Cell culture platform, cell culture method and uses thereof

Abstract

The present disclosure provides a method for preparing a cell culture (e.g., a tumor spheroid), a method for evaluating a cancer therapeutic agent, and a method for treating cancer. The method for preparing the cell culture includes the steps of: (a) providing a cell culture article having a surface coated with a polyelectrolyte multilayer and, optionally, an absorbent polymer; (b) seeding a plurality of cancer cells on the surface, the plurality of cancer cells being obtained from a body fluid sample of a cancer patient; and (c) culturing the cancer cells under a suitable medium for a sufficient time to generate a cell culture, the cell culture comprising a three-dimensional (3D) cell culture comprising a plurality of tumor spheroids attached to the coated surface.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 252,268, filed October 5, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] From basic science to preclinical drug discovery applications, including studies of tumor biology, neurodegenerative diseases, and drug toxicity, researchers are increasingly interested in 3D spheroid models. Three-dimensional (3D) cell culture methods are increasingly being used to generate complex tissue and tumor models.

[0003] There are many variations in the spheroids formed using commercially available 3D cell culture methods and products, which may affect the readout. For example, non-adherent techniques widely used for 3D cell culture, including Ultra-Low Attachment (ULA) plates and hanging drop techniques, have not proven suitable because these methods usually create spheroids via cell aggregation. Such spheroids generally maintain their original heterogeneity and harbor multiple cells with various characteristics, so there is a need to better understand the cellular heterogeneity. When tens of thousands of cells aggregate into a spheroid (i.e., a spherical mass), an extensive central necrotic core forms over several hours, preventing cell proliferation due to the lack of nutrients and oxygen beyond a depth of 200 μm. Extensive central necrosis is a rare phenomenon in real cancer.

[0004] Alternatively, Matrigel is a commonly used embedded substrate for tissue-based cell growth such as organoid formation, but it has limitations in ex vivo 3D spheroid-based applications due to poor focusing, inefficient compound diffusion, and difficult sample isolation.

[0005] Standardizing spheroid formation is crucial to generate uniform 3D cell cultures and obtain reproducible results from spheroid-based assays for drug screening and therapeutic guidelines for cancer patients. Currently, the techniques and methods to reliably culture primary cancer cells, especially circulating tumor cells, from the majority of patients remain a challenge.

[0006] Therefore, there is a need for the development of improved cell culture platforms and methods that can reliably generate 3D cell cultures from cancer cells, in particular circulating tumor cells taken from blood samples of cancer patients. Summary of the Invention

[0007] The present disclosure provides improved cell culture platforms and methods for preparing cell cultures, particularly 3D cell cultures (e.g., spheroids). The platforms disclosed herein include cell culture articles whose surfaces are coated with polyelectrolyte multilayers (PEMs) and, optionally, absorbent polymers.

[0008] The present disclosure also relates to the use of cell cultures, particularly cancer cell cultures (e.g., tumor spheroids) prepared using the cell culture platform disclosed herein, for in vitro drug screening and evaluation of therapeutic agents for cancer. Methods of treating cancer are also provided.

[0009] Accordingly, one aspect of the present disclosure provides a method of preparing a cell culture (e.g., a 3D culture), comprising the steps of: (a) providing a cell culture article having a surface coated with a polyelectrolyte multilayer and, optionally, an absorbable polymer; (b) seeding a plurality of cancer cells on the surface; and (c) culturing the plurality of cancer cells under a suitable medium for a sufficient time to produce a cell culture, wherein the cell culture is a 3D cell culture comprising a plurality of tumor spheroids attached to the coated surface.

[0010] In some embodiments, the plurality of cancer cells is obtained from a fluid sample of a cancer patient. The fluid sample may be serum, plasma, whole blood, urine, or ascites. In some embodiments, the plurality of cancer cells may comprise circulating tumor cells, wherein the circulating tumor cells comprise cancer cells from a solid tumor.

[0011] In some embodiments, each tumor spheroid is generated via single cell proliferation.

[0012] In some embodiments, the average diameter of each tumor spheroid is about 50 μM to about 150 μM.

[0013] In some embodiments, the seeding in step (b) is at least 1000 cells / cm on the substrate. 2 This involves plating the cells at a density of less than 100 μg / ml.

[0014] In some embodiments, the coating surface described herein comprises a polyelectrolyte multilayer and an absorbent polymer. Examples of 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(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (p-HEMA), and derivatives thereof.

[0015] In some embodiments, the suitable media described herein comprises a Rho-associated protein kinase (ROCK) inhibitor, hi some embodiments, the ROCK inhibitor is of a formula having an isoquinoline, 4-amidopyridine, or 4-amidopyrrolopyridine scaffold.

[0016] In another aspect, the disclosure provides a method for evaluating a therapeutic agent for cancer, comprising the steps of: (a) preparing a cell culture (e.g., a tumor spheroid) according to the method described above; (b) optionally incubating 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) determining the cancer patient as responsive to the therapeutic agent if the therapeutic agent is effective on the cell culture, and determining the cancer patient as not responsive to the therapeutic agent if the therapeutic agent is not effective on the cell culture.

