U.S. patent application number 17/566166 was filed with the patent office on 2022-06-30 for cell culture systems, methods and uses thereof.
This patent application is currently assigned to Academia Sinica. The applicant listed for this patent is Academia Sinica. Invention is credited to Ying-Chih CHANG.
Application Number | 20220204943 17/566166 |
Document ID | / |
Family ID | 1000006107607 |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220204943 |
Kind Code |
A1 |
CHANG; Ying-Chih |
June 30, 2022 |
CELL CULTURE SYSTEMS, METHODS AND USES THEREOF
Abstract
The present disclosure provides a surface coating comprising a
hydrophilic polymer and polyelectrolyte multilayers. Also provided
is a cell culture system comprising a cell culture article having a
surface coated with the surface coating. Uses and methods of
preparing the surface coatings and systems are provided as
well.
Inventors: |
CHANG; Ying-Chih; (Atherton,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
|
TW |
|
|
Assignee: |
Academia Sinica
Taipei
TW
|
Family ID: |
1000006107607 |
Appl. No.: |
17/566166 |
Filed: |
December 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
63132934 |
Dec 31, 2020 |
|
|
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63252268 |
Oct 5, 2021 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0693 20130101;
C12N 2537/10 20130101; C12N 2539/10 20130101; C12N 2533/32
20130101; C12N 2533/40 20130101; C12N 2513/00 20130101; G01N
33/5088 20130101 |
International
Class: |
C12N 5/09 20060101
C12N005/09; G01N 33/50 20060101 G01N033/50 |
Claims
2. The composition of claim 1, wherein the hydrophilic polymer is
selected from the group consisting of poly(vinyl alcohol) (PVA),
poly(ethylene glycol) (PEG), PEG-acrylate, polyvinylpyrrolidone
(PVP), polyethyleneimine (PEI), poly-L-lactide (PLLA),
poly-D-lactide (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA),
poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid)
(PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl
methacrylate) (p-HEMA) and derivatives thereof.
3. The composition of claim 2, wherein the hydrophilic polymer is
PVA, PEG, PEG-acrylate, PVP, PEI, PMMA or a derivative thereof.
4. The composition of claim 2, wherein the hydrophilic polymer is
PVA, PEG or PEG-acrylate.
5. The composition of claim 1, wherein the hydrophilic polymer is
crosslinked to the surface of the cell culture article.
6. The composition of claim 1, wherein the hydrophilic polymer is
free of crosslink.
7. The composition of claim 1, wherein the polycation is a
poly(amino acid).
8. The composition of claim 1, wherein the polyanion is a
poly(amino acid).
9. The composition of claim 1, wherein the polycation is selected
from the group consisting of poly(L-lysine) (PLL), poly(L-arginine)
(PLA), poly(L-ornithine) (PLO), poly(L-histidine) (PLH), and a
combination thereof.
10. The composition of claim 9, wherein the polycation is PLL, PLA,
PLO or PLH.
11. The composition of claim 1, wherein the polyanion is
poly(L-glutamic acid) (PLGA), poly(L-aspartic acid) (PLAA), or a
combination thereof.
12. The composition of claim 1, wherein the bilayer of
(polycation/polyanion) is selected from the group consisting of
PLL/PLGA, PLL/PLAA, PLA/PLGA, PLA/PLAA, PLO/PLGA, PLO/PLAA,
PLH/PLGA, PLH/PLAA, and a combination thereof.
13. The composition of claim 1, wherein the polyelectrolyte
multilayers are formed via layer-by-layer assembly.
14. The composition of claim 1, wherein the polyelectrolyte
multilayers comprise n bilayers, wherein n is an integer number
ranging from 1 to 30, and wherein the outermost layer is a
polycation or an polyanion.
15. The composition of claim 1, wherein the polyelectrolyte
multilayers comprise n bilayers of (polycation/polyanion) and an
additional layer of polyanion, wherein n is an integer number
ranging from 1 to 30, and wherein the outermost layer is
polyanion.
16. The composition of claim 1, wherein the polyelectrolyte
multilayers comprise n bilayers of (polyanion/polycation) and an
additional layer of polycation, wherein n is an integer number
ranging from 1 to 30, and wherein the outermost layer is
polycation.
17. The composition of claim 1, wherein the cell culture article is
coated by a method comprising the step of: a) providing a cell
culture article having a surface, wherein the surface is
hydrophobic, b) modifying the hydrophobic surface with a treatment,
c) applying a hydrophilic polymer to the modified surface, and d)
sequentially depositing on the hydrophilic polymer alternating
layers of polycations and polyanions.
18. The composition of claim 17, wherein the treatment is a plasma
treatment, corona discharge or UV ozone treatment.
19. The composition of claim 17, wherein the treatment is a
hydrosilylation.
20. The composition of claim 17, wherein the hydrophobic surface is
irradiated or hydrophilized after the treatment.
21. The composition of claim 17, wherein the surface is
hydrophilized after applying the absorbent polymer to the modified
surface.
22. The composition of claim 17, wherein the hydrophilic polymer is
PVA, PEG or PEG-acrylate.
23. The composition of claim 17, wherein the hydrophilic polymer is
crosslinked to the surface.
24. The composition of claim 17, wherein the surface is free of
crosslink.
25. A cell culture system, comprising a cell culture article having
a surface coated with the composition of claim 1.
26. The cell culture system of claim 25, further comprising cells
and/or culture media.
27. A method for culturing cells, the method comprising: a)
providing a cell culture article having a surface coated with the
composition of claim 1, b) seeding cells on the coated surface, and
c) culturing the cells under a suitable medium for a sufficient
period of time to form one or more spheroids.
28. The method of claim 27, wherein the one or more spheroids are
generated via single cell proliferation; or wherein the one or more
spheroids have an average diameter between 50 .mu.M and 150
.mu.M.
29. The method of claim 27, wherein the seeding comprises plating
the cells at a density less than 1000 cells per cm.sup.2 on the
substrate.
30. A method for obtaining and optionally characterizing
single-cell-derived spheroid, the method comprising: a) culturing a
heterogeneous population of cells on a surface coated with the
composition of claim 1 to obtain at least one single-cell-derived
spheroid disposed on the surface, and b) optionally, analyzing the
single-cell-derived spheroid to thereby obtain at least one
characteristic of the single cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application No. 63/132,934, filed Dec. 31, 2020
and U.S. Provisional Patent Application No. 63/252,268, filed Oct.
5, 2021, the disclosure of which is hereby incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The interest in 3D spheroid models is growing among
researchers, from basic science to preclinical drug discovery
applications, including studies in tumor biology, neurodegenerative
diseases, and drug toxicity. Three-dimensional (3D) cell culture
methods are increasingly used to generate complex tissue or tumor
models.
[0003] There is a lot of variation in the spheroids formed using 3D
cell culture methods and products available on the market, and this
may impact their read-out. For instance, the widely used
non-adherent techniques for 3D cell culture, including Ultra Low
Attachment (ULA) plate and hanging drop method, have not proven
suitable because these methods usually generate spheroids via cell
agglomeration. Such spheroids generally maintain their original
heterogeneity and harbor multiple cells with various
characteristics, requiring a better understanding of cellular
heterogeneity. When tens-of-thousands cells are aggregated into a
spheroid (i.e., a mass with spherical shape), an extensive central
necrotic core may form over a few hours due to the lack of nutrient
and oxygen penetration, and thus hinders cell proliferation.
Extended central necrosis is a rare phenomenon in real cancers.
[0004] Alternatively, Matrigel is a commonly used embedded
substrate for tissue-based cell growth, such as organoid formation.
But out of focus, inefficient compound diffusion, and difficulty in
sample isolation limits its application for ex vivo 3D
spheroid-based applications.
[0005] Standardizing spheroid formation is critical to generating
uniform 3D cell culture and obtaining reproducible results from
spheroid-based assays and drug screening. Therefore, there is a
need for the development of new cell culture systems and methods
that can reliably form single cell-derived spheroids.
SUMMARY OF THE INVENTION
[0006] The present disclosure provides a surface coating for
coating a cell culture article. The surface coating described
herein comprises a hydrophilic polymer and polyelectrolyte
multilayers. The substrate provided herein is advantageous for
hydration preservation. It can prevent the cell culture substrate
from undesirable surface cracks caused by prolonged storage at
ambient temperature. In some embodiments, the surface coating
provided herein enables the formation of single-cell derived
spheroids derived from single cells. Also provided is a cell
culture system comprising the cell culture article. Uses and
methods of preparing the surface coatings and systems are provided
as well.
[0007] Accordingly, one aspect of the present disclosure provides a
composition for coating a surface of a cell culture article. The
composition described herein comprises a) a hydrophilic polymer, in
which the hydrophilic polymer is deposited on a surface of the cell
culture article, and b) polyelectrolyte multilayers, in which the
hydrophilic polymer is in direct contact with a polycation or an
polyanion of the polyelectrolyte multilayers.
[0008] The cell culture article described herein may be made of any
suitable plastics or polymers such as polyethylene, polypropylene,
polymethylpentene, cyclic olefin polymer, cyclic olefin copolymer,
polyvinyl chloride, polyurethane, polyester, polyamide,
ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer,
ethylene-acrylic acid copolymer, ethylene-methyl acrylate
copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl
methacrylate copolymer, polyacrylic acid, polymethacrylic acid,
methyl polyacrylate, and methyl polymethacrylate, or derivatives of
these or the like.
[0009] The surface coating described herein may be dehydrated or
hydrated. In some embodiments, the surface coating is in a
dehydrated state. In some embodiments, the surface coating is in a
hydrated state.
[0010] Suitable hydrophilic polymers include, but are not limited
to, poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG),
PEG-acrylate, polyvinylpyrrolidone (PVP), polyethyleneimine (PEI),
poly-L-lactide (PLLA), poly-D-lactide (PDLA),
poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA),
poly(lactic-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate)
(PMMA), poly(hydroxyethyl methacrylate) (p-HEMA) and derivatives
thereof.
[0011] In some embodiments, the hydrophilic polymer is PVA, PEG,
PVP, PEI, PMMA or a derivative thereof. In some embodiments, the
absorbent polymer is PVA. In some embodiments, the hydrophilic
polymer is PEG or PEG-acrylate such as PEGMA, PEGDMA or PEGDA. In
some embodiments, the hydrophilic polymer is PLA or a derivative
such as PLLA, PDLA or PLDLLA. In some embodiments, the hydrophilic
polymer is PGA or a derivative such as PLGA. In some embodiments,
the hydrophilic polymer is PMAA or a derivative such as pHEMA.
[0012] In certain embodiments, the volume of the hydrophilic
polymer (e.g. PVA) is 0.01-10% of the total volume of the surface
coating.
[0013] The polyelectrolyte multiplayers described herein comprise
at least one layer pair (referred as "bilayer") comprising a
cationic polyelectrolyte (referred as "polycation") and an
polyelectrolyte (referred as "polyanion"). In some embodiments, the
polycation is a poly(amino acid). In some embodiments, the
polyanion is a poly(amino acid). In some embodiments, the
polycation and the polyanion are poly(amino acid)s. The poly(amino
acid)s described herein may comprise L and/or D amino-acid forms.
As described herein, the polyelectrolyte multiplayers can be formed
by depositing polycations and polyanions in an alternative fashion
via layer-by-layer assembly.
[0014] In some embodiments, the polyelectrolyte multilayers having
a formula of (polycation/polyanion)n comprise n bilayers of
polycations and polyanions, wherein n is an integer number ranging
from 1 to 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments,
n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18,
13-17, or 14-16.
[0015] In some embodiments, the polyelectrolyte multilayers having
a formula of polyanion(polycation/polyanion)n comprise n+1 layers
of polyanions and n layers of polycations, wherein n is an integer
number ranging from 1 to 30. In some embodiments, n is 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In
some embodiments, n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0016] In some embodiments, the polyelectrolyte multilayers having
a formula of polycation(polyanion/polycation)n comprise n+1 layers
of polycations and n layers of polyanions, wherein n is an integer
number ranging from 1 to 30. In some embodiments, n is 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In
some embodiments, n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0017] In some embodiments, the polycation is poly(L-lysine) (PLL),
poly(L-arginine) (PLA), poly(L-ornithine) (PLO), poly(L-histidine)
(PLH), or a combination thereof. In a preferred embodiment, the
polycation is PLL.
[0018] In preferred embodiments, the polyanion is poly(L-glutamic
acid) (PLGA), poly(L-aspartic acid) (PLAA), or a combination
thereof. In a preferred embodiment, the polyanion is PLGA.
[0019] In some embodiments, said polyelectrolyte multilayers
comprise at least one layer pair (i.e., bilayer) of
polycation/polycation selecting from the group consisting of
PLL/PLGA, PLL/PLAA, PLA/PLGA, PLA/PLAA, PLO/PLGA, PLO/PLAA,
PLH/PLGA, PLH/PLAA, and a combination thereof.
[0020] In some embodiments, the bilayer described herein comprises
a combination of PLL and PLGA. In some embodiments, the bilayer
described herein comprises a combination of PLO and PLGA. In some
embodiments, the bilayer described herein comprises a combination
of PLH and PLGA. In some embodiments, the bilayer described herein
comprises a combination of PLA and PLGA.
[0021] In some embodiments, the bilayer described herein comprises
a combination of PLL and PLAA. In some embodiments, the bilayer
described herein comprises a combination of PLO and PLAA. In some
embodiments, the bilayer described herein comprises a combination
of PLH and PLAA. In some embodiments, the bilayer described herein
comprises a combination of PLA and PLAA.
