Cell culture systems, methods and applications

Abstract

This invention provides a surface coating comprising a multilayer of a hydrophilic polymer and a polyelectrolyte. It also provides a cell culture system comprising a cell culture article with the surface coating said above. Uses and methods of preparing these surface coatings and systems are also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefits to U.S. Provisional Patent Application No. 63 / 132,934, filed December 31, 2020, and U.S. Provisional Patent Application No. 63 / 252,268, filed October 5, 2021, the disclosures of which are hereby incorporated herein by reference in their entirety. Background Technology

[0003] From basic science to preclinical drug discovery applications, including research in tumor biology, neurodegenerative diseases, and drug toxicity, researchers' interest in 3D spherical models continues to grow. Three-dimensional (3D) cell culture methods are increasingly being used to generate complex tissue or tumor models.

[0004] The spheroids formed using 3D cell culture methods and commercially available products vary considerably, which can affect readout results. For example, non-adhesion techniques widely used in 3D cell culture, including ultra-low adhesion (ULA) discs and hanging drop methods, have proven unsuitable because these methods typically produce spheroids through cell aggregation. Such spheroids generally retain their original heterogeneity and contain a variety of cells with different characteristics, requiring a better understanding of cellular heterogeneity. When tens of thousands of cells aggregate into spheroids (i.e., spherical clumps), extensive central necrosis forms within hours due to lack of nutrients and oxygen permeability, thus hindering cell proliferation. In true cancer, extensive central necrosis is a rare phenomenon.

[0005] Additionally, Matrigel is commonly used as an embedding matrix for tissue-based cell growth, such as organoid formation. However, defocusing, inefficient compound diffusion, and difficulties in sample separation limit its in vitro applications based on 3D spheroids.

[0006] Standardization of spheroid formation is crucial for producing uniform 3D cell cultures and obtaining reproducible results from spheroid-based analyses and drug screening. Therefore, there is a need to develop new cell culture systems and methods capable of reliably forming single-cell-derived spheroids. Summary of the Invention

[0007] This invention provides a surface coating for applying cell culture articles. The surface coating described herein comprises a multilayer of hydrophilic polymer and polyelectrolyte. The matrix provided herein facilitates hydration preservation. It prevents unwanted surface cracking of the cell culture matrix due to prolonged storage at ambient temperatures. In some embodiments, the surface coating provided herein enables the formation of single-cell-derived spheroids derived from single cells. This invention also provides a cell culture system comprising cell culture articles. The invention further provides uses and methods of preparation of these surface coatings and systems.

[0008] Therefore, one aspect of the present invention provides a composition for coating the surface of a cell culture article. The composition described herein comprises a) a hydrophilic polymer, wherein the hydrophilic polymer is deposited on the surface of the cell culture article, and b) a polyelectrolyte multilayer, wherein the hydrophilic polymer is in direct contact with the polycationic or polyanionic polyelectrolyte multilayer.

[0009] The cell culture products described herein may be made of any suitable plastic or polymer, such as polyethylene, polypropylene, polymethylpentene, cyclic olefin polymers, cyclic olefin copolymers, 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, polymethyl acrylate and polymethyl methacrylate or their derivatives or analogs.

[0010] 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.

[0011] 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 acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (p-HEMA) and their derivatives.

[0012] 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.

[0013] In some embodiments, the volume of the hydrophilic polymer (e.g., PVA) is 0.01-10% of the total volume of the surface coating.

[0014] The polyelectrolyte multilayers described herein comprise at least one layer pair (referred to as a “bilayer”) comprising a cationic polyelectrolyte (referred to as a “polycation”) and a polyelectrolyte (referred to as a “polyanion”). In some embodiments, the polycation is a poly(amino acid). In some embodiments, the polycation and polyanion are both poly(amino acid). The poly(amino acid) described herein may comprise L and / or D amino acid forms. As described herein, the polyelectrolyte multilayers can be formed by layer-by-layer assembly through alternating deposition of the polycation and polyanion.

[0015] In some embodiments, it has the formula (polycationic / polyanionic). n The polyelectrolyte multilayer comprises a bilayer of n polycations and polyanions, where n is an integer 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 the 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, it has the form of polyanionic (polycationic / polyanionic) n The polyelectrolyte multilayer comprises n+1 layers of polyanionic polymers and n layers of polycationic polymers, where n is an integer 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 the 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, it has the form of polycationic (polyanionic / polycationic) n The polyelectrolyte multilayer comprises n+1 layers of polycations and n layers of polyanions, where n is an integer 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 the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0018] 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.

[0019] In a preferred embodiment, the polyanion is poly(L-glutamic acid) (PLGA), poly(L-aspartic acid) (PLAA), or a combination thereof. In one preferred embodiment, the polyanion is PLGA.

[0020] In some embodiments, these polyelectrolyte multilayers comprise at least one polycationic / polycationic layer pair (i.e., bilayer) selected from the group consisting of: PLL / PLGA, PLL / PLAA, PLA / PLGA, PLA / PLAA, PLO / PLGA, PLO / PLAA, PLH / PLGA, PLH / PLAA, and combinations thereof.

[0021] In some embodiments, the dual-layer described herein includes a combination of PLL and PLGA. In some embodiments, the dual-layer described herein includes a combination of PLO and PLGA. In some embodiments, the dual-layer described herein includes a combination of PLH and PLGA. In some embodiments, the dual-layer described herein includes a combination of PLA and PLGA.

[0022] In some embodiments, the dual-layer described herein comprises a combination of PLL and PLAA. In some embodiments, the dual-layer described herein comprises a combination of PLO and PLAA. In some embodiments, the dual-layer described herein comprises a combination of PHL and PLAA. In some embodiments, the dual-layer described herein comprises a combination of PLA and PLAA.

[0023] In some embodiments, the thickness of the polyelectrolyte multilayer described herein may be in the range of 30 nm to 30 μm. In some embodiments, the thickness of the surface coating is in the range of 100 nm to 20 μm. In some embodiments, the thickness of the surface coating is 200, 400, 600, or 800 nm. In some embodiments, the thickness of the surface coating is 1, 5, 10, 15, or 20 μm.

[0024] Compared to known culture methods, the surface coating of this invention improves the proliferation rate of various cell types, including but not limited to tumor cells, pluripotent and multipotent stem cells and precursor cells, hematopoietic cells, and immune cells. Furthermore, the highly water-retaining surface coating offers the advantage of preventing unwanted surface cracks caused by dehydration due to prolonged storage at ambient temperatures.

[0025] In another embodiment, the present invention provides a method for coating cell culture articles. The method herein comprises the steps of: (a) providing a cell culture article having a hydrophobic surface; (b) modifying the hydrophobic surface by treatment; (c) applying a hydrophilic polymer to the modified surface; and (d) sequentially depositing alternating layers of polycationic and polyanionic polymers on the hydrophilic polymer.

[0026] In some embodiments, the treatments described herein are plasma treatment, corona discharge, or UV ozone treatment. In some embodiments, the hydrophobic surfaces described herein are irradiated or hydrophilized after treatment. In some embodiments, the hydrophobic surfaces are hydrophilized after a hydrophilic polymer (e.g., PVA) is applied to the surface. In some embodiments, the hydrophilic polymer (e.g., PVA) is covalently linked (i.e., bonded) to the surface. Crosslinking agents may be used to promote crosslinking (i.e., bonding). Exemplary crosslinking agents include, but are not limited to, maleic acid, formaldehyde, glutaraldehyde, butyraldehyde, sodium borate, or combinations thereof.

[0027] This invention provides a cell culture system comprising a cell culture article having a matrix configured for culturing cells and having a surface coating of the present invention. 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 live cells. In some embodiments, the cell culture system further comprises a culture medium.

[0028] In some embodiments, the cell culture system disclosed herein can efficiently and scalably proliferate cells, particularly single-cell or low-density cells (e.g., cells with an abundance of less than 1000 per milliliter), into 3D, making it possible to form 3D cell cultures from difficult-to-form cell types that cannot be formed on current market platforms (e.g., ultra-low adhesion (ULA) discs, hanging droplets).

[0029] As revealed in this article, one or more parameters of the polyelectrolyte multilayer and the culture medium can be selected by the user based on one or more microenvironment selection criteria of the cell.

[0030] The cell culture system disclosed herein not only enables cell attachment and growth but also allows for the harvesting of live cultured cells (e.g., 3D cell cultures, tissues, and organs). The inability to harvest live cells is a significant drawback of current market platforms, making it difficult to establish and maintain a sufficient number of cells for production. According to one embodiment of the invention, live cells can be harvested from the cell culture system, including 80% to 100% live, or about 85% to about 99% live, or about 90% to about 99% live. For example, in the harvested cells, at least 80% are live, at least 85% are live, at least 90% are live, at least 91% are live, at least 92% are live, at least 93% are live, at least 94% are live, at least 95% are live, at least 96% are live, at least 97% are live, at least 98% are live, or at least 99% are live. In some embodiments, cell dissociation enzymes, such as trypsin, TrypLE, or Accutase, may be used to release cells from the surface coating. In a preferred embodiment, cells may be released from the surface coating without the use of cell dissociation enzymes.

[0031] In another embodiment, the present invention provides a method for culturing cells using the cell culture articles disclosed herein. The method for culturing cells comprises the steps of: a) providing a cell culture article with a surface coating of the present invention; b) seeding cells on the coated surface; and c) culturing the cells in a suitable culture medium for a sufficient period of time to form one or more spheroids. In some embodiments, the spheroids generated herein are adhered to a matrix. In some embodiments, the spheroids generated herein are partially adhered to a matrix. In some embodiments, the spheroids are derived from single cells via single-cell proliferation. The cultured cells (e.g., cultured and harvested cells) can be used for a variety of applications, such as analysis and characterization, drug screening, isolation of single-cell derived pure lines, generation of cell banks, and generation of animal models.

[0032] 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.

[0033] As described herein, cultured cells include maintenance and / or proliferating cells. In some embodiments, cultured cells include maintenance cells. In some embodiments, cultured cells include proliferating cells. In some embodiments, cultured cells may further include differentiated cells.

[0034] 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).

[0035] In some embodiments, the cells are tumor cells, and the cultured cells are tumor spheroids. Tumor spheroids may be derived from cell lines, tumor tissue, or liquid biopsy. In some embodiments, the tumor spheroids described herein are derived from circulating tumor cells (CTCs) isolated from blood samples obtained from cancer patients. In some embodiments, the blood sample described herein is whole blood. The blood sample may be obtained through liquid biopsy. In some embodiments, the cancer patient described herein is a human cancer patient with metastatic cancer. In some embodiments, the blood sample is obtained from the cancer patient before, during, and / or after therapeutic treatment.