[0017] The effect of the therapeutic agent on the cell culture can be determined by performing a luminescence- and / or fluorescence-based cell-based assay and / or a biochemical assay. In some embodiments, the effect of the therapeutic agent on the cell culture can be determined by performing a luminescence-based cell viability assay. In some embodiments, the effect of the therapeutic agent can be determined by performing one or more assays to determine the size, morphology, physical properties, biological properties, and / or kinetic properties of the cells in the single-cell-derived spheroids. The assay results can provide treatment guidelines for cancer patients.

[0018] In some embodiments, the immune cells described herein comprise autologous immune cells obtained from the peripheral blood of a cancer patient. In some embodiments, the autologous immune cells comprise autologous immune cells expanded ex vivo.

[0019] Therapeutic agents described herein include, but are not limited to, chemotherapeutic agents, immune checkpoint inhibitors, nucleic acid medicines, therapeutic cell compositions, and combinations thereof.

[0020] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor and the cell culture is incubated with a plurality of immune cells (e.g., autologous immune cells obtained from peripheral blood). The immune checkpoint inhibitor described herein may be a PD-1 inhibitor, a PD-L1 inhibitor, or a CLTA-4 inhibitor. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0021] In some embodiments, the therapeutic agent is a therapeutic cell composition. In some embodiments, the therapeutic cell composition described herein is a T cell, a natural killer (NK) cell, or a dendritic cell. 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.

[0022] In some embodiments, the therapeutic agent comprises one or more chemotherapeutic agents. One or more of the chemotherapeutic agents described herein may be a cytotoxic or cytostatic chemotherapeutic agent.

[0023] In some embodiments, the therapeutic agent comprises a nucleic acid medicine.

[0024] In some embodiments, the cell culture (e.g., 3D culture) may be in direct contact with the outermost layer of the polyelectrolyte multilayer described herein. The outermost layer may be a polycation or a polyanion. 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 may be poly(L-glutamic acid) (PLGA) or poly(L-aspartic acid) (PLAA).

[0025] In some embodiments, the polyelectrolyte multilayer comprises n bilayers, n being an integer between 1 and 30, and the outermost layer is a polycation or a polyanion. The substrate of claim 16, wherein the polyelectrolyte multilayer comprises n bilayers of a polycation and a polyanion, and an additional layer of a polyanion, n being an integer between 1 and 30, and the outermost layer is a polycation or a polyanion.

[0026] In another aspect, the disclosure provides a method for treating cancer, the method comprising: (a) evaluating a therapeutic agent for a cancer patient according to the methods described herein; and (b) administering a therapeutically effective amount of the therapeutic agent to a cancer patient that is responsive to the therapeutic agent.

[0027] The therapeutic agents described herein may be one or more chemotherapeutic agents, immune checkpoint inhibitors, nucleic acid medicines, therapeutic cell compositions, or combinations thereof. [Brief description of the drawings]

[0028] [Figure 1] 1A-C show representative images showing the time course of CTC cultures generated on surfaces according to one embodiment of the present disclosure. (A) CTC cultures generated from CTCs obtained from a blood sample of a breast cancer patient were cultured on surfaces for 7 and 14 days. Scale bar: 50 μm; (B) CTC cultures generated from CTCs obtained from a blood sample of a head and neck squamous cell carcinoma (HNSCC) patient were cultured on surfaces for 12, 15, and 38 days. Scale bar: 50 μm; (C) CTC cultures generated from CTCs obtained from a blood sample of a colorectal cancer (CRC) patient were cultured on surfaces for 2, 13, and 27 days. Scale bar: 50 μm. [Figure 2A] Figure 2A-E shows the correlation between drug sensitivity of CTC cultures and clinical response. (A) Overview flow chart of clinical sample classification in CTC-originated multi-cancer drug test. (B) Test of CTC-derived spheroids established from freshly collected blood specimens. CTC spheroids were successfully established in a total of 29 out of 33 specimens (88%) from many cancer types including breast cancer (9 / 13), colon cancer (9 / 9), head and neck cancer (4 / 4), urothelial cancer (3 / 3), gastric cancer (1 / 1), and lung cancer (1 / 1). (C) Waterfall plot of cell viability test based on clinical drug response of CTC-spheroids performed paired assay on the cell culture platform of the present invention. Grey bars indicate clinically resistant group, white bars indicate clinically sensitive group. (D) Dot plot distribution of mean ± SEM of cell viability of drug test obtained from clinically resistant and sensitive groups. These groups were compared by t-test. Each point / box represents an individual paired clinical response. (E) ROC curves of clinical drug test results. The dotted line represents AUCROC0.5, indicating no predictive value. CI is confidence interval. [Figure 2B] This is a continuation of Figure 2A. [Figure 2C] This is a continuation of Figure 2B. [Figure 2D] This is a continuation of Figure 2C. [Figure 2E] This is a continuation of Figure 2D. [Figure 3A] Figure 3A-F shows the in vitro drug test results of CTC-derived spheroids and the clinical pathology results of clinical patients. (A) Drug cytotoxicity assay results of CTC-derived spheroids from patient A. Six chemotherapy drugs were tested in this assay: oxaliplatin, docetaxel, doxorubicin, irinotecan, mitomycin-C, and 5-FU. (B and D) Images of the morphology of CTC-derived spheroids at 3 days after treatment with the drug panel. Scale bar: 20 μm. (C and E) Normalized viability at 3 days after treatment of CTC-derived spheroids with the indicated drug panel. (F) Contrast-enhanced CT images of patient B before and after treatment with cisplatin / gemcitabine. [Figure 3B] This is a continuation of Figure 3A. [Figure 3C] This is a continuation of Figure 3B. [Figure 3D] This is a continuation of Figure 3C. [Figure 3E] This is a continuation of Figure 3D. [Figure 3F] Continuation of Figure 3E. [Figure 4A] 1 is a flow chart illustrating a procedure for determining the efficacy of a personalized immune cell therapy on a surface of the present disclosure (labeled a PEM plate). [Figure 4B] Representative time-lapse images showing the destruction of HCT116 spheroids by NK-92MI and PDNK cells, respectively, on a surface of the present disclosure. The average viability of HCT116 spheroids after 24 hours of co-culture was analyzed. PDNK cells from three separate individuals were used, and the number of HCT116 spheroids employed for viability analysis in each independent experiment ranged from 15 to 37. Arrowheads indicate HCT116 spheroids, arrows indicate NK cells, ns indicates no significant difference. Scale bar: 50 mm. [Figure 4C]Representative time-lapse images showing the difference in cytotoxicity between autologous PDNK and NK-92MI cells against CTC spheroids on the RCE platform. Interaction events between CTC spheroids and NK cells during co-culture are shown. The viability of CTC spheroids from three patients was examined after 24 h of co-culture with different NK cells. Between 13 and 58 of the CTC spheroids were used in each independent experiment to determine viability. Arrowheads indicate CTC spheroids, arrows indicate NK cells. Scale bar: 50 mm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description The present disclosure provides an improved cell culture platform and method for preparing cell cultures, particularly 3D cell cultures (e.g., spheroids). The surfaces described herein can induce the formation of 3D cell cultures, making it possible to form 3D cell cultures of certain primary cells, such as CTCs, that are difficult to grow using other cell culture methods available on the market. Currently, techniques and methods for reliably culturing CTCs from a majority of patients remain a challenge. The present disclosure provides an improved platform and method that allows for reliable and consistent growth of CTCs. Successful establishment of CTC cultures will provide valuable insights into the metastatic process and treatment response of individual patients.