[0022] In some embodiments, the polyelectrolyte multilayers
described herein may have a thickness ranging from 30 nm to 30
.mu.m. In some embodiments, the surface coating has a thickness
ranging from 100 nm to 20 .mu.m. In some embodiments, the surface
coating has a thickness of 200, 400, 600 or 800 nm. In some
embodiments, the surface coating has a thickness of 1, 5, 10, 15 or
20 .mu.m.
[0023] Compared with conventional culture methods, the surface
coating of the present disclosure offers an improved proliferation
rate for a variety of cells including, but not limited to, tumor
cells, pluripotent and multipotent stem and progenitor cells,
hematopoietic cells and immune cells. In addition, the surface
coating with elevated water retention offers an advantage to
prevent the surface coating from undesirable surface cracks caused
by dehydration due to prolonged storage at ambient temperature.
[0024] In another aspect, the present invention provides methods
for coating a cell culture article. The method described herein
comprises the steps of: (a) providing a cell culture article having
a hydrophobic surface; (b) modifying the hydrophobic surface with a
treatment; (c) applying a hydrophilic polymer to the modified
surface; and (d) sequentially depositing on the hydrophilic polymer
alternating layers of polycations and polyanions.
[0025] In some embodiments, the treatment described herein is a
plasma treatment, corona discharge or UV ozone treatment. In some
embodiments, the hydrophobic surface described herein is irradiated
or hydrophilized after the treatment. In some embodiments, the
hydrophobic surface is hydrophilized after applying the hydrophilic
polymer (e.g., PVA) to the surface. In some embodiments, the
hydrophilic polymer (e.g., PVA) is covalently linked (i.e.,
conjugated) to the surface. A cross-linking agent may be used to
facilitate the crosslinking (i.e., conjugation). Exemplary
cross-linking agents include, but are not limited to, maleic acid,
formaldehyde, glutaraldehyde, butanal (butyraldehyde), sodium
borate, or a combination thereof.
[0026] In another aspect, the present invention provides a cell
culture system comprising a cell culture article having a substrate
with the inventive surface coating configured to culture cells. In
some embodiments, the cell culture system further comprises cells.
In some embodiments, the cells are adapted to be human cells. In
some embodiments, the cells are adapted to be living cells. In some
embodiments, the cell culture system further comprises culture
media.
[0027] In some embodiments, the cell culture system disclosed
herein enables an efficient and scalable multiplication of cells,
in particular, single cells or low-density cells (e.g., cells with
an abundance of less than 1000 in one milliliter) into 3D, making
it possible to form 3D cell culture on difficult cell types that
did not form on current platforms in the market (e.g., Ultra Low
Attachment (ULA) plate, Hanging-Drop).
[0028] As disclosed herein, one or more parameters of the
polyelectrolyte multilayers and the culture medium may be selected
by the user, based on one or more microenvironment selection
criteria for the cells.
[0029] The cell culture system disclosed herein enables not only
cell attachment and growth, but also the viable harvest of cultured
cells (e.g. 3D cell culture, tissue and organs). The inability to
harvest viable cells is a significant drawback in current platforms
on the market, and it leads to difficulty in building and
sustaining a sufficient number of cells for production capacity.
According to an aspect of embodiments of this disclosure, it is
possible to harvest viable cells from the cell culture system,
including between 80% to 100% viable, or about 85% to about 99%
viable, or about 90% to about 99% viable. For example, of the cells
that are harvested, at least 80% are viable, at least 85% are
viable, at least 90% are viable, at least 91% are viable, at least
92% are viable, at least 93% are viable, at least 94% are viable,
at least 95% are viable, at least 96% are viable, at least 97% are
viable, at least 98% are viable, or at least 99% are viable. In
some embodiments, cells can be released from the surface coating
with using a cell dissociation enzyme, for example, trypsin,
TrypLE, or Accutase. In preferred embodiments, cells can be
released from the surface coating without using a cell dissociation
enzyme.
[0030] In another aspect, the present invention provides methods
for culturing cells using the cell culture article disclosed
herein. The method for culturing cells comprises the steps of: a)
providing a cell culture article having a surface coated with the
surface coating of the present disclosure; b) seeding cells on the
coated surface; c) culturing the cells under a suitable medium for
a sufficient period of time to form one or more spheroids. In some
embodiments, the spheroids generated herein are adhered to the
substrate. In some embodiments, the spheroids generated herein are
semi-attached to the substrate. In some embodiments, the spheroids
are derived from single cells via single cell proliferation. The
cultured cells (e.g., cultured and harvested cells) may be used for
various applications such as analysis and characterization,
screening drugs, isolating single-cell derived clone, generating
cell banks, and generating animal models.
[0031] As described herein, the cells are living cells. In some
embodiments, the cells are mammalian cells. In some embodiments,
the cells are tissue cells, immune cells, endothelial cells, stem
cells, epithelial cells, mesenchymal cells, mesothelial cells,
tumor cells or tumor-associated cells.
[0032] As described herein, culturing the cells comprise
maintaining and/or proliferating cells. In some embodiments,
culturing the cells comprises maintaining cells. In some
embodiments, culturing the cells comprises proliferating cells. In
some embodiments, culturing the cells may further comprise
differentiating cells.
[0033] In some embodiments, the cells are stem cells such as
mesenchymal stem cells (MSCs) or pluripotent stem cells (PSCs)
including embryonic stem cells (ESCs) and induced pluripotent stem
cells (iPSCs).
[0034] In some embodiments, the cells are tumor cells, and the
cultured cells are tumor spheroids. The tumor spheroids may be
derived from a cell line, a tumor tissue or a liquid biopsy. In
some embodiments, tumor spheroids described herein are derived from
circulating tumor cells (CTCs) isolated from a blood sample
obtained from a cancer patient. In some embodiments, the blood
sample described herein is a whole blood. The blood sample can be
obtained by liquid biopsy. In some embodiments, the cancer patient
described herein is a human cancer patient having a metastatic
cancer. In some embodiments, the blood sample is obtained from the
cancer patient before, during, and/or after therapeutic
treatment.
[0035] Another aspect of the present disclosure provides a method
of preparing a single-cell derived spheroid in vitro, the method
comprising the steps of: (a) providing a cell culture system
comprising the substrate of the present disclosure; (b) isolating
cells (e.g. tumor cells and/or tumor-associated cells) from a
sample to provide isolated cells; (c) seeding the isolated cells on
the substrate; and (d) culturing the cells under a suitable medium
for a time sufficient to produce one or more spheroids, wherein the
one or more spheroids are single-cell derived.
[0036] Another aspect of the present disclosure provides methods
for isolating single cell derived clones, each composed of a
homogenous cell population that is genetically identical.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1A is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are not
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 4 bilayers of
301 and 302. The outermost layer is 301. 102 is in direct contact
with 302.
[0038] FIG. 1B is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 4 bilayers of
301 and 302. The outermost layer is 301. 102 is in direct contact
with 302.
[0039] FIG. 2A is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are not
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 5 layers of
302 and 4 layers of 301. The outermost layer is 302. 102 is in
direct contact with 302.
[0040] FIG. 2B is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 5 layers of
302 and 4 layers of 301. The outermost layer is 302. 102 is in
direct contact with 302.
[0041] FIG. 3A is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are not
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 4 bilayers of
301 and 302. The outermost layer is 302. 102 is in direct contact
with 301.
[0042] FIG. 3B is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 4 bilayers of
301 and 302. The outermost layer is 302. 102 is in direct contact
with 301.
[0043] FIG. 4A is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are not
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 5 layers of
301 and 4 layers of 302. The outermost layer is 301. 102 is in
direct contact with 301.
[0044] FIG. 4B is a side cross-sectional view of an embodiment of
the surface coating of the present disclosure. A hydrophilic
polymer 102 is deposited on a surface of the well 101 in the cell
culture article 202. 102 and the surface of the well 101 are
crosslinked. 301 is a polyanion. 302 is a polycation. 103 is an
embodiment of polyelectrolyte multiplayers including 5 layers of
301 and 4 layers of 302. The outermost layer is 301. 102 is in
direct contact with 301.
[0045] FIG. 5 illustrates of an embodiment of the surface
modification of a cell culture article. A tissue culture plate made
of polystyrene plastic is first treated by an ozone plasma,
followed by addition of a photo-activated azidophenyl-PVA to the
modified surface to form a PVA-crosslinked polystyrene plate.
[0046] FIG. 6 illustrates of an embodiment of the surface
modification of a cell culture article. A tissue culture plate made
of polytetrafluoroethylene (PTFE) is first treated by plasma gas,
followed by depositing PVA onto the modified surface of the PTFE
plate. A cross-linking agent, glutaraldehyde (GA), is applied to
crosslink PVA to PTFE to form a PVA-crosslinked PTFE plate.
[0047] FIG. 7 shows the time-lapse microscope observation of HCT116
colorectal cancer cells cultured on the surface coating of the
invention on day 0, 1, 2, 3, 4 and 5 during the growth of the
cancer cells supplied with complete DMEM medium. (Image
photographed by Leica DMI6000B time-lapse microscope under
10.times. objective).
[0048] FIGS. 8A-8E show the results of ex vivo cultivation using
the culture platform of the invention, and the formation of
spheroids (after 7-14 days) derived from (A) lung cancer cell lines
A549, H1299, PC-9 and H1975; (B) liver cancer cell lines SNU-398,
SNU-475, PLC/PRF/S, Hep3B and Huh? (C) breast cancer cell lines
MDA-MB-231 and CGBC01; (D) colorectal cancer cell lines HCT116,
HCT15 and WiDr; and (E) human tongue squamous carcinoma cell line
SAS, ovarian cancer cell line SK-OV-3, and cell line T24 derived
from a human urinary bladder cancer patient
[0049] FIGS. 9A-9C show the representative time-dependent images of
CTC-derived spheroid cultivation on the culture platform of the
invention. (A) CTCs were isolated from a blood sample of a breast
cancer patient; CTC-derived spheroids formed after 14 days. (B)
CTCs were isolated from a blood sample of a head&neck cancer
patient; CTC-derived spheroids formed after 38 days. (C) CTCs were
isolated from a blood sample of a colorectal cancer patient;
CTC-derived spheroids formed after 13-27 days. Scale bar: 50
.mu.m.
[0050] FIGS. 10A-10B show the images of tumor spheroids derived
from primary colorectal tumor tissues obtained from colorectal
cancer (CRC) patient. Tumor spheroids were generated on the culture
platform of the invention after (A) 2 weeks and (B) 4 weeks.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The present disclosure relates to a new generation of
scaffold-free 3D cell culture technology and uses thereof. In some
embodiments, provided is a new composition for surface coating.
Also provided is a cell culture system comprising a surface coating
that is useful for cell culture, in particular, 3D cell culture.
The surface coating can induce the formation of highly uniform 3D
cell culture, making it possible to form 3D cell culture on
difficult primary cell types that did not form on any other low
attachment surface. Compared with conventional culture methods, the
surface coating described herein offers an improved proliferation
rate for a variety of cells including, but not limited to, tumor
cells, pluripotent and multipotent stem and progenitor cells,
hematopoietic cells and immune cells. In certain embodiments, the
surface coating comprises a hydrophilic polymer (e.g. PVA), and one
or more pairs of polyelectrolytes.
Surface Coatings
[0052] The surface coating of the present disclosure comprises a
hydrophilic polymer and polyelectrolyte multilayers. In some
instances, the surface coating is as illustrated in FIG. 1. As show
in FIG. 1, 104 indicates an illustrative surface coating. The
hydrophilic polymer 102 is deposited on the top surface of the well
201 of a cell culturing plate 202. Polyelectrolyte multilayers 103
is deposited on top of the hydrophilic polymer layer 102.
[0053] Without being bound to any particular theory, it is believed
that the surface coating enables robust multiplication or stable
maintenance of cells (e.g., rare cells extracted from blood,
low-density cells, or single cells) seeded on the surface coating
with or without the substrate for an extended period, for example,
over 48 hours, over 72 hours, over 96 hours, over 5 days, over 6
days, over 7 days, or in one to several weeks (e.g., 1, 2, 3, 4, 5,
6, or more weeks).
[0054] 1) Hydrophilic Polymers
[0055] Hydrophilic polymers described herein are hydrophilic
absorbent polymers ("absorbent polymers") that water soluble and
may swell as a result of uptake and retention of aqueous solutions.
A non-limiting list of hydrophilic absorbent polymers that may be
used with the present invention includes hydrophilic and
biocompatible grades of the following polymers and their
derivatives: poly(vinyl alcohol) (PVA), ethylene vinyl alcohol
co-polymers (typically non-biodegradable materials which degree of
hydrophilicity depends on distribution of ethylene (hydrophobic)
and vinyl alcohol (hydrophilic) groups), co-polymers of polyvinyl
alcohol and ethylene vinyl alcohol, polyacrylate compositions,
polyurethane compositions, poly(ethylene glycol) (PEG), otherwise
known as poly(oxyethylene) (POE) and poly(ethylene oxide) (PEO),
and its derivatives including but not limited to polyethylene
glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate
(PEGDMA) and polyethylene glycol diacrylate (PEGDA);
nitrogen-containing materials such as polyacrylamide (without
acrylamide toxic residuals), polyvinylpyrrolidone, polyvinylamine,
and polyethyleneimine; electrically charged materials such as
poly(lactic acid) also known as polylactide in various forms (e.g.
poly-L-lactide (PLLA) and its derivatives, poly-D-lactide (PDLA)
and its derivatives, poly(L-lactide-co-D,L-lactide) (PLDLLA) and
its derivatives), poly(glycolic acid) (PGA) also known as
polyglycolide, co-polymers of lactic acid and glycolic acid
poly(lactic-co-glycolic acid) (PL-co-GA), co-polymers of PLA and/or
PGA with PEG; polymethacrylic acid; poly(hydroxyethyl methacrylate)
(poly-HEMA), among other absorbent, hydrophilic and biocompatible
materials known in the art.