[0036] Another aspect of the present invention provides a method for preparing single-cell-derived spheroids in vitro, the method comprising the steps of: (a) providing a cell culture system comprising the matrix of the present invention; (b) separating cells (e.g., tumor cells and / or tumor-associated cells) from a sample to provide isolated cells; (c) seeding the isolated cells onto the matrix; and (d) culturing the cells in a suitable culture medium for a sufficient time to produce one or more spheroids, wherein the one or more spheroids are single-cell-derived.

[0037] Another aspect of the present invention provides a method for isolating single-cell-derived pure lines, each pure line consisting of a homogeneous population of genetically identical cells.

[0038] Simple Explanation of the Diagram

[0039] Figure 1A This is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 and the surface of pores 101 are not cross-linked. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising four bilayers of 301 and 302. The outermost layer is 301. 102 is in direct contact with 302.

[0040] Figure 1B This is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 is cross-linked with the surface of pores 101. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising four bilayers of 301 and 302. The outermost layer is 301. 102 is in direct contact with 302.

[0041] Figure 2AThis is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 and the surface of pores 101 are not cross-linked. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising five layers of 302 and four layers of 301. The outermost layer is 302. 102 and 302 are in direct contact.

[0042] Figure 2B This is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 is cross-linked with the surface of pores 101. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising five layers of 302 and four layers of 301. The outermost layer is 302. 102 and 302 are in direct contact.

[0043] Figure 3A This is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 and the surface of pores 101 are not cross-linked. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising four bilayers of 301 and 302. The outermost layer is 302. 102 is in direct contact with 301.

[0044] Figure 3B This is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 is cross-linked with the surface of pores 101. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising four bilayers of 301 and 302. The outermost layer is 302. 102 is in direct contact with 301.

[0045] Figure 4A This is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 and the surface of pores 101 are not cross-linked. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising five layers of 301 and four layers of 302. The outermost layer is 301. 102 is in direct contact with 301.

[0046] Figure 4BThis is a side cross-sectional view of an embodiment of the surface coating of the present invention. Hydrophilic polymer 102 is deposited on the surface of pores 101 in the cell culture article 202. 102 is cross-linked with the surface of pores 101. 301 is a polyanionic polymer. 302 is a polycationic polymer. 103 is an embodiment of a polyelectrolyte multilayer comprising five layers of 301 and four layers of 302. The outermost layer is 301. 102 is in direct contact with 301.

[0047] Figure 5 This describes an embodiment of surface modification of cell culture products. Tissue culture discs made of polystyrene plastic are first treated with ozone plasma, followed by the addition of photoactivated azidophenyl-PVA to the modified surface to form PVA-crosslinked polystyrene discs.

[0048] Figure 6 This describes an example of surface modification of cell culture products. Tissue culture discs made of polytetrafluoroethylene (PTFE) are first treated with plasma gas, followed by the deposition of PVA on the modified surface of the PTFE discs. The PVA is then crosslinked to the PTFE using the crosslinking agent glutaraldehyde (GA) to form PVA-crosslinked PTFE discs.

[0049] Figure 7 The images show time-lapse microscopy observations of HCT116 colorectal cancer cells cultured on the surface coating of this invention at days 0, 1, 2, 3, 4, and 5 of the cancer cell growth process in a fully DMEM culture medium. (Images were taken with a Leica DMI6000B time-lapse microscope at 10x objective).

[0050] Figures 8A-E The results demonstrate the formation of spheroids (after 7-14 days) of cells 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 cell carcinoma cell line SAS, ovarian cancer cell line SK-OV-3, and cell line T24 derived from human bladder cancer patients using the culture platform of the present invention for in vitro culture.

[0051] Figure 9A-CRepresentative time-dependent images showcasing the culture of CTC-derived spheroids on the culture platform of this invention. (A) CTCs isolated from blood samples of breast cancer patients; CTC-derived spheroids formed after 14 days. (B) CTCs isolated from blood samples of head and neck cancer patients; CTC-derived spheroids formed after 38 days. (C) CTCs isolated from blood samples of colorectal cancer patients; CTC-derived spheroids formed after 13–27 days. Scale bar: 50 μm.

[0052] Figure 10A-B Images of tumor spheroids derived from primary colorectal tumor tissue obtained from a patient with colorectal cancer (CRC) are shown. Tumor spheroids were generated on the culture platform of this invention after (A) 2 weeks and (B) 4 weeks. Detailed Implementation

[0053] This invention relates to next-generation scaffold-free 3D cell culture technology and its uses. In some embodiments, a novel composition for a surface coating is provided. A cell culture system comprising a surface coating suitable for cell culture, particularly 3D cell culture, is also provided. The surface coating can induce the formation of highly homogeneous 3D cell cultures, making it possible to form 3D cell cultures from difficult primary cell types that cannot form on any other low-adhesion surface. Compared to known culture methods, the surface coating described herein improves the proliferation rate of a variety of cells, including but not limited to tumor cells, pluripotent and multipotent stem cells and precursor cells, hematopoietic cells, and immune cells. In some embodiments, the surface coating comprises a hydrophilic polymer (e.g., PVA) and one or more pairs of polyelectrolytes.

[0054] Surface coating

[0055] The surface coating of the present invention comprises a hydrophilic polymer and a polyelectrolyte multilayer. In some cases, the surface coating is as shown in FIG1. ​​In FIG1, 104 indicates an illustrative surface coating. The hydrophilic polymer 102 is deposited on the top surface of the well 201 of the cell culture dish 202. The polyelectrolyte multilayer 103 is deposited on top of the hydrophilic polymer layer 102.

[0056] Without being bound by any particular theory, Xianxin surface coatings enable cells seeded on the surface coating (such as rare cells, low-density cells, or single cells extracted from blood) to robustly proliferate or stably remain for extended periods of time, with or without a matrix, such as more than 48 hours, more than 72 hours, more than 96 hours, more than 5 days, more than 6 days, more than 7 days, or for 1 to several weeks (e.g., 1, 2, 3, 4, 5, 6 or more weeks).

[0057] 1) Hydrophilic polymers

[0058] The hydrophilic polymers described herein are hydrophilic absorbent polymers (“absorbent polymers”), which are water-soluble and swell due to absorption and retention of aqueous solutions. A non-limiting list of hydrophilic absorbent polymers that can be used in this invention includes the following polymers and their derivatives with hydrophilicity and biocompatibility levels: poly(vinyl alcohol) (PVA), ethylene-vinyl alcohol copolymers (typically non-biodegradable materials whose hydrophilicity depends on the distribution of ethylene (hydrophobic) and vinyl alcohol (hydrophilic) groups), polyvinyl alcohol and copolymers of ethylene-vinyl alcohol, polyacrylate compositions, polyurethane compositions, poly(ethylene glycol) (PEG) or poly(ethylene oxide) (POE) and poly(ethylene oxide) (PEO), and their derivatives, including but not limited to polyethylene glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate (PEGDMA), and polyethylene glycol diacrylate (PEGDA); nitrogen-containing materials, such as poly… Acrylamide (without acrylamide toxic residues), polyvinylpyrrolidone, polyethyleneamine, and polyethyleneimine; charged materials, such as various forms of poly(lactic acid), also known as polylactide (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 polyglycolic acid, copolymers of lactic acid and glycolic acid, poly(lactic acid-co-glycolic acid) (PL-co-GA), copolymers of PLA and / or PGA with PEG; polymethacrylic acid; poly(hydroxyethyl methacrylate) (poly-HEMA), and other absorbent, hydrophilic, and biocompatible materials known in this art.

[0059] In some embodiments, the hydrophilic absorbent polymer is selected from the group consisting of: poly(vinyl alcohol) (PVA), copolymers of ethylene vinyl alcohol, polyvinyl alcohol and copolymers of 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), polyethyleneamine (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 acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), and poly(hydroxyethyl methacrylate) (p-HEMA).

[0060] In some embodiments, the hydrophilic absorbent polymer is selected from the group consisting of: PVA, PEG, PEG-acrylate, polylactide, PMMA, p-HEMA, combinations thereof, or derivatives 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.

[0061] In some embodiments, the average molecular weight of the hydrophilic polymer is 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, or about 10,000 g / mol. / mol to about 175,000 g / mol, about 10,000 g / mol to about 150,000 g / mol, about 10,000 g / mol to about 125,000 g / mol, about 10,000 g / mol to about 100,000 g / mol, about 10,000 g / mol to about 75,000 g / mol, about 10,000 g / mol to about 50,000 g / mol, about 10,000 g / mol to about 25,000 g / mol, about 20,000 g / mol to about 150,000 g / mol, or about 50,000 g / mol to about 150,000 g / mol.

[0062] In some cases, the hydrophilic polymer is deposited directly onto the surface of the target substrate. In other cases, 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 substrate surface. In some cases, an additional layer (also referred to herein as the innermost layer) is formed between the hydrophilic polymer layer and the substrate surface.

[0063] In some embodiments, the hydrophilic polymer is PVA. The average molecular weight of PVA can range from about 10,000 g / mol to about 125,000 g / mol. In some cases, the average molecular weight of PVA is about 10,000 g / mol to about 100,000 g / mol, about 10,000 g / mol to about 75,000 g / mol, about 10,000 g / mol to about 50,000 g / mol, about 20,000 g / mol to about 125,000 g / mol, about 20,000 g / mol to about 100,000 g / mol, about 20,000 g / mol to about 75,000 g / mol, about 20,000 g / mol to about 50,000 g / mol, about 50,000 g / mol to about 125,000 g / mol, or about 50,000 g / mol to about 100,000 g / mol.

[0064] In some cases, PVA is deposited directly onto the surface of the target substrate. In other cases, 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, an additional layer is formed between the PVA layer and the substrate surface.

[0065] In some embodiments, the hydrophilic polymer is PEG. In some cases, 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, or 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.

[0066] In some cases, the PEG used in this paper is discrete PEG (dPEG). Discrete PEG can be a polymeric PEG containing more than one repeating ethylene oxide unit. In some cases, discrete PEG contains 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some cases, dPEG contains 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.