[0030] Cell culture products of the present invention The platforms disclosed herein include cell culture articles having a surface coated with a polyelectrolyte multilayer (PEM) and optionally an absorbent polymer. In some embodiments, the surface is coated with a PEM. In some embodiments, the surface is coated with a PEM and an absorbent polymer. The PEMs disclosed herein are made up of alternating layers of oppositely charged polymers (i.e., polyelectrolytes). The oppositely charged polymers described herein are combinations of positively charged polyelectrolytes (also referred to herein as polycations) and negatively charged polyelectrolytes (also referred to herein as polyanions).

[0031] Examples of polycations include, but are not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA), poly(L-ornithine) (PLO), poly(L-histidine) (PLH), polyethyleneimine (PEI), poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), N,N-diethylaminoethyl methacrylate (DEAEMA), and combinations thereof. In some examples, the polycation is PLL. In some examples, the polycation is PLO. In some examples, the polycation is PLH. In some examples, the polycation is PLA.

[0032] Examples of 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 examples, the polyanion is PLGA. In some examples, the polyanion is PLAA.

[0033] Polyelectrolyte multilayers can be formed by alternating layers of polycations and polyanions via layer-by-layer assembly. The polyelectrolyte multilayers described herein include at least one bilayer that includes a polycation layer and a polyanion layer.

[0034] In some embodiments, the PEM may include from about 1 to about 100 bilayers. In some embodiments, the PEM may include from about 1 to about 50 bilayers. In some embodiments, the PEM may include from about 1 to about 30 bilayers. In some embodiments, the PEM may include from about 1 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. In some embodiments, the number of bilayers is 3. In some embodiments, the number of bilayers is 4. In some embodiments, the number of bilayers is 5. In some embodiments, the number of bilayers is 6. In some embodiments, the number of bilayers is 7. In some embodiments, the number of bilayers is 8. In some embodiments, the number of bilayers is 9. In some embodiments, the number of bilayers is 10. In some embodiments, the number of bilayers is 11. In some embodiments, the number of bilayers is 12. In some embodiments, the number of bilayers is 13. In some embodiments, the number of bilayers is 14. In some embodiments, the number of bilayers is 15. In some embodiments, the number of bilayers is 16. In some embodiments, the number of bilayers is 17. In some embodiments, the number of bilayers is 18. In some embodiments, the number of bilayers is 19. In some embodiments, the number of bilayers is 20.

[0035] In some embodiments, the polyelectrolyte multilayers described herein include one or more bilayers of positively charged polyelectrolytes and negatively charged polyelectrolytes, where the polycation is selected from PLL, PLO, PLH, and PLA, and the polyanion is selected from PLGA and PLAA. In some embodiments, the number of sets is 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 1-10, 1-8, 1-5, 3-20, 5-20, 10-20, 11-19, 12-18, 13-17, or 14-16.

[0036] 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 is 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.

[0037] 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 is 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.

[0038] 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 is 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.

[0039] In some embodiments, the polyelectrolyte multilayers described herein include one or more bilayers of PLA and PLGA. In some embodiments, the number of PLA and PLGA bilayers is 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.

