TW202330917A - Cell culture platforms, methods and uses thereof - Google Patents

Abstract

The present disclosure provides methods of preparing cell cultures (e.g. tumor spheroids), methods of evaluating a therapeutic agent for cancer, and methods of cancer treatment.

Description

Cell culture platform, method and use thereof

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

There are many differences in the formation of spheroids using 3D cell culture methods and commercially available products, and this may affect their readout. For example, non-adhesive techniques widely used in 3D cell culture, including ultra-low attachment (ULA) plates and hanging drop methods, have proven inappropriate because these methods typically generate spheroids through cell aggregation . Such spheroids often maintain their original heterogeneity and contain multiple cells with different characteristics, which will require a better understanding of cellular heterogeneity. When tens of thousands of cells aggregate into spheroids (ie, masses with a spherical shape), extensive central necrotic nuclei develop within hours due to lack of nutrient and oxygen penetration at depths greater than 200 µm , and thus hinder cell proliferation. Extensive central necrosis is a rare phenomenon in true cancer.

In addition, Matrigel is an embedding substrate commonly used for tissue-based cell growth, such as organoid formation. However, out-of-focus sample observation, inefficient compound diffusion, and difficulty in sample separation limit its application in ex vivo based on 3D spheroids.

Standardizing spheroid formation is critical to generating uniform 3D cell cultures and obtaining reproducible results from spheroid-based assays for drug screening and treatment guidance in cancer patients. Currently, techniques and methods for reliably culturing primary cancer cells, and in particular circulating tumor cells, from the majority of patients remain a challenge.

Therefore, there is a need to develop improved cell culture platforms and methods that can reliably generate 3D cell cultures from cancer cells, in particular circulating tumor cells, extracted from blood samples of cancer patients.

The present invention provides improved cell culture platforms and methods for preparing cell cultures, in particular 3D cell cultures (eg, spheroids). The platforms disclosed herein comprise cell culture articles having surfaces coated with polyelectrolyte multilayers (PEM) and optionally absorbent polymers.

The present invention also relates to the use of cell cultures prepared using the cell culture platform disclosed herein, in particular, cancer cell cultures (eg, tumor spheroids), for use in in vitro ( in vitro ) medicine Screening and evaluation of cancer therapeutics. Cancer treatments are also provided.

Accordingly, one aspect of the invention provides a method for preparing a cell culture (e.g., a 3D culture), wherein the method comprises the steps of: (a) providing a cell culture article having a polyelectrolyte-coated multilayer and optionally the surface of the selected absorbent polymer; (b) seeding the surface with a plurality of cancer cells; and (c) culturing the plurality of cancer cells in a suitable medium for a time sufficient to produce a cell culture, wherein the cell culture Contains 3D cell cultures comprising a plurality of tumor spheroids adhered to a coated surface.

In some embodiments, the plurality of cancer cell lines are obtained from a body fluid sample of a cancer patient. Body fluid samples may be serum, plasma, whole blood, urine or ascitic fluid. In some embodiments, the plurality of cancer cells may comprise circulating tumor cells, wherein the circulating tumor cells comprise cancer cells from solid tumors.

In some embodiments, each tumor spheroid system is generated via single cell proliferation.

In some embodiments, the average diameter of each tumor spheroid is about 50 μM to about 150 μM.

In some embodiments, seeding in step (b) comprises spreading cells onto the substrate at a density of less than 1000 cells/cm2.

In some embodiments, the coated surfaces described herein comprise polyelectrolyte multilayers and absorbent polymers. Exemplary absorbent 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(ethanol acid) (PGA), poly(lactic-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (p-HEMA) and its derivatives.

In some embodiments, suitable media described herein comprise a Rho-associated protein kinase (ROCK) inhibitor. In some embodiments, the structural formula of the ROCK inhibitor has an isoquinoline, 4-amidopyridine or 4-amidopyrrolopyridine skeleton.

In another aspect, the invention provides a method for evaluating a cancer therapeutic, wherein the method comprises the steps of: (a) preparing cell cultures (eg, tumor spheroids) according to the methods described above; ( b) optionally culturing the cell culture with a plurality of immune cells; (c) contacting the cell culture with a therapeutic agent; (d) evaluating the effect of the therapeutic agent on the cell culture; and (e) when the therapeutic agent acts on the cells The cancer patient is determined to be responsive to the therapeutic agent when the culture is effective, or non-responsive to the therapeutic agent when the therapeutic agent is ineffective on the cell culture.

The effect of therapeutic agents on cell cultures can be determined by performing luminescence and/or fluorescence-based cell-based and/or biochemical assays. In some embodiments, the effect of a therapeutic agent on a cell culture is determined by performing a luminescence-based cell viability assay. In some embodiments, the effect of a therapeutic agent can be determined by performing one or more assays for measuring the size, morphology, physical properties, biological and/or kinetic properties. The results of the assay can provide treatment guidelines for cancer patients.

In some embodiments, the immune cells described herein comprise autologous immune cells from peripheral blood of a cancer patient. In some embodiments, the autologous immune cells comprise ex vivo expanded autologous immune cells.

The therapeutic agents described herein may include, but are not limited to, chemotherapeutic drugs, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, and combinations thereof.

In some embodiments, the therapeutic agent is an immune checkpoint inhibitor, and the cell culture is incubated with a plurality of immune cells (eg, autologous immune cells from peripheral blood). The immune checkpoint inhibitors described herein can be PD-1 inhibitors, PD-L1 inhibitors or CLTA-4 inhibitors. Examples of immune checkpoint inhibitors may include, but are not limited to, nivolumab, pembrolizumab, cemiplimab, atezolizumab , avelumab, durvalumab and ipilimumab.

In some embodiments, the therapeutic agent is a therapeutic cellular composition. In some embodiments, the therapeutic cell compositions described herein are T cells, natural killer (NK) or dendritic cells. In some embodiments, the therapeutic cell composition is a chimeric antigen receptor T (CAR-T) cell or a chimeric antigen receptor-natural killer (CAR-NK) cell.

In some embodiments, the therapeutic agent comprises one or more chemotherapeutic drugs. One or more chemotherapeutic drugs described herein may be cytotoxic or cytostatic chemotherapeutic drugs.

In some embodiments, the therapeutic agent comprises a nucleic acid drug.

In some embodiments, cell cultures (eg, 3D cultures) can be in direct contact with the outermost layers of the polyelectrolyte multilayers described herein. The outermost layer can be polycationic or polyanionic. In some embodiments, the polycation is selected from the group consisting of poly(L-lysine) (PLL), poly(L-arginine) (PLA), poly(L-ornithine) (PLO ) or poly(L-histidine) (PLH) and combinations thereof. In some embodiments, the polyanion can be poly(L-glutamic acid) (PLGA) or poly(L-aspartic acid) (PLAA).

In some embodiments, the polyelectrolyte multilayer comprises n bilayers, where n is an integer ranging from 1 to 30, and wherein the outermost layer is a polycation or polyanion. The substrate of technical scheme 16, wherein the polyelectrolyte multilayer comprises n polycations/polyanions and additional polyanion layers, wherein n is an integer in the range of 1 to 30, and wherein the outermost layer is polycation or polyanion anion.

In another aspect, the present invention provides a method for treating cancer, wherein the method comprises the steps of: (a) evaluating a therapeutic agent for a cancer patient according to the methods described herein; A therapeutically effective dose of a therapeutic agent is administered to a cancer patient who responds to the drug.

The therapeutic agents described herein can be one or more chemotherapeutic drugs, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, or combinations thereof.

Cross-reference to related applications

This application claims priority and rights to U.S. Provisional Patent Application Serial No. 63/252,268, filed October 05, 2021, the disclosure of which is incorporated herein by reference in its entirety.

The present invention provides improved cell culture platforms and methods for preparing cell cultures, in particular 3D cell cultures (eg, spheroids). The surfaces described herein can induce the formation of 3D cell cultures, making it possible to cultivate 3D cell cultures of certain primary cells (such as CTCs) that are difficult to proliferate with other commercially available cell culture methods. Currently, techniques and methods for reliably culturing CTCs from a majority of patients remain a challenge. The present invention provides improved platforms and methods for achieving reliable and consistent expansion of CTCs. When CTC cultures can be successfully generated, it will provide valuable insight into the metastatic process and treatment response of individual patients. Cell culture articles of the present invention (Cell Culture Articles)

The platforms disclosed herein comprise cell culture articles having surfaces coated with polyelectrolyte multilayers (PEM) and optionally absorbent polymers. In some embodiments, the surface is coated with PEM. In some embodiments, the surface is coated with PEM and absorbent polymer. The PEMs disclosed herein comprise multiple alternating layers of oppositely charged polymers (ie, polyelectrolytes). The oppositely charged polymers described herein comprise combinations of positively charged polyelectrolytes (also referred to herein as polycations) and negatively charged polyelectrolytes (also referred to herein as polyanions).

Exemplary polycations include, but are not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA), poly(L-ornithine) (PLO), poly(L- Histidine) (PLH), polyethyleneimine (PEI), poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)methacrylate Ethyl ester (DMAEMA), N,N-diethylaminoethyl methacrylate (DEAEMA), and combinations thereof. In some cases, the polycation based PLL. In some cases, the polycation is PLO. In some cases, the polycation is PLH. In some cases, the polycation is PLA.