[0067] In some 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 other cases, 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, or from about 0% v / v. 0.1% to about 8% v / v, about 0.1% to about 7% v / v, about 0.1% to about 6% v / v, about 0.1% to about 5% v / v, about 0.1% to about 4% v / v, about 0.1% to about 3% v / v, about 1% to about 10% v / v, about 1% to about 9% v / v, about 1% to about 8% v / v, about 1% to about 7% v / v, about 1% to about 6% v / v, about 1% to about 5% v / v, about 1% to about 4% v / v, about 2% to about 10% v / v, or about 5% to about 10% v / v. In some cases, the volume of the hydrophilic polymer (such as 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.

[0068] In some cases, the weight of the hydrophilic polymer (e.g., PVA or PEG) relative to the total weight of the surface coating is from about 1% to about 50%. In some cases, the weight of the hydrophilic polymer (e.g., PVA or PEG) relative to the total weight of the surface coating is from about 1% to about 40%. In some cases, the weight of the hydrophilic polymer (e.g., PVA or PEG) relative to the total weight of the surface coating is from about 1% to about 30%. In some cases, the weight of the hydrophilic polymer (e.g., PVA or PEG) relative to the total weight of the surface coating is from about 1% to about 20%. In some cases, the weight of the hydrophilic polymer (e.g., PVA or PEG) relative to the total weight of the surface coating is from about 1% to about 10%.

[0069] 2) Polyelectrolyte multilayer

[0070] In some embodiments, the surface coating comprises a polyelectrolyte multilayer (PEM). The PEM described herein comprises a plurality of alternating layers of polymers (i.e., polyelectrolytes) with opposite charges. The oppositely charged polymers described herein comprise a combination of positively charged polyelectrolytes (also referred to herein as polycations) and negatively charged polyelectrolytes (also referred to herein as polyanions).

[0071] 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[α-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), N,N-diethylaminoethyl methacrylate (DEAEMA), and combinations thereof. In some cases, the polycation is PLL. In some cases, the polycation is PLO. In some cases, the polycation is PLH. In some cases, the polycation is PLA.

[0072] 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(styrene sulfonic acid) (PSS), poly(N-isopropylacrylamide) (NIPAM), poly(2-acryloylamino-2-methyl-1-propanesulfonic acid) (PAMPS), and combinations thereof. In some cases, the polyanion is PLGA. In some cases, the polyanion is PLAA.

[0073] Polyelectrolyte multilayers can be formed by layer-by-layer assembly through the alternating deposition of polycationic and polyanionic layers. The polyelectrolyte multilayers described herein include at least one bilayer comprising a polycationic layer and a polyanionic layer.

[0074] In some embodiments, the PEM may include about 1 double layer to about 100 double layers. In some embodiments, the PEM may include about 1 double layer to about 50 double layers. In some embodiments, the PEM may include about 1 double layer to about 30 double layers. In some embodiments, the PEM may include about 1 double layer to about 20 double layers. In some embodiments, the number of double layers 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 double layers is in the 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 double layers is 3. In some embodiments, the number of double layers is 4. In some embodiments, the number of double layers is 5. In some embodiments, the number of double layers is 6. In some embodiments, the number of double layers is 7. In some embodiments, the number of double layers is 8. In some embodiments, the number of double layers is 9. In some embodiments, the number of double layers is 10. In some embodiments, the number of double layers is 11. In some embodiments, the number of double layers is 12. In some embodiments, the number of double layers is 13. In some embodiments, the number of double layers is 14. In some embodiments, the number of double layers is 15. In some embodiments, the number of double layers is 16. In some embodiments, the number of double layers is 17. In some embodiments, the number of double layers is 18. In some embodiments, the number of double layers is 19. In some embodiments, the number of double layers is 20.

[0075] In some embodiments, the polyelectrolyte multilayer described herein comprises one or more bilayers of positively charged polyelectrolytes and negatively charged polyelectrolytes, wherein 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 groups is in the range of 1 to 100, 3 to 60, 3 to 50, or 3 to 30. In some embodiments, the number of groups 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 groups 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 groups is in the 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 multilayer described herein comprises one or more bilayers of PLL and PLGA. In some embodiments, the number of bilayers of PLL and PLGA is in the range of 1 to 100, 3 to 60, 3 to 50, or 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 the 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 multilayer described herein comprises one or more bilayers of PLO and PLGA. In some embodiments, the number of PLO and PLGA bilayers ranges from 1 to 100, 3 to 60, 3 to 50, or 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 ranges from 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 multilayer described herein comprises one or more bilayers of PLH and PLGA. In some embodiments, the number of PLH and PLGA bilayers ranges from 1 to 100, 3 to 60, 3 to 50, or 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 ranges from 1 to 10, 1 to 8, 1 to 5, 3 to 20, 5 to 20, 10 to 20, 11 to 19, 12 to 18, 13 to 17, or 14 to 16.

[0079] In some embodiments, the polyelectrolyte multilayer described herein comprises one or more double layers of PLA and PLGA. In some embodiments, the number of double layers of PLA and PLGA is in the range of 1 to 100, 3 to 60, 3 to 50, or 3 to 30. In some embodiments, the number of double layers 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 double layers 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 double layers is in the 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 multilayer described herein comprises one or more bilayers of PLL and PLAA. In some embodiments, the number of bilayers of PLL and PLAA is in the range of 1 to 100, 3 to 60, 3 to 50, or 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 the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0081] In some embodiments, the polyelectrolyte multilayer described herein comprises one or more bilayers of PLO and PLAA. In some embodiments, the number of PLO and PLAA bilayers ranges from 1 to 100, 3 to 60, 3 to 50, or 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 ranges from 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0082] In some embodiments, the polyelectrolyte multilayer described herein comprises one or more bilayers of PLH and PLAA. In some embodiments, the number of bilayers of PLH and PLAA is in the range of 1 to 100, 3 to 60, 3 to 50, or 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 the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0083] In some embodiments, the polyelectrolyte multilayer described herein comprises one or more double layers of PLA and PLAA. In some embodiments, the number of double layers of PLA and PLAA ranges from 1 to 100, 3 to 60, 3 to 50, or 3 to 30. In some embodiments, the number of double layers 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 double layers 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 double layers ranges from 1 to 10, 1 to 8, 1 to 5, 3 to 20, 5 to 20, 10 to 20, 11 to 19, 12 to 18, 13 to 17, or 14 to 16.

[0084] The thickness of the PEM as a thin film can be in a wide range, for example, from about 30 nm to about 30 μm or from about 100 nm to about 20 μm. In some embodiments, the thickness is from about 100 nm to about 500 nm, from about 500 nm to about 1 μm, or from about 1 μm to about 10 μm. In some embodiments, the thickness is about 200, 400, 600, 800 nm, or any number between the two. In some embodiments, the thickness is about 1, 5, 10, 15, or 20 μm, or any number between the two.

[0085] Many methods can be used to characterize PEM. In some embodiments, these methods may include ellipsometer method (thickness), quartz crystal microbalance with dissipation monitoring (mass adsorption, 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).

[0086] In some embodiments, PEM can be deposited by transferring a polyanionic or polycationic solution, either as a mixture or sequentially, into / onto a dish using a pipette.

[0087] In some embodiments, PEM is formed on the surface by dip coating. In dip coating, the matrix is ​​immersed in a polyelectrolyte solution for a certain period of time (typically 10-15 minutes), followed by multiple rinses and immersion in a second polyelectrolyte solution of the opposite charge. This process is repeated until the desired number of layers is achieved.

[0088] In some embodiments, PEM is formed on the surface by spraying. In some embodiments, the polyelectrolyte may be sprayed onto the surface for 3-10 seconds, followed by a 10-30 second rest / drying period, the surface is washed with water for 3-20 seconds, followed by another 10-second rest period, and the cycle is repeated with a polyelectrolyte of the opposite charge.

[0089] In some embodiments, PEM is formed on the surface by spin coating. Spin coating is a highly controlled method for solution-based coatings of a system. A typical spin coating procedure involves spin coating for 10-15 seconds, rinsing at least once with spin-coating water for 15-30 seconds, and repeating the procedure with a polyelectrolyte of opposite charge. A washing step may not be necessary in spin coating.

[0090] 3) Surface coating construction

[0091] Another aspect of the invention is characterized by a method for coating cell culture articles using the compositions 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 by treatment; (c) applying a hydrophilic polymer to the modified surface; and (d) sequentially depositing alternating layers of polycationic and polyanionic polymers on the hydrophilic polymer.

[0092] In some embodiments, the treatments described herein are plasma treatment, corona discharge, or UV ozone treatment. In some embodiments, the hydrophobic surfaces described herein are irradiated or hydrophilized after treatment. In some embodiments, the hydrophobic surfaces are hydrophilized after a hydrophilic polymer (e.g., PVA) is applied to the surface. In some embodiments, the hydrophilic polymer (e.g., PVA) is covalently linked (i.e., bonded) to the surface. Crosslinking agents may be used to promote crosslinking (i.e., bonding). Exemplary crosslinking agents include, but are not limited to, maleic acid, formaldehyde, glutaraldehyde, butyraldehyde, sodium borate, or combinations thereof.

[0093] As described herein, a surface is hydrophilic if the contact angle of a water droplet on the surface is less than 90 degrees (the contact angle is defined as the angle through the interior of the water droplet). Examples include hydrophilic surfaces with contact angles from 90 degrees to 0 degrees; those skilled in the art will immediately understand that all ranges and values ​​between explicitly defined limits are taken into account, such as any of the following which may be used as upper or lower limits: 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2, 0 degrees.

[0094] In some embodiments, the matrix described herein comprises (polyanionic / polycationic) n / PVA, wherein the polyanionic / polycationic polyatomy 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 in the range of 1 to 20, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, n is in the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0095] In some embodiments, the matrix described herein comprises (polycationic / polyanionic) n / PEG, wherein the polycationic / polyanionic poly(pLC) 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 in the range of 1 to 20, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, n is in the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0096] In some embodiments, the matrix described herein comprises polycationic (polyanionic / polycationic) materials. n / PEG-acrylate, wherein the polyanionic / polycationic acrylate 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 in the range of 1 to 20, specifically 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 the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0097] In some embodiments, the matrix described herein comprises polyanionic (polycationic / polyanionic) compounds. n / PVP, wherein the polycationic / polyanionic polypeptide 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 in the range of 1 to 20, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, n is in the range of 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0098] The surface coating described herein may 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, "dehydrated state" and "hydrated state" each refer to the volume of an aqueous solution (e.g., water) relative 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 the 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 more of the total volume of the surface coating.

