the tested organ is a liver
and the different cell types comprise parenchymal cells, ito cells,
kuffer cells and endothelial cells.
[0038] The various features described above are intended to be
illustrative and the method should not be interpreted as being
limited to the described embodiments. For example, a cell culture
tool having many more wells may be used as part of the method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1A is a perspective view of a conventional multi-well
culture plate;
[0040] FIG. 1B is a cross-section view of the conventional
multi-well culture plate shown in FIG 1A;
[0041] FIG. 2A is a perspective view of a cell culture tool;
[0042] FIG. 2B is a cross-section view of the cell culture tool
shown in FIG. 2A;
[0043] FIG. 3A is a perspective view of another embodiment of a
cell culture tool;
[0044] FIG. 3B is a cross-section view of the cell culture tool
shown in FIG. 3A;
[0045] FIG. 4A is a perspective view of a further embodiment of a
cell culture tool;
[0046] FIG. 4B is a cross-section view of the cell culture tool
shown in FIG. 4A;
[0047] FIG. 5A is a perspective view of a cell culture tool with
separate chambers and multiple wells per chamber;
[0048] FIG. 5B is a cross-section view of the cell culture tool
shown in FIG. 5A;
[0049] FIG. 6 shows part of an organ system of an animal;
[0050] FIG. 7 is a flow diagram of evaluating metabolism of an
exogenous substance by multiple cell types;
[0051] FIG. 8 is a flow diagram of evaluating the toxicity of an
exogenous substance on multiple cell types;
[0052] FIG. 9 is a flow diagram of establishing a therapeutic index
of a drug; and
[0053] FIG. 10 shows a cell culture tool with an insert tray.
[0054] Reference numerals in the drawings correspond to numbers in
the Detailed Description for ease of reference.
DETAILED DESCRIPTION
[0055] Implementations of the tool 200, 300, 400 embodying the
current invention are shown in FIGS. 2A-5B and FIG. 10. The tool
200, 300, 400 includes multiple wells within each a type of cells
can be cultured, but each well can be overfilled or flooded, so
that the cells in the different wells can share a common medium.
This is achieved by configuring each well as an indentation inside
a larger plate (FIGS. 2A and 2B), placing short partitions inside a
larger plate (FIGS. 3A and 3B), or placing small inserts inside a
larger plate (FIGS. 4A and 4B). However, this invention can be
applied to any multi-well format with any number of wells per
plate.
[0056] Referring to FIGS. 2A and 2B, a multi-well tool 200 of the
present invention comprises a body 205 having a substantially
planar top surface 210, and an outer wall 215 extending from the
body 205. Six wells 220 are formed in the body 205 by depressions
in the top surface 210. Each well 220 has a containing wall 225
that may slant downward from or be perpendicular to the flat
surface 210.
[0057] The overall dimensions of the tool 200 may be about 12.60 cm
long and 8.40 cm wide. The body 205 may have a height of 0.20 cm,
with the outer wall 215 extending upward from the flat surface 210
approximately 0.15 cm. The height of each containing wall 225 may
be 0.05 cm. The wells 220 are configured in a regular array and are
separated by approximately 0.02 cm. In another implementation (not
shown), the wells are equi-distant from each other by positioning
the wells around a circumference of a circle. The dimensions of the
tool 200 are merely illustrative, however, the tool 200 is
configured to allow overfilling of each well 220 in order to
interconnect the wells 220 in a common fluid media while preventing
the cells in the individual wells 220 from drowning.
[0058] Referring to FIGS. 3A and 3B, a multi-well tool 300 includes
a body 305 having a planar top surface 310, surrounded by an outer
wall 315. Partitions 320 are positioned on the top surface 310 to
divide the space bounded by the outer wall 315 into six wells 325.
The outer wall 315 extends upward 0.15 cm from the top surface 310
and the height of the partitions is approximately 0.05 cm. Thus,
each well 325 can be overfilled to interconnect the wells 325 in a
fluid medium.