Exemplary polyanions include, but are not limited to, poly-L-glutamic acid (PLGA), poly-L-aspartic acid (PLAA), poly(acrylic acid), poly(methacrylic acid) (PMAA), poly( Styrenesulfonic acid) (PSS), poly(N-isopropylacrylamide) (NIPAM), poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), and combinations thereof . In some cases, the polyanion is PLGA. In some instances, the polyanion is PLAA.

Polyelectrolyte multilayers can be formed by layer-by-layer assembly by depositing polycations and polyanions in an alternating manner. The polyelectrolyte multilayers described herein include at least one bilayer comprising a polycation layer and a polyanion layer.

In some embodiments, a PEM can include from about 1 bilayer to about 100 bilayers. In some embodiments, a PEM can include from about 1 bilayer to about 50 bilayers. In some embodiments, the PEM can include from about 1 bilayer to about 30 bilayers. In some embodiments, the PEM can include from about 1 bilayer to about 20 bilayers. In some embodiments, the number of bilayers is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 . In some embodiments, the number of bilayers is 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range. In some embodiments, the number of bilayers is three. In some embodiments, the number of bilayers is four. In some embodiments, the number of bilayers is five. In some embodiments, the number of bilayers is six. In some embodiments, the number of bilayers is seven. In some embodiments, the number of bilayers is eight. In some embodiments, the number of bilayers is nine. In some embodiments, the number of bilayers is ten. In some embodiments, the number of bilayers is eleven. In some embodiments, the number of bilayers is twelve. In some embodiments, the number of bilayers is 13. In some embodiments, the number of bilayers is fourteen. In some embodiments, the number of bilayers is 15. In some embodiments, the number of bilayers is sixteen. In some embodiments, the number of bilayers is seventeen. In some embodiments, the number of bilayers is 18. In some embodiments, the number of bilayers is nineteen. In some embodiments, the number of bilayers is twenty.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of positively charged polyelectrolytes and negatively charged polyelectrolytes, wherein the polycation is selected from the group consisting of PLL, PLO, PLH, and PLA, And the polyanion is selected from PLGA and PLAA. In some embodiments, the number of sets ranges from 1-100, 3-60, 3-50, or 3-30. In some embodiments, the number of sets is greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of sets is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the number of sets is between 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLL and PLGA. In some embodiments, the number of bilayers of PLL and PLGA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLO and PLGA. In some embodiments, the number of bilayers of PLO and PLGA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLH and PLGA. In some embodiments, the number of bilayers of PLH and PLGA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLA and PLGA. In some embodiments, the number of bilayers of PLA and PLGA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLL and PLAA. In some embodiments, the number of bilayers of PLL and PLAA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLO and PLAA. In some embodiments, the number of bilayers of PLO and PLAA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLH and PLAA. In some embodiments, the number of bilayers of PLH and PLAA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the polyelectrolyte multilayers described herein comprise one or more bilayers of PLA and PLAA. In some embodiments, the number of bilayers of PLA and PLAA ranges from 1-100, 3-60, 3-50, or 3-30. 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16 within the range.

In some embodiments, the surfaces described herein are coated with (polyanion/polycation) _n , wherein the polyanion/polycation is selected from the group consisting of PLGA/PLL, PLAA/PLL, PLGA/PLA, PLAA/PLA, PLGA/ PLO, PLAA/PLO, PLGA/PLH, and PLAA/PLH. In some embodiments, n is an integer ranging from 1 to 30, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some embodiments, n is in the range of 1-5, 5-15, 5-20, 10-20, 10-25, 10-30, 15-20, 15-25, or 15-30.

In some embodiments, the surfaces described herein are coated with polycations (polyanions/polycations) _n , wherein the polyanions/polycations are selected from the group consisting of PLGA/PLL, PLAA/PLL, PLGA/PLA, PLAA/PLA, PLGA/PLO, PLAA/PLO, PLGA/PLH, and PLAA/PLH. In some embodiments, n is an integer ranging from 1 to 30, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some embodiments, n is in the range of 1-5, 5-15, 5-20, 10-20, 10-25, 10-30, 15-20, 15-25, or 15-30.

In some embodiments, the surfaces described herein are coated with polyanions (polycations/polyanions) _n , wherein the polycations/polyanions are selected from the group consisting of PLL/PLGA, PLL/PLAA, PLA/PLGA, PLA/PLAA, PLO/PLGA, PLO/PLAA, PLH/PLGA, and PLH/PLAA. In some embodiments, n is an integer ranging from 1 to 30, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some embodiments, n is in the range of 1-5, 5-15, 5-20, 10-20, 10-25, 10-30, 15-20, 15-25, or 15-30.

The thickness of the PEM as a thin film can be in a wide range, for example in the range of about 30 nm to about 30 μm or 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 value therebetween. In some embodiments, the thickness is about 1, 5, 10, 15, or 20 μm, or any value therebetween.

In some embodiments, PEM can be deposited by pipetting a solution of polyanions or polycations as a mixture or sequentially into/on a Petri dish. Each of the polycations and polyanions described herein can be dissolved in the aqueous solutions used in the present invention. The aqueous solution is free or substantially free of organic solvents. It is understood that some small amount of organic solvent may be present in the aqueous solution, for example as a result of some organic solvent remaining in the polymer after polymerization. As used herein, "substantially free" as it relates 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% organic solvent. Each of the polycation and polyanion can be dissolved in the aqueous solution in any concentration suitable for coating purposes.

In some embodiments, a PEM can be formed by dip coating on the surface of a cell culture article. In dip coating, the article is immersed in a polyelectrolyte solution for a period of time (typically 10-15 minutes), followed by multiple rinses and immersion in a second polyelectrolyte solution of opposite charge. Repeat this process until the desired number of layers is achieved.

In some embodiments, the PEM can be formed by spraying on the surface of the cell culture preparation. In some embodiments, the polyelectrolyte can be sprayed onto the surface for 3-10 seconds, followed by a rest/draining period of 10-30 seconds, and the surface is washed by spraying water for 3-20 seconds , followed by a resting period of 10 seconds, and the cycle was repeated with the oppositely charged polyelectrolyte.

In some embodiments, a PEM can be formed on the surface of a cell culture article by spin coating. Spin coating is a highly controlled method for solution-based coating of systems. A typical spin coating procedure involves spinning for 10-15 seconds, rinsing with "spin" water at least once for 15-30 seconds, and repeating the procedure with an oppositely charged polyelectrolyte. Wash steps may not be required in spin coating.

There are a variety of methods that can be used to characterize PEMs. In some embodiments, such methods may include ellipsometry (thickness), quartz crystal microbalance with dissipation monitoring (mass adsorption, viscoelasticity), contact angle analysis (surface energy), Fourier transform infrared spectroscopy ( Fourier transform infrared spectroscopy) (functional groups), X-ray photoelectron spectroscopy (chemical composition), scanning electron microscopy (surface structure) and atomic force microscopy (roughness/surface structure).

As described herein, a surface is hydrophilic if the contact angle of a water droplet on the surface is less than 90 degrees (contact angle is defined as the angle through the interior of the water droplet). Embodiments include hydrophilic surfaces having a contact angle of 90 degrees to 0 degrees; the skilled artisan will immediately understand that all ranges and values between well-defined limits are contemplated, for example any of the following may be used as an upper or lower limit: 90 , 80, 70, 60, 50, 40, 30, 20, 10, 5, 2, 0 degrees.

In some embodiments, the surface may also be coated with an absorbent polymer. In some cases, the absorbent polymer can be the innermost layer of the coating attached to the surface of the cell culture article. In some embodiments, the absorbent polymer is physically crosslinked to the surface of the load. In some embodiments, the absorbent polymer is chemically crosslinked to the surface of the load.

The absorbent polymers described herein are hydrophilic absorbent polymers. A non-limiting list of absorbent polymers that can be used with the present invention includes hydrophilic and biocompatible grades of the following polymers and their derivatives: poly(vinyl alcohol) (PVA), ethylene vinyl alcohol copolymer (usually Non-biodegradable materials whose hydrophilicity depends on the distribution of ethylene (hydrophobic) and vinyl alcohol (hydrophilic) groups), copolymers of polyvinyl alcohol and ethylene vinyl alcohol, polyacrylate compositions, polyurethane ester compositions, poly(ethylene glycol) (PEG) (also known as poly(ethylene oxide) (POE) and poly(ethylene oxide) (PEO)) and derivatives thereof, including but not limited to poly Ethylene glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate (PEGDMA), and polyethylene glycol diacrylate (PEGDA); materials containing nitrogen, such as polyacrylamide (acryl-free amine toxic residues), polyvinylpyrrolidone, polyvinylamine, and polyethyleneimine; charged materials such as poly(lactic acid), also known as various forms of polylactide (such as poly-L-lactide (PLLA ) and its derivatives, poly-D-lactide (PDLA) and its derivatives, poly(L-lactide-co-D,L-lactide) (PLDLLA) and its derivatives), poly( Glycolic acid) (PGA) (also known as polyglycolide), copolymers of lactic acid and glycolic acid poly(lactic-co-glycolic acid) (PL-co-GA), PLA and/or copolymers of PGA and PEG ; polymethacrylic acid; poly(hydroxyethyl methacrylate) (poly-HEMA), and other absorbent, hydrophilic, and biocompatible materials known in the art.