[0056] In some embodiments, the hydrophilic absorbent polymer is
selected from the group consisting of poly(vinyl alcohol) (PVA),
copolymers of ethylene vinyl alcohol, copolymers of polyvinyl
alcohol and ethylene vinyl alcohol, polyacrylate compositions,
polyurethane compositions, poly(ethylene glycol) (PEG),
PEG-acrylate, polyethylene glycol methacrylate (PEGMA),
polyethylene glycol dimethacrylate (PEGDMA), polyethylene glycol
diacrylate (PEGDA), polyacrylamide (PAM), polyvinylpyrrolidone
(PVP), polyvinylamine (PVAm), polyethyleneimine (PEI),
poly-L-lactide (PLLA), poly-D-lactide (PDLA),
poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA),
poly(lactic-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate)
(PMMA) and poly(hydroxyethyl methacrylate) (p-HEMA).
[0057] In some embodiments, the hydrophilic absorbent polymer is
selected from the group consisting of PVA, PEG, PEG-acrylate,
polylactide, PMMA, p-HEMA, a combination or a derivative thereof.
In some embodiments, the absorbent polymer is PVA or a derivative
thereof. In some embodiments, the absorbent polymer is PEG or
PEG-acrylate such as PEGMA, PEGDMA or PEGDA. In some embodiments,
the absorbent polymer is polylactide or a derivative such as PLLA,
PDLA or PLDLLA. In some embodiments, the absorbent polymer is PGA
or a derivative such as PLGA. In some embodiments, the absorbent
polymer is PMAA or a derivative such as pHEMA.
[0058] In some embodiments, the hydrophilic polymer has an average
molecular weight of from about 2,500 g/mol to about 200,000 g/mol.
In some cases, the average molecular weight of the hydrophilic
polymer is from about 5,000 g/mol to about 175,000 g/mol, from
about 5,000 g/mol to about 150,000 g/mol, from about 5,000 g/mol to
about 125,000 g/mol, from about 5,000 g/mol to about 100,000 g/mol,
from about 5,000 g/mol to about 75,000 g/mol, from about 5,000
g/mol to about 50,000 g/mol, from about 5,000 g/mol to about 25,000
g/mol, from about 5,000 g/mol to about 10,000 g/mol, from about
10,000 g/mol to about 175,000 g/mol, from about 10,000 g/mol to
about 150,000 g/mol, from about 10,000 g/mol to about 125,000
g/mol, from about 10,000 g/mol to about 100,000 g/mol, from about
10,000 g/mol to about 75,000 g/mol, from about 10,000 g/mol to
about 50,000 g/mol, from about 10,000 g/mol to about 25,000 g/mol,
from about 20,000 g/mol to about 150,000 g/mol, or from about
50,000 g/mol to about 150,000 g/mol.
[0059] In some instances, the hydrophilic polymer is deposited
directly onto the surface of a target substrate. In other
instances, the hydrophilic polymer is deposited indirectly onto the
surface. In some cases, one or more additional layers (e.g., 1, 2,
3, 4, 5, or more layers) are formed between the hydrophilic polymer
layer and the surface of the substrate. In some cases, one
additional layer (also referred to herein as the innermost layer)
is formed between the hydrophilic polymer layer and the surface of
the substrate.
[0060] In some embodiments, the hydrophilic polymer is PVA. PVA can
have an average molecular weight ranging from about 10,000 g/mol to
about 125,000 g/mol. In some instances, PVA has an average
molecular weight of from about 10,000 g/mol to about 100,000 g/mol,
from about 10,000 g/mol to about 75,000 g/mol, from about 10,000
g/mol to about 50,000 g/mol, from about 20,000 g/mol to about
125,000 g/mol, from about 20,000 g/mol to about 100,000 g/mol, from
about 20,000 g/mol to about 75,000 g/mol, from about 20,000 g/mol
to about 50,000 g/mol, from about 50,000 g/mol to about 125,000
g/mol, or from about 50,000 g/mol to about 100,000 g/mol.
[0061] In some instances, the PVA is deposited directly onto the
surface of a target substrate. In other instances, the PVA is
deposited indirectly onto the surface. In some cases, one or more
additional layers (e.g., 1, 2, 3, 4, 5, or more layers) are formed
between the PVA layer and the surface. In some cases, one
additional layer is formed between the PVA layer and the surface of
the substrate.
[0062] In some embodiment, the hydrophilic polymer is PEG. In some
instances, the average molecular weight of PEG is about 200, 300,
400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450,
1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,
2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250,
4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000,
12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
[0063] In some instances, the PEG utilized herein is a discrete PEG
(dPEG). A discrete PEG can be a polymeric PEG comprising more than
one repeating ethylene oxide units. In some cases, the discrete PEG
comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating
ethylene oxide units. In some cases, the dPEG comprises 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26,
28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide
units.
[0064] In certain embodiments, the volume of the hydrophilic
polymer (e.g. PVA or PEG) is from about 0.01% to about 10% of the
total volume of the surface coating. In some instances, the
hydrophilic polymer is from about 0.01% to about 9% v/v, from about
0.01% to about 8% v/v, from about 0.01% to about 7% v/v, from about
0.01% to about 6% v/v, from about 0.01% to about 5% v/v, from about
0.01% to about 4% v/v, from about 0.01% to about 3% v/v, from about
0.01% to about 2% v/v, from about 0.01% to about 1% v/v, from about
0.1% to about 10% v/v, from about 0.1% to about 9% v/v, from about
0.1% to about 8% v/v, from about 0.1% to about 7% v/v, from about
0.1% to about 6% v/v, from about 0.1% to about 5% v/v, from about
0.1% to about 4% v/v, from about 0.1% to about 3% v/v, from about
1% to about 10% v/v, from about 1% to about 9% v/v, from about 1%
to about 8% v/v, from about 1% to about 7% v/v, from about 1% to
about 6% v/v, from about 1% to about 5% v/v, from about 1% to about
4% v/v, from about 2% to about 10% v/v, or from about 5% to about
10% v/v, of the total volume of the surface coating. In some cases,
the volume of the hydrophilic polymer (e.g. PVA or PEG) is about
0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%,
about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about
9%, or about 10% of the total volume of the surface coating.
[0065] In some instances, the weight of the hydrophilic polymer
(e.g. PVA or PEG) per total weight of the surface coating is from
about 1% to about 50%. In some instances, the weight of the
hydrophilic polymer (e.g. PVA or PEG) per total weight of the
surface coating is from about 1% to about 40%. In some instances,
the weight of the hydrophilic polymer (e.g. PVA or PEG) per total
weight of the surface coating is from about 1% to about 30%. In
some instances, the weight of the hydrophilic polymer (e.g. PVA or
PEG) per total weight of the surface coating is from about 1% to
about 20%. In some instances, the weight of the hydrophilic polymer
(e.g. PVA or PEG) per total weight of the surface coating is from
about 1% to about 10%.
[0066] 2) Polyelectrolyte Multilayers
[0067] In certain embodiments, the surface coating comprises
polyelectrolyte multilayers (PEMs). PEMs described herein comprise
a plurality of alternating layers of oppositely charged polymers
(i.e., polyelectrolytes). The oppositely charged polymers described
herein comprise a combination of a positively charged
polyelectrolyte (also referred to herein as a polycation) and a
negatively charged polyelectrolyte (also referred to herein as a
polyanion).
[0068] Exemplary polycations include, but are not limited to,
poly(L-lysine) (PLL), poly(L-arginine) (PLA), poly(L-ornithine)
(PLO), poly(L-histidine) (PLH), polyethyleneimine (PEI),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid] (PAGA),
2-(dimethylamino)ethyl methacrylate (DMAEMA), N,N-Diethylaminoethyl
methacrylate (DEAEMA), and a combination thereof. In some
instances, the polycation is PLL. In some instances, the polycation
is PLO. In some instances, the polycation is PLH. In some
instances, the polycation is PLA.
[0069] Exemplary polyanions include, but are not limited to,
poly-L-glutamic acid (PLGA), poly-L-aspartic acid (PLAA),
poly(acrylic acid), poly(methacrylic acid) (PMAA),
poly(styrenesulfonic acid) (PSS), poly(N-isopropylacrylamide)
(NIPAM), poly(-acrylamido-2-methyl-1-propane sulfonic acid)
(PAMPS), and a combination thereof. In some instances, the
polyanionis PLGA. In some instances, the polyanion is PLAA.
[0070] Polyelectrolyte multilayers may be formed by depositing
polycations and polyanions in an alternative fashion via
layer-by-layer assembly. Polyelectrolyte multilayers described
herein include at least one bilayer including a polycation layer
and a polyanion layer.
[0071] In some embodiments, the PEMs may include from about 1
bilayers to about 100 bilayers. In some embodiments, the PEMs may
include from about 1 bilayers about 50 bilayers. In some
embodiments, the PEMs may include from about 1 bilayers to about 30
bilayers. In some embodiments, the PEMs may include from about 1
bilayers to about 20 bilayers. In some embodiments, the number of
bilayers is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20. In some embodiments, the number of bilayers is in
a range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17,
or 14-16. In some embodiments, the number of bilayers is 3. In some
embodiments, the number of bilayers is 4. In some embodiments, the
number of bilayers is 5. In some embodiments, the number of
bilayers is 6. In some embodiments, the number of bilayers is 7. In
some embodiments, the number of bilayers is 8. In some embodiments,
the number of bilayers is 9. In some embodiments, the number of
bilayers is 10. In some embodiments, the number of bilayers is 11.
In some embodiments, the number of bilayers is 12. In some
embodiments, the number of bilayers is 13. In some embodiments, the
number of bilayers is 14. In some embodiments, the number of
bilayers is 15. In some embodiments, the number of bilayers is 16.
In some embodiments, the number of bilayers is 17. In some
embodiments, the number of bilayers is 18. In some embodiments, the
number of bilayers is 19. In some embodiments, the number of
bilayers is 20.
[0072] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of positively
charged polyelectrolyte(s) and negatively charged
polyelectrolyte(s), in which the polycation is selected from PLL,
PLO PLH, and PLA, and the polyanion is selected from PLGA and PLAA.
In some embodiments, the number of sets ranges from 1 to 100, 3 to
60, from 3 to 50, or from 3 to 30. In some embodiments, the number
of sets is greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20. In some embodiments, the number of sets
is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20. In some embodiments, the number of sets is in a range of 1-10,
1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.
[0073] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLL and PLGA. In
some embodiments, the number of bilayers of PLL and PLGA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0074] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLO and PLGA. In
some embodiments, the number of bilayers of PLO and PLGA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0075] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLH and PLGA. In
some embodiments, the number of bilayers of PLH and PLGA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0076] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLA and PLGA. In
some embodiments, the number of bilayers of PLA and PLGA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0077] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLL and PLAA. In
some embodiments, the number of bilayers of PLL and PLAA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0078] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLO and PLAA. In
some embodiments, the number of bilayers of PLO and PLAA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0079] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLH and PLAA. In
some embodiments, the number of bilayers of PLH and PLAA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0080] In some embodiments, the polyelectrolyte multilayers
described herein comprise one or more bilayers of PLA and PLAA. In
some embodiments, the number of bilayers of PLA and PLAA ranges
from 1 to 100, 3 to 60, from 3 to 50, or from 3 to 30. In some
embodiments, the number of bilayers is greater than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some
embodiments, the number of bilayers is 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the
number of bilayers is in a range of 1-10, 1-8, 1-5, 3-20, 5-20,
10-20, 11-19, 12-18, 13-17, or 14-16.
[0081] The thickness of the PEM as a thin film may be in a broad
range, for example, in a range from about 30 nm to about 30 .mu.m,
or from about 100 nm to about 20 .mu.m. In some embodiments, the
thickness is about 100 nm to about 500 nm, about 500 nm to about 1
.mu.m, or about 1 .mu.m to about 10 .mu.m. In some embodiments, the
thickness is about 200, 400, 600, 800 nm, or any number in between.
In some embodiments, the thickness is about 1, 5, 10, 15 or 20
.mu.m, or any number in between.
[0082] A number of methodologies are available for characterizing
PEMs. In some embodiments, the methodologies may comprise
ellipsometry (thickness), quartz crystal microbalance with
dissipation monitoring (mass adsorbed, viscoelasticity), contact
angle analysis (surface energy), Fourier transform infrared
spectroscopy (functional groups), X-ray photoelectron spectroscopy
(chemical composition), scanning electron microscopy (surface
structure), and atomic force microscopy (roughness/surface
structure).
[0083] In some embodiments, PEMs may be deposited by pipetting
polyanion or polycation solutions into/onto the dish, either as a
mixture or sequentially.
[0084] In some embodiments, a PEM is formed on the surface by dip
coating. In dip coating, the substrate is immersed in a
polyelectrolyte solution for a set amount of time (usually 10-15
min), followed by multiple rinses and immersion in a second
polyelectrolyte solution of opposite charge. This process is
repeated until the desired number of layers is achieved.