[0059] The partitions 320 may be bonded to the top surface 310 and
the outer wall 315. In another implementation, the partitions 320
may be removable.
[0060] Referring to FIGS. 4A and 4B, a multi-well tool 400 includes
a body 405 having a planar top surface 410, surrounded by an outer
wall 415. Inserts 420 are placed on the flat surface 410, with each
insert defined by a bottom 425 and a containing wall 430. The
height of the containing wall is about 0.05 cm and the height of
the outer wall extends 0.15 cm from the top surface 410. In other
implementations, the inserts 420 may comprise cups, dishes, or a
tray that may be removed from the top surface 310.
[0061] The multi-well plates as described in FIGS. 2A-4B can be
grouped to form a cell culture tray 500 as a single body 505 with
multiple compartments or chambers 510 (FIGS. 5A and 5B), each
compartment 510 having multiple wells 515, to allow experimentation
with different cell selections, liquid medium, or a different
exogenous substance in each compartment. Limiting walls 520
surrounding each compartment 510 are higher than the containing
walls 525 of the individual wells 515 within that compartment 510,
with the limiting walls 520 having a height of 0.20 cm and each
well 515 inside the larger body 505 having a height of 0.04 cm.
[0062] The tool 200-500 may be formed of various suitable
materials. In one implementation, the tool 200-500 is formed of a
substantially rigid, water-insoluble, fluid-impervious, typically
thermoplastic material substantially chemically non-reactive with
the fluids to be employed in the assays to be carried out with the
tool 200-500. The term "substantially rigid" as used herein is
intended to mean that the material will resist deformation or
warping under a light mechanical or thermal load, which deformation
would prevent maintenance of the substantially planar surface,
although the material may be somewhat elastic. Suitable materials
include, for example, polystyrene or polyvinyl chloride with or
without copolymers, polyethylenes, polystyrenes,
polystyrene-acrylonitrile, polypropylene, polyvinylidine chloride,
and similar materials. Polystyrene is a material that can be used
as it is the common polymer used for cell culture vessels, inasmuch
as it characterized by very low, non-specific protein binding,
making it suitable for use with samples, such as, for example,
blood, viruses and bacteria, incorporating one or more proteins of
interest. Glass is also a suitable material, being used routinely
in cell culture vessels and can be washed and sterilized after each
use.
[0063] The cell culture tool can be used to test drug metabolism.
As shown in FIG. 6, the major organs that are known to metabolize
drugs are the liver 610, intestines and kidneys 620, whereas other
organs such as the heart 630, spleen 640, lungs 650, and blood
vessels 660 also possess specific metabolizing pathways. Referring
to FIG. 7, method of using the cell culture tool includes
evaluating metabolism of an exogenous substance by multiple cell
types 700. Using the tool, the cells from major organs including
the liver, intestines, kidneys, heart, spleen, lungs, and brain are
placed in the multiple well plate, with cells from each organ
placed separately in individual wells (operation 710). For example,
in the six-well format, liver cells are placed in well 1,
intestines in well 2, kidneys in well 3, heart in well 4, spleen in
well 5 and lungs in well 6. Each cell type can be cultured
(operation 720) using different attachment substrate and culture
medium, for example, liver cells are best cultured on collagen and
require supplementation with insulin and dexamethasone, spleen
cells are cultured in agar suspension, etc. After each cell type is
established, the plate can be "flooded" by overfilling each well
(operation 730), with the cells from the different wells sharing a
common liquid medium. The exogenous substance, such as, for
example, a drug, a drug candidate, an environmental pollutant, or a
natural product, can be added to the medium (operation 740) and
incubated for specific time periods (operation 750). After
incubation, the medium can be collected for the examination of the
extent of metabolism (how much of the parent substance is
remaining), or metabolic fate (what are the identities of the
metabolites), using established analytical methods (operation
760).