In some embodiments, the absorbent polymer is selected from the group consisting of poly(vinyl alcohol) (PVA), copolymers of ethylene vinyl alcohol, copolymers of polyvinyl alcohol and ethylene vinyl alcohol, polyacrylate compositions , polyurethane composition, poly(ethylene glycol) (PEG), PEG-acrylate, polyethylene glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate (PEGDMA), Polyethylene glycol diacrylate (PEGDA), polyacrylamide (PAM), polyvinylpyrrolidone (PVP), polyvinylamine (PVAm), polyethyleneimine (PEI), poly-L-lactide (PLLA), poly-D-lactide (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid- co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA) and poly(hydroxyethyl methacrylate) (p-HEMA).

In some embodiments, the absorbent polymer is selected from the group consisting of PVA, PEG, PEG-acrylate, polylactide, PMMA, p-HEMA, combinations 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.

In some embodiments, the average molecular weight of the absorbent 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, about 5,000 g/mol to about 75,000 g/mol, about 5,000 g/mol to about 50,000 g/mol, about 5,000 g/mol to about 25,000 g/mol, about 5,000 g /mol to about 10,000 g/mol, about 10,000 g/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.

In certain embodiments, the volume of the absorbent polymer (eg, PVA or PEG) is from about 0.01% to about 10% of the total volume of the surface coating. In some cases, about 0.01% to about 9% v/v, about 0.01% to about 8% v/v, about 0.01% to about 7% v/v, About 0.01% to about 6% v/v, about 0.01% to about 5% v/v, about 0.01% to about 4% v/v, about 0.01% to about 3% v/v, about 0.01% to about 2 % v/v, about 0.01% to about 1% v/v, about 0.1% to about 10% v/v, about 0.1% to about 9% v/v, about 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 weight of absorbent polymer (eg, PVA or PEG) per total weight of the surface coating is from about 1% to about 50%. In some cases, the weight of absorbent polymer per total weight of the surface coating is from about 1% to about 10%, 20%, 30%, or 40%.

In some embodiments, the absorbent polymer can be in direct contact with the surface of the cell culture article. In some cases, the absorbent polymer is deposited directly on the surface of the cell culture product. In some cases, the absorbent polymer is deposited indirectly on the surface of the cell culture product. In some cases, the absorbent polymer is physically cross-linked to the surface of the cell culture article. In some cases, the absorbent polymer is chemically crosslinked to the surface of the cell culture article.

In some embodiments, the absorbent polymer may be in direct contact with the PEM. In some embodiments, the absorbent polymer is physically crosslinked with the polyanion and/or polycation of the PEM. In some embodiments, the absorbent polymer is chemically crosslinked with the polyanion and/or polycation of the PEM.

In some embodiments, the surface further comprises a filler. In some cases, the filler comprises a mineral filler such as, but not limited to, silica, alumina, calcium carbonate, or silicone.

The surfaces disclosed herein not only enable cell attachment and growth, but also enable living collections of cultured cells (eg, 3D cell cultures). According to some embodiments of the present invention, the cell culture substrate can be used to collect viable cells, including 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the collected 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 alive, at least 94% alive, at least 95% alive, at least 96% alive, at least 97% alive, at least 98% alive, or at least 99% alive. In some embodiments, cells can be released from a cell culture system with or without the use of a cell dissociation enzyme such as trypsin, TrypLE, or Accutase.

Cell culture articles disclosed herein include supports made of any suitable material, such as silicon, plastic, glass, elastomers, and the like. In some embodiments, the payload is made of elastomer. The elastomers described herein may be silicone elastomers. In some embodiments, the silicone elastomer is polydimethylsiloxane (PDMS).

In certain embodiments, the support is made of a glass material (such as soda lime glass, pyrex glass, vycor glass, quartz glass). In some embodiments, the carrier is made of plastic or polymer such as polyethylene, polypropylene, polymethylpentene, cycloolefin polymers, cycloolefin copolymers, polyvinyl chloride, polyurethane Ester, polyester, polyamide, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-acrylic acid copolymer, ethylene-methyl acrylate copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl Methyl acrylate copolymer, polyacrylic acid, polymethacrylic acid, polymethyl acrylate and polymethyl methacrylate or derivatives or the like. In certain embodiments, the load is made of a material comprising at least one of: polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene , polyvinyl chloride, polyethylene oxide, polypyrrole and polypropylene oxide. Method and use thereof 1) Method for preparing cell culture

In one aspect, the invention provides a method of preparing a cell culture using the cell culture substrate of the invention. The method disclosed herein comprises the steps of: (a) providing a cell culture substrate as described herein having a surface comprising a polyelectrolyte multilayer and optionally an absorbent polymer; (b) seeding the surface with a plurality of cells; and (c) culturing the plurality of cells in a suitable medium for a time sufficient to produce a cell culture on the surface. In preferred embodiments, the cell culture is adhered to the surface. In some embodiments, the cell culture comprises a three-dimensional (3D) cell culture. 3D cell cultures can be in the form of spheroids. In some embodiments, the spheroid systems described herein are produced by single cell proliferation. In some embodiments, the cell culture comprises one or more single cell-derived spheroids. In preferred embodiments, single cell-derived spheroids adhere to surfaces. In some embodiments, the single cell-derived spheroid system is semi-attached or loosely attached to the surface.

In some embodiments, seeding the plurality of cells in step (b) comprises spreading the cells on the surface at a density of 1 cell to 10 cells/cm2. In some embodiments, seeding the plurality of cells in step (b) comprises spreading the cells on the surface at a density of 10 cells to 100 cells/cm2. In some embodiments, seeding the plurality of cells in step (b) comprises spreading the cells on the surface of the substrate at a density of 100 cells to 1000 cells/cm2. In some embodiments, seeding the plurality of cells in step (b) comprises spreading the cells on the surface of the substrate at a density of 200 cells to 5000 cells/cm2.

In some cases, the spheroids described herein can comprise from about 8 to about 1000 cells. In some cases, the spheroid comprises about 8 to about 800 cells, about 8 to about 500 cells, about 8 to about 400 cells, about 8 to about 300 cells, about 8 to about 200 cells, about 8 to about 100 cells, about 10 to about 1000 cells, about 10 to about 800 cells, about 10 to about 500 cells, about 10 to about 400 cells, about 10 to about 300 cells, about 10 to about 800 cells about 200 cells, about 10 to about 100 cells, about 50 to about 1000 cells, about 50 to about 800 cells, about 50 to about 500 cells, about 50 to about 400 cells, about 50 to about 300 cells 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.

In some embodiments, the spheroids described herein can have a diameter of about 40 μm to about 200 μm. In some embodiments, the spheroids may have a diameter of about 50 µm to about 150 µm. In some cases, the spheroids may have a thickness of about 50 µm to about 120 µm, about 50 µm to about 100 µm, about 50 µm to about 80 µm, about 50 µm to about 60 µm, about 80 µm to about 150 µm, A diameter of about 80 µm to about 120 µm, about 80 µm to about 100 µm, about 100 µm to about 200 µm, about 100 µm to about 150 µm, or about 100 µm to about 120 µm.

In some embodiments, cells are cultured for a period of 2-8 days (eg, 2, 3, 4, 5, 6, 7, or 8 days). In other embodiments, the cells are cultured for a period of 7-14 days (eg, 7, 8, 9, 10, 11, 12, 13, or 14 days). In other embodiments, the cells are cultured for a period of 1-4 weeks (eg, 1, 2, 3, or 4 weeks).

In some embodiments, the plurality of cells described herein comprises cancer cells. In some embodiments, the cancer cell line is obtained from a biological sample of a cancer patient. In some embodiments, the biological sample is a bodily fluid sample. The bodily fluid samples described herein can be serum, plasma, whole blood, urine or ascitic fluid. In some embodiments, the bodily fluid sample is a blood sample, and the cancer cells include circulating tumor cells (CTCs) and/or tumor-associated cells. CTCs can be isolated from blood samples. There are currently several strategies available for CTC isolation. Enrichment is a critical step in isolating live CTCs from the rest of the blood components, such as platelets, red and white blood cells, which achieves the concentration of CTCs and thus facilitates the detection process. The enrichment step can be performed by three different types of techniques: protein expression based techniques, physical property based techniques and function based techniques. Examples of strategies for isolation of live circulating tumor cells (CTCs) include, but are not limited to, RosetteSep®, CTC-iChip, Ficoll®, Ficoll-Pacque®, Lymphoprep®, Percoll®, MetaCell®, Parsortix®, Collagen Adhesion Matrix Analysis Method (CAM) and EPithelial ImmunoSPOT Analysis Method (EPISPOT).

Any suitable medium can be used in the methods of the exemplary embodiments. Exemplary 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); supplemented with B27 Supplement is a mixture of Dulbecco's Modified Eagle's Medium (DMEM), Epidermal Growth Factor (EGF) and Basic Fibroblast Growth Factor (bFGF).