[0085] In some embodiments, the PEM is formed on the surface by
spray coating. In some embodiments, a polyelectrolyte may be
sprayed onto the surface for 3-10 sec followed by a rest/draining
period of 10-30 sec, washing of the surface with a water spray for
3-20 sec, an additional rest period of 10 sec, and repeating the
cycle with a polyelectrolyte of opposite charge.
[0086] In some embodiments, the PEM is formed on the surface by
spin coating. Spin coating is a highly controlled method for
solution-based coating of a system. A typical spin coating
procedure includes spin coating for 10-15 sec, rinsing at least
once by "spin coating" water for 15-30 sec and repeating the
procedure with the oppositely charged polyelectrolyte. The wash
step may not be necessary in spin coating.
[0087] 3) Surface Coating Construction
[0088] Another aspect of the present disclosure features a method
for coating a cell culture article using the composition described
herein. The method described herein comprises the steps of: (a)
providing a cell culture article having a hydrophobic surface; (b)
modifying the hydrophobic surface with a treatment; (c) applying a
hydrophilic polymer to the modified surface; and (d) sequentially
depositing on the hydrophilic polymer alternating layers of
polycations and polyanions.
[0089] In some embodiments, the treatment described herein is a
plasma treatment, corona discharge or UV ozone treatment. In some
embodiments, the hydrophobic surface described herein is irradiated
or hydrophilized after the treatment. In some embodiments, the
hydrophobic surface is hydrophilized after applying the hydrophilic
polymer (e.g., PVA) to the surface. In some embodiments, the
hydrophilic polymer (e.g., PVA) is covalently linked (i.e.,
conjugated) to the surface. A cross-linking agent may be used to
facilitate the crosslinking (i.e., conjugation). Exemplary
cross-linking agents include, but are not limited to, maleic acid,
formaldehyde, glutaraldehyde, butanal (butyraldehyde), sodium
borate, or a combination thereof.
[0090] As described herein, a surface is hydrophilic if a contact
angle for a water droplet on the surface is less than 90 degrees
(the contact angle is defined as the angle passing through the drop
interior). Embodiments include hydrophilic surfaces with a contact
angle from 90 to 0 degrees; Artisans will immediately appreciate
that all ranges and values between the explicitly stated bounds are
contemplated, with, e.g., any of the following being available as
an upper or lower limit: 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2,
0 degrees.
[0091] In some embodiments, the substrate described herein
comprises (polyanion/polycation)n/PVA, wherein the
polyanion/polycation is selected from PLGA/PLL, PLAA/PLL, PLGA/PLA,
PLAA/PLA, PLGA/PLO, PLAA/PLO, PLGA/PLH and PLAA/PLH, and n is an
integer number ranging from 1 to 20, optionally 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some
embodiments, n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20,
11-19, 12-18, 13-17, or 14-16.
[0092] In some embodiments, the substrate described herein
comprises (polycation/polyanion)n/PEG, wherein the
polycation/polyanion is selected from PLL/PLGA, PLL/PLAA, PLA/PLGA,
PLA/PLAA, PLO/PLGA, PLO/PLAA, PLH/PLGA and PLH/PLAA, and n is an
integer number ranging from 1 to 20, optionally 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some
embodiments, n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20,
11-19, 12-18, 13-17, or 14-16.
[0093] In some embodiments, the substrate described herein
polycation (polyanion/polycation).sub.n/PEG-acrylate, wherein the
polyanion/polycation is selected from PLGA/PLL, PLAA/PLL, PLGA/PLA,
PLAA/PLA, PLGA/PLO, PLAA/PLO, PLGA/PLH and PLAA/PLH, and n is an
integer number ranging from 1 to 20, optionally 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some
embodiments, n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20,
11-19, 12-18, 13-17, or 14-16.
[0094] In some embodiments, the substrate described herein
polyanion (polycation/polyanion)n/PVP, wherein the
polycation/polyanion is selected from PLL/PLGA, PLL/PLAA, PLA/PLGA,
PLA/PLAA, PLO/PLGA, PLO/PLAA, PLH/PLGA and PLH/PLAA, and n is an
integer number ranging from 1 to 20, optionally 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some
embodiments, n is in a range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20,
11-19, 12-18, 13-17, or 14-16.
[0095] The surface coating described herein can be dehydrated or
hydrated. In some embodiments, the surface coating is in a
dehydrated state. In other embodiments, the surface coating is in a
hydrated state. As used herein, a "dehydrated state" and a
"hydrated state" each refers to a volume of an aqueous solution
(e.g., water) in reference to the total volume of the surface
coating. In the dehydrated state, the volume of the aqueous
solution (e.g., water) is less than 20%, less than 15%, less than
10%, less than 5%, less than 1%, or less than 0.5% of the total
volume of the surface coating. In a hydrated state, the volume of
the aqueous solution (e.g., water) is at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, or higher of the total
volume of the surface coating.
[0096] In some embodiments, the surface coating described herein
comprises an aqueous solution (e.g., water). In some cases, the
aqueous solution (e.g., water) is from about 1% to about 60% by
weight of the total weight of the surface coating. In some cases,
the aqueous solution (e.g., water) is from about 1% to about 50% by
weight, from about 1% to about 40% by weight, from about 1% to
about 30% by weight, from about 1% to about 20% by weight, from
about 10% to about 60% by weight, from about 10% to about 50% by
weight, from about 10% to about 40% by weight, from about 10% to
about 30% by weight, from about 10% to about 20% by weight, from
about 20% to about 60% by weight, from about 20% to about 50% by
weight, from about 20% to about 40% by weight, or from about 30% to
about 60% by weight of the total weight of the surface coating.
[0097] In some embodiments, the surface coating further comprises a
filler. In some instances, the filler comprises a mineral filler
such as but not limited to silica, alumina, calcium carbonate, or
silicone resin.
[0098] Each of polycations and polyanions, and absorbent polymer
may be dissolved in an aqueous solution for use in the present
disclosure. The aqueous solution is free, or substantially free, of
organic solvents. It will be understood that some minor amounts of
organic solvents may be present in the aqueous solution, for
example as a result some organic solvent remaining in the polymer
after polymerization. As used herein, "substantially free," as it
relates to an organic solvent in an aqueous solution, means that
the aqueous solution comprises less than 1% of the organic solvent
by weight. In many embodiments, the aqueous solution contains less
than 0.8%, less than 0.5%, less than 0.2% or less that 0.1% of an
organic solvent.
[0099] Each of polycations and polyanions, and absorbent polymer
may be dissolved in an aqueous solution at any suitable
concentration for the purposes of coating.
Cell Culture Systems
[0100] The cell culture system of the present disclosure comprises
a cell culture article having a surface coated with the surface
coating described herein.
[0101] The cell culture article described herein can be made of any
suitable plastic and the like. In certain embodiments, the cell
culture article is made of a material comprising at least one of
polystyrene, polyethylene terephthalate, polycarbonate,
polyvinylpyrrolidone, polybutadiene, polyvinylchloride,
polyethylene oxide, polypyrroles, and polypropylene oxide.
[0102] In some embodiments, the cell culture system further
comprises cells. In some embodiments, the cells are derived from
cell lines. In some embodiments, the cells are mammalian cells. In
some embodiments, the cells are human cells. In some embodiments,
the cells are tissue cells, immune cells, endothelial cells, stem
cells, epithelial cells, mesenchymal cells, mesothelial cells,
cancer cells or tumor-associated cells. In some embodiments, the
cell culture system further comprises a culture media.
[0103] The cell culture systems disclosed herein enable not only
cell attachment and growth, but also the viable harvest of cultured
cells (e.g. 3D cell culture, tissue and organs). According to some
embodiments of the present disclosure, the cell culture systems can
be used to harvest viable cells, including between 80% to 100%
viable, or about 85% to about 99% viable, or about 90% to about 99%
viable. For example, of the cells that are harvested, at least 80%
are viable, at least 85% are viable, at least 90% are viable, at
least 91% are viable, at least 92% are viable, at least 93% are
viable, at least 94% are viable, at least 95% are viable, at least
96% are viable, at least 97% are viable, at least 98% are viable,
or at least 99% are viable. In some embodiments, cells can be
released from the cell culture systems with or without using a cell
dissociation enzyme, for example, trypsin, TrypLE, or Accutase.
Methods and Uses Thereof
Methods for Culturing Cells
[0104] Without being bound to any particular theory, it is believed
that the surface coating disclosed herein enables robust
multiplication and/or stable maintenance of cells. The present
disclosure thus provides a method for culturing cells. The method
comprises the steps of: (a) providing a cell culture article having
a surface coated with the surface coating of the present
disclosure; (b) seeding cells on the coated surface; and (c)
culturing the cells under a suitable medium. In some embodiments,
the cells are cultured for a sufficient period of time to form
spheroids. In preferred embodiments, the spheroids are 3D
spheroids. In some embodiments, the spheroids described herein are
generated via single cell proliferation. In some embodiments, the
spheroids described herein are generated via single cell
proliferation without cell agglomeration. In some embodiments, the
spheroids have uniform size.
[0105] In some embodiments, the cells described herein may be
derived from a cell line, a tissue biopsy or a liquid biopsy. In
some embodiments, the cells are mammalian cells. In some
embodiments, the cells are human cells. In some embodiments, the
cells are tissue cells, immune cells, endothelial cells, stem
cells, epithelial cells, mesenchymal cells, mesothelial cells,
cancer cells or tumor-associated cells.
[0106] In some embodiments, the cells described herein are stem
cells such as mesenchymal stem cells (MSCs) or pluripotent stem
cells (PSCs) including embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs).
[0107] In some embodiments, the cells described herein are cancer
cells. Exemplary cancer described herein includes, but is not
limited to, acute lymphatic cancer, acute myeloid leukemia,
alveolar rhabdomycosarcoma, bone cancer, brain cancer, breast
cancer, cancer of the anus, anal canal or anorectum cancer, cancer
of the eye, cancer of the intrahepatic bile duct cancer, cancer of
the joints, cancer of the neck, gallbladder or pleura cancer,
cancer of the nose, nasal cavity or middle ear, cancer of the oral
cavity, cancer of the vulva, chronic lymphatic leukemia, chronic
myeloid cancer, colon cancer, esophageal cancer, cervical cancer,
gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma,
hypopharynx cancer, kidney cancer, larynx cancer, liver cancer,
lung cancer, malignant mesothelioma, melanoma, multiple myeloma,
nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,
pancreatic cancer, peritoneum cancer, omentum and mesentary cancer,
pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin
cancer, small intestine cancer, soft tissue cancer, stomach cancer,
testicular cancer, thyroid cancer, ureter cancer, and urinary
bladder cancer.
[0108] In some embodiments, the cells described herein are
tumor-associated cells. Exemplary tumor-associated cells include,
but are not limited to, tumor cell clusters, tumor infiltrating
lymphocytes (TILs), cancer associated macrophage-like cells
(CAMLs), tumor-associated macrophages (TAMs), tumor-associated
monocyte/macrophage lineage cells (MMLCs), cancer stem cells, tumor
microemboli, tumor-associated stromal cells (TASC),
tumor-associated myeloid cells (TAMCs), tumor-associated regulatory
T cells (Treg), cancer-associated fibroblasts (CAFs), tumor-derived
endothelial cells (TECs), tumor-associated neutrophils (TAN),
tumor-associated platelets (TAP), tumor-associated immune cells
(TAI), myeloid-derived suppressor cells (MDSC), and a combination
thereof
[0109] Exemplary cells include low-density cells, single cells,
rare cells, or a combination thereof. Low-density cells can be
cells when seeded, are less than 5000 per cm.sup.2 on the
substrate, e.g., no more than about any of 1, 5, 10, 20, 50, 100,
200, 300, 500, 1000, 2000, 3000, 4000, or 4500 per cm.sup.2 on the
substrate.
[0110] In some embodiments, seeding the isolated cells in step (c)
comprises plating the cells at a density of between one cell and 10
cells per cm.sup.2 on the substrate surface (i.e. cell growth
surface). In some embodiments, seeding the isolated cells in step
(c) comprises plating the cells at a density of between 10 cells
and 100 cells per cm.sup.2 on the substrate surface. In some
embodiments, seeding the isolated cells in step (c) comprises
plating the cells at a density of between 100 cells and 1000 cells
per cm.sup.2 on the substrate surface.
[0111] In some embodiments, the cells are cultured for a period of
time ranging from about 2 days to about 5 weeks, such as from about
3 to about 14 days, for example about 7 days. In some embodiments,
the cells are cultured for 3 days and the spheroids have an average
diameter ranging from about 40 .mu.m to about 200 .mu.m.
[0112] Any suitable culture medium can be employed in the methods
of exemplary embodiments. Exemplary culture medium includes, but is
not limited to, Dulbecco's modified Eagle's medium (DMEM),
epidermal growth factor (EGF) and/or basic fibroblast growth factor
(bFGF), a mixture of Dulbecco's modified Eagle's medium (DMEM),
supplemented with B27 supplement, epidermal growth factor (EGF) and
basic fibroblast growth factor (bFGF).
Method of Preparing Single-Cell-Derived Spheroids
[0113] In another aspect, the present disclosure provides a
provides a method of preparing a single-cell derived spheroid, the
method comprising the steps of: (a) providing a cell culture
article having a surface coated with the surface coating of the
present disclosure; (b) seeding cells on the coated surface; and
(c) culturing the cells under a suitable medium for a sufficient
period of time to form spheroids, in which the spheroids are
single-cell derived. The spheroids described herein are generated
via single cell proliferation. In some embodiments, the spheroids
have uniform size. In some embodiments, the single-cell-derived
clones are semi-attached or loosely attached on the substrate of
the present disclosure.