[0099] 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 wt% to about 60 wt% of the total weight of the surface coating. In some cases, the aqueous solution (e.g., water) is from about 1 wt% to about 50 wt%, from about 1 wt% to about 40 wt%, from about 1 wt% to about 30 wt%, from about 1 wt% to about 20 wt%, from about 10 wt% to about 60 wt%, from about 10 wt% to about 50 wt%, from about 10 wt% to about 40 wt%, from about 10 wt% to about 30 wt%, from about 10 wt% to about 20 wt%, from about 20 wt% to about 60 wt%, from about 20 wt% to about 50 wt%, from about 20 wt% to about 40 wt%, or from about 30 wt% to about 60 wt% of the total weight of the surface coating.

[0100] In some embodiments, the surface coating further comprises filler. In some cases, the filler comprises mineral filler, such as, but not limited to, silica, alumina, calcium carbonate, or polysiloxane.

[0101] Each of the polycationic and polyanionic polymers and the absorbent polymers is soluble in an aqueous solution for use in this invention. The aqueous solution is free of or substantially free of organic solvents. It should be understood that small amounts of organic solvents may be present in the aqueous solution, for example, as a result of some organic solvents remaining in the polymer after polymerization. As used herein, “substantially free of” when referring to organic solvents in an aqueous solution means that the aqueous solution contains less than 1% by weight of organic solvents. In many embodiments, the aqueous solution contains less than 0.8%, less than 0.5%, less than 0.2%, or less than 0.1% of organic solvents.

[0102] Each of the polycationic and polyanionic polymers and the absorbent polymers can be dissolved in an aqueous solution at any suitable concentration for coating purposes.

[0103] Cell culture system

[0104] The cell culture system of the present invention comprises a cell culture article having a surface coating as described herein.

[0105] The cell culture articles described herein can be made of any suitable plastic and the like. In some embodiments, the cell culture articles are made of a material comprising at least one of the following: polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0106] In some embodiments, the cell culture system further comprises cells. In some embodiments, the cells are derived from a cell line. 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 medium.

[0107] The cell culture system disclosed herein can not only achieve cell attachment and growth, but also harvest live cultured cells (e.g., 3D cell cultures, tissues, and organs). According to some embodiments of the invention, the cell culture system can be used to harvest live cells, including those that are 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, in the harvested cells, 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, cell dissociation enzymes, such as trypsin, TrypLE, or Accutase, may or may not be used to release cells from the cell culture system.

[0108] Its methods and uses

[0109] Methods for culturing cells

[0110] Without being bound by any particular theory, it is believed that the surface coatings disclosed herein enable robust cell proliferation and / or stable maintenance. Therefore, the present invention provides a method for culturing cells. The method comprises the steps of: (a) providing a cell culture article with the surface coating of the present invention coated on its surface; (b) seeding cells on the coated surface; and (c) culturing the cells in a suitable culture medium. In some embodiments, the cells are cultured for a sufficient period to form spheroids. In a preferred embodiment, 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 aggregation. In some embodiments, the spheroids have a uniform size.

[0111] In some embodiments, the cells described herein may be derived from cell lines, tissue sections, or liquid biopsies. 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.

[0112] 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).

[0113] In some embodiments, the cells described herein are cancer cells. Exemplary cancers described herein include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder or pleural cancer, nasal, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer.

[0114] 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 (CAML), tumor-associated macrophages (TAMs), tumor-associated monocyte / macrophage lineage cells (MMLCs), cancer stem cells, tumor microemboli, tumor-associated stromal cells (TASCs), tumor-associated bone marrow cells (TAMCs), tumor-associated regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), tumor-derived endothelial cells (TECs), tumor-associated neutrophils (TANs), tumor-associated platelets (TAPs), tumor-associated immune cells (TAIs), bone marrow-derived suppressor cells (MDSCs), and combinations thereof.

[0115] Exemplary cells include low-density cells, single cells, rare cells, or combinations thereof. Low-density cells may be fewer than 5,000 cells per square centimeter on the substrate at the time of inoculation, for example, no more than about 1, 5, 10, 20, 50, 100, 200, 300, 500, 1,000, 2,000, 3,000, 4,000, or 4,500 cells per square centimeter on the substrate.

[0116] In some embodiments, inoculating the isolated cells in step (c) involves seeding a plate of cells at a density of one to ten cells per square centimeter onto the matrix surface (i.e., the cell growth surface). In some embodiments, inoculating the isolated cells in step (c) involves seeding a plate of cells at a density of 10 to 100 cells per square centimeter onto the matrix surface. In some embodiments, inoculating the isolated cells in step (c) involves seeding a plate of cells at a density of 100 to 1000 cells per square centimeter onto the matrix surface.

[0117] 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 average diameter of the spheroids is in the range of about 40 μm to about 200 μm.

[0118] Any suitable culture medium may be used in the methods of the exemplary embodiments. Exemplary culture media include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), epidermal growth factor (EGF) and / or basic fibroblast growth factor (bFGF), and a mixture of Dulbecco's modified Eagle's medium (DMEM) supplemented with B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

[0119] Methods for preparing single-cell derived spheroids

[0120] In another embodiment, the present invention provides a method for preparing single-cell-derived spheroids, the method comprising the steps of: (a) providing a cell culture article with a surface coating of the present invention; (b) seeding cells on the coated surface; and (c) culturing the cells in a suitable culture medium for a sufficient period of time to form spheroids, wherein these spheroids are single-cell-derived. The spheroids described herein are generated via single-cell proliferation. In some embodiments, the spheroids have a uniform size. In some embodiments, single-cell-derived homologs are semi-attached or loosely attached to the matrix of the present invention.

[0121] In some embodiments, the cells are derived from a cell line. 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.

[0122] 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).

[0123] In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are isolated from human primary tumor tissue. In some embodiments, the cancer cells are isolated from blood samples from cancer patients. The exemplary cancers described herein include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder or pleural cancer, nasal, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer.

[0124] 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 (CAML), tumor-associated macrophages (TAMs), tumor-associated monocyte / macrophage lineage cells (MMLCs), cancer stem cells, tumor microemboli, tumor-associated stromal cells (TASCs), tumor-associated bone marrow cells (TAMCs), tumor-associated regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), tumor-derived endothelial cells (TECs), tumor-associated neutrophils (TANs), tumor-associated platelets (TAPs), tumor-associated immune cells (TAIs), bone marrow-derived suppressor cells (MDSCs), and combinations thereof.

[0125] Exemplary cells include low-density cells, single cells, rare cells, or combinations thereof. Low-density cells may be fewer than 5,000 cells per square centimeter on the substrate at the time of inoculation, for example, no more than about 1, 5, 10, 20, 50, 100, 200, 300, 500, 1,000, 2,000, 3,000, 4,000, or 4,500 cells per square centimeter on the substrate.

[0126] In some embodiments, inoculating the isolated cells in step (c) involves seeding a plate of cells at a density of one to ten cells per square centimeter onto the matrix surface (i.e., the cell growth surface). In some embodiments, inoculating the isolated cells in step (c) involves seeding a plate of cells at a density of 10 to 100 cells per square centimeter onto the matrix surface. In some embodiments, inoculating the isolated cells in step (c) involves seeding a plate of cells at a density of 100 to 1000 cells per square centimeter onto the matrix surface.

[0127] In some embodiments, the culture step occurs over a period of 2-8 days (e.g., 2, 3, 4, 5, 6, 7, or 8 days). In other embodiments, the culture 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 culture 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 average diameter of the spheroids is in the range of about 40 μm to about 200 μm.

[0128] In the methods of the exemplary embodiments, any suitable culture medium may be used. Exemplary culture media include, but are not limited to, Dalberg modified Eagle's medium (DMEM), epidermal growth factor (EGF) and / or basic fibroblast growth factor (bFGF), or a mixture of Dalberg modified Eagle's medium (DMEM) supplemented with B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

[0129] In some embodiments, the single-cell-derived spheroids have a diameter of less than 200 μm. In some embodiments, the single-cell-derived spheroids have a diameter of less than 150 μm. In some embodiments, the single-cell-derived spheroids have a diameter of about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 μm.

[0130] In some embodiments, single-cell-derived spheroids can be used to screen therapeutic agents. In some embodiments, the method of screening therapeutic agents includes: (a) applying a test substance to the single-cell-derived spheroids generated therefrom; and (b) evaluating the effect of the test substance on the single-cell-derived spheroids. In some embodiments, the effect of the test substance is analyzed using an imaging system, for example, analyzing the biochemical activity and / or expression level of a gene or protein.

[0131] In some embodiments, the single-cell-derived spheroids produced are tumor spheroids. In some embodiments, the test substance described herein is a chemotherapeutic agent, such as a cytotoxic or cell-inhibitory chemotherapeutic agent. 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.

[0132] 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 average diameter of at least one 3D spheroid is in the range of about 40 μm to about 200 μm.

[0133] In some embodiments, this document provides a library of single-cell-derived spheroids (e.g., tumor spheroids) produced according to any of the culture methods employing the cell culture system described herein. In some embodiments, this document provides a library of single-cell-derived spheroids (e.g., tumor spheroids) derived according to any of the culture methods employing the cell culture system described herein.

[0134] Methods for isolating pure lines derived from single cells

[0135] With the adoption of genome editing technology in routine laboratories, single-cell derived pure lines are becoming increasingly important. Limiting dilution, a traditional method for isolating single cells, relies on the statistical probability of pure lineage, which changes significantly with small variations in the protocol. While extremely inefficient at isolating single cells, this technique maintains cell viability. Conversely, flow cytometry can efficiently provide single-cell pure lines but negatively impacts cell viability. These platforms typically begin with a suspension containing a large number of cells, which are then "individualized" by random confinement within microstructures. These methods are impractical when the cell population is small due to considerable cell loss during mixing and / or transfer. The method of the present invention provides an efficient alternative for isolating viable single-cell pure lines. In some embodiments, the method does not require confining cells individually within microstructures.

[0136] In some embodiments, this document provides a method for isolating single-cell-derived pure lines. The method comprises: 1) culturing a heterogeneous cell population using a cell culture article with the composition of the present invention coated on its surface to obtain a plurality of cell pure lines comprising single-cell-derived pure lines; and 2) isolating single-cell-derived pure lines from the cell culture article.

[0137] In some embodiments, the heterogeneous cell population comprises adherent cells. In some embodiments, the heterogeneous cell population comprises non-adhesive cells. In some embodiments, the heterogeneous cell population comprises cells isolated from a cell line. In some embodiments, the heterogeneous cell population comprises cells isolated from an individual's liquid biopsy. In some embodiments, the heterogeneous cell population comprises cells isolated from an individual's tissue biopsy. In some embodiments, the heterogeneous cell population comprises genetically engineered cells. In some embodiments, the heterogeneous cell population comprises cells engineered to contain gene mutations. In some embodiments, the heterogeneous cell population comprises cells engineered to contain heterologous nucleotide sequences.