Media for culturing cancer cells (e.g., CTCs) may contain epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF2), other growth factors such as fibroblast growth factor-10 (FGF10), Granulocyte-macrophage colony-stimulating factor (GM-CSF), insulin, and insulin-like growth factor 1 (IGF-1). In some examples, R-reactive 1 protein can be included to maintain the survival and growth of epithelial cells via WNT/β-catenin signaling. In some embodiments, when serum-free medium is used, B27 and N2 supplements can be added with or without insulin, transferrin, and selenium (ITS) supplementation. In some embodiments, small molecule inhibitors of specific signaling pathways may be included in the culture medium to promote CTC growth. For example, A83-01 and ALX-270-445 (ALK5 inhibitor II) can specifically inhibit the function of type 1 TGFβ receptor kinase (ALK5), thereby preventing epithelial to mesenchymal transition and promoting CTC survival and proliferation. SB202190, a p38 MAPK inhibitor, prevents apoptosis induced by p38 activation. In some embodiments, the culture medium may comprise a Rho-associated protein kinase (ROCK) inhibitor. The structural formula of the ROCK inhibitors described herein can have an isoquinoline, 4-amidopyridine, or 4-amidopyrrolopyridine skeleton. In some embodiments, the ROCK inhibitor is an isoquinoline-based ROCK inhibitor, such as Fasudil, hydroxyfasudil, H-1152P, Ripasudil, or derivative. In some embodiments, the ROCK inhibitor is a 4-amidopyridine-based ROCK inhibitor, such as Y27632, Y32885, or a derivative thereof. In some embodiments, the ROCK inhibitor is a 4-amidopyrrolopyridine based ROCK inhibitor, such as Y30141, Y39983 or a derivative thereof. The structural formulas of Fasudil, Hydroxyfasudil, H-1152P, Resudil, Y30141, Y39983, Y27632 and Y32885 are shown below.

In some embodiments, the biological sample can be a human primary tumor sample. A primary tumor sample can comprise primary tumor cells or metastatic tumor cells from a patient. In some embodiments, cancer cells are obtained by: mincing a primary tumor sample in medium supplemented with serum; treating the minced primary tumor sample with an enzyme; and enzymatically treating the Tumor spheroids were collected from the samples. In some embodiments, the minced primary tumor samples are treated with enzymes, the amount of enzyme used and/or the action time must be sufficient to partially decompose the above samples, and the preferred action time is between 10 minutes and 60 minutes , the more optimal condition is to act at a temperature of 25° C. to 39° C. for 15 minutes to 45 minutes.

Exemplary cancers described herein include, but are not limited to, acute lymphocarcinoma, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or anorectal cancer, eye cancer, intrahepatic Cancer of the cholangiocarcinoma, cancer of the joints, cancer of the neck, cancer of the gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic bone marrow cancer, cancer of the large intestine, cancer of the esophagus, cancer of the cervix, gastrointestinal Carcinoid tumor, glioma, Hodgkin lymphoma, hypopharyngeal carcinoma, renal carcinoma, laryngeal carcinoma, liver carcinoma, lung carcinoma, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma , non-Hodgkin lymphoma (non-Hodgkin 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, ureter cancer and bladder cancer.

In some embodiments, the plurality of cells may comprise tumor-associated cells. Exemplary tumor-associated cells include, but are not limited to, tumor cell clusters, tumor-infiltrating lymphocytes (TILs), cancer-associated macrophage-like cells (CAMLs), tumor-associated macrophages (TAMs), tumor-associated monocytes/ Macrophage lineage cells (MMLC), cancer stem cells, tumor microemboli, tumor-associated stromal cells (TASC), tumor-associated myeloid cells (TAMC), tumor-associated regulatory T cells (Treg), cancer-associated fibroblasts (CAF ), tumor-derived endothelial cells (TEC), tumor-associated neutrophils (TAN), tumor-associated platelets (TAP), tumor-associated immune cells (TAI), myeloid-derived suppressor cells (MDSC), and combinations thereof.

In certain embodiments, cancer cell cultures described herein comprise one or more cancer cell spheroids (eg, tumor spheroids). The tumor spheroids described herein can be used to evaluate cancer therapeutics. 2) Methods for evaluating therapeutic agents

Another aspect of the invention provides methods for evaluating therapeutic agents for use in cancer patients. The method comprises the steps of: (a) preparing a cell culture (e.g., a CTC culture) according to the methods described herein; (b) optionally culturing the cell culture with a plurality of immune cells; (c) allowing the cells contacting the culture with the therapeutic agent; (d) assessing the effect of the therapeutic agent on the cell culture; and (e) determining that the cancer patient responds to the therapeutic agent when the therapeutic agent is effective on the cell culture; or when the therapeutic agent is effective on the cell culture When the drug is not effective, it is determined that the cancer patient does not respond to the therapeutic agent.

In some embodiments, the effects of therapeutic agents are analyzed by performing luminescence- and/or fluorescence-based cell-based and/or biochemical assays. In some embodiments, the effect of a therapeutic agent is analyzed by performing a luminescence-based cell viability assay. In some embodiments, the effect of a therapeutic agent can be analyzed by performing one or more assays to measure the size, morphology, physical properties, biological properties of cells in tumor spheroids and/or kinetic properties. In some embodiments, the effects of therapeutic agents can be further analyzed based on one or more assays for the biochemical activity of one or more genes or one or more proteins in tumor spheroids and/or performance. The results of the assay can provide treatment guidelines for cancer patients.

In some embodiments, the plurality of immune cells described herein comprises autologous immune cells from peripheral blood. In some embodiments, the autologous immune cells comprise ex vivo expanded autologous immune cells. In some embodiments, the immune cells comprise autologous natural killer (NK) cells isolated from peripheral blood and expanded ex vivo. In other embodiments, the immune cells comprise allogeneic natural killer (NK) cells isolated from donors and expanded ex vivo. In other embodiments, the immune cells comprise natural killer (NK) cells differentiated and expanded in vitro from human induced potent stem cells (iPSCs) or embryonic stem cells (ESCs). The step of cultivating tumor spheroids with multiple immune cells can form a tumor cell-immune cell co-culture, which can realize the in vitro recapitulation of the tumor microenvironment that mimics the in vivo environment, so that the in vivo delivery of therapeutic agents can be evaluated. Effectiveness in the treatment of cancer over time.

Suitable therapeutic agents include, but are not limited to, chemotherapeutic drugs, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, and combinations thereof.

In some embodiments, the therapeutic agent is a cytotoxic or cytostatic chemotherapeutic drug. The methods described herein comprise the step of contacting tumor spheroids with a cytotoxic or cytostatic chemotherapeutic drug in the presence or absence of a plurality of immune cells. Chemotherapeutic drugs can be alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, darax Dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites Drugs (such as fluorouracil (fluorouracil, 5-FU), gemcitabine (gemcitabine), methotrexate (methotrexate), cytarabine (cytosine arabinoside), fludarabine (fludarabine) and floxuridine (floxuridine)), Antimitotic agents (including taxanes such as paclitaxel and decetaxel and vinca alkaloids such as vincristine, vinblastine, vinblastine vinorelbine and vindesine), anthracycline (including doxorubicin, daunorubicin, valrubicin, adamycin (idarubicin) and epirubicin (epirubicin), as well as actinomycins, such as actinomycin D (actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), topoisomerase inhibitors (including camptothecins, such as camptothecin, irinotecan, and topotecan, and epitope Podophyllotoxin derivatives such as amsacrine, etoposide, etoposide phosphate, and teniposide), antibodies against vascular endothelial growth factor (VEGF) (such as bevacizumab (AVASTIN®), other anti-VEGF compounds); anti-PD-1 (anti-planned death-1) therapeutics such as antibodies or compounds (e.g., nivolumab); Lilidomide (THALOMID®) and its derivatives, such as lenalidomide (REVLIMID®); endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors, such as Sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib (Nexavar®), erlotinib (Tarceva®), pazopanib, aspirin Axitinib and lapatinib; transforming growth factor-alpha or transforming growth factor-beta inhibitors and antibodies against epidermal growth factor receptor, such as panitumumab (VECTIBIX®) and western medicine Tuximab (cetuximab, ERBITUX®).

In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. The methods described herein comprise the step of contacting tumor spheroids with an immune checkpoint inhibitor in the presence of a plurality of immune cells. Immune checkpoint inhibitors can 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, CHK 1, 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. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor selected from a PD-1 inhibitor, a PD-L1 inhibitor, and a CLTA-4 inhibitor. The immune checkpoint inhibitors described herein are selected from the group consisting of nivolumab, pembrolizumab, simiprizumab, atezolizumab, avelumab, Valirumab and ipilimumab.

In some embodiments, the therapeutic agent is a nucleic acid drug. The methods described herein comprise the step of contacting tumor spheroids with a nucleic acid drug in the presence or absence of a plurality of immune cells. The nucleic acid drug can be DNA, DNA plastids, 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 plastid comprising a nucleotide sequence encoding a gene selected from the group consisting of: GM-CSF, IL-12, IL-6, IL-4, IL-12 , TNF, IFNy, IFNa and combinations thereof.

In some embodiments, the therapeutic agent is a therapeutic cellular composition. The methods described herein comprise the step of contacting tumor spheroids with a therapeutic cell composition in the absence of a plurality of immune cells. Exemplary therapeutic cell compositions include, but are not limited to, T cells, natural killer (NK) cells and dendritic cells, chimeric antigen receptor T (CAR-T) cells, and chimeric antigen receptor-natural killer ( CAR-NK) cells. 3) Method of using immunotherapy to treat cancer

Another aspect of the invention provides methods of treating cancer using immunotherapy for cancer patients. The method comprises the steps of: (a) evaluating a therapeutic agent for immunotherapy of a cancer patient according to the methods described herein; and (b) administering a therapeutically effective dose of the therapeutic agent to the cancer patient responding to the therapeutic agent.