[0114] In some embodiments, the cells are derived from cell lines.
In some embodiments, the cells are mammalian cells. In some
embodiments, the cells are human cells. In some embodiments, the
cells are tissue cells, immune cells, endothelial cells, stem
cells, epithelial cells, mesenchymal cells, mesothelial cells,
cancer cells or tumor-associated cells.
[0115] In some embodiments, the cells are stem cells such as
mesenchymal stem cells (MSCs) or pluripotent stem cells (PSCs)
including embryonic stem cells (ESCs) and induced pluripotent stem
cells (iPSCs).
[0116] In some embodiments, the cells are cancer cells. In some
embodiments, the cells are cancer cells. In certain embodiments,
the cancer cells are isolated from human primary tumor tissue. In
certain embodiments, the cancer cells are isolated from a blood
sample of a cancer patient. Exemplary cancer described herein
includes, but is not limited to, acute lymphatic cancer, acute
myeloid leukemia, alveolar rhabdomycosarcoma, bone cancer, brain
cancer, breast cancer, cancer of the anus, anal canal or anorectum
cancer, cancer of the eye, cancer of the intrahepatic bile duct
cancer, cancer of the joints, cancer of the neck, gallbladder or
pleura cancer, cancer of the nose, nasal cavity or middle ear,
cancer of the oral cavity, cancer of the vulva, chronic lymphatic
leukemia, chronic myeloid cancer, colon cancer, esophageal cancer,
cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin
lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver
cancer, lung cancer, malignant mesothelioma, melanoma, multiple
myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,
pancreatic cancer, peritoneum cancer, omentum and mesentary cancer,
pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin
cancer, small intestine cancer, soft tissue cancer, stomach cancer,
testicular cancer, thyroid cancer, ureter cancer, and urinary
bladder cancer.
[0117] In some embodiments, the cells are tumor-associated cells.
Exemplary tumor-associated cells include, but are not limited to,
tumor cell clusters, tumor infiltrating lymphocytes (TILs), cancer
associated macrophage-like cells (CAMLs), tumor-associated
macrophages (TAMs), tumor-associated monocyte/macrophage lineage
cells (MMLCs), cancer stem cells, tumor microemboli,
tumor-associated stromal cells (TASC), tumor-associated myeloid
cells (TAMCs), tumor-associated regulatory T cells (Treg),
cancer-associated fibroblasts (CAFs), tumor-derived endothelial
cells (TECs), tumor-associated neutrophils (TAN), tumor-associated
platelets (TAP), tumor-associated immune cells (TAI),
myeloid-derived suppressor cells (MDSC), and a combination
thereof
[0118] Exemplary cells include low-density cells, single cells,
rare cells, or a combination thereof. Low-density cells can be
cells when seeded, are less than 5000 per cm.sup.2 on the
substrate, e.g., no more than about any of 1, 5, 10, 20, 50, 100,
200, 300, 500, 1000, 2000, 3000, 4000, or 4500 per cm.sup.2 on the
substrate.
[0119] In some embodiments, seeding the isolated cells in step (c)
comprises plating the cells at a density of between one cell and 10
cells per cm.sup.2 on the substrate surface (i.e. cell growth
surface). In some embodiments, seeding the isolated cells in step
(c) comprises plating the cells at a density of between 10 cells
and 100 cells per cm.sup.2 on the substrate surface. In some
embodiments, seeding the isolated cells in step (c) comprises
plating the cells at a density of between 100 cells and 1000 cells
per cm.sup.2 on the substrate surface.
[0120] In some embodiments, the culturing step occurs over a period
of 2-8 days (e.g., 2, 3, 4, 5, 6, 7, or 8 days). In other
embodiments, the culturing step culturing step occurs over a period
of 7-14 days (e.g., 7, 8, 9, 10, 11, 12, 13, or 14 days). In other
embodiments, the culturing step culturing step occurs over a period
of 1-4 weeks (e.g., 1, 2, 3, or 4 weeks). In some embodiments, the
cells are cultured for 3 days and the spheroids have an average
diameter ranging from about 40 .mu.m to about 200 .mu.m.
[0121] Any suitable culture medium can be employed in the methods
of exemplary embodiments. Exemplary culture medium includes, but is
not limited to, Dulbecco's modified Eagle's medium (DMEM),
epidermal growth factor (EGF) and/or basic fibroblast growth factor
(bFGF), a mixture of Dulbecco's modified Eagle's medium (DMEM),
supplemented with B27 supplement, epidermal growth factor (EGF) and
basic fibroblast growth factor (bFGF).
[0122] In some embodiments, the size of a single-cell derived
spheroid less than 200 .mu.m in diameter. In some embodiments, the
size of a single-cell derived spheroid less than 150 .mu.m in
diameter. In some embodiments, the size of a single-cell derived
spheroid is about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or 140
.mu.m in diameter.
[0123] In certain embodiments, the single-cell derived spheroid may
be used for screening a therapeutic agent. In certain embodiments,
a method of screening a therapeutic agent comprises: (a) applying a
test substance to the single-cell derived spheroid generated
thereof; and (b) evaluating an effect of the test substance on the
single-cell derived spheroid. In some embodiments, the effect of
the test substance is analyzed with an imaging system, e.g., to
analyze the biochemical activity and/or the expression levels of a
gene or a protein.
[0124] In some embodiments, the single-cell derived spheroid
generated thereof is a tumor spheroid. In some embodiments, the
test substance described herein is a chemotherapeutic drug, such as
a cytotoxic or cytostatic chemotherapeutic drug. In some
embodiments, the therapeutic agent is an immune checkpoint
inhibitor, such as an immune checkpoint inhibitor. In some
embodiments, the therapeutic agent is a nucleic acid drug. In some
embodiments, the therapeutic agent is a therapeutic cell
composition, including, but not limited to, T cells, natural killer
(NK) cells, and dendritic cells.
[0125] In some embodiments, the cells are cultured for a period of
time ranging from about 2 days to about 5 weeks, such as from about
3 to about 14 days, for example about 7 days. In some embodiments,
the cells are cultured for 3 days and the at least one 3D spheroid
has an average diameter ranging from about 40 .mu.m to about 200
.mu.m.
[0126] In some aspects, provided herein is a single-cell-derived
spheroid (e.g., tumor spheroid) generated according to any one of
the culture methods employing the cell culture systems described
herein. In some aspects, there is provided a library of
single-cell-derived spheroids (e.g., tumor spheroids) derived
according to any one of the culture methods employing the cell
culture systems described herein.
Method of Isolating Single-Cell-Derived Clones
[0127] Single-cell-derived clone has gained increasing importance
as genome editing techniques have entered routine laboratory
practice. Limiting dilution, the traditional method for isolating
single cells, relies on statistical probabilities for monoclonality
that can vary significantly with slight changes to protocols. The
technique, while highly inefficient at isolating single cells,
preserves cell viability. Conversely, flow cytometry can provide
single cell clones with high efficiency but negatively affects cell
viability. A common trait of these platforms is that they generally
start with a suspension containing a large number of cells that are
`individualized` by random confinement in microstructures. Both of
these methods are impractical when the cell population is small as
they generate considerable cell loss during mixing and/or transfer.
The method of the invention provides an efficient alternative for
isolating viable single cell clones. In some embodiments, the
method does not require individual confinement of cells in
microstructures.
[0128] In some embodiments, there is provided herein a method of
isolating a single-cell-derived clone. The method described herein
comprises: 1) culturing a heterogeneous population of cells using a
cell culture article having a surface coated with the composition
of the present disclosure to obtain a plurality of cell clones
comprising a single-cell-derived clone; and 2) isolating the
single-cell-derived clone from the cell culture article.
[0129] In some embodiments, the heterogeneous population of cells
comprises adherent cells. In some embodiments, the heterogeneous
population of cells comprises non-adherent cells. In some
embodiments, the heterogeneous population of cells comprises cells
isolated from a cell line. In some embodiments, the heterogeneous
population of cells comprises cells isolated from a liquid biopsy
of a subject. In some embodiments, the heterogeneous population of
cells comprises cells isolated from a tissue biopsy of a subject.
In some embodiments, the heterogeneous population of cells
comprises cells that have been genetically engineered. In some
embodiments, the heterogeneous population of cells comprises cells
that have been engineered to comprise a genetic mutation. In some
embodiments, the heterogeneous population of cells comprises cells
that have been engineered to comprise a heterologous nucleotide
sequence.
[0130] In some embodiments, the single-cell-derived clones are
semi-attached or loosely attached on the coated surface disclosed
herein.
[0131] In some embodiments, no cell debris is observed in the cell
culture system after 7 or more days of cultivation.
[0132] In some embodiments, the culturing step occurs over a period
of 2-8 days (e.g., 2, 3, 4, 5, 6, 7, or 8 days). In other
embodiments, the culturing step culturing step occurs over a period
of 7-14 days (e.g., 7, 8, 9, 10, 11, 12, 13, or 14 days). In other
embodiments, the culturing step culturing step occurs over a period
of 1-4 weeks (e.g., 1, 2, 3, or 4 weeks).
[0133] In some embodiments, the single-cell-derived clone forms a
single-cell-derived spheroid. In some embodiments, the
single-cell-derived clone has a diameter of from about 40 .mu.m to
about 200 .mu.m. In some embodiments, the single-cell-derived clone
has a diameter of from about 50 .mu.m to about 150 .mu.m. In some
cases, the single-cell-derived clone has a diameter of from about
50 .mu.m to about 120 .mu.m, from about 50 .mu.m to about 100
.mu.m, from about 50 .mu.m to about 80 .mu.m, from about 50 .mu.m
to about 60 .mu.m, from about 80 .mu.m to about 150 .mu.m, from
about 80 .mu.m to about 120 .mu.m, from about 80 .mu.m to about 100
.mu.m, from about 100 .mu.m to about 200 .mu.m, from about 100
.mu.m to about 150 .mu.m, or from about 100 .mu.m to about 120
.mu.m.
[0134] In some instances, the single-cell-derived clones form a
single-cell-derived spheroid. In some cases, the spheroid comprises
from about 8 to about 1000 cells. In some cases, the spheroid
comprises from about 8 to about 800 cells, from about 8 to about
500 cells, from about 8 to about 400 cells, from about 8 to about
300 cells, from about 8 to about 200 cells, from about 8 to about
100 cells, from about 10 to about 1000 cells, from about 10 to
about 800 cells, from about 10 to about 500 cells, from about 10 to
about 400 cells, from about 10 to about 300 cells, from about 10 to
about 200 cells, from about 10 to about 100 cells, from about 50 to
about 1000 cells, from about 50 to about 800 cells, from about 50
to about 500 cells, from about 50 to about 400 cells, from about 50
to about 300 cells, from about 50 to about 200 cells, from about
100 to about 1000 cells, from about 100 to about 800 cells, from
about 100 to about 500 cells, from about 100 to about 400 cells,
from about 100 to about 300 cells, from about 300 to about 1000
cells, from about 300 to about 800 cells, from about 300 to about
500 cells, from about 500 to about 1000 cells, or from about 500 to
about 800 cells.
[0135] In some embodiments, at least 10% of the cells disposed on
the coated surface forms single-cell-derived spheroids. In some
embodiments, at least 20% of the cells disposed on the coated
surface forms single-cell-derived spheroids. In some embodiments,
at least 30% of the cells disposed on the coated surface forms
single-cell-derived spheroids. In some embodiments, at least 40% of
the cells disposed on the coated surface forms single-cell-derived
spheroids. In some embodiments, at least 50% of the cells disposed
on the coated surface forms single-cell-derived spheroids. In some
embodiments, at least 60% of the cells disposed on the coated
surface forms single-cell-derived spheroids. In some embodiments,
at least 70% of the cells disposed on the coated surface forms
single-cell-derived spheroids.
[0136] In some embodiments, the method described herein further
comprises analyzing the single-cell-derived clone, thereby
obtaining a characteristic of the single cell. In some instances,
the step of analyzing the single-cell-derived clone comprises
subjecting the single-cell-derived clone to sequencing analysis. In
some embodiments, the analyzing step comprises performing a
genotyping analysis. In some embodiments, the genotyping analysis
is a PCR-based analysis. In some embodiments, the genotyping
analysis is an array hybridization-based analysis. In some
embodiments, the analyzing step comprises analyzing a copy number
variation. In some embodiments, the analyzing step comprises
analyzing a genetic mutation. In some embodiments, the analyzing
step comprises analyzing a single nucleotide polymorphism.
[0137] In some embodiments, the step of analyzing the
single-cell-derived clone comprises subjecting the
single-cell-derived clone to proteomic analysis. Exemplary
proteomic analysis include gel electrophoresis such as
polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel
electrophoresis, or capillary electrophoresis; high-performance
liquid chromatography (HPLC); and affinity chromatography.
[0138] In some embodiments, the characteristic of the single cell
comprises one or more of: a genotype, an epigenetic profile, an
expression level of a gene or a protein, a response to a drug, a
drug resistance profile, or a metastatic potential.
[0139] In some aspects, provided herein is a single-cell-derived
clone generated according to any one of the culture methods
employing the cell culture systems described herein. In some
aspects, there is provided a library of single-cell-derived clones
derived according to any one of the culture methods employing the
cell culture systems described herein.