[0040] In some embodiments, the polyelectrolyte multilayers described herein include one or more bilayers of PLL and PLAA. In some embodiments, the number of bilayers of PLL and PLAA is 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.

[0041] In some embodiments, the polyelectrolyte multilayers described herein include one or more bilayers of PLO and PLAA. In some embodiments, the number of bilayers of PLO and PLAA is 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.

[0042] In some embodiments, the polyelectrolyte multilayers described herein include one or more bilayers of PLH and PLAA. In some embodiments, the number of bilayers of PLH and PLAA is 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.

[0043] In some embodiments, the polyelectrolyte multilayers described herein include one or more bilayers of PLA and PLAA. In some embodiments, the number of bilayers of PLA and PLAA is 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.

[0044] In some embodiments, the surfaces described herein are (polyanion / polycation) n and the polyanion / polycation is selected from PLGA / PLL, PLAA / PLL, PLGA / PLA, PLAA / PLA, PLGA / PLO, PLAA / PLO, PLGA / PLH, and PLAA / PLH. In some embodiments, n is an integer from 1 to 30, and 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 1 to 5, 5 to 15, 5 to 20, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, or 15 to 30.

[0045] In some embodiments, the surfaces described herein are provided with polycations (polyanions / polycations). nand the polyanion / polycation is selected from PLGA / PLL, PLAA / PLL, PLGA / PLA, PLAA / PLA, PLGA / PLO, PLAA / PLO, PLGA / PLH, and PLAA / PLH. In some embodiments, n is an integer from 1 to 30, and 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 1 to 5, 5 to 15, 5 to 20, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, or 15 to 30.

[0046] In some embodiments, the surfaces described herein are polyanionic (polycation / polyanion) n and the polycation / polyanion is selected from PLL / PLGA, PLL / PLAA, PLA / PLGA, PLA / PLAA, PLO / PLGA, PLO / PLAA, PLH / PLGA, and PLH / PLAA. In some embodiments, n is an integer from 1 to 30, and 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 1 to 5, 5 to 15, 5 to 20, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, or 15 to 30.

[0047] The thickness of the PEM as a thin film may range over a wide range, for example, from about 30 nm to about 30 μm, or from about 100 nm to about 20 μm. In some embodiments, the thickness is from about 100 nm to about 500 nm, from about 500 nm to about 1 μm, or from about 1 μm to about 10 μm. In some embodiments, the thickness is about 200 nm, about 400 nm, about 600 nm, about 800 nm, or any value therebetween. In some embodiments, the thickness is about 1, 5, 10, 15, 20 μm, or any value therebetween.

[0048] In some embodiments, the PEM may be deposited by pipetting the polyanion and polycation solutions into / onto the dish, either as a mixture or sequentially. For use in the present disclosure, each of the polycations and polyanions described herein may be dissolved in an aqueous solution. The aqueous solution is free or substantially free of organic solvents. It will be understood that some organic solvents may be present in the aqueous solution, for example, due to organic solvents remaining in the polymer after polymerization. As used herein, "substantially free" with respect to organic solvents in an aqueous solution means that the aqueous solution contains less than 1% by weight of organic solvent. In many embodiments, the aqueous solution contains less than 0.8%, less than 0.5%, less than 0.2%, or less than 0.1% of organic solvent. Each of the polycations and polyanions may be dissolved in the aqueous solution at any concentration suitable for coating purposes.

[0049] In some embodiments, PEMs can be applied to the surface of cell culture articles by dip coating, where an article is immersed in a polyelectrolyte solution for a period of time (usually 10-15 minutes), rinsed several times, and then immersed in a second polyelectrolyte solution of the opposite charge. The process is repeated until the desired number of layers is achieved.

[0050] In some embodiments, the PEM can be applied to the surface of the cell culture article by spray coating. In some embodiments, the polyelectrolyte can be sprayed onto the surface for 3-10 seconds, followed by a 10-30 second rest / draining period, the surface can be washed with a water spray for 3-20 seconds, followed by an additional 10 second rest period, and the cycle can be repeated with a polyelectrolyte of opposite charge.

[0051] In some embodiments, the PEMs can be formed on the surface of the cell culture article by spin coating. Spin coating is a highly controlled method for solution-based coating systems. A typical spin coating procedure involves spin coating for 10-15 seconds, at least one rinse with "spin coating" water for 15-30 seconds, and repeating the procedure with an oppositely charged polyelectrolyte. A wash step may not be necessary with spin coating.

[0052] Many methodologies are available for characterizing PEMs, in some embodiments, methodologies may include ellipsometry (thickness), quartz crystal microbalance with dissipation monitoring (adsorbed mass, viscoelasticity), contact angle analysis (surface energy), Fourier transform infrared spectroscopy (functional groups), X-ray photoelectron spectroscopy (chemical composition), scanning electron microscopy (surface structure), and atomic force microscopy (roughness / surface structure).

[0053] As described herein, a surface is hydrophilic if the contact angle for a drop of water on the surface is less than 90 degrees (the contact angle is defined as the angle passing through the interior of the drop of water). Embodiments include hydrophilic surfaces with contact angles between 90 degrees and 0 degrees. One of ordinary skill in the art will readily appreciate that all ranges and values ​​within this defined boundary are contemplated, e.g., any of the following may be set as upper or lower limits: 90 degrees, 80 degrees, 70 degrees, 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20 degrees, 10 degrees, 5 degrees, 2 degrees, 0 degrees.