In certain embodiments, therapeutic agents for immunotherapy include immune checkpoint inhibitors. In certain embodiments, therapeutic agents for immunotherapy comprise therapeutic cell compositions. In certain embodiments, therapeutic agents for immunotherapy comprise nucleic acid drugs. In certain embodiments, cytotoxic or cytostatic chemotherapeutic drugs are also administered to cancer patients. 4) Method for preparing single cell-derived spheroids in vitro

Another aspect of the invention provides a method for the in vitro preparation of single cell-derived spheroids, the method comprising the steps of: (a) providing a cell culture substrate having a polyelectrolyte-containing multilayer as described herein and a surface of an optional absorbent polymer; (b) isolating cells (eg, tumor cells and/or tumor-associated cells) from a sample (eg, liquid biopsy sample, needle biopsy, tissue section) to provide isolated cells; (c) seeding the isolated cells on the substrate; and (d) expanding the cells by culturing in a cell culture substrate under a suitable medium for a time sufficient to produce one or more spheroids, wherein one or more Each of the various spheroids was single cell derived.

One or more spheroid systems described herein are generated via single cell proliferation. In preferred embodiments, one or more spheroid systems described herein are produced by single cell proliferation without cell aggregation. In some embodiments, the one or more spheroids are of uniform size.

In some embodiments, seeding the isolated cells in step (c) comprises spreading the cells on the surface (ie, the cell growth surface) at a density of 1 cell to 10 cells/cm2. In some embodiments, seeding the isolated cells in step (c) comprises spreading the cells on the surface at a density of 10 cells to 100 cells/cm2. In some embodiments, seeding the isolated cells in step (c) comprises spreading the cells on the surface at a density of 100 cells to 1000 cells/cm2.

In some embodiments, the isolated cells are cancer cells. In certain embodiments, cancer cells are isolated from human primary tumor tissue. In certain embodiments, cancer cells are isolated from a blood sample of a cancer patient.

In some embodiments, the medium comprises Dulbecco's Modified Eagle's Medium (DMEM). In some embodiments, the culture medium comprises epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF). In some embodiments, the culture medium comprises a mixture of Dulbecco's Modified Eagle's Medium (DMEM), Epidermal Growth Factor (EGF), and Basic Fibroblast Growth Factor (bFGF) supplemented with B27 supplement.

In some embodiments, the cell culture systems of the present invention are capable of maintaining the size of single cell-derived spheroids less than 200 μm in diameter to prevent cell necrosis and induce cell division into smaller cells as they continue to proliferate over time. of spheroids. In some embodiments, the size of the single cell-derived spheroids is less than 150 μm in diameter. In some embodiments, the size of the single cell-derived spheroid is about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 μm in diameter. In contrast to platforms routinely used on the market such as hanging drops or U-shaped ULA dishes, no cell debris resulting from apoptosis/necrosis was observed in single cell-derived spheroids after 7 days of culture. 5) Methods for screening therapeutic agents

In another aspect, the invention provides methods for screening for therapeutic agents. The method comprises: (a) providing single cell-derived spheroids containing a population of cancer cells, wherein the single cell-derived spheroid system is prepared using the cell culture substrate disclosed herein; (b) applying a test substance to the single cell cell-derived spheroids; and (c) assessing the effect of the test substance on single cell-derived spheroids.

In certain embodiments, the isolated cells are stem cell-like cells. Such stem-like cells can be identified and isolated using markers known in the art.

In some embodiments, the single cell-derived spheroid system comprises tumor spheroids of a population of cancer stem-like cells. In some embodiments, the population of cancer stem-like cells comprises at least about 1 x ¹⁰⁴ cancer stem-like cells. In some embodiments, the population of cancer stem-like cells comprises at least about 1 x ¹⁰³ cancer stem-like cells. In some embodiments, the population of cancer stem-like cells comprises at least about 5-900 cancer stem-like cells, such as at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 cancer stem-like cells. In some embodiments, the single cell-derived spheroids prepared herein exhibit high stem cell properties and epithelial to mesenchymal transition (EMT) potential.

The cancer stem-like cells described herein can be derived from cancer cells and induced after being cultured in the cell culture systems described herein. In some embodiments, the cancer stem-like cells are cultured in serum-free stem cell medium. In some embodiments, the cancer stem cells are cultured in complete cancer stem cell medium.

The cancer stem-like cell (CSC) population generated herein is suitable for in vitro drug screening of CSC-targeted cancer therapy and cancer immunotherapy. 6) Method for screening therapeutic agents selectively targeting tumor stem cell-like cells

In another aspect, the present invention provides methods for screening therapeutic agents that selectively target cancer stem-like cells. The method comprises: (a) providing a single cell-derived spheroid comprising a population of cancer stem-like cells, wherein the single cell-derived spheroid system is prepared using a cell culture substrate having a surface disclosed herein; (b) incorporating The test substance is applied to the single cell-derived spheroids; and (c) the effect of the test substance on the single cell-derived spheroids is evaluated. 7) Methods for inhibiting cancer metastasis and preventing, alleviating or treating cancer

In another aspect, the present invention provides methods for inhibiting cancer metastasis and preventing, alleviating or treating cancer. The method comprises: administering to a cancer patient an effective amount of a therapeutic agent selectively targeting cancer stem-like cells. The therapeutic agent is screened and selected by the methods described above.

In another aspect, the present invention provides compositions comprising single cell-derived spheroids produced in vitro, wherein the single cell-derived spheroid system is prepared by the methods described above.

In some embodiments according to any of the methods described above, the cancer cells comprise cells derived from a primary cell culture of a tumor sample, such as a tissue or bodily fluid sample, obtained from surgery, a biopsy, or blood from an individual Obtain, such as a liquid biopsy, biopsy needle, or Pap smear. In some embodiments, the sample is a blood, saliva or urine sample. In some embodiments, the sample is from a cancer patient, such as a cancer patient with metastatic cancer or a cancer patient before, during or after therapeutic treatment. In some embodiments, the cells of the primary cell culture are circulating cancer cells isolated from a patient's blood sample. In some embodiments, the primary cell culture comprises a patient-derived xenograft (PDX). In some embodiments according to any of the methods described above, the cancer cells comprise cells of a cancer cell line.

In some embodiments according to any of the methods described above, the test substance is a chemotherapeutic drug, such as a cytotoxic or cytostatic chemotherapeutic drug. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor, such as an immune checkpoint inhibitor. In some embodiments, the therapeutic agent is a nucleic acid drug. In some embodiments, the therapeutic agent is a composition of therapeutic cells including, but not limited to, T cells, natural killer (NK) cells, and dendritic cells.

In some embodiments according to any of the cell culturing methods described above, the cells are cultured for a period in the range of about 2 days to about 5 weeks, such as about 3 to about 14 days, eg 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.

In some embodiments according to any of the methods described above, the thickness of the polyelectrolyte multilayer is in the range of about 30 nm to about 30 μm, including about 100 nm to about 20 μm, such as about 500 nm. Certain Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter that is claimed. In this application, the use of the singular includes the plural unless expressly stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting.

As used herein, ranges and amounts can be expressed "about" a particular value or range. About also includes exact quantities. Therefore, "about 5 µL" means "about 5 µL" and "5 µL". In general, the term "about" includes an amount that would be expected to be within experimental error.

The use of each subheading herein is for organizational purposes only and should not be construed as limiting the subject matter described.

As used herein, the term "comprising" is intended to mean that the method includes the recited steps or elements, but does not exclude other steps or elements. "Consisting essentially of" shall mean leaving the claim open to include only steps or elements that do not materially affect the basic and novel characteristics of the claimed method. "Consisting of" shall mean excluding any elements or steps not specified in the scope of the patent application. Embodiments defined by each of these variations of terms are within the scope of this invention.

As used herein, the term "positively charged polyelectrolyte" encompasses a plurality of monomeric units or non-polymeric molecules comprising two or more positive charges. In some cases, a positively charged polyelectrolyte also encompasses a plurality of monomeric units or non-polymeric molecules comprising positively charged groups, neutrally charged groups, or negatively charged groups, wherein the net charge is positive.

As used herein, the term "cationic polymer" encompasses a plurality of monomeric units or non-polymeric molecules. In some cases, cationic polymers are synthetic polymers. In other cases, the cationic polymers are natural polymers.

As used herein, the term "cationic polypeptide" refers to a polypeptide comprising two or more positive charges. In some instances, cationic polypeptides comprise positively charged amino acid residues, negatively charged residues, and polar residues, but the net charge of the polypeptide is positive. In some cases, the cationic polypeptide is 8 to 100 amino acids in length. In some cases, the length of the 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.

As used herein, the term "negatively charged polyelectrolyte" encompasses a plurality of monomeric units or non-polymeric molecules comprising two or more negative charges. In some cases, a negatively charged polyelectrolyte also encompasses a plurality of monomeric units or non-polymeric molecules comprising positively charged groups, neutrally charged groups, or negatively charged groups, wherein the net charge is negative.