Generation of Patient-Derived Tumoroid Xenografts
[0140] In another aspect, the present disclosure provides a method
for generating patient-derived tumoroid-based xenograft.
Patient-derived tumoroids can be generated via in vitro growth of
tumor cells derived from patient blood, tissue (e.g. bladder,
stomach, breast, pancreas, colon, or lung) or cell lines.
Patient-derived tumoroid-based xenografts can be established by the
direct transfer of tumoroids into highly immunodeficient mice and
then maintained by passaging from mouse to mouse. The xenograft
model is useful for biomedical translational research, and once
validated, it can be used as a translational preclinical model for
efficacy screening in cancer drug development.
[0141] In some embodiments, provided herein is a method of
generating a patient-derived tumor xenograft animal model,
comprising: a) culturing a plurality of cells comprising tumor
cells derived from a patient using any of the cell culture systems
provided herein to obtain a plurality of tumoroids; b) isolating a
tumoroid from the 3D cell culture system; and 3) inoculating the
isolated tumoroid into a non-human animal, thereby generating the
patient-derived tumor xenograft animal model.
[0142] In some embodiments, the plurality of tumor cells are
obtained from a primary tissue of the patient. In some embodiments,
the plurality of tumor cells are obtained from the blood of the
patient. In some embodiments, the tumor cells derived from a
patient are grown as animal model primary xenografts prior to
culturing on the 3D cell culture system.
[0143] In some embodiments, the non-human animal is
immunodeficient. In some embodiments, the non-human animal is a
mouse.
[0144] In some embodiments, the tumor cells are circulating tumor
cells (CTCs) derived from a solid tumor. In other instances, the
tumor cells are CTCs derived from a hematologic malignancy. In some
embodiments, the patient has a metastatic cancer.
[0145] In some embodiments, the culturing step comprises expansion
of the tumor cells by 10 to 100-fold (e.g., 10-, 20-, 30-, 40-,
50-, 60-, 70-, 80-, 90-, or 100-fold) within one week.
[0146] Methods that employ non-adherent conditions for 3D cell
culture include the hanging drop method (Kelm et al. Biotechnol.
Bioeng. 83, 173-180 (2003)), rotating bioreactor (Zhau, et al. In
Vitro Cell. Dev. Biol. Anim. 33, 375-380 (1997)), magnetic
levitation (Souza et al. Nat. Nanotechnol. 5, 291-296 (2010)).
However, some of the most widely used non-adherent techniques do
not represent a true 3D cell culture that mimics tumor formation in
vivo. When tens-of-thousands cells are aggregated into a spheroid
(i.e., a mass with spherical shape) such as in a hanging drop,
reactor or U-bottom plates, an extensive central necrotic core
forms over a few hours due to the lack of nutrient and oxygen
penetration beyond a 200 .mu.m depth. Extended central necrosis is
a rare phenomenon in real cancers. This nonphysiologically-relevant
cancer representation is exacerbated by the lack of progressive
tumor development via cell division and the lack of interaction
with an appropriate extracellular matrix (ECM). In some
embodiments, the 3D cell culture systems provided herein are able
to maintain the size of a cell spheroid/tumoroid around 100 .mu.m
and induce its division to a smaller spheroid as cells continue to
proliferate over time.
[0147] In some embodiments, the tumoroid has a diameter from about
50 .mu.M to about 150 .mu.M. In some embodiments, the tumoroid has
a diameter from about 100 .mu.M to about 150 .mu.M. In some cases,
the tumoroid has a diameter of about 50 .mu.M, about 60 .mu.M,
about 70 .mu.M, about 80 .mu.M, about 90 .mu.M, about 100 .mu.M,
about 110 .mu.M, about 120 .mu.M, about 130 .mu.M, about 140 .mu.M,
or about 150 .mu.M. In some embodiments, the average size of the
tumoroids is about 150 .mu.M after 8 days of culturing.
[0148] In some embodiments, the culturing step occurs over a period
of 7-14 days (e.g., 7, 8, 9, 10, 11, 12, 13, or 14 days). In some
embodiments, the size of the tumoroid cultured for more than 7 days
is maintained within the range of from about 50 .mu.M to about 150
.mu.M in diameter. In some cases, the tumoroid has a diameter of
about 50 .mu.M, about 60 .mu.M, about 70 .mu.M, about 80 .mu.M,
about 90 .mu.M, about 100 .mu.M, about 110 .mu.M, about 120 .mu.M,
about 130 .mu.M, about 140 .mu.M, or about 150 .mu.M.
[0149] In some embodiments, the plurality of cells further
comprises tumor-associated cells derived from the patient. In some
embodiments, the tumor-associated cells comprise tumor-associated
stromal cells. In some embodiments, the plurality of tumoroids
comprise tumor cells and tumor-associated cells. In some
embodiments, the tumoroid is derived from a single cell.
[0150] In some embodiments, the cancer is a solid tumor or a
hematologic malignancy. In some cases, the cancer is colorectal,
breast, pancreatic, head and neck, bladder, ovarian, stomach, or
prostate cancer.
[0151] In some embodiments, the method of making a patient-derived
tumoroid provides tumoroids at a yield ratio (e.g., yield from
CTCs) of between 0.5 and 1. In some embodiments, the method of
making a patient-derived tumoroid provides tumoroids at a yield
ratio (e.g., yield from CTCs) of between 0.6 and 1. In some
embodiments, the method of making a patient-derived tumoroid
provides tumoroids at a yield ratio (e.g., yield from CTCs) of
between 0.7 and 1. In some embodiments, the method of making a
patient-derived tumoroid provides tumoroids at a yield ratio (e.g.,
yield from CTCs) of between 0.8 and 1.
[0152] In some aspects, there is provided herein a patient-derived
tumor xenograft model prepared according any one of the methods
employing the cell culture systems described herein. In some
embodiments, the patient-derived tumor xenograft model is capable
of spontaneous metastasis. In some embodiments, the xenograft model
can be propagated in vivo by injecting patient-derived tumor cells
isolated from one xenograft model into another non-human
animal.
[0153] In some aspects, there is provided herein a method of
analyzing the in vivo activity of a therapeutic agent (e.g., an
anti-cancer drug), comprising administering the therapeutic agent
(e.g., the anti-cancer drug) to the patient-derived tumor xenograft
animal model prepared according to any one of the methods described
herein, and analyzing the effect of the therapeutic agent on the
patient-derived tumor xenograft animal model.
[0154] In some aspects, there is provided herein a biobank
comprising a plurality of different tumoroids prepared according to
any one of the methods employing the cell culture systems described
herein.
Generation of 3D Co-Cultures In Vitro
[0155] In another aspect, the present disclosure provides a method
for generating 3D co-culture tumor model derived from tumor cells
and tumor-associated stromal or immune cells (e.g. fibroblasts,
endothelial cells and immune cells), in particular, patient-derived
3D co-culture tumor model derived from patient-derived tumor cells
and tumor-associated stromal cells (e.g. fibroblasts, endothelial
cells and/or immune cells). 3D co-culture tumor model provides an
improved mimicry of in vivo tumor microenvironments that is useful
for basic research on tumor microenvironment and establishment of
in vitro drug screening models for cancer therapy and cancer
immunotherapy.
[0156] In some embodiments, provided herein is a method for
generating a 3D co-culture tumor model derived from tumor cells and
tumor-associated stromal or immune cells (e.g. fibroblasts,
endothelial cells and/or immune cells), wherein the method
comprises a) culturing a plurality of cells comprising tumor cells
and tumor-associated stromal cells on any of the 3D culture systems
provided herein to obtain a plurality tumoroids comprising both
tumor cells and tumor-associated stromal cells. In some
embodiments, the tumor cells and tumor-associated stromal cells
form direct cell-cell contacts within the tumoroids. In some
embodiments, the method comprises aggregating tumor cells and
tumor-associated stromal cells (e.g., by culturing the mixed cell
population.
[0157] In some embodiments, the stromal cells comprise fibroblasts,
endothelial cells, or mesenchymal stem cells. In some embodiments,
the immune cells comprise myeloid-derived suppressor cells (MDSCs),
tumor associated macrophages, neutrophils, tumor-infiltrating
lymphocytes, T cells, B cells, dendritic cells, or any other
tumor-associated immune cells.
[0158] In some embodiments, the 3D co-culture tumor models provided
herein provide a valuable tool to study the cytotoxic effect of
anticancer drugs on normal cells. In some embodiments, a method
provided herein comprises evaluating the effect of an anticancer
drug on tumor cells (e.g., highly proliferative cells) vs. on
normal, non-tumor cells within the co-culture.
[0159] Generation of 3D Cell Culture for Hepatocytes
[0160] In another aspect, the present disclosure provides a method
for generating 3D cell culture model for hepatocytes. 3D cell
culture exhibits superior liver-specific functions over the
conventional 2D cell culture in evaluating hepatobiliary drug
disposition and drug-induced hepatotoxicity due to the in vivo-like
physiological condition recapitulated by 3D model. 3D liver cell
culture is useful for tissue engineering and drug development.
[0161] Once hepatocytes are isolated from the liver and are grown
in conventional primary cultures, the activity of these important
enzymes is rapidly lost. This loss is particularly prominent for
rat hepatocytes which lose 80% of their CYP activity in the first
24 hours of culture (Paine, A J, In: Berry, M N et al. (eds.), The
Hepatocyte Review, Kluwer Academic Publishers, Netherlands, pp.
411-420, 2000).
[0162] In some embodiments, provided herein is a method for
culturing hepatocytes on any one of the 3D culture systems
described herein, wherein the hepatocytes maintain one or more
liver-specific gene expressions and functions of primary
hepatocytes, such as albumin secretion, viral infectivity, and/or
cytochrome 3 P450 (CYP) enzyme activity. CYPs are a family of
enzymes, localized to the cytoplasmic side of the endoplasmic
reticulum of the liver cell, that catalyze the oxidation of organic
compounds, resulting in increased water solubility which promotes
excretion from the cell. In some embodiments, the hepatocytes
cultured on any of the 3D culture systems described herein maintain
gene expression and function of one or more genes involved in
normal drug metabolism for a culturing period of at least 5 days,
at least 7 days, at least 10 days, at least 14 days, or at least 21
days.
[0163] In some embodiments, the 3D cell culture platform is used
for culture of primary hepatocytes. In some embodiments, the
primary hepatocytes are isolated from a liver biopsy of a human or
other mammal. In some embodiments, the 3D cell culture platform is
used for culture of fetal liver cells.
[0164] In some embodiments, the 3D cell culture platform is used
for culturing an immortalized cell line. In some embodiments, the
cell line expresses one or more Phase 1/II xenobiotic drug
metabolism genes and/or hepatocyte-specific transcripts. In some
embodiments, the cells cultured on any of the 3D culture systems
described herein maintain gene expression and function of one or
more genes involved in normal drug metabolism for a culturing
period of at least 5 days, at least 7 days, at least 10 days, at
least 14 days, or at least 21 days. In some embodiments, the cells
are HepG2, Huh7, or HepaRG.
[0165] In some embodiments, provided herein is a method of
culturing hepatocytes using any one of the 3D cell culture systems
described herein, wherein the hepatocytes maintain hepatic
functions in vitro, such as the expression of critical cytochrome
P450 (CYP) drug metabolizing enzymes. In some embodiments, the
hepatocytes maintain hepatic function over an extended period of
time, such as at least 5 days, at least 7 days, at least 10 days,
at least 15 days, or at least 20 days.
[0166] Culturing hepatocytes using conventional 3D culture systems
results in formation of a necrotic core in the cell spheroids due
to hypoxia in the center of spheroids greater than 200 (Hussein et
al. "Three dimensional culture of HepG2 liver cells on a rat
decellularized liver matrix for pharmacological studies." J Biomed
Mater Res B Appl Biomater, 2016. 104(2): p. 263-73). In some
embodiments, the 3D cell culture systems provided herein are able
to maintain the size of a cell spheroid (e.g., a liver cell
spheroid) around 100 .mu.m and induce its division to a smaller
spheroid as cells continue to proliferate over time.
[0167] In some embodiments, the cell spheroid has a diameter of
from about 50 .mu.M to about 150 .mu.M. In some embodiments, the
cell spheroid has a diameter of from about 100 .mu.M to about 150
.mu.M. In some cases, the cell spheroid has a diameter of about 50
.mu.M,about 60 .mu.M,about 70 .mu.M, about 80 about 90 .mu.M,about
100 .mu.M,about 110 .mu.M,about 120 .mu.M,about 130 .mu.M, about
140 .mu.M, or about 150 .mu.M. In some embodiments, the average
size of the cell spheroids is 150 .mu.M after 8 days of culturing.
In some embodiments, the size of the cell spheroid cultured for
more than 7 days is maintained within the range of from about 50
.mu.M to about 150 .mu.M.
[0168] In some embodiments, the method comprises culturing liver
cells and nonparenchymal cells (NPCs) on any of the 3D culture
systems provided herein. In some embodiments, the nonparenchymal
cells include bile duct epithelial cells, liver sinusoidal
endothelial cells (LSEC), hepatic stellate cells (HSC) and/or
Kupffer 8 cells (KC).
[0169] In another embodiment, this invention provides a method for
evaluating the metabolism of an agent that is metabolized by
mammalian liver cells in vivo, comprising (a) culturing mammalian
hepatocytes using any of the 3D culture systems described herein;
(b) adding the agent being evaluated to the hepatocyte culture in
the culture vessels for a period of time sufficient for enzymes of
the hepatocytes to metabolize the agent and converting it to one of
more metabolites thereof; (c) identifying the presence of, or
measuring the concentration of, the one or more metabolites in the
medium or cells of the culture, thereby evaluating the metabolism
of the agent.