[0138] In some embodiments, single-cell-derived pure lines are semi-attached or loosely attached to the coated surface disclosed herein.

[0139] In some embodiments, no cell debris was observed in the cell culture system after 7 days or more of culture.

[0140] In some embodiments, the culture step occurs over a period of 2 to 8 days (e.g., 2, 3, 4, 5, 6, 7, or 8 days). In other embodiments, the culture step occurs over a period of 7 to 14 days (e.g., 7, 8, 9, 10, 11, 12, 13, or 14 days). In other embodiments, the culture step occurs over a period of 1 to 4 weeks (e.g., 1, 2, 3, or 4 weeks).

[0141] In some embodiments, the single-cell-derived homologous lines form single-cell-derived spheroids. In some embodiments, the diameter of the single-cell-derived homologous lines is from about 40 μm to about 200 μm. In some embodiments, the diameter of the single-cell-derived homologous lines is from about 50 μm to about 150 μm. In some cases, the diameter of the single-cell-derived homologous lines is from about 50 μm to about 120 μm, from about 50 μm to about 100 μm, from about 50 μm to about 80 μm, from about 50 μm to about 60 μm, from about 80 μm to about 150 μm, from about 80 μm to about 120 μm, from about 80 μm to about 100 μm, from about 100 μm to about 200 μm, from about 100 μm to about 150 μm, or from about 100 μm to about 120 μm.

[0142] In some cases, single-cell-derived pure lines form single-cell-derived spheroids. In some cases, the spheroids contain approximately 8 to approximately 1000 cells. In some cases, the spheroids contain approximately 8 to approximately 800 cells, approximately 8 to approximately 500 cells, approximately 8 to approximately 400 cells, approximately 8 to approximately 300 cells, approximately 8 to approximately 200 cells, approximately 8 to approximately 100 cells, approximately 10 to approximately 1000 cells, approximately 10 to approximately 800 cells, approximately 10 to approximately 500 cells, approximately 10 to approximately 400 cells, approximately 10 to approximately 300 cells, approximately 10 to approximately 200 cells, approximately 10 to approximately 100 cells, approximately 50 to approximately 1000 cells, approximately 50 to approximately 800 cells. One cell, about 50 to about 500 cells, about 50 to about 400 cells, about 50 to about 300 cells, about 50 to about 200 cells, about 100 to about 1000 cells, about 100 to about 800 cells, about 100 to about 500 cells, about 100 to about 400 cells, about 100 to about 300 cells, about 300 to about 1000 cells, about 300 to about 800 cells, about 300 to about 500 cells, about 500 to about 1000 cells, or about 500 to about 800 cells.

[0143] In some embodiments, at least 10% of the cells disposed on the coated surface form single-cell-derived spheroids. In some embodiments, at least 20% of the cells disposed on the coated surface form single-cell-derived spheroids. In some embodiments, at least 30% of the cells disposed on the coated surface form single-cell-derived spheroids. In some embodiments, at least 40% of the cells disposed on the coated surface form single-cell-derived spheroids. In some embodiments, at least 50% of the cells disposed on the coated surface form single-cell-derived spheroids. In some embodiments, at least 60% of the cells disposed on the coated surface form single-cell-derived spheroids. In some embodiments, at least 70% of the cells disposed on the coated surface form single-cell-derived spheroids.

[0144] In some embodiments, the method described herein further includes analyzing single-cell-derived homologous lines to obtain the characteristics of single cells. In some cases, the step of analyzing single-cell-derived homologous lines includes performing sequencing analysis on the single-cell-derived homologous lines. In some embodiments, the analysis step includes performing 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 analysis step includes analyzing duplicate number variation. In some embodiments, the analysis step includes analyzing gene mutations. In some embodiments, the analysis step includes analyzing single nucleotide polymorphisms.

[0145] In some embodiments, the step of analyzing a single-cell-derived homologous line includes protein plasmid analysis of the single-cell-derived homologous line. Exemplary protein plasmid analyses 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.

[0146] In some embodiments, the characteristics of a single cell include one or more of the following: genotype, epigenetic profile, expression level of a gene or protein, response to a drug, drug resistance profile, or metastatic potential.

[0147] In some respects, this document provides a single-cell-derived pure line produced according to any of the culture methods using the cell culture system described herein. In some respects, a library of single-cell-derived pure lines derived according to any of the culture methods using the cell culture system described herein is provided.

[0148] The generation of patient-derived tumor-like xenografts

[0149] In another embodiment, the present invention provides a method for generating patient-derived tumor-like xenografts. The patient-derived tumor-like tissue can be generated through in vitro growth of tumor cells derived from a patient's blood, tissue (e.g., bladder, stomach, breast, pancreas, colon, or lung), or cell line. The patient-derived tumor-like xenograft can be established by directly transferring the tumor-like tissue into highly immunodeficient mice and subsequently maintaining it through succession between mice. The xenograft model can be used for biomedical transfer studies and, once validated, can serve as a preclinical transfer model for efficacy screening in cancer drug development.

[0150] In some embodiments, this document provides a method for generating a patient-derived tumor xenograft animal model, comprising: a) culturing a plurality of cells containing tumor cells derived from a patient using any of the cell culture systems provided herein to obtain a plurality of tumor-like cells; b) isolating the tumor-like cells from a 3D cell culture system; and c) inoculating the isolated tumor-like cells into a non-human animal to generate a patient-derived tumor xenograft animal model.

[0151] In some embodiments, a plurality of tumor cells are obtained from the patient's primary tissue. In some embodiments, a plurality of tumor cells are obtained from the patient's blood. In some embodiments, tumor cells derived from the patient are grown as primary xenografts in an animal model prior to culture on a 3D cell culture system.

[0152] In some embodiments, the non-human animal is immunodeficient. In some embodiments, the non-human animal is a mouse.

[0153] In some embodiments, the tumor cells are circulating tumor cells (CTCs) derived from solid tumors. In other embodiments, the tumor cells are CTCs derived from hematologic malignancies. In some embodiments, the patient has metastatic cancer.

[0154] In some embodiments, the culture step includes expanding tumor cells 10 to 100 times (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times) within one week.

[0155] Methods for 3D cell culture using non-adhesive conditions include the hanging drop method (Kelm et al., Biotechnol. Bioeng. 83, 173-180 (2003)), rotating bioreactors (Zhau et al., In VitroCell. Dev. Biol. Anim. 33, 375-380 (1997)), and magnetic levitation (Souza et al., Nat. Nanotechnol. 5, 291-296 (2010)). However, some of the most widely used non-adhesive techniques do not represent true 3D cell culture that mimics in vivo tumor formation. When tens of thousands of cells aggregate into spherical masses (i.e., spherical clumps) in structures such as hanging drops, reactors, or U-shaped trays, extensive central necrotic nuclei form within hours due to the lack of nutrient and oxygen permeation to a depth exceeding 200 μm. In true cancer, extensive central necrosis is a rare phenomenon. This non-physiologically related cancer presentation is exacerbated by the lack of progressive tumor development via cell division and the absence of interaction with the appropriate extracellular matrix (ECM). In some embodiments, the 3D cell culture system provided herein is able to maintain the size of cell spheroids / tumor-like structures at approximately 100 μm and induce them to divide into smaller spheroids as the cells continue to proliferate over time.

[0156] In some embodiments, the tumor-like structure has a diameter of about 50 μm to about 150 μm. In some embodiments, the tumor-like structure has a diameter of about 100 μm to about 150 μm. In some cases, the tumor-like structure has a diameter of about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, or about 150 μm. In some embodiments, after 8 days of culture, the average size of the tumor-like structure is about 150 μm.

[0157] In some embodiments, the culture 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 tumor-like structures cultured for more than 7 days is maintained in the range of about 50 μm to about 150 μm in diameter. In some cases, the diameter of the tumor-like structures is about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, or about 150 μm.

[0158] 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 tumor-like cells comprise tumor cells and tumor-associated cells. In some embodiments, the tumor-like cells are derived from a single cell.

[0159] In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some cases, the cancer is colorectal cancer, breast cancer, pancreatic cancer, head and neck cancer, bladder cancer, ovarian cancer, stomach cancer, or prostate cancer.

[0160] In some embodiments, the method of preparing patient-derived tumor-like structures provides tumor-like structures at a yield ratio (e.g., yield from CTCs) between 0.5 and 1. In some embodiments, the method of preparing patient-derived tumor-like structures provides tumor-like structures at a yield ratio (e.g., yield from CTCs) between 0.6 and 1. In some embodiments, the method of preparing patient-derived tumor-like structures provides tumor-like structures at a yield ratio (e.g., yield from CTCs) between 0.7 and 1. In some embodiments, the method of preparing patient-derived tumor-like structures provides tumor-like structures at a yield ratio (e.g., yield from CTCs) between 0.8 and 1.

[0161] In some embodiments, this document provides patient-derived tumor xenograft models prepared according to any of the methods employing the cell culture systems described herein. In some embodiments, the patient-derived tumor xenograft models are capable of spontaneous metastasis. In some embodiments, the xenograft models can be viable in vivo by injecting patient-derived tumor cells isolated from one xenograft model into another non-human animal.

[0162] In some cases, this article provides a method for analyzing the in vivo activity of a therapeutic agent (e.g., an anticancer drug), comprising administering the therapeutic agent (e.g., an anticancer drug) to a patient-derived tumor xenograft animal model prepared according to any of the methods described herein, and analyzing the effect of the therapeutic agent on the patient-derived tumor xenograft animal model.

[0163] In some cases, this article provides a biobank containing a plurality of different types of tumors prepared according to any of the methods employing the cell culture system described herein.

[0164] Generation of in vitro 3D co-cultures

[0165] In another embodiment, the present invention provides a method for generating 3D co-cultured tumor models derived from tumor cells and tumor-associated stromal or immune cells (e.g., fibroblasts, endothelial cells, and immune cells), particularly patient-derived 3D co-cultured tumor models derived from patient-derived tumor cells and tumor-associated stromal cells (e.g., fibroblasts, endothelial cells, and / or immune cells). These 3D co-cultured tumor models provide an improved simulation of the in vivo tumor microenvironment and can be used for basic research on the tumor microenvironment and for establishing in vitro drug screening models for cancer therapies and cancer immunotherapies.