[0054] In some embodiments, the surface may be further coated with an absorbent polymer. In some cases, the absorbent polymer may be the innermost layer of a coating attached to the surface of the cell culture article. In some embodiments, the absorbent polymer is physically crosslinked to the surface of the support. In some embodiments, the absorbent polymer is chemically crosslinked to the surface of the support.

[0055] The absorbent polymers described herein are hydrophilic absorbent polymers. A non-limiting list of absorbent polymers that can be used in the present invention includes hydrophilic and biocompatible grades of the following polymers and their derivatives: polyvinyl alcohol (PVA), ethylene vinyl alcohol copolymers (typically non-biodegradable materials whose degree of hydrophilicity depends on the distribution of ethylene (hydrophobic) and vinyl alcohol (hydrophilic) groups), copolymers of polyvinyl alcohol and ethylene vinyl alcohol, polyacrylate compositions, polyurethane compositions, poly(ethylene glycol) (PEG), also known as poly(oxyethylene) (POE) and poly(ethylene oxide) (PEO), and its derivatives, including but not limited to polyethylene glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate (PEGDMA) and polyethylene glycol diacrylate (PEGDA); Nitrogen-containing materials such as polyacrylamide (free of acrylamide toxic residues), polyvinylpyrrolidone, polyvinylamine, polyethyleneimine; electrostatically charged materials such as polylactic acid, also known as polylactide in its various forms, and its derivatives, e.g., poly-L-lactide (PLLA) and its derivatives, poly-D-lactide (PDLA) and its derivatives, poly(L-lactide-co-D,L-lactide) (PLDLLA) and its derivatives), polyglycolic acid (PGA), also known as polyglycolide, poly(lactic acid-co-glycolic acid) (PL-co-GA), which is a copolymer of lactic acid and glycolic acid, copolymers of PLA and / or PGA with PEG; polymethacrylic acid; poly(hydroxyethyl methacrylate) (poly-HEMA), among other absorbable, hydrophilic, and biocompatible materials known in the art.

[0056] In some embodiments, the absorbable polymer is selected from the group consisting of poly(vinyl alcohol) (PVA), copolymers of ethylene vinyl alcohol, copolymers of polyvinyl alcohol and ethylene vinyl alcohol, polyacrylate compositions, polyurethane compositions, poly(ethylene glycol) (PEG), PEG-acrylate, polyethylene glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate (PEGDMA), polyethylene glycol diacrylate (PEGDA), polyacrylamide (PAM), polyvinylpyrrolidone (PVP), polyvinylamine (PVAm), polyethyleneimine (PEI), poly-L-lactide (PLLA), poly-D-lactide (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), and poly(hydroxyethyl methacrylate) (p-HEMA).

[0057] In some embodiments, the absorbable polymer is selected from the group consisting of PVA, PEG, PEG-acrylate, polybutyric acid, PMMA, p-HEMA, combinations or derivatives thereof. In some embodiments, the absorbable polymer is PVA or a derivative thereof. In some embodiments, the absorbable polymer is PEG or a PEG-acrylate such as PEGMA, PEGDMA or PEGDA. In some embodiments, the absorbable polymer is polylactic acid or a derivative such as PLLA, PDLA or PLDLLA. In some embodiments, the absorbable polymer is PGA or a derivative such as PLGA. In some embodiments, the absorbable polymer is PMAA or a derivative such as pHEMA.

[0058] In some embodiments, the average molecular weight of the absorbent polymer is about 2,500 g / mol to about 200,000 g / mol. In some cases, the average molecular weight of the hydrophilic polymer is about 5,000 g / mol to about 175,000 g / mol, about 5,000 g / mol to about 150,000 g / mol, about 5,000 g / mol to about 125,000 g / mol, 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, or about 10,000 g / mol. mol to about 175,000 g / mol, about 10,000 g / mol to about 150,000 g / mol, about 10,000 g / mol to about 125,000 g / mol, about 10,000 g / mol to about 100,000 g / mol, about 10,000 g / mol to about 75,000 g / mol, about 10,000 g / mol to about 50,000 g / mol, about 10,000 g / mol to about 25,000 g / mol, about 20,000 g / mol to about 150,000 g / mol, or about 50,000 g / mol to about 150,000 g / mol.

[0059] In some embodiments, the volume of absorbent polymer (e.g., PVA or PEG) is about 0.01% to about 10% of the total volume of the surface coating. In some examples, the absorbent polymer is 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.

[0060] In some examples, the weight of absorbent polymer (e.g., PVA or PEG) per total weight of the surface coating is about 1% to about 50%. In some examples, the weight of absorbent polymer per total weight of the surface coating is about 1% to about 10%, 20%, 30%, or 40%.

[0061] In some embodiments, the absorbent polymer may be in direct contact with the surface of the cell culture article. In some instances, the absorbent polymer is directly layered onto the surface of the cell culture article. In some instances, the absorbent polymer is indirectly layered onto the surface of the cell culture article. In some instances, the absorbent polymer is physically crosslinked to the surface of the cell culture article. In some instances, the absorbent polymer is chemically crosslinked to the surface of the cell culture article.