Generation of Stem Cell Spheroids In Vitro
[0170] In another aspect, the present disclosure provides a method
for generating stem cell spheroids in vitro. Stem cells described
herein comprise mesenchymal stem cells (MSCs) and pluripotent stem
cells (PSCs) including embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs). These stem cell spheroids are
promising candidates for cell therapies, tissue engineering, high
throughput pharmacology screens, and toxicity testing. These
applications require large numbers of high quality cells; however,
scalable production of MSCs and PSCs has been a challenge. 3D
culture system provided herein can allow efficient stem cell
proliferation and differentiation of MSCs and PSCs.
[0171] In some embodiments, provided herein is a method of 3D cell
culture using any one of the 3D culture systems provided herein,
wherein the method increases stemness properties and/or
proliferation rate of a cell population. In some embodiments, the
cells are mesenchymal stem cells or pluripotent stem cells. In some
embodiments, the cells are embryonic stem cells or induced
pluripotent stem cells.
[0172] In some embodiments, the method increases expression of one
or more stemness-associated genes, For example, cell-surface CD133
represents one of the biomarkers for stem cell characterization and
is associated with multiple cellular characters such as stemness,
regeneration, differentiation, and metabolism of diverse cell
lineages. In some embodiments, the method increases expression of
CD133 in the cells. In some embodiments, the method induces CD133
expression in a population of CD133.sup.- cells.
[0173] In some embodiments, the method maintains expression of one
or more stemness-associated genes for the duration of a culturing
period. In some embodiments, the culturing period is at least 7
days, at least 10 days, at least 14 days, or at least 21 days.
[0174] Kits
[0175] In certain embodiments, disclosed herein is a kit or article
of manufacture that comprises a 3D cell culture system described
herein. In some instances, the kit is for use in isolating a single
cell-derived clone. In some instances, the kit further comprises a
package, or container that is compartmentalized to receive one or
more containers such as vials, tubes, and the like, each of the
container(s) comprising one of the separate elements to be used in
a method described herein. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. In one
embodiment, the containers are formed from a variety of materials
such as glass or plastic.
[0176] In some cases, the kit further comprises labels listing
contents and/or instructions for use, and package inserts with
instructions for use, e.g., instructions for culturing a
heterogeneous population of cells using a 3D culture system
described herein to obtain a plurality of cell clones comprising a
single-cell-derived clone. A set of instructions will also
typically be included.
[0177] Certain Terminology
[0178] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs. It
is to be understood that the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive of any subject matter claimed. In this
application, the use of the singular includes the plural unless
specifically stated otherwise. It must be noted that, as used in
the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly
dictates otherwise. In this application, the use of "or" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes,"
and "included," is not limiting.
[0179] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.L" means "about 5 .mu.L" and also "5
.mu.L." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0180] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0181] As used herein, the term "comprising" is intended to mean
that the methods include the recited steps or elements, but do not
exclude others. "Consisting essentially of" shall mean rendering
the claims open only for the inclusion of steps or elements, which
do not materially affect the basic and novel characteristics of the
claimed methods. "Consisting of" shall mean excluding any element
or step not specified in the claim. Embodiments defined by each of
these transition terms are within the scope of this disclosure.
[0182] As used herein, the term "positively charged
polyelectrolyte" encompasses a plurality of monomer units or a
non-polymeric molecule that comprises two or more positive charges.
In some instances, the positively charged polyelectrolyte also
encompasses a plurality of monomer units or a non-polymeric
molecule that comprise charge positive groups, charge neutral
groups, or charge negative groups, with a net charge of being
positive.
[0183] As used herein, the term "cationic polymer" encompasses a
plurality of monomer units or a non-polymeric molecule. In some
instances, the cationic polymer is a synthetic polymer. In other
instances, the cationic polymer is a natural polymer.
[0184] As used herein, the term "cationic polypeptide" refers to a
polypeptide comprising two or more positive charges. In some
instances, the cationic polypeptide comprises positively charged
amino acid residues, negatively charged residues, and polar
residues but the net charge of the polypeptide is positive. In some
cases, the cationic polypeptide is from 8 to 100 amino acids in
length. In some cases, the cationic polypeptide is from 8 to 80, 8
to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, 8 to 15, 10 to 100, 10
to 80, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 100, 20 to
80,20 to 50,20 to 40,20 to 30,30 to 100,30 to 80, 30 to 50, 40 to
100, 40 to 80, or 50 to 100 amino acids in length.
[0185] As used herein, the term "negatively charged
polyelectrolyte" encompasses a plurality of monomer units or a
non-polymeric molecule that comprises two or more negative charges.
In some instances, the negatively charged polyelectrolyte also
encompasses a plurality of monomer units or a non-polymeric
molecule that comprise charge positive groups, charge neutral
groups, or charge negative groups, with a net charge of being
negative.
[0186] As used herein, the term "anionic polymer" encompasses a
plurality of monomer units or a non-polymeric molecule. In some
instances, the anionic polymer is a synthetic polymer. In other
instances, the anionic polymer is a natural polymer.
[0187] As used herein, the term "anionic polypeptide" refers to a
polypeptide comprising two or more negative charges. In some
instances, the anionic polypeptide comprises positively charged
amino acid residues, negatively charged residues, and polar
residues but the net charge of the polypeptide is negative. In some
cases, the anionic polypeptide is from 8 to 100 amino acids in
length. In some cases, the anionic polypeptide is from 8 to 80, 8
to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, 8 to 15, 10 to 100, 10
to 80, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 100, 20 to
80,20 to 50,20 to 40,20 to 30,30 to 100,30 to 80, 30 to 50, 40 to
100, 40 to 80, or 50 to 100 amino acids in length.
[0188] As used herein, the term "hydrophilic polymer" encompasses a
plurality of monomer units or a non-polymeric molecule that
comprise one or more hydrophilic groups. In some instances, the
hydrophilic polymer is permeable to an aqueous solution. In other
instances, the hydrophilic polymer is impermeable or does not
absorb the aqueous solution. In some cases, the hydrophilic polymer
encompasses a non-reactive polymer, or a polymer that does not
contain a reactive group, e.g., a group that forms covalent bonds
with another compound.
[0189] As used herein, the term "polymer" includes both homo- and
copolymers, branched and unbranched, and natural or synthetic
polymers.
[0190] As used herein, the term "article" refers to a cell culture
article, such as a sheet, film, tube, plate, dish, or a biomedical
device. In some instances, a biomedical device is any article that
is designed to be used while either in or on tissue (e.g.,
mammalian tissue) or fluid, preferably in or on human tissue or
fluids. Exemplary devices include, but are not limited to, cell
culturing dishes, cell culture plates, bioreactors, and the
like.
[0191] As used herein, immune cells encompass neutrophils,
eosinophils, basophils, mast cells, monocytes, macrophages,
dendritic cells, natural killer cells, and lymphocytes (B cells and
T cells).
[0192] Endothelial cells are cells that line the interior surface
of blood vessels and lymphatic vessels. Exemplary endothelial cells
include high endothelial venules (HEV), endothelium of the bone
marrow, and endothelium of the brain.
[0193] Epithelial cells are cells that line the outer surfaces of
organs and blood vessels, and the inner surfaces of cavities within
internal organs. Exemplary epithelial cells include squamous
epithelium, cuboidal epithelium, and columnar epithelium.
[0194] As used herein, the term "stem cell" encompasses an adult
stem cell and an embryonic stem cell. Exemplary stem cells include
hematopoietic stem cells, mesenchymal stem cells (MSCs), neural
stem cells, epithelial stem cells, skin stem cells, embryonic stem
cells (ESCs), and induced pluripotent stem cells (iPSCs).
[0195] As used herein, the term "chemically defined medium" refers
to an in vitro culture medium in which all of the chemical
components are known. A chemically defined medium can include a
basal media (such as DMEM, F12, or RPMI 1640, containing amino
acids, vitamins, inorganic salts, buffers, antioxidants and energy
sources), which is supplemented with recombinant albumin,
chemically defined lipids, recombinant insulin and/or zinc,
recombinant transferrin or iron, selenium and an antioxidant thiol
such as 2-mercaptoethanol or 1-thioglycerol.
[0196] As used herein, the term "enriched medium" refers to an in
vitro culture medium in which a basal media is further supplemented
with growth factors, vitamins, and essential nutrients.
[0197] As used herein, the term "semi-attached" and "loosely
attached" are used interchangeably and in reference to cultured
cells refer to cells that can be detached from the surface of a
substrate with gentle agitation, or gentle mechanical force. In
some instances, the cells can be detached without the need for a
cell dissociation enzyme.
[0198] As used herein, the terms "single-cell-derived spheroid"
refers to a cluster of cells grown ex vivo and formed in 3D format,
which cluster is grown from a single cell disposed on the surface
coating.
[0199] In some embodiments, therapeutic agents include, but are not
limited to, a chemotherapeutic drug, an immune checkpoint
inhibitor, a nucleic acid drug, a therapeutic cell composition, or
a combination thereof
[0200] In some embodiments, the therapeutic agent is a cytotoxic or
cytostatic chemotherapeutic drug. The chemotherapeutic drug can be
alkylating agents (such as cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine,
lomustine, carmustine, procarbazine, chlorambucil and ifosfamide),
antimetabolites (such as fluorouracil (5-FU), gemcitabine,
methotrexate, cytosine arabinoside, fludarabine, and floxuridine),
antimitotics (including taxanes such as paclitaxel and decetaxel
and vinca alkaloids such as vincristine, vinblastine, vinorelbine,
and vindesine), anthracyclines (including doxorubicin,
daunorubicin, valrubicin, idarubicin, and epirubicin, as well as an
actinomycin such as actinomycin D), cytotoxic antibiotics
(including mitomycin, plicamycin, and bleomycin), topoisomerase
inhibitors (including camptothecins such as camptothecin,
irinotecan, and topotecan as well as derivatives of
epipodophyllotoxins such as amsacrine, etoposide, etoposide
phosphate, and teniposide), antibodies to vascular endothelial
growth factor (VEGF) such as bevacizumab (AVASTIN.RTM.), other
anti-VEGF compounds; anti-PD-1 (anti-programmed death-1)
therapeutics such as antibodies or compounds (e.g., Nivolumab);
thalidomide (THALOMID.RTM.) and derivatives thereof such as
lenalidomide (REVLIMID.RTM.); endostatin; angiostatin; receptor
tyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT.RTM.);
tyrosine kinase inhibitors such as sorafenib (Nexavar.RTM.),
erlotinib (Tarceva.RTM.), pazopanib, axitinib, and lapatinib;
transforming growth factor-a or transforming growth factor-.beta.
inhibitors, and antibodies to the epidermal growth factor receptor
such as panitumumab (VECTIBIX.RTM.) and cetuximab
(ERBITUX.RTM.).
[0201] In some embodiments, the therapeutic agent is an immune
checkpoint inhibitor. The immune checkpoint inhibitor can be CD137,
CD134, PD-1, KIR, LAG-3, PD-1, PDL2, CTLA-4, B7.1, B7.2, B7-DC,
B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, BTLA, LIGHT, HVEM,
GALS, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK 1, CHK2, A2aR,
TGF-.beta., PI3K.gamma., GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG,
CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, CD40, and a
combination thereof
[0202] In some embodiments, the therapeutic agent is a nucleic acid
drug. The nucleic acid drug can be DNA, DNA plasmid, nDNA, mtDNA,
gDNA, RNA, siRNA, miRNA, mRNA, piRNA, antisense RNA, snRNA, snoRNA,
vRNA, and a combination thereof. In some embodiments, the
therapeutic nucleic acid is a DNA plasmid comprising a nucleotide
sequence encoding a gene selected from the group consisting of
GM-CSF, IL-12, IL-6, IL-4, IL-12, TNF, IFN.gamma., IFN.alpha., and
a combination thereof.
[0203] In some embodiments, the therapeutic agent is a therapeutic
cell composition. Exemplary therapeutic cell compositions include,
but are not limited to T cells, natural killer (NK) cells and
dendritic cells.
[0204] In some embodiments, the therapeutic agent is a therapeutic
antigen-binding molecule composition. Exemplary therapeutic
antigen-binding molecule compositions include, but are not limited
to monoclonal antibody, bispecific antibody, multispecific
antibody, scFv, Fab, VHH/VH, etc.
[0205] In some cases, the therapeutic agent comprises a first-line
therapy. As used herein, "first-line therapy" comprises a primary
treatment for a subject with a cancer. In some instances, the
cancer is a primary cancer. In other instances, the cancer is a
metastatic or recurrent cancer. In some cases, the first-line
therapy comprises chemotherapy. In other cases, the first-line
treatment comprises radiation therapy. A skilled artisan would
readily understand that different first-line treatments may be
applicable to different type of cancers.
[0206] In some cases, the therapeutic agent comprises a second-line
therapy, a third-line therapy, or a fourth-line therapy.
[0207] As used herein, the terms "individual(s)", "subject(s)" and
"patient(s)" mean any mammal. In some embodiments, the mammal is a
human. In some embodiments, the mammal is a non-human. None of the
terms require or are limited to situations characterized by the
supervision (e.g. constant or intermittent) of a health care worker
(e.g. a doctor, a registered nurse, a nurse practitioner, a
physician's assistant, an orderly or a hospice worker).