[0064] Referring to FIG. 8, another method 800 of using the cell
culture tool includes evaluating the toxicity of an exogenous
substance on multiple cell types. The major organs that are
susceptible to drug toxicity are the liver, intestines, kidneys,
heart, spleen, lungs, and brain. Using the tool, the cells from the
liver, intestines, kidneys, heart, spleen, lungs, brains and blood
vessels, are placed in the multiple well plate (operation 810).
Cells from each organ are placed in individual wells. For example,
in an eight-well format, liver cells are placed in well 1,
intestines in well 2, kidneys in well 3, heart in well 4, spleen in
well 5, lungs in well 6, brain in well 7, and blood vessels in well
8. Each cell type can be cultured using a different attachment
substrate and culture medium (operation 820), for example, liver
cells are best cultured on collagen and require supplementation
with insulin and dexamethasone, spleen cells are cultured in agar
suspension, etc. After each cell type is established, the plate can
be "flooded" by overfilling each well, with the cells from the
different wells sharing a common liquid medium (operation 830). The
exogenous substance, such as, for example, a drug, a drug
candidate, an environmental pollutant, or a natural product, is
added to the medium (operation 840). The mixture is then incubated
for specific time periods (operation 850). After incubation, the
medium can be removed, and each individual cell type can be
evaluated for toxicity (operation 860) morphologically, such as,
for example, microscopic analysis, and by a biochemical analysis,
such as, for example, lysed with detergent for the measurement of
ATP content of the cells in each individual well.
[0065] The cell culture tool can also be used to evaluate drug
efficacy and safety. In drug discovery, intact cells are used as
indicators of drug efficacy. For example, liver cells are used to
evaluate the effect of a drug on cholesterol synthesis in order to
develop a novel inhibitor of cholesterol synthesis as a drug to
lower the cholesterol level in patients with high levels of
cholesterol. A culture can be applied with cells from multiple
organs as described above to evaluate the effects of a drug
candidate on cholesterol synthesis in multiple organs. The method
can be used to evaluate efficacy, metabolism and toxicity
simultaneously using the culture system.
[0066] For example, a "therapeutic index" of a potential new drug
to treat high cholesterol levels can be evaluated by using liver
cells as indicator cells to determine the effectiveness and
toxicity of the drug. Efficacy can be measured in the presence of
metabolism of all key cell types, thereby mimicking an in vivo
situation where metabolism may lower the efficacy (or increase the
efficacy) of the new drug.
[0067] Referring to FIG. 9, a method 900 of establishing a
therapeutic index of a drug includes depositing cells in separate
wells of the multi-well plate (operation 910), depositing a harmful
agent, such as, for example, tumor cells, in another of the wells
(operation 920), interconnecting the wells with a fluid medium
(operation 930), and adding a drug to the fluid medium (operation
940).
[0068] Safety is evaluated by determining the effect of the drug on
the various organ cells (operation 950). If the drug damages any of
the organ cells, the drug doseage is deemed to exceed a safe level
(operation 960). If the healthy cells are intact, the effect of the
drug to reduce the harmful agent is examined. If the harmful agent
is reduced, the result is recorded as an effective dose level
(operation 970). The dose of the drug is then increased (operation
980) and the process is repeated.
[0069] The tool also may be used in a high throughput screening
(HTS) process to allow evaluation of a large number of potential
drug candidates. In this method, a robotic system is utilized with
multi-well plates to perform experimentation. By using a
multi-compartment tool as described herein, HTS with co-cultured
multiple cell types can be performed for efficacy, toxicity, and
metabolism as described above.
[0070] Still a further method includes evaluation of co-culture
conditions. Some cell types can enhance the culturing of an
otherwise difficult to culture cell type. This is routinely
performed by trial and error. Using the HTS format, the effects of
different cell types on the growth of a difficult to culture cell
can be examined. For example, to evaluate which cells are best to
maintain the differentiation of cultured liver cells, liver cells
can be co-cultured with cell type 1 (e.g. endothelial cells) in
compartment 1; cell type 2 (e.g. 3T3 cells) in compartment 2, and
so on. At the end of co-culturing, the properties of the liver
cells can be evaluated without complications by the co-cultured
cells.