[0166] In some embodiments, this document provides a method for generating a 3D co-cultured tumor model derived from tumor cells and tumor-associated stromal cells 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 of tumor-like cells comprising 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 tumor-like cells. In some embodiments, the method comprises aggregating tumor cells and tumor-associated stromal cells (e.g., by culturing a mixed cell population).

[0167] In some embodiments, the stromal cells comprise fibroblasts, endothelial cells, or mesenchymal stem cells. In some embodiments, the immune cells comprise bone marrow-derived suppressor cells (MDSCs), tumor-associated macrophages, neutrophils, tumor-infiltrating lymphocytes, T cells, B cells, dendritic cells, or any other tumor-associated immune cells.

[0168] In some embodiments, the 3D co-culture tumor models provided herein offer a valuable tool for studying the cytotoxic effects of anticancer drugs on normal cells. In some embodiments, the methods provided herein include assessing the effects of anticancer drugs on tumor cells (e.g., highly proliferating cells) and normal non-tumor cells in a co-culture.

[0169] Production of 3D cell cultures of hepatocytes

[0170] In another embodiment, the present invention provides a method for generating a 3D cell culture model of hepatocytes. 3D cell culture demonstrates superior liver-specific function compared to known 2D cell cultures in assessing hepatobiliary drug administration and drug-induced hepatotoxicity, attributed to the 3D model's reproduction of in vivo physiological conditions. 3D hepatocyte culture can be used for tissue engineering and drug development.

[0171] Once hepatocytes are isolated from the liver and grown in a known primary culture, the activity of these important enzymes is rapidly lost. This loss is particularly pronounced in rat hepatocytes, which lose 80% of their CYP activity within 24 hours before culture (Paine, AJ, In: Berry, MN et al. (eds.), The Hepatocyte Review, Kluwer Academic Publishers, Netherlands, pp. 411-420, 2000).

[0172] In some embodiments, this document provides a method for culturing hepatocytes on any of the 3D culture systems described herein, wherein the hepatocytes maintain the expression and function of one or more liver-specific genes of primary hepatocytes, such as albumin secretion, viral infectivity, and / or cytochrome 3P450 (CYP) enzyme activity. CYP is a family of enzymes located on the cytoplasmic side of the endoplasmic reticulum of hepatocytes, catalyzing the oxidation of organic compounds, increasing water solubility, thereby promoting cellular excretion. In some embodiments, hepatocytes cultured on any of the 3D culture systems described herein maintain the expression and function of one or more genes involved in normal drug metabolism for a culture period of at least 5 days, at least 7 days, at least 10 days, at least 14 days, or at least 21 days.

[0173] In some embodiments, the 3D cell culture platform is used to culture primary hepatocytes. In some embodiments, the primary hepatocytes are isolated from the livers of humans or other mammals via biopsy. In some embodiments, the 3D cell culture platform is used to culture fetal hepatocytes.

[0174] In some embodiments, the 3D cell culture platform is used to culture immortalized cell lines. In some embodiments, the cell lines express one or more phase 1 / II xenobiotic drug metabolism genes and / or hepatocyte-specific transcripts. In some embodiments, 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 culture 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.

[0175] In some embodiments, this document provides a method for culturing hepatocytes using any of the 3D cell culture systems described herein, wherein the hepatocytes maintain liver function in vitro, such as expressing key cytochrome P450 (CYP) drug-metabolizing enzymes. In some embodiments, the hepatocytes maintain liver function for 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.

[0176] When hepatocytes are cultured using known 3D culture systems, hypoxia at the center of spheroids larger than 200 μm leads to the formation of necrotic nuclei within the spheroids (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): 263-73). In some embodiments, the 3D cell culture system provided herein is able to maintain the size of cell spheroids (e.g., hepatocyte spheroids) at approximately 100 μm and induce them to divide into smaller spheroids as the cells continue to proliferate over time.

[0177] In some embodiments, the diameter of the cell spheroids is from about 50 μm to about 150 μm. In some embodiments, the diameter of the cell spheroids is from about 100 μm to about 150 μm. In some cases, the diameter of the cell spheroids is about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, or about 150 μm. In some embodiments, the average size of the cell spheroids is 150 μm after 8 days of culture. In some embodiments, the size of the cell spheroids cultured for more than 7 days remains in the range of about 50 μm to about 150 μm.

[0178] In some embodiments, the method comprises culturing hepatocytes and non-parenchymal cells (NPCs) on any of the 3D culture systems provided herein. In some embodiments, non-parenchymal cells include bile duct epithelial cells, hepatic sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and / or Kupffer 8 cells (KCs).

[0179] In another embodiment, the present invention provides a method for evaluating the metabolism of a drug by which mammalian hepatocytes are metabolized in vivo, comprising (a) culturing mammalian hepatocytes using any of the 3D culture systems described herein; (b) adding the drug to be evaluated to the hepatocyte culture in a culture vessel for a period of time sufficient to allow enzymes in the hepatocytes to metabolize the drug and convert it into one or more metabolites; and (c) identifying the presence of or measuring the concentration of the one or more metabolites in the culture medium or cells of the culture, thereby evaluating the metabolism of the drug.

[0180] Production of live in vitro stem cell spheroids

[0181] In another embodiment, the present invention provides a method for generating stem cell spheroids in vitro. The stem cells described herein include 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 therapy, tissue engineering, high-throughput pharmacology screening, and toxicity testing. These applications require large quantities of high-quality cells; however, scalable production of MSCs and PSCs has been a challenge. The 3D culture system provided herein allows for efficient stem cell proliferation and differentiation of MSCs and PSCs.

[0182] In some embodiments, this document provides a method for 3D cell culture using any of the 3D culture systems provided herein, wherein the method increases the stem cell characteristics and / or proliferation rate of the 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.

[0183] In some embodiments, the method increases the expression of one or more stem cell characteristic-related genes. For example, cell surface CD133 represents one of the biomarkers for stem cell characterization and is associated with a variety of cellular characteristics, such as stem cell characteristics, regeneration, differentiation, and metabolism of different cell lineages. In some embodiments, the method increases the expression of CD133 in cells. In some embodiments, the method induces CD133 expression in a CD133 cell population.

[0184] In some embodiments, the method maintains the expression of one or more stem cell characteristic-related genes during culture. In some embodiments, the culture period is at least 7 days, at least 10 days, at least 14 days, or at least 21 days.

[0185] Set

[0186] In some embodiments, this document discloses a kit or article of manufacture comprising the 3D cell culture system described herein. In some cases, the kit is used to isolate single-cell-derived pure lines. In some cases, the kit further comprises a package or container partitioned to receive one or more containers (such as vials, tubes, and the like), each container containing one of the various elements to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed of a variety of materials, such as glass or plastic.

[0187] In some cases, the kit further includes a label listing the contents and / or instructions for use, and a packaging insert with instructions for use, such as instructions for culturing heterogeneous cell populations using the 3D culture system described herein to obtain multiple cell lines containing single-cell-derived pure lines. A set of instructions will also typically be included.

[0188] Specific terms

[0189] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that the foregoing general description and the following detailed description are illustrative and explanatory only and do not limit any of the claimed subject matter. In this application, the singular is used to include plural unless otherwise specifically stated. It must be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “described” as used in this specification and the appended claims include plural indicators. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0190] As used herein, ranges and quantities may be expressed as “about” a specific value or range. “About” also includes exact quantities. Therefore, “about 5 μL” means “about 5 μL” and also “5 μL”. Generally, the term “about” includes quantities expected to be within experimental error.

[0191] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0192] As used herein, the term "comprising" is intended to mean that a method includes the described steps or elements, but does not exclude others. "consisting substantially of" is intended to mean that the scope of the claim is open only to including steps or elements that do not materially affect the fundamental and novel features of the claimed method. "Constitutes of" is intended to exclude any elements or steps not specified in the scope of the claim. Embodiments defined by each of these transitional terms are within the scope of this invention.

[0193] As used herein, the term "positively charged polyelectrolyte" encompasses a plurality of monomer units or nonpolymeric molecules containing two or more positively charged groups. In some cases, a positively charged polyelectrolyte may also encompass a plurality of monomer units or nonpolymeric molecules containing positively charged, neutrally charged, or negatively charged groups, with a net positive charge.

[0194] As used herein, the term "cationic polymer" encompasses multiple monomer units or non-polymerized molecules. In some cases, cationic polymers are synthetic polymers. In other cases, cationic polymers are natural polymers.

[0195] As used herein, the term "cationic polypeptide" refers to a polypeptide containing two or more positively charged residues. In some cases, a cationic polypeptide contains positively charged amino acid residues, negatively charged residues, and polar residues, but the net charge of the polypeptide is positive. In some cases, the length of a cationic polypeptide is 8 to 100 amino acids. In some cases, the length of a cationic polypeptide is 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.

[0196] As used herein, the term "negatively charged polyelectrolyte" encompasses a plurality of monomer units or nonpolymeric molecules containing two or more negatively charged groups. In some cases, negatively charged polyelectrolytes also encompass a plurality of monomer units or nonpolymeric molecules containing positively charged, neutrally charged, or negatively charged groups, with a net negative charge.

[0197] As used herein, the term "anionic polymer" encompasses multiple monomer units or nonpolymeric molecules. In some cases, anionic polymers are synthetic polymers. In others, anionic polymers are natural polymers.

[0198] As used herein, the term "anionic polypeptide" refers to a polypeptide containing two or more negatively charged residues. In some cases, anionic polypeptides contain positively charged amino acid residues, negatively charged residues, and polar residues, but the net charge of the polypeptide is negative. In some cases, the length of anionic polypeptides is 8 to 100 amino acids. In some cases, the length of anionic polypeptides is 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.

[0199] As used herein, the term "hydrophilic polymer" encompasses a plurality of monomeric units or nonpolymeric molecules containing one or more hydrophilic groups. In some cases, hydrophilic polymers are permeable to aqueous solutions. In others, hydrophilic polymers are impermeable or do not absorb aqueous solutions. In some cases, hydrophilic polymers encompass nonreactive polymers, or polymers that do not contain reactive groups (e.g., groups that form covalent bonds with another compound).

[0200] As used herein, the term “polymer” includes homopolymers and copolymers, branched and unbranched polymers, and natural or synthetic polymers.

[0201] As used herein, the term "article" refers to cell culture articles, such as sheets, membranes, tubes, trays, dishes, or biomedical devices. In some cases, a biomedical device is any article designed for use in or on tissues (e.g., mammalian tissues) or body fluids, preferably in or on human tissues or body fluids. Exemplary devices include, but are not limited to, cell culture dishes, cell culture trays, bioreactors, and the like.