EXAMPLES
[0208] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1
Construction of the Surface Coating of the Invention
[0209] (i) PVA or PEG Coated Polystyrene Plate
[0210] A tissue culture plate made of polystyrene plastic is first
treated by exposing the polystyrene plate to a plasma gas to modify
the hydrophobic plastic surface to make it more hydrophilic,
followed by depositing a hydrophilic polymer (e.g. PVA or PEG) onto
the modified surface of the polystyrene plate to form a PVA or
PEG-coated polystyrene plate.
[0211] (ii) PVA-Crosslinked Polystyrene Plate
[0212] A tissue culture plate made of polystyrene plastic is first
treated by exposing the polystyrene plate to an ozone plasma to
modify the hydrophobic plastic surface to make it more hydrophilic,
followed by addition of a photo-activated azidophenyl-PVA to the
plasma treated surface of the polystyrene plate to form a
PVA-crosslinked polystyrene plate (shown in FIG. 5).
Azidophenyl-derivatized poly(vinyl alcohol) (AzPh-PVA) can be
synthesized by coupling -OH groups of PVA to 4-azidobenzoic acid,
as reported (J. Nanosci. Nanotechnol., 2009, 9, 230-239).
[0213] (iii) PVA-Crosslinked PTFE Plate
[0214] A tissue culture plate made of polytetrafluoroethylene
(PTFE) is first treated by exposing the PTFE plate to a plasma gas
to modify the hydrophobic plastic surface to make it more
hydrophilic, followed by depositing a hydrophilic polymer (e.g. PVA
or PEG) onto the modified surface of the PTFE plate. A
cross-linking agent, glutaraldehyde (GA), is applied to crosslink
PVA to PTFE to form a PVA-crosslinked PTFE plate (shown in FIG.
6)
[0215] Buildup of Polyelectrolyte Multilayers
[0216] PLL (MW 150K-300K), PLGA (MW 50K-100K), PLO (0.01%)
solution, PLH (MW 5K-25K), PLA (MW 15K-70K) are commercially
available from Sigma-Aldrich (St. Louis, Mo., USA). Both polycation
and polyanion are dissolved in Tris-HCl buffer (pH 7.4) and
deposited onto the PVA or PEG coated surface after rinsing with
Tris-HCl buffer. Each layer of polycation or polyanion is deposited
and incubated for 10 min, followed by washing with Tris-HCl buffer
3 times for 2, 1, and 1 min. The PLL/PLGA, PLO/PLGA, PLH/PLGA and
PLA/PLGA multilayer films can be fabricated by layer-by-layer
self-assembly onto the PVA or PEG coated surface as follows.
[0217] PLL/PLGA Multilayers
[0218] In some embodiments, the polyelectrolyte multilayers are
PLL/PLGA multilayers that can be constructed by sequentially
depositing PLL and PLGA on a surface of (i) PVA or PEG-coated
polystyrene plate, (ii) PVA-crosslinked polystyrene plate, or (iii)
PVA-crosslinked PTFE plate. Each depositing step comprises adding
the PLL or PLGA solution to the plate surface, incubated for 10 min
and washed 3 times for 2, 1, and 1 min.
[0219] In one embodiment, the surface coating composed of
(PLGA/PLL).sub.3/PVA is constructed. In one embodiment, the surface
coating composed of (PLGA/PLL).sub.5/PVA is constructed. In one
embodiment, the surface coating composed of (PLGA/PLL).sub.10/PVA
is constructed. In one embodiment, the surface coating composed of
(PLGA/PLL).sub.15/PVA is constructed. In one embodiment, the
surface coating composed of PLL(PLGA/PLL).sub.3/PVA is constructed.
In one embodiment, the surface coating composed of PLL
(PLGA/PLL).sub.5/PVA is constructed. In one embodiment, the surface
coating composed of PLL (PLGA/PLL).sub.10/PVA is constructed. In
one embodiment, the surface coating composed of PLL
(PLGA/PLL).sub.15/PVA is constructed.
[0220] PLO/PLGA Multilayers
[0221] In some embodiments, the polyelectrolyte multilayers are
PLO/PLGA multilayers that can be constructed by sequentially
depositing PLO and PLGA on a surface of (i) PVA or PEG-coated
polystyrene plate, (ii) PVA-crosslinked polystyrene plate, or (iii)
PVA-crosslinked PTFE plate. Each depositing step comprises adding
the PLO or PLGA solution to the plate surface, incubated for 10 min
and washed 3 times for 2, 1, and 1 min.
[0222] In one embodiment, the surface coating composed of
(PLGA/PLO).sub.3/PVA is constructed. In one embodiment, the surface
coating composed of (PLGA/PLO).sub.5/PVA is constructed. In one
embodiment, the surface coating composed of (PLGA/PLO).sub.10/PVA
is constructed. In one embodiment, the surface coating composed of
(PLGA/PLO).sub.15/PVA is constructed. In one embodiment, the
surface coating composed of PLO(PLGA/PLO).sub.3/PVA is constructed.
In one embodiment, the surface coating composed of
PLO(PLGA/PLO)5/PVA is constructed. In one embodiment, the surface
coating composed of PLO(PLGA/PLO).sub.10/PVA is constructed. In one
embodiment, the surface coating composed of
PLO(PLGA/PLO).sub.15/PVA is constructed.
[0223] PLH/PLGA Multilayers
[0224] In some embodiments, the polyelectrolyte multilayers are
PLH/PLGA multilayers that can be constructed by sequentially
depositing PLH and PLGA on a surface of (i) PVA or PEG-coated
polystyrene plate, (ii) PVA-crosslinked polystyrene plate, or (iii)
PVA-crosslinked PTFE plate. Each depositing step comprises adding
the PLH or PLGA solution to the plate surface, incubated for 10 min
and washed 3 times for 2, 1, and 1 min.
[0225] In one embodiment, the surface coating composed of
(PLGA/PLH).sub.3/PVA is constructed. In one embodiment, the surface
coating composed of (PLGA/PLH).sub.5/PVA is constructed. In one
embodiment, the surface coating composed of (PLGA/PLH).sub.10/PVA
is constructed. In one embodiment, the surface coating composed of
(PLGA/PLH).sub.15/PVA is constructed. In one embodiment, the
surface coating composed of PLH(PLGA/PLH).sub.3/PVA is constructed.
In one embodiment, the surface coating composed of
PLH(PLGA/PLH).sub.5/PVA is constructed. In one embodiment, the
surface coating composed of PLH(PLGA/PLH).sub.10/PVA is
constructed. In one embodiment, the surface coating composed of
PLH(PLGA/PLH).sub.15/PVA is constructed.
[0226] PLA/PLGA Multilayers
[0227] In some embodiments, the polyelectrolyte multilayers are
PLA/PLGA multilayers that can be constructed by sequentially
depositing PLA and PLGA on a surface of (i) PVA or PEG-coated
polystyrene plate, (ii) PVA-crosslinked polystyrene plate, or (iii)
PVA-crosslinked PTFE plate. Each depositing step comprises adding
the PLA or PLGA solution to the plate surface, incubated for 10 min
and washed 3 times for 2, 1, and 1 min.
[0228] In one embodiment, the surface coating composed of
(PLGA/PLA).sub.3/PVA is constructed. In one embodiment, the surface
coating composed of (PLGA/PLA).sub.5/PVA is constructed. In one
embodiment, the surface coating composed of (PLGA/PLA).sub.10/PVA
is constructed. In one embodiment, the surface coating composed of
(PLGA/PLA).sub.15/PVA is constructed. In one embodiment, the
surface coating composed of PLA(PLGA/PLA).sub.3/PVA is constructed.
In one embodiment, the surface coating composed of
PLA(PLGA/PLA).sub.5/PVA is constructed. In one embodiment, the
surface coating composed of PLA(PLGA/PLA).sub.10/PVA is
constructed. In one embodiment, the surface coating composed of
PLA(PLGA/PLA).sub.15/PVA is constructed.
[0229] Quartz Crystal Microbalance-Dissipation (QCM-D)
Measurement
[0230] QCM experiments were performed under Q-Sense E4 (Biolin
Scientific AB/Q-sence, Sweden). The silicon oxide (SiO.sub.2)
coated quartz crystal chips (AT-cut quartz crystals, f0=5 MHz) were
cleaned in 0.1M sodium dodecyl sulfate, followed by rinsing with
Milli-Q water, drying under nitrogen, and exposing to oxygen plasma
for 20 seconds. For QCM-D measurement, the chamber was stabilized
to 25 degree C. and all measurements were recorded at the third
overtone (15 MHz). To simulate the serial surface coating, the
concentration and the washing conditions of each coating step in
the QCM-D chamber are identical. About 1% bovine serum albumin
(BSA, Millipore, Bedford, Mass.) was used for non-specific
adsorption investigation and was introduced to chambers on the
surface.
[0231] Surface Chemical Analysis
[0232] The chemical composition of the surface coating of the
present disclosure was analyzed by X-ray photoelectron spectroscopy
(XPS;VersaProbe III, PHI) with C60 (10 kV, 10 nA) etching on
silicon wafer. The pass energy used was 93.9 eV at steps of 0.5 eV.
The relative atomic concentrations of carbon, nitrogen, oxygen and
silicon were measured in the layer of samples to a maximum
thickness of 10 nm.
[0233] Surface Roughness Measurement by Using Atomic-Force
Microscope (AFM)
[0234] The roughness of the surface coating of the present
disclosure was measured using atomic force microscope
(AFM;Nanowizard 3, JPK instrument) with tapping mode. Silicon
cantilevers with a resonant frequency of 134 kHz were utilized for
the experiments.
Example 2
Formation of Single-Cell Derived Spheroids
[0235] FIG. 7 shows the time-lapse microscope observation of HCT116
colorectal cancer cells cultured on the surface coating of the
present disclosure on day 0, 1, 2, 3, 4 and 5 during the growth of
the cancer cells supplied with complete DMEM medium. (Image
photographed by Leica DMI6000B time-lapse microscope under
10.times. objective).
Example 3
Generation of Cell Line-Derived Tumor Spheroids
[0236] The surface coating of the present disclosure provides a
biocompatible multilayer coated surface that enables cell adhesion
for cell proliferation, and also provides non-fouling
characteristic for spheroid formation directly on the surface. The
cell culture system comprising the surface coating of the present
disclosure was tested with various cancer cell lines and resulted
in the successful cultivation and formation of spheroids derived
from various cancer cell lines (shown in FIG. 8A-E). FIGS. 8A-8E
show the results of ex vivo cultivation using the culture platform
of the invention, and the formation of spheroids (after 7-14 days)
derived from (A) lung cancer cell lines A549, H1299, PC-9 and
H1975; (B) liver cancer cell lines SNU-398, SNU-475, PLC/PRF/S,
Hep3B and Huh7 (C) breast cancer cell lines MDA-MB-231 and CGBC01;
(D) colorectal cancer cell lines HCT116, HCT15 and WiDr; and (E)
human tongue squamous carcinoma cell line SAS, ovarian cancer cell
line SK-OV-3, and cell line T24 derived from a human urinary
bladder cancer patient. These cancer cells were grown on the
culture system of the invention for 7 to 14 days (the number of
seeding cells is about 1000).
Example 4
Generation of CTC-Derived Tumor Spheroids
[0237] The cell culture system comprising the surface coating of
the present disclosure was tested with various patient-derived CTCs
and resulted in the successful cultivation and formation of
spheroids derived from patient-derived CTCs (shown in FIG. 9A-C).
FIGS. 9A-9C show the representative time-dependent images of
CTC-derived spheroid cultivation on the culture platform of the
invention. (A) CTCs were isolated from a blood sample of a breast
cancer patient; CTC-derived spheroids formed after 14 days. (B)
CTCs were isolated from a blood sample of a head&neck cancer
patient; CTC-derived spheroids formed after 38 days. (C) CTCs were
isolated from a blood sample of a colorectal cancer patient;
CTC-derived spheroids formed after 13-27 days. Scale bar: 50
.mu.m.
[0238] The spheroids generated thereof may further benefit for
future diagnosis and guidance in medical treatment and application,
ex: non-invasive early cancer detection, personal medicine
guidance, pre- and post-treatment drug resistance investigation,
cell activity evaluation for immune cell-based cancer therapy, and
provide substantial material to elucidate the mechanism
participated in cancer progression by using the ex vivo cultivated
patient-derived primary CTC cells.
Example 5
Generation of Tissue-Derived Tumor Spheroids
[0239] Primary tissue cells derived from animal model primary
xenografts were cultured on the cell culture system of the
invention for 7 days. FIGS. 10A-10B show the images of tumor
spheroids derived from primary colorectal tumor tissues obtained
from colorectal cancer (CRC) patient. Tumor spheroids were
generated on the culture platform of the invention after 2 weeks
and 4 weeks. The results indicated that the cell culture platform
of the invention is capable of forming tumoroids from primary
tissue cells.
[0240] While preferred embodiments of the present disclosure 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 the
disclosure. It should be understood that various alternatives to
the embodiments of the disclosure described herein may be employed
in practicing the disclosure. It is intended that the following
claims define the scope of the disclosure and that methods and
structures within the scope of these claims and their equivalents
be covered thereby. A composition for coating a cell culture
article, the composition comprising: [0241] a) a hydrophilic
polymer, wherein the hydrophilic polymer is deposited on a surface
of the cell culture article, and [0242] b) polyelectrolyte
multilayers, wherein the hydrophilic polymer is in direct contact
with a polycation or an polyanion of the polyelectrolyte
multilayers.
* * * * *