As used herein, the term "anionic polymer" encompasses a plurality of monomeric units or non-polymeric molecules. In some cases, the anionic polymer is a synthetic polymer. In other instances, the anionic polymers are natural polymers.

As used herein, the term "anionic polypeptide" refers to a polypeptide comprising two or more negative charges. In some instances, anionic polypeptides comprise positively charged amino acid residues, negatively charged residues, and polar residues, but the net charge of the polypeptide is negative. In some cases, anionic polypeptides are 8 to 100 amino acids in length. In some cases, the length of the anionic 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.

As used herein, the term "polymer" includes homopolymers and copolymers, branched and unbranched, and natural or synthetic polymers.

As used herein, the term "tumor" refers to a neoplasm, i.e., an abnormal growth of cells or tissue and is understood to include benign, i.e., non-cancerous growths, and malignant, i.e., cancerous growths. Sexual growths, including primary or metastatic cancerous growths.

Examples of neoplasms include, but are not limited to, mesothelioma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), skin cancer (e.g., melanoma), gastric cancer, liver cancer, colorectal cancer, breast cancer, pancreatic cancer , prostate cancer, blood cancer, bone cancer, bone marrow cancer and other cancers.

As used herein, the term "tumor spheroid" or "tumor cell spheroid" refers to an aggregate of tumor cells that constitutes small clumps or clumps of tumor cells. In some embodiments, the diameter of the tumor spheroid is less than about 3 cm, less than about 2 cm, less than about 1 cm, less than about 5 mm, less than about 2.5 mm, less than about 1 mm, less than about 100 μm, less than about 50 μm, less than about 25 μm, less than about 10 μm, or less than about 5 μm. In some embodiments, the diameter of the tumor spheroid is 10 μm to 500 μm. In some embodiments, the diameter of the tumor spheroid is 40 μm to 100 μm. In some embodiments, the diameter of the tumor spheroid is 40 μm to 70 μm.

As used herein, immune cells encompass neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cell). example

These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. Example 1 : Construction of the surface of the present invention ( construction )

To construct surface coatings with polyelectrolyte multilayers, polycation/polyanion deposition was performed using plasma-treated tissue culture dishes (TCP, polystyrene). Plasma treatment exposes the surface of the TCP disk to an oxygen plasma. Oxygen plasma treatment generates free radical species of surface silanol groups (Si-OH), alcoholic hydroxyl groups (C-OH) and carboxylic acids (COOH) on the surface, and these species allow polyanions (or polyanions) Hydrogen bonding with activated TCP surface.

To create polyelectrolyte multilayers, polycations and polyanions were dissolved in Tris-HCl buffer (pH 7.4) and deposited onto the TCP surface after rinsing with Tris-HCl buffer. Each polycation or polyanion layer was deposited and incubated, followed by washing with Tris-HCl buffer. In some examples, the polycation is poly-L-lysine (PLL) and the polyanion is poly-L-glutamic acid (PLGA). In some examples, the polycation is PLO and the polyanion is PLGA. In some examples, the polycation is PLH and the polyanion is PLGA. In some examples, the polycation is PLA and the polyanion is PLGA. PLL (MW 150K-300K), PLGA (MW 50K-100K), PLO (0.01%) solution, PLH (MW 5K-25K), PLA (MW 15K-70K) can be purchased from Sigma-Aldrich (St. Louis, MO , USA). Polycations and polyanions were dissolved in Tris-HCl buffer (pH 7.4) and deposited onto TCP disks after rinsing with Tris-HCl buffer. Each polycation or polyanion layer was deposited and incubated for 10 minutes, followed by three washes with Tris-HCl buffer for 2, 1 and 1 minute. PLL/PLGA, PLO/PLGA, PLH/PLGA, and PLA/PLGA multilayer films can be fabricated by layer-by-layer self-assembly as follows.

In some examples, the polyelectrolyte multilayer is a PLL/PLGA multilayer, which can be constructed by sequentially depositing PLL and PLGA on a disc. Each deposition step consisted of adding PLL or PLGA solution to the disc surface, incubating for 10 minutes and washing 3 times for 2, 1 and 1 minute. In one embodiment, a substrate composed of (PLGA/PLL) ₃ is constructed. In one embodiment, a substrate coating consisting of (PLGA/PLL) ₅ is constructed. In one embodiment, a substrate composed of (PLGA/PLL) ₁₀ is constructed. In one embodiment, a substrate composed of (PLGA/PLA) ₁₅ is constructed.

In some examples, the polyelectrolyte multilayer is a PLO/PLGA multilayer, which can be constructed by sequentially depositing PLO and PLGA on a disc. Each deposition step consisted of adding PLO or PLGA solution to the disc surface, incubating for 10 minutes and washing 3 times for 2, 1 and 1 minute. In one embodiment, a substrate composed of (PLGA/PLO) ₃ is constructed. In one embodiment, a substrate coating consisting of (PLGA/PLO) ₅ is constructed. In one embodiment, a substrate composed of (PLGA/PLO) ₁₀ is constructed. In one embodiment, a substrate composed of (PLGA/PLO) ₁₅ is constructed.

In some examples, the polyelectrolyte multilayer is a PLH/PLGA multilayer, which can be constructed by sequentially depositing PLH and PLGA on a disc. Each deposition step consisted of adding PLH or PLGA solution to the disc surface, incubating for 10 minutes and washing 3 times for 2, 1 and 1 minute. In one embodiment, a substrate composed of (PLGA/PLH) ₃ is constructed. In one embodiment, a substrate coating consisting of (PLGA/PLH) ₅ is constructed. In one embodiment, a substrate composed of (PLGA/PLH) ₁₀ is constructed. In one embodiment, a substrate composed of (PLGA/PLH) ₁₅ is constructed.

In some examples, the polyelectrolyte multilayer is a PLA/PLGA multilayer, which can be constructed by sequentially depositing PLA and PLGA on a disc. Each deposition step consisted of adding PLA or PLGA solution to the disc surface, incubating for 10 minutes and washing 3 times for 2, 1 and 1 minute. In one embodiment, a substrate composed of (PLGA/PLA) ₃ is constructed. In one embodiment, a substrate coating composed of (PLGA/PLA) ₅ is constructed. In one embodiment, a substrate composed of (PLGA/PLA) ₁₀ is constructed. In one embodiment, a substrate composed of (PLGA/PLA) ₁₅ is constructed. Example 2 : Comparison of Cell Culture Platforms for Spheroid Formation

To create in vitro 3D models useful for clinical applications, such as personalized drug testing, various culture platforms were evaluated. As a suspension culture platform, ultra-low attachment (ULA) discs (Corning) achieve spheroid formation based on cell aggregation, resulting in extremely low yields of spheroid formation. In our experiments, less than 1% of the number of cells seeded and cultured on ULA dishes could form spheroids. As a comparison, discs coated with layer-by-layer polyelectrolyte multilayers (PEMs) were evaluated for in vitro culture of spheroids. Using the HCT116 colorectal cancer cell line as an example, 78.7±2.7% of the cells seeded and cultured on the PEM-coated dish could proliferate and form cell spheroids after culturing in DMEM complete medium for 5-7 days, while Only 0.2±0.1% of cells seeded and cultured on ULA dishes formed spheroids. To assess whether the medium achieves spheroid formation on various culture platforms, a spheroid formation test was performed using serum-free medium (SPH medium) provided with growth factors. The results showed that after culturing for 5-7 days, 62.2±2.3% of the cells could proliferate and form cell spheroids inoculated and cultured on the PEM-coated culture dish, while only 0.7±0.1% of the cells inoculated and cultured on the ULA dish % of cells formed spheroids. Similar to ULA discs, discs coated with polyHEMA resulted in lower yields of spheroid formation. Matrigel, a commonly used embedding substrate for tissue-based cell cultures, was also tested. The results indicated that the resulting spheroids were embedded in Matrigel, making it difficult to analyze and image the spheroids as the images were blurred or out of focus. Example 3 : Form live spheroids on the culture platform of the present invention

Liquid biopsies make it possible to obtain immediate cancer samples directly from blood. But the rarity of circulating tumor cells (CTCs) impairs the ex vivo application of the entire approach for clinical use. Several literatures have demonstrated the feasibility of using circulating tumor cells for individual drug screening or testing, but few of them provide non-biased 3D cultured models for clinical applications. To test the feasibility of PEM-coated dishes for expansion of rare cells, very low density of HCT116 cells were seeded onto the surface of PEM-coated dishes and imaged under a time-lapse microscope. Single cells displayed semi-adherent and continuous proliferation on the surface of the PEM-coated culture dish, forming spheroids with a diameter of approximately 70 μm after 7 days. Thus, single cell-derived spheroids on PEM-coated culture dishes facilitate subsequent clinical applications by utilizing rare cell samples. Furthermore, where higher densities of cells were seeded onto the surface of PEM-coated culture dishes, the cells maintained growth from individual single cells into spheroids. But when two spheroids get close enough to each other, cells undergo spheroid fusion to form a larger spheroid. To establish a 3D spheroid-based rare cell expansion technique for clinical use, uniformity of spheroid size and maintenance of viable spheroids is required to eliminate potential bias during in vitro testing. A total of 300 cells were seeded onto wells with the surface coating of the invention in 96-well plates to test the uniformity of size of spheroids grown on the cell culture platform of the invention. Cells proliferate and form spheroids. The size of the spheroids was verified by an Opera Phenix high-throughput confocal sieving system (Perkin Elmer). On day 5, the average number of spheroids per well was 320±54 and the average spheroid diameter in each well was 69±34 μm. The results demonstrate high spheroid forming capacity and uniform spheroid size formed on the PEM coated culture dishes. Spheroids grown on the UL platform or the cell culture platform of the present invention were isolated and stained by LIVE/DEAD assay (Invitrogen) to test cell viability. Analysis of the fluorescence intensity of EthD1 showed that there were significantly more dead spheroid cells cultured on the UL platform compared to spheroids cultured on the cell culture platform of the present invention. Furthermore, spheroids cultured on PEM-coated culture dishes exhibited high cell viability after freeze-thaw cycle storage, indicating efficient cell survival after the cryopreservation process. Furthermore, confocal microscopy scans revealed tight cell-cell interactions in spheroids cultured on PEM-coated culture dishes, rather than loose cell-cell interactions in aggregated spheroids generated on ULA dishes. cell contact. Therefore, uniform and living single cell-derived spheroids may be useful for clinical applications, such as personalized drug testing. Example 4 : Patient-derived spheroids from clinical samples