[0062] In some embodiments, the absorbent polymer may be in direct contact with the PEM. In some embodiments, the absorbent polymer is physically crosslinked to the polyanion and / or polycation of the PEM. In some embodiments, the absorbent polymer is chemically crosslinked to the polyanion and / or polycation of the PEM.

[0063] In some embodiments, the surface further comprises a filler. In some examples, the filler comprises a mineral filler, such as, but not limited to, silica, alumina, calcium carbonate, or silicone resin.

[0064] The surfaces disclosed herein allow for cell attachment and proliferation as well as harvesting of live cultured cells (e.g., 3D cell culture). According to some embodiments of the present disclosure, the cell culture substrate can be used to harvest live cells (e.g., 80% to 100% live cells, or about 85% to about 99% live cells, or about 90% to about 99% live cells, etc.). For example, of the cells harvested, at least 80% are live cells, at least 85% are live cells, at least 90% are live cells, at least 91% are live cells, at least 92% are live cells, at least 93% are live cells, at least 94% are live cells, at least 95% are live cells, at least 96% are live cells, at least 97% are live cells, at least 98% are live cells, or at least 99% are live cells. In some embodiments, cells can be released from the cell culture system with or without the use of cell dissociation enzymes, such as trypsin, TrypLE, Accutase, etc.

[0065] The cell culture articles disclosed herein include supports made of any suitable material, such as silicon, plastic, glass, elastomers, etc. In some embodiments, the supports are made of elastomers. The elastomers described herein may be silicone elastomers. In some embodiments, the silicone elastomer is polydimethylsiloxane (PDMS).

[0066] In some embodiments, the support is made of a glass material such as soda lime glass, Pyrex glass, Vycor glass, quartz glass, etc. In some embodiments, the support is made of a plastic or polymer such as polyethylene, polypropylene, polymethylpentene, cyclic olefin polymers, cyclic olefin copolymers, polyvinyl chloride, polyurethane, polyester, polyamide, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-acrylic acid copolymer, ethylene-methyl acrylate copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl methacrylate copolymer, polyacrylic acid, polymethacrylic acid, polymethyl acrylate, and polymethyl methacrylate, or derivatives thereof. In some embodiments, a support is used made of a material comprising at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0067] Methods and uses thereof 1) Preparation of cell cultures In one aspect, the present disclosure provides a method of preparing a cell culture using the cell culture substrate of the present disclosure. The method disclosed herein includes the steps of (a) providing a cell culture substrate having a surface comprising a polyelectrolyte multilayer as described herein and optionally a resorbable polymer, (b) seeding a plurality of cells on the surface, and (c) culturing the plurality of cells under a suitable medium for a sufficient time to generate a cell culture on the surface. In a preferred embodiment, the cell culture is adherent to the surface. In some embodiments, the cell culture comprises a three-dimensional (3D) cell culture. The 3D cell culture may be in the form of a spheroid. In some embodiments, the spheroids described herein are generated via single cell growth. In some embodiments, the cell culture comprises one or more single cell-derived spheroids. In a preferred embodiment, the single cell-derived spheroids are adherent to the surface. In some embodiments, the single cell-derived spheroids are semi-adherent or loosely adherent to the surface.

[0068] In some embodiments, the seeding of the plurality of cells in step (b) is at a surface density of between 1 cell and 10 cells / cm. 2 In some embodiments, the seeding of the plurality of cells in step (b) comprises plating the cells at a density of between 10 cells / cm and 100 cells / cm on the surface. 2 In some embodiments, the seeding of the plurality of cells in step (b) comprises plating the cells at a density of between 100 cells / cm and 1000 cells / cm on the substrate surface. 2 In some embodiments, the seeding of the plurality of cells in step (b) comprises plating the cells at a density of between 200 cells / cm and 5000 cells / cm on the substrate surface. 2 This involves plating the cells at a density of 100-200 μg / ml.

[0069] In some cases, the spheroids described herein may contain about 8 to about 1000 cells. In some cases, the spheroids may contain 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 200 cells, about 10 to about 100 cells, about 50 to about 1000 cells, about 50 to about 800 cells, about 10 to about 100 cells, about 50 to about 800 cells, about 10 to about 100 cells, about 50 to about 1000 cells, about 50 to about 800 cells, about 10 to about 1 ... cells, about 50 to about 500 cells, about 50 to about 400 cells, about 50 to about 300 cells, about 50 to about 200 cells, about 100 to about 1000 cells, about 100 to about 800 cells, about 100 to about 500 cells, about 100 to about 400 cells, about 100 to about 300 cells, about 300 to about 1000 cells, about 300 to about 800 cells, about 300 to about 500 cells, about 500 to about 1000 cells, or about 500 to about 800 cells.

[0070] In some embodiments, the diameter of the spheroids described herein may be about 40 μm to about 200 μm. In some embodiments, the diameter of the spheroids may be about 50 μm to about 150 μm. In some cases, the diameter of the spheroids may be 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, 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.

[0071] In some embodiments, the cells are cultured for 2-8 days (e.g., 2, 3, 4, 5, 6, 7, or 8 days). In other embodiments, the cells are cultured for 7-14 days (e.g., 7, 8, 9, 10, 11, 12, 13, or 14 days). In other embodiments, the cells are cultured for 1-4 weeks (e.g., 1, 2, 3, 4 weeks).