[0071] Referring to FIG. 10, a cell culture tool 1000 is shown with
an adaptation to measure drug absorption. The cell culture tool
1000 comprises a body 1105 having a substantially planar top
surface 1110 surrounded by an outer wall 1115. Six wells 1120 are
formed in the body 1105 by depressions in the top surface 1110.
Each well 1120 has a containing wall 1125 that is perpendicular to
the flat surface 1110.
[0072] An insert tray 1130 rests on a lip 1135 at the top of the
outer wall 1115. The insert tray 1130 includes a chamber 1138 with
a porous membrane 1145 that is positioned inside the outer wall
1115.
[0073] Intestinal cells 1140 are placed at the bottom of the
chamber 1138 proximate to the membrane. When the tool 1000 is
filled, the fluid level rises through the membrane 1140 and a drug
1150 is added to the chamber 1138. The drug 1150 is "absorbed" when
it permeates the membrane 1140 to interact with the cells 1120.
Thus, the amount of absorption can be measured to simulate
absorption of the drug within the intestines.
[0074] Using the cell culture tool an in vitro model of a single
organ can be co-cultured using multiple cell types from a single
organ in a chamber. These in vitro models can be used in of
biochemistry, physiology, pharmacology, and toxicological
studies.
[0075] Examples of the different types of single organ models
include:
[0076] 1. In vitro lung model.
[0077] The lung organ consists of multiple cell types. For example,
there are capillary cells lining the pulmonary blood vessels,
epithelial cells lining the small airways, and epithelial cells
lining the large airways. Current technologies in cell culture
allow culturing of a single cell type or a mixture of cell types.
The use of single cell types does not consider differential
effects, for example, evaluation of the effects of an inhaled
toxicant on the multiple cell types. The use of mixed culture
renders it difficult to examine effects of a particular substance
on a specific cell type as they are physically mixed.
[0078] The use of the cell culture tool co-culture system allows
placement of a single cell type in an individual well. For example,
the use of a cell culture tool with 6 wells in a chamber allows the
placement of pulmonary microvascular endothelial cells in two
wells, small airway epithelial cells in two wells and bronchial
epithelial cells in two wells. A toxicant can be introduced into
the wells, for example, cigarette smoke condensate, to examine its
effect on the lung capillary endothelium, small airway epithelium
and bronchial epithelium.
[0079] 2. In vitro eye chamber model.
[0080] Multiple cell types from the eye can be tested in the cell
culture tool such as retinal cells, corneal cells and conjunctiva
cells for the assessment of pharmacological and toxicity of
substances in the eye. This can be used, for example, in the
discovery of drugs for the treatment of eye diseases.
[0081] 3. In vitro liver model.
[0082] Multiple cell types from the liver in the cell culture tool
such as parenchymal cells, ito cells, kuffer cells and endothelial
cells can be cultured to evaluate the pharmacology, physiology and
toxicity of various substances to the liver.
[0083] 4. In vitro kidney model.
[0084] Multiple cell types from the kidney can be cultured in the
cell culture tool such as renal proximal tubule cells, distal
proximal tubule cells and glomeruli cells to construct an in vitro
model of the multicellular kidney for toxicity and pharmacological
studies.
[0085] Since certain changes may be made in the above apparatus and
process without departing from the scope of the invention herein
involved, it is intended that all matter contained in the above
description or shown in the accompanying drawing shall be
interpreted in an illustrative and not in a limiting sense. For
example, advantageous results still could be achieved if steps of
the disclosed techniques were performed in a different order and/or
if components in the disclosed systems were combined in a different
manner and/or replaced or supplemented by other components.
Accordingly, other implementations are within the scope of the
following claims.
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