To generate patient-derived spheroids for clinical use, patient-derived samples from tissue resection, core-needle biopsy, and phlebotomy blood draw were collected for use in In vitro spheroid culture. In some examples, cancer cell lines are obtained from tumor tissue. In some examples, cancer cell lines are obtained from blood samples. Viable CTCs can be isolated using methods known in the art. All viable cells can be seeded onto the surface of PEM-coated culture dishes to generate patient-derived spheroids within 2 to 4 weeks. Figures 1A- 1C illustrate representative time-dependent images of CTC cultures produced on surfaces according to one embodiment of the invention. ( A ) CTCs from blood samples from breast cancer patients, CTC cultures produced on the surface after 7 and 14 days of culture; ( B ) CTCs from blood samples from patients with head and neck squamous cell carcinoma (HNSCC) CTCs produced on the surface after 12, 15 and 38 days of culture; ( C ) CTCs in blood samples from colorectal cancer (CRC) patients after 2, 13 and 27 days of culture , CTC cultures generated on surfaces. Scale bar: 50 μm. Example 5 : High agreement between in vitro drug testing and clinical response of CTC- derived spheroids

Patient-derived CTC cultures were successfully generated in vitro from 33 blood samples obtained from 31 cancer patients recruited in the study. Samples were obtained from those diagnosed with breast cancer (n=13), colorectal cancer (n=11), head and neck cancer (n=4), urothelial cancer (n=3), lung cancer (n=1) and gastric cancer (n=1) =1) patients. These blood samples were collected and cultured on the surface of the cell culture platform of the present invention.

The rate of successful generation of CTC cultures was calculated to be 88% (29 out of 33 samples). 4 samples from breast cancer patients did not have sufficient sample size for drug testing.

We then examined the correlation between the cell viability of each drug tested in CTC cultures and the clinical response in patients. A total of 167 in vitro drug tests from 29 CTC cultures were generated on the surface of the culture platform of the present invention. For comparison, 42 treatment outcomes were obtained from matched clinical patients. Of the 42 treatment outcomes with known clinical responses, 35 CTC cultures and paired patient treatments were classified as clinically resistant and 7 additional patient treatments were classified as clinically sensitive based on their own treatment response as described by physicians .

Significantly different responses of cell viability to chemotherapeutic drugs were found between the resistant and sensitive groups of CTC cultures ( Fig. 2D , calculated by Mann-Whitney t-test, p=0.0159 ). With a sufficient judgment threshold, the CTC cultures from the sensitive group exhibited significantly lower cell viability than the CTC cultures from the resistant group. The receiver operating characteristic (ROC) curve generated from this window showed a high AUC value of 0.939 [confidence interval (CI), 0.86 to 1.01; Figure 2E ]. Data indicate that CTC cultures exhibit highly correlated drug responses and clinical treatment outcomes when tested by in vitro drug assays using the same clinical therapeutic drugs. Importantly, the assay requires only ^1-10 cells for initial seeding, and subsequent spheroids can be generated within 3 weeks for therapeutic drug screening. Example 6 : In vitro CTC culture drug response profile correlates with clinical treatment outcome

To validate the in vitro drug assay results, we tested single or combination therapeutics in CTC cultures derived from patient blood samples. In patient A, he had been previously treated with oxaliplatin, docetaxel, 5-FU and showed disease progression after clinical treatment. The designed in vitro drug assay was completed in a time-dependent and dose-dependent manner using a panel of used and potential drug candidates including oxaliplatin, docetaxel, 5-FU, cranberry, Mitomycin-c and irinotecan ( Figure 3A ). Regarding cell morphology, the diameter of CTC spheroids derived from patient A's blood sample was approximately 30-40 μm, with intact spheroid morphology in the untreated mock group. After 3 days of treatment with oxaliplatin and 5-FU, CTC spheroids were smaller in diameter than the mock group and became leaky in appearance. In the case of cranberry treatment, CTC cultures displayed a distinctly disrupted morphology and indicated cellular debris around the cells ( FIG. 3C ). On the other hand, Patient A exhibited high spheroid viability of more than 50% after 5-FU and docetaxel treatment, but less than 4% under cranberry treatment. These results are highly consistent with clinical treatment outcomes for therapeutic resistance to clinically used drugs. Therefore, for the next line of treatment, cranberry is a relevant promising drug candidate among the unused drugs (mitomycin-c and irinotecan) ( FIG. 3B ). Additional in vitro drug testing was also performed on Patient B with urothelial carcinoma. CTC cultures from patient B treated with the cisplatin/gemcitabine combination were smaller in size and cell viability than spheroids in the untreated mock group or spheroids treated with single agent ( FIGS. 3C and 3D ). Same as the actual situation, under the diagnosis of CT imaging, the patient also showed a positive clinical response and a significant shrinkage of the tumor mass after the cisplatin/gemcitabine combination therapy ( Fig . 3F ) . In conclusion, the results of CTC-based in vitro drug susceptibility assays showed high agreement with clinical patient responses. These results demonstrate the feasibility of the drug testing platform for clinical use to predict the clinical outcome of drug treatment response. Example 8 : Co-culture of patient-derived tumor spheroids with autologous NK cells from peripheral blood

Peripheral blood can be used as a source of natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) contain 10-12% of circulating NK cells. Patient-derived NK (PDNK) cells were isolated from peripheral blood of colorectal cancer patients and then expanded ex vivo two weeks after isolation.

Tumor spheroids derived from colorectal cancer patients were generated on the surface of the present invention. To examine the cytotoxicity of PDNK cells on patient-derived tumor spheroids, tumor spheroids were co-cultured with PDNK cells for 24 hours. Patient-derived tumor spheroids remained intact and showed no morphological changes after 24 hours of incubation, indicating that PDNK cells are not cytotoxic against patient-derived tumor spheroids. The results indicated that autologous PDNK cells could approach the patient-derived tumor spheroids on the surface of the present invention, but they did not recognize the patient-derived tumor spheroids as targets for killing.

As a comparison, NK-92MI cell line was used as a side-by-side control group of PDNK cells. Patient-derived tumor spheroids were co-cultured with NK-92MI cells. NK-92MI cells were observed to recognize and then rapidly surround patient-derived tumor spheroids within 2 hours. Then, NK-92MI cells further destroyed the patient-derived tumor spheroids, and fragments of the patient-derived tumor spheroids were observed on the surface of the present invention. The analysis showed that NK-92MI cells were significantly more cytotoxic against patient-derived tumor spheroids than autologous PDNK cells. Analysis is performed by cell-based assays based on luminescence or biochemical assays based on luminescence or fluorescence. Methods and Materials

Immunofluorescence staining and microscopy imaging . For immunofluorescence staining, cells were fixed with 4% paraformaldehyde in PBS buffer for 30 minutes, permeabilized with 0.1% triton X-100 in PBS buffer for an additional 30 minutes, and treated with 5% BSA blocked for 1 hour. Next, cells were stained with the large intestine-specific marker rabbit anti-human pan-cytokeratin (panCK, Abcam, Cambridge, UK), followed by nuclear staining with goat anti-rabbit 647 secondary antibody and DAPI (Invitrogen). Unstained and immunostained cells were photographed under a Nikon Ti Eclipse inverted fluorescent microscope.

Capture and release of circulating tumor cells (CTC) from clinical patients . From Chang Gung Memorial Hospital (Linkou, Taoyuan), Taipei Veterans General Hospital (Taipei Veterans General Hospital, Taipei, Taiwan) and National Taiwan University Hospital (National Taiwan University Hospital, Taipei, Taiwan) obtained peripheral blood and tissue samples from patients with breast cancer, colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma. Tissues from surgical resections or biopsy biopsies were preserved in ice-cold DMEM medium and transferred immediately after surgical removal. A total of 2 mL whole blood samples were collected from each patient by ethylene-diamine-tetra-acetic acid (EDTA) vacutainers (BD Biosciences) and used for CTC capture and release on the CTC capture platform.