[0072] In some embodiments, the plurality of cells described herein comprises cancer cells. In some embodiments, the cancer cells are obtained from a biological sample of a cancer patient. In some embodiments, the biological sample is a bodily fluid sample. The bodily fluid sample described herein can be serum, plasma, whole blood, urine, or ascites. In some embodiments, the bodily fluid sample is a blood sample, and the cancer cells comprise circulating tumor cells (CTCs) and / or tumor-associated cells. CTCs can be isolated from blood samples. Several strategies are currently available for the isolation of CTCs. Enrichment is a key step when it comes to isolating live CTCs from the remaining blood components such as platelets, red blood cells, and white blood cells, and increasing the concentration of CTCs can facilitate 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 isolating viable circulating tumor cells (CTCs) include, but are not limited to, RosetteSep®, CTC-iChip, Ficoll®, Ficoll-Pacque®, Lymphoprep®, Percoll®, MetaCell®, Parsortix®, Collagen adhesion matrix assay (CAM), and EPithelial ImmunoSPOT assay (EPISPOT).

[0073] Any suitable culture medium may be employed in the methods of the exemplary embodiments, including, but not limited to, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with epidermal growth factor (EGF) and / or basic fibroblast growth factor (bFGF), and a mixture of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with B27 supplement, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

[0074] Media for culturing cancer cells (e.g., CTCs) may include additional growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF, or FGF2), fibroblast growth factor-10 (FGF10), granulocyte macrophage colony-stimulating factor (GM-CSF), insulin, and insulin-like growth factor 1 (IGF-1). In some instances, R-spondin 1 protein may be included to maintain epithelial cell survival and proliferation via WNT / β-catenin signaling. In some embodiments, when serum-free culture media is used, B27 and N2 supplements may be added with or without supplementation with insulin, transferrin, and selenium (ITS). In some embodiments, small molecule inhibitors for certain signaling pathways may be included in the media to promote CTC proliferation. For example, A83-01 and ALX-270-445 (ALK5 inhibitor II) may specifically inhibit the function of TGFβ type 1 receptor kinase (ALK5) to prevent epithelial to mesenchymal transition and promote survival and proliferation of CTCs. SB202190, a p38 MAPK inhibitor, may prevent apoptosis due to p38 activation. In some embodiments, the culture medium may include a Rho-associated protein kinase (ROCK) inhibitor. The ROCK inhibitors described herein may be of a structural formula having an isoquinoline, 4-amidopyridine or 4-amidopyrrolopyridine backbone. In some embodiments, the ROCK inhibitor is an isoquinoline-based ROCK inhibitor, such as fasudil, hydroxyfasudil, H-1152P, ripasudil or derivatives thereof. In some embodiments, the ROCK inhibitor is a 4-amidopyridine-based ROCK inhibitor, such as Y27632, Y32885 or derivatives 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, ripasudil, Y30141, Y39983, Y27632, and Y32885 are shown below. [ka] TIFF2024535500000003.tif56149

[0075] In some embodiments, the biological sample may be a human primary tumor sample. The primary tumor sample may include primary tumor cells or metastatic tumor cells of a patient. In some embodiments, the cancer cells may be obtained by disintegrating the primary tumor sample in serum-supplemented medium, treating the disintegrated primary tumor sample with an enzyme, and harvesting tumor spheroids from the enzyme-treated sample. In some embodiments, the disintegrated primary tumor sample is treated with a sufficient amount of enzyme and / or for a sufficient time to effect partial digestion of the disintegrated primary tumor sample, preferably, the treatment is at 25° C. to 39° C. for 10 minutes to 60 minutes, more preferably, 15 minutes to 45 minutes.

[0076] Examples of cancers described herein include acute lymphatic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, cancer of the intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, cancer of the nose, nasal cavity or middle ear cancer, and / or pulmonary cancer. ear), oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, pleural 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.

[0077] In some embodiments, the plurality of cells may include tumor-associated cells, including, but not limited to, tumor cell clusters, tumor-infiltrating lymphocytes (TILs), cancer-associated macrophage-like cells (CAMLs), tumor-associated macrophages (TAMs), tumor-associated monocyte / macrophage lineage cells (MMLCs), cancer stem cells, tumor microemboli, tumor-associated stromal cells (TASCs), tumor-associated myeloid cells (TAMCs), tumor-associated regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), tumor-derived endothelial cells (TECs), tumor-associated neutrophils (TANs), tumor-associated platelets (TAPs), tumor-associated immune cells (TAIs), myeloid-derived suppressor cells (MDSCs), and combinations thereof.

[0078] In some embodiments, the cancer cell cultures described herein include one or more cancer cell spheroids (e.g., tumor spheroids). The tumor spheroids described herein can be used to evaluate therapeutic agents for cancer.

[0079] 2) Methods for evaluating therapeutic agents Another aspect of the disclosure features a method for evaluating a therapeutic agent for a cancer patient, the method including: (a) preparing a cell culture (e.g., a CTC culture) according to the methods described herein; (b) optionally incubating 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) determining the cancer patient as responsive to the therapeutic agent if the therapeutic agent is effective on the cell culture, or determining the cancer patient as not responsive to the therapeutic agent if the therapeutic agent is not effective on the cell culture.