[0202] As used in this article, immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells).

[0203] Endothelial cells are cells that line the inner surface of blood vessels and lymphatic vessels. Exemplary endothelial cells include high endothelial venules (HEVs) in bone marrow and brain endothelium.

[0204] Epithelial cells are cells that line the outer surface of organs and blood vessels, as well as the inner surface of the lumens of internal organs. Exemplary epithelial cells include squamous epithelial cells, cubic epithelial cells, and columnar epithelial cells.

[0205] As used herein, the term "stem cell" encompasses both adult stem cells and embryonic stem cells. 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).

[0206] As used herein, the term "chemically defined culture medium" refers to a live in vitro culture medium in which all chemical components are known. Chemically defined culture media may include basal media (such as DMEM, F12, or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) supplemented with recombinant albumin, chemically defined lipids, recombinant insulin and / or zinc, recombinant transferrin or iron, selenium, and antioxidant thiols, such as 2-mercaptoethanol or 1-thioglycerol.

[0207] As used in this article, the term "enriched medium" refers to an in vitro culture medium in which the basal medium is further supplemented with growth factors, vitamins and essential nutrients.

[0208] As used herein, the terms “semi-attached” and “loosely attached” are used interchangeably, and in relation to cultured cells, it refers to cells that can be separated from the matrix surface by gentle agitation or gentle mechanical force. In some cases, cells can be separated without the need for cell dissociation enzymes.

[0209] As used herein, the term "single-cell-derived spheroid" refers to a cluster of cells grown in vitro and formed in a 3D manner, the cluster being formed by the growth of single cells placed on a surface coating.

[0210] In some embodiments, therapeutic agents include, but are not limited to, chemotherapeutic agents, immune checkpoint inhibitors, nucleic acid agents, therapeutic cell compositions, or combinations thereof.

[0211] In some embodiments, the therapeutic agent is a cytotoxic or cell-inhibiting chemotherapeutic agent. The chemotherapeutic agent may be an alkylating agent (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), an antimetabolite (such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosine), etc. Arabinoside, fludarabine, and fluxuridine; antimitotic agents (including taxanes such as paclitaxel and decetaxel) and vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine); anthracyclines (including doxorubicin, daunorubicin, and valrubicin). Actinomycin D, idarubicin, and epirubicin, as well as actinomycin D, cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), topoisomerase inhibitors (including camptothecin, irinotecan, and topootecan, and derivatives of podophyllotoxin, such as amsacrine, etoposide, etoposide phosphate, and teniposide), and antibodies against vascular endothelial growth factor (VEGF), such as bevacizumab. Other anti-VEGF compounds; anti-PD-1 (anti-planned death-1) therapeutics, such as antibodies or compounds (e.g., nivolumab); thalidomide. ) and its derivatives, such as lenalidomide, Endothelial somatostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors, such as sunitinib. Tyrosine kinase inhibitors, such as sorafenib; ), erlotinib ), pazopanib, axitinib, and lapatinib; inhibitors of transforming growth factor-α or transforming growth factor-β and antibodies against epidermal growth factor receptors, such as panitumumab, ) and cetuximab ).

[0212] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors may be CD137, CD134, PD-1, KIR, LAG-3, PD-L1, 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, GAL9, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK1, CHK2, A2aR, TGF-β, PI3Kγ, GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG, CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, CD40, and combinations thereof.

[0213] In some embodiments, the therapeutic agent is a nucleic acid drug. The nucleic acid drug may be DNA, DNA plasmid, nDNA, mtDNA, gDNA, RNA, siRNA, miRNA, mRNA, piRNA, antisense RNA, snRNA, snoRNA, vRNA, and combinations thereof. In some embodiments, the therapeutic nucleic acid is a DNA plasmid containing nucleotide sequences encoding genes selected from the following groups: GM-CSF, IL-12, IL-6, IL-4, IL-12, TNF, IFNy, IFNa, and combinations thereof.

[0214] 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.

[0215] In some embodiments, the therapeutic agent is a therapeutic antigen-binding molecular composition. Exemplary therapeutic antigen-binding molecular compositions include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, scFV, Fab, VHH / VH, etc.

[0216] In some cases, treatment options include first-line therapy. As used in this article, "first-line therapy" refers to the initial treatment of an individual with cancer. In some cases, the cancer is primary. In others, the cancer is metastatic or recurrent. In some cases, first-line therapy includes chemotherapy. In others, it includes radiation therapy. Those familiar with this technique will easily understand that different first-line treatments can be applied to different types of cancer.

[0217] In some cases, treatments include second-line, third-line, or fourth-line therapies.

[0218] As used herein, the terms “individual(s)”, “subject(s)”, and “patient” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of these terms require or limit a situation characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, caregiver, or hospice worker).

[0219] Example

[0220] These examples are provided for illustrative purposes only and do not limit the scope of the patent applications presented herein. Example 1: Construction of the surface coating of the present invention

[0221] (i) PVA or PEG coated polystyrene discs

[0222] First, the tissue culture discs made of polystyrene plastic are treated by exposing the polystyrene discs to plasma gas to modify the hydrophobic plastic surface, making it more hydrophilic. Then, a hydrophilic polymer (such as PVA or PEG) is deposited on the modified surface of the polystyrene discs to form PVA or PEG-coated polystyrene discs.

[0223] (ii) PVA crosslinked polystyrene discs

[0224] First, tissue culture discs made of polystyrene plastic were treated: the polystyrene discs were exposed to ozone plasma to modify the hydrophobic plastic surface, making it more hydrophilic. Subsequently, photoactivated azidophenyl-PVA was added to the plasma-treated surface of the polystyrene discs to form PVA-crosslinked polystyrene discs. Figure 5 (As shown in the figure). Azide-phenyl-derived poly(vinyl alcohol) (AzPh-PVA) can be synthesized by coupling the -OH group of PVA with 4-azidobenzoic acid, as reported (J. Nanosci. Nanotechnol., 2009, 9, 230-239).

[0225] (iii) PVA crosslinked PTFE disc

[0226] First, tissue culture discs made of polytetrafluoroethylene (PTFE) are treated: the PTFE discs are exposed to plasma gas to modify the hydrophobic plastic surface, making it more hydrophilic. Subsequently, a hydrophilic polymer (e.g., PVA or PEG) is deposited onto the modified surface of the PTFE discs. The PVA is then crosslinked to the PTFE using the crosslinking agent glutaraldehyde (GA) to form PVA-crosslinked PTFE discs. Figure 6 (as shown in the image).

[0227] Polyelectrolyte multilayer stacking

[0228] PLL (MW 150K-300K), PLGA (MW 50K-100K), PLO (0.01%) solution, PLH (MW 5K-25K), and PLA (MW 15K-70K) were available from Sigma-Aldrich (St. Louis, MO, USA). Both polycationic and polyanionic polymers were dissolved in Tris-HCl buffer (pH 7.4) and deposited onto PVA or PEG-coated surfaces after rinsing with Tris-HCl buffer. Each polycationic or polyanionic layer was deposited and incubated for 10 minutes, followed by three washes with Tris-HCl buffer for 2, 1, and 1 minutes respectively. PLL / PLGA, PLO / PLGA, PLH / PLGA, and PLA / PLGA multilayer films can be fabricated by layer-by-layer self-assembly on PVA or PEG-coated surfaces.

[0229] PLL / PLGA multilayer

[0230] In some embodiments, the polyelectrolyte multilayer is a PLL / PLGA multilayer, which can be constructed by sequentially depositing PLL and PLGA on the surface of (i) a PVA or PEG-coated polystyrene disk, (ii) a PVA-crosslinked polystyrene disk, or (iii) a PVA-crosslinked PTFE disk. Each deposition step includes adding a PLL or PLGA solution to the disk surface, incubating for 10 minutes and washing three times for 2, 1, and 1 minutes respectively.

[0231] In one embodiment, a surface coating composed of (PLGA / PLL)3 / PVA is constructed. In another embodiment, a surface coating composed of (PLGA / PLL)5 / PVA is constructed. In yet another embodiment, a surface coating composed of (PLGA / PLL)... 10 A surface coating composed of PVA. In one embodiment, a coating composed of (PLGA / PLL) is constructed. 15 A surface coating composed of PLL (PLGA / PLL)3 / PVA is constructed. In one embodiment, a surface coating composed of PLL (PLGA / PLL)5 / PVA is constructed. In another embodiment, a surface coating composed of PLL (PLGA / PLL)5 / PVA is constructed. 10 A surface coating composed of PLL (PLGA / PLL) is used in one embodiment. 15 A surface coating composed of PVA.

[0232] PLO / PLGA multilayer

[0233] In some embodiments, the polyelectrolyte multilayer is a PLO / PLGA multilayer, which can be constructed by sequentially depositing PLO and PLGA on the surface of (i) a PVA or PEG-coated polystyrene disk, (ii) a PVA-crosslinked polystyrene disk, or (iii) a PVA-crosslinked PTFE disk. Each deposition step includes adding a PLO or PLGA solution to the disk surface, incubating for 10 minutes and washing three times for 2, 1, and 1 minutes respectively.

[0234] In one embodiment, a surface coating composed of (PLGA / PLO)3 / PVA is constructed. In another embodiment, a surface coating composed of (PLGA / PLO)5 / PVA is constructed. In yet another embodiment, a surface coating composed of (PLGA / PLO)3 / PVA is constructed. 10 A surface coating composed of PVA. In one embodiment, a coating composed of (PLGA / PLO) is constructed. 15A surface coating composed of PLO(PLGA / PLO)3 / PVA is constructed in one embodiment. In another embodiment, a surface coating composed of PLO(PLGA / PLO)5 / PVA is constructed. In yet another embodiment, a surface coating composed of PLO(PLGA / PLO)3 / PVA is constructed. 10 A surface coating composed of PVA is used in one embodiment. 15 A surface coating composed of PVA.

[0235] PLH / PLGA multilayer

[0236] In some embodiments, the polyelectrolyte multilayer is a PLH / PLGA multilayer, which can be constructed by sequentially depositing PLH and PLGA on the surface of (i) a PVA or PEG-coated polystyrene disk, (ii) a PVA-crosslinked polystyrene disk, or (iii) a PVA-crosslinked PTFE disk. Each deposition step includes adding a PLH or PLGA solution to the disk surface, incubating for 10 minutes and washing three times for 2, 1, and 1 minutes respectively.