Spheroid Formation on the Culture Platform of the Invention . Samples of cells to be cultured on the culture platform of the invention were collected in suspension medium. Cell suspension medium was added to each well. Cells can be cultured in a humidified 37°C incubator with 5% CO ₂ . Change half of the medium once or twice a week. Depending on cell density and desired spheroid size, cultured spheroids (tumoroids) were maintained in the culture platform of the invention for more than two weeks.

Statistical Analysis . All statistical analyzes were performed using GraphPad Prism software (version 6.0c, La Jolla, CA). A student's t-test was performed for statistical analysis and the p-value was set at 0.05 to investigate statistically significant differences between the two groups. All representative results are presented as mean ± SEM based on at least three repeated experiments.

Figures 1A- 1C show representative time-dependent images of CTC cultures produced on surfaces according to one embodiment of the invention. ( A ) CTCs obtained from blood samples of breast cancer patients, cultured for 7 and 14 days, and CTC cultures produced on the surface; ( B ) CTCs obtained from blood samples of head and neck squamous cell carcinoma patients, cultured for 12, CTC cultures generated on the surface after 15 and 38 days; ( C ) CTC cultures obtained from colorectal cancer patient blood samples cultured for 2, 13 and 27 days, and CTC cultures generated on the surface. Scale bar: 50 μm.

Figures 2A- 2E show the correlation between drug sensitivity and clinical response of CTC cultures. ( A ) General flowchart of clinical sample distribution for multiple cancer drug testing of CTC origin. ( B ) Attempt to generate CTC-derived spheroids from freshly collected blood samples. A total of 29 (88%) of 33 samples successfully generated CTC-spheroids, including breast cancer (9/13), colorectal cancer (9/9), head/neck cancer (4/4), urinary tract cancer Epithelial cancer (3/3), gastric cancer (1/1) and lung cancer (1/1). ( C ) Waterfall plot of clinical drug response based cell viability assay of paired CTC-spheroids tested on the cell culture platform of the present invention. Gray bars represent the clinically resistant population and white bars represent the clinically sensitive population. ( D ) Dot plot distribution of mean ± SEM of cell viability for drug testing from clinically resistant and clinically sensitive populations. Groups were compared using a t-test. Each dot/square represents an individual paired clinical response. ( E ) ROC curve of clinical drug test results. Dashed line represents _AUCROC of 0.5, which indicates non-predicted value. CI, confidence interval.

Figures 3A- 3F show the results of in vitro drug testing of CTC - derived spheroids and clinicopathological results in clinical patients. ( A ) Results of drug cytotoxicity assay of Patient A-derived CTC-derived spheroids. Six chemotherapeutic drugs were tested in this assay, including oxaliplatin, docetaxel, doxorubicin, irinotecan, mitomycin -C) and 5-FU. ( B and D ) Images of the morphology of CTC-derived spheroids at day 3 after drug group treatment. Scale bar = 20 μm. ( C and E ) Normalized survival of CTC-derived spheroids at day 3 post-treatment with the indicated drug groups. ( F ) Contrast-enhanced CT images of patient B before and after cisplatin/gemcitabine treatment.

Figure 4A shows a flow diagram illustrating the procedure for determining the efficacy of personalized immune cell therapy on a surface of the present invention (denoted as a PEM disc).

Figure 4B shows representative time-lapse images showing destruction of HCT116 spheroids by NK-92MI and PDNK cells, respectively, on the surface of the present invention. The mean survival of HCT116 spheroids was analyzed after 24 hours of co-cultivation. PDNK cells derived from three individuals were used and the number of HCT116 spheroids used in each independent experiment for viability analysis ranged from 15-37. Arrows, HCT116 spheroids; arrowheads, NK cells. ns, not significant. Scale bar, 50 mm.

Figure 4C shows representative time-lapse images illustrating the differential cytotoxicity of autologous PDNK cells and NK-92MI cells against CTC spheroids on the RCE platform. Interaction between CTC spheroids and NK cells during co-culture is shown. After co-cultivation with different NK cells for 24 hours, the survival rate of CTC spheroids derived from three patients was examined respectively. For determination of survival, 13-58 CTC spheroids were used in each independent experiment. Arrowheads, CTC spheroids; arrowheads, NK cells. Scale bar, 50 mm.

Claims (31)

A method for preparing a cell culture comprising: (a) provision of cell culture articles having surfaces coated with polyelectrolyte layers and optionally absorbent polymers; (b) seeding the surface with a plurality of cancer cells obtained from a fluid sample of a cancer patient; and (c) culturing the cancer cells in a suitable medium for a time sufficient to produce cell cultures, wherein the cell cultures comprise three-dimensional (3D) cell cultures comprising a plurality of cells adhered to the coated surface. of tumor spheroids. A method for evaluating a cancer therapeutic comprising: (a) prepare cell culture according to the method as claim item 1; (b) optionally culturing such cell cultures with a plurality of immune cells; (c) contacting the cell culture with a therapeutic agent; (d) assess the effect of the therapeutic agent on the cell cultures; and (e) determining that the cancer patient responds to the therapeutic agent when the therapeutic agent is effective on the cell cultures; or determining that the cancer patient responds to the therapeutic agent when the therapeutic agent is ineffective on the cancer cell cultures Unresponsive. The method of claim 1, wherein the suitable medium comprises a Rho-related protein kinase (ROCK) inhibitor, wherein the ROCK inhibitor has a structural formula of isoquinoline, 4-amidopyridine (4-amidopyridine) or 4- 4-amidopyrrolopyridine skeleton. The method of claim 1, wherein the surface is coated with a polyelectrolyte multilayer and an absorbent polymer, wherein the absorbent polymer is selected from the group consisting of: poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), PEG-acrylate, polyvinylpyrrolidone (PVP), polyethyleneimine (PEI), poly-L-lactide (PLLA), poly-D-lactide (PDLA), poly( L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PL-co-GA), poly(methyl Methyl acrylate) (PMMA), poly(hydroxyethyl methacrylate) (p-HEMA) and their derivatives. The method of claim 2, wherein the therapeutic agent is selected from the group consisting of one or more chemotherapeutic drugs, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, and combinations thereof. The method of claim 5, wherein the therapeutic agent comprises an immune checkpoint inhibitor, wherein the cell cultures are further cultured with a plurality of immune cells. The method according to claim 6, wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors and CLTA-4 inhibitors. The method of claim 7, wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, Atezolizumab, avelumab, durvalumab, and ipilimumab. The method of claim 5, wherein the therapeutic agent comprises a therapeutic cell composition. The method according to claim 9, wherein the therapeutic cell composition comprises T cells, natural killer cells (NK) or dendritic cells. The method according to claim 9, wherein the therapeutic cell composition comprises chimeric antigen receptor T (CAR-T) cells or chimeric antigen receptor-natural killer (CAR-NK) cells. The method according to claim 5, wherein the therapeutic agent comprises one or more chemotherapeutic drugs. The method according to claim 12, wherein the one or more chemotherapeutic drugs are cytotoxic or cytostatic chemotherapeutic drugs. The method according to claim 5, wherein the therapeutic agent comprises a nucleic acid drug. The method of claim 1, wherein the polyelectrolyte multilayers are formed by layer-by-layer assembly. The method of claim 1, wherein the cell cultures are in direct contact with the outermost layers of the polyelectrolyte multilayers. The method according to claim 16, wherein the outermost layer is polycation or polyanion. The method of claim 17, wherein the polycation is selected from the group consisting of poly(L-lysine) (PLL), poly(L-arginine) (PLA), poly(L-ornithine) ) (PLO) or poly(L-histidine) (PLH) and combinations thereof. The method according to claim 17, wherein the polyanion is poly(L-glutamic acid) (PLGA) or poly(L-aspartic acid) (PLAA). The substrate of claim 1, wherein the polyelectrolyte multilayers comprise n double layers of polycations and polyanions, wherein n is an integer ranging from 1 to 30. The substrate of claim 1, wherein the polyelectrolyte multilayers comprise n double layers of polycations and polyanions, and additional polyanion layers, wherein n is an integer ranging from 1 to 30. The substrate of claim 1, wherein the polyelectrolyte multilayers comprise n double layers of polycations and polyanions, and additional polycation layers, wherein n is an integer ranging from 1 to 30. The method according to claim 1, wherein the body fluid sample comprises serum, plasma, whole blood, urine or ascites. The method according to claim 1, wherein the body fluid sample comprises a blood sample. The method of claim 1, wherein the plurality of cancer cells comprise circulating tumor cells (CTCs). The method of claim 1, wherein each of the tumor spheroids is produced by single cell proliferation. The method of claim 1, wherein the average diameter of each of the tumor spheroids is between about 50 μM and about 150 μM. The method according to claim 1, wherein the seeding in step (b) comprises spreading cells on the substrate at a density of less than 1000 cells/cm2. The method of claim 2, wherein the effect of the therapeutic agent on the cell cultures is determined by performing cell-based assays and/or biochemical assays for assaying the cell cultures The size, shape, physical properties, biological properties and/or kinetic properties of an object. A method for treating cancer comprising: (a) evaluating therapeutic agents for cancer patients according to the method as claimed in claim 2; and (b) administering a therapeutically effective amount of the therapeutic agent to a cancer patient who responds to the therapeutic agent. The method of claim 30, wherein the therapeutic agent is selected from the group consisting of one or more chemotherapeutic drugs, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, and combinations thereof.