[0237] In one embodiment, a surface coating composed of (PLGA / PLH)3 / PVA is constructed. In another embodiment, a surface coating composed of (PLGA / PLH)5 / PVA is constructed. In yet another embodiment, a surface coating composed of (PLGA / PLH)3 / PVA is constructed. 10 A surface coating composed of PVA. In one embodiment, a coating composed of (PLGA / PLH) is constructed. 15 A surface coating composed of PLH(PLGA / PLH)3 / PVA is constructed. In one embodiment, a surface coating composed of PLH(PLGA / PLH)5 / PVA is constructed. In another embodiment, a surface coating composed of PLH(PLGA / PLH)3 / PVA is constructed. 10 A surface coating composed of PVA is used in one embodiment. In another embodiment, a PLH (PLGA / PLH) coating is constructed. 15 A surface coating composed of PVA.

[0238] PLA / PLGA multilayer

[0239] In some embodiments, the polyelectrolyte multilayer is a PLA / PLGA multilayer, which can be constructed by sequentially depositing PLA and PLGA on the surface of (i) a PVA or PEG-coated polystyrene disk, (ii) a PVA-crosslinked polystyrene disk, or (iii) a PVA-crosslinked PTFE disk. Each deposition step includes adding a PLA or PLGA solution to the disk surface, incubating for 10 minutes and washing three times for 2, 1, and 1 minutes respectively.

[0240] In one embodiment, a surface coating composed of (PLGA / PLA)3 / PVA is constructed. In another embodiment, a surface coating composed of (PLGA / PLA)5 / PVA is constructed. In yet another embodiment, a surface coating composed of (PLGA / PLA)3 / PVA is constructed. 10 A surface coating composed of PVA / PLA is constructed in one embodiment. 15 A surface coating composed of PLA (PLGA / PLA)3 / PVA is constructed. In one embodiment, a surface coating composed of PLA (PLGA / PLA)5 / PVA is constructed. In another embodiment, a surface coating composed of PLA (PLGA / PLA)5 / PVA is constructed. 10 A surface coating composed of PLA (PLA / PVA) is constructed in one embodiment. 15 A surface coating composed of PVA.

[0241] Dissipative Quartz Crystal Microbalance (QCM-D) Measurement

[0242] QCM experiments were conducted in a Q-Sense E4 (Biolin Scientific AB / Q-Sense, Sweden). The silica (SiO2)-coated quartz crystal chip (AT-cut quartz crystal, f0 = 5 MHz) was cleaned in 0.1 M sodium dodecyl sulfate, followed by rinsing with Milli-Q water, drying under nitrogen, and exposure to oxygen plasma for 20 seconds. For QCM-D measurements, the chamber was stabilized at 25°C, and all measurements were recorded at the third harmonic (15 MHz). To simulate continuous surface coating, the concentration and washing conditions were identical for each coating step in the QCM-D chamber. Approximately 1% bovine serum albumin (BSA, Millipore, Bedford, MA) was used for nonspecific adsorption studies and introduced onto the surface within the chamber.

[0243] Surface chemical analysis

[0244] The chemical composition of the surface coating of the present invention is obtained by performing C on a silicon wafer. 60 The analysis was performed using X-ray photoelectron spectroscopy (XPS; VersaProbe III, PHI) on a 10kV, 10nA etching layer. The pass voltage was 93.9 eV, and the step size was 0.5 eV. The relative atomic concentrations of carbon, nitrogen, oxygen, and silicon in the sample layer were measured, with a maximum thickness of 10 nm.

[0245] Surface roughness was measured using atomic force microscopy (AFM).

[0246] The surface roughness of the coating of this invention was measured using an atomic force microscope (AFM; Nanowizard 3, JPK instrument) in tapping mode. A silicon cantilever with a resonant frequency of 134 kHz was used in the experiment.

[0247] Example 2: Formation of single-cell-derived spheroids

[0248] Figure 7 The images show time-lapse microscopy observations of HCT116 colorectal cancer cells cultured on the surface coating of this invention at days 0, 1, 2, 3, 4, and 5 of the cancer cell growth process in a fully DMEM culture medium. (Images were taken with a Leica DMI6000B time-lapse microscope at 10x objective).

[0249] Example 3: Generation of cell line-derived tumor spheroids

[0250] The surface coating of this invention provides a biocompatible multilayer coating surface that enables cell adhesion for cell proliferation and also provides a non-fouling feature for the direct formation of spheroids on the surface. Cell culture systems incorporating the surface coating of this invention were tested with various cancer cell lines, resulting in successful culture and formation of spheroids derived from various cancer cell lines. Figures 8A-E (as shown in the image). Figures 8A-E This document demonstrates the results of in vitro culture and 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 cell carcinoma cell line SAS, ovarian cancer cell line SK-OV-3, and cell line T24 derived from human bladder cancer patients on the culture system of this invention. These cancer cells were grown for 7 to 14 days on the culture system of this invention (with a cell number of approximately 1000). Example 4: Generation of CTC-derived tumor spheroids

[0251] The cell culture system incorporating the surface coating of this invention was tested with various patient-derived CTCs, and the result was successful culture and formation of spheroids derived from patient-derived CTCs. Figure 9A-C (as shown in the image). Figure 9A-CRepresentative time-dependent images showcasing the culture of CTC-derived spheroids on the culture platform of this invention. (A) CTCs isolated from blood samples of breast cancer patients; CTC-derived spheroids formed after 14 days. (B) CTCs isolated from blood samples of head and neck cancer patients; CTC-derived spheroids formed after 38 days. (C) CTCs isolated from blood samples of colorectal cancer patients; CTC-derived spheroids formed after 13–27 days. Scale bar: 50 μm.

[0252] The resulting spheroids can further benefit future diagnosis and guidance in medical treatment and applications, such as: non-invasive early cancer detection, personalized medication guidance, pre- and post-treatment drug resistance surveys, cell activity assessment for immune cell-based cancer therapies, and provide a wealth of material to elucidate the mechanisms involved in cancer progression by using in vitro cultured patient-derived primary CTC cells.

[0253] Example 5: Formation of tissue-derived tumor spheroids

[0254] Primary tissue cells derived from primary xenografts in animal models were cultured for 7 days on the cell culture system of this invention. Figure 10A-B Images of tumor spheroids derived from primary colorectal tumor tissue obtained from a patient with colorectal cancer (CRC) are shown. Tumor spheroids were generated on the culture platform of this invention after 2 and 4 weeks. The results indicate that the cell culture platform of this invention can generate tumor-like structures from primary tissue cells.

[0255] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will arise for those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims are intended to define the scope of the invention and therefore encompass the methods and structures within the scope of these claims and their equivalents.

Claims

1. A composition for coating cell culture products, said composition comprising: a) A polymer selected from the group consisting of: poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), PEG-acrylate, polyvinylpyrrolidone (PVP), poly-L-lactide (PLLA), poly-D-lactide (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (p-HEMA), and derivatives thereof, wherein the polymer is cross-linked to the surface of the cell culture article, and b) A polyelectrolyte multilayer comprising polycations and polyanions, wherein the polycation is a poly(amino acid) and the polyanion is a poly(amino acid), wherein the polymer is in direct contact with the polycations or polyanions of the polyelectrolyte multilayer.

2. The composition of claim 1, wherein the polymer is PVA, PEG, PEG-acrylate, PVP, PMMA or a derivative thereof.

3. The composition of claim 1, wherein the polymer is PVA, PEG, or PEG-acrylate.

4. 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 combinations thereof.

5. The composition of claim 1, wherein the polyanion is poly(L-glutamic acid) (PLGA), poly(L-aspartic acid) (PLAA), or a combination thereof.

6. The composition of claim 1, wherein the polyelectrolyte multilayer comprises at least one layer pair consisting of a polycation and a polyanion (polycation / polyanion), wherein the layer pair is selected from the group consisting of: PLL / PLGA, PLL / PLAA, PLA / PLGA, PLA / PLAA, PLO / PLGA, PLO / PLAA, PLH / PLGA, PLH / PLAA, and combinations thereof.

7. The composition of claim 1, wherein the polyelectrolyte multilayers are formed by layer-by-layer assembly.

8. The composition of claim 1, wherein the polyelectrolyte multilayer comprises n layer pairs, wherein n is an integer in the range of 1 to 30, and wherein the outermost layer is a polycationic or polyanionic layer.

9. The composition of claim 1, wherein the polyelectrolyte multilayer comprises n (polycationic / polyanionic) layer pairs and an additional polyanionic layer, wherein n is an integer in the range of 1 to 30, and wherein the outermost layer is polyanionic.

10. The composition of claim 1, wherein the polyelectrolyte multilayer comprises n (polyanionic / polycationic) layer pairs and an additional polycationic layer, wherein n is an integer in the range of 1 to 30, and wherein the outermost layer is polycationic.

11. A method for preparing a composition according to any one of claims 1 to 10, the method comprising the following steps: a) Provide cell culture products with hydrophobic surfaces, b) Modifying the hydrophobic surface by treatment, wherein the treatment is plasma treatment, corona discharge, UV ozone treatment, or hydrosilylation. c) Crosslinking the polymer to the modified surface, wherein the polymer is selected from the group consisting of: PVA, PEG, PEG-acrylate, PVP, PLLA, PDLA, PLDLLA, PGA, PL-co-GA, PMMA, and p-HEMA. d) Sequentially depositing alternating layers of polycationic and polyanionic polymers on the polymer to form a composition as claimed in any one of claims 1 to 10.

12. The method of claim 11, wherein the polymer is PVA, PEG, or PEG-acrylate.

13. A cell culture system comprising a cell culture article having a surface coated with a composition of any one of claims 1 to 10.

14. The cell culture system of claim 13, further comprising cells and / or culture medium.

15. A method for culturing cells, the method comprising: a) Providing a cell culture article with a surface coated with the composition of claim 1, b) Inoculate cells onto the coated surface, and c) Culture these cells in a suitable culture medium for a sufficient period of time to form one or more spheroids.

16. The method of claim 15, wherein the one or more spherical bodies are produced via single-cell proliferation.

17. The method of claim 15 or 16, wherein the average diameter of the one or more spheres is between 50 μm and 150 μm.

18. The method of claim 15 or 16, wherein the inoculation comprises inoculating the cells onto a substrate at a density of less than 1,000 cells per square centimeter.

19. A method for obtaining and, as appropriate, characterizing single-cell-derived spheroids, the method comprising: a) Culturing a heterogeneous cell population on a surface coated with the composition of claim 1 to obtain at least one single-cell-derived spheroid disposed on said surface, and b) As appropriate, analyze the single-cell-derived spheroids to obtain at least one characteristic of the single cell.

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