[0080] In some embodiments, the effect of the therapeutic agent is analyzed by performing a luminescence- and / or fluorescence-based cell-based assay and / or a biochemical assay. In some embodiments, the effect of the therapeutic agent is analyzed by performing a luminescence-based cell viability assay. In some embodiments, the effect of the therapeutic agent can be analyzed by performing one or more assays to determine the size, morphology, physical properties, biological properties, and / or kinetic properties of cells in the tumor spheroids. In some embodiments, the effect of the therapeutic agent can be further analyzed based on one or more assays to analyze the biochemical activity and / or expression level of one or more genes or one or more proteins in the tumor spheroids. The assay results can provide treatment guidelines for cancer patients.

[0081] In some embodiments, the plurality of immune cells described herein comprises autologous immune cells derived 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 a donor and expanded ex vivo. In other embodiments, the immune cells comprise natural killer (NK) cells differentiated from human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) and expanded ex vivo. Incubating the tumor spheroids with the plurality of immune cells can form a tumor cell-immune cell co-culture, which allows for the ex vivo recreation of a tumor microenvironment that mimics what occurs in vivo so that the efficacy of a therapeutic agent can be evaluated when administered in vivo in treating cancer.

[0082] Suitable therapeutic agents include, but are not limited to, chemotherapeutic agents, immune checkpoint inhibitors, nucleic acid medicines, therapeutic cell compositions, and combinations thereof.

[0083] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic chemotherapeutic agent. The methods described herein include contacting the tumor spheroids with a cytotoxic or cytostatic chemotherapeutic agent in the presence or absence of a plurality of immune cells. The chemotherapeutic agent may be an alkylating agent (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), antimetabolites (taxanes such as paclitaxel and dextaxel, vincristine, and the like), and / or cyclosporine (such as cyclosporine, cyclosporine, and the like). vinca alkaloids (such as vinostine, vinblastine, vinorelbine, and vindesine), anthracyclines (such as doxorubicin, daunorubicin, barbicin, idarubicin, epirubicin, and actinomycins (such as actinomycin D), cytotoxic antibiotics (such as mitomycin, plicamycin, and bleomycin), topoisomerase inhibitors (camptothecins such as camptothecin, irinotecan, and topotecan, as well as amsacrine and etoposide) , epipodophyllotoxin derivatives such as etoposide phosphate, teniposide, etc.), antibodies against vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®) and other anti-VEGF compounds; anti-PD-1 (anti-programmed death-1) therapeutics such as antibodies or compounds (e.g., nivolumab); thalidomide (THALOMID®) and its derivatives (lenalidomide (REVLIMID®) etc.); endostatin; angiostatin; sunitinib (SUTE receptor tyrosine kinase (RTK) inhibitors such as rifenib (NT®); tyrosine kinase inhibitors such as sorafenib (Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib; transforming growth factor-α or transforming growth factor-β inhibitors, antibodies against the epidermal growth factor receptor such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

[0084] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. The methods described herein include contacting the tumor spheroid with an immune checkpoint inhibitor in the presence of a plurality of immune cells. The immune checkpoint inhibitor may be CD137, CD134, PD-1, KIR, LAG-3, PD-L1, PDL2, CTLA-4, B7.1, B7.2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, BTLA, LIGHT, HVEM, GAL9, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK1, CHK2, A2aR, TGF-β, PI3Kγ, GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG, CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, CD40, and combinations thereof. 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, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0085] In some embodiments, the therapeutic agent is a nucleic acid drug. The methods described herein include contacting tumor spheroids with a nucleic acid drug in the presence or absence of a plurality of immune cells. The nucleic acid drug may be DNA, a DNA plasmid, nDNA, mtDNA, gDNA, RNA, siRNA, miRNA, mRNA, piRNA, antisense RNA, snRNA, snoRNA, vRNA, and combinations thereof. In some embodiments, the therapeutic nucleic acid is a DNA plasmid 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.

[0086] In some embodiments, the therapeutic agent is a therapeutic cell composition. The methods described herein include contacting tumor spheroids with a therapeutic cell composition in the absence of a plurality of immune cells. Examples of 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.

[0087] 3) Cancer treatment using immunotherapy Another aspect of the disclosure features a method of treating cancer with immunotherapy in a cancer patient, the method including (a) evaluating a therapeutic agent for immunotherapy in a cancer patient according to the methods described herein, and (b) administering a therapeutically effective amount of the therapeutic agent to a cancer patient that is responsive to the therapeutic agent.

[0088] In some embodiments, the immunotherapeutic therapeutic comprises an immune checkpoint inhibitor. In some embodiments, the immunotherapeutic therapeutic comprises a therapeutic cell composition. In some embodiments, the immunotherapeutic therapeutic comprises a nucleic acid medicine. In some embodiments, the cancer patient is further administered a cytotoxic or cytostatic chemotherapeutic agent.

[0089] 4) Method for preparing single-cell-derived spheroids in vitro Another aspect of the present disclosure provides a method of preparing single-cell-derived spheroids in vitro, the method comprising: (a) providing a cell culture substrate having a surface comprising a polyelectrolyte multilayer as described herein and optionally an absorbent polymer; (b) isolating cells (e.g., tumor cells and / or tumor-associated cells) from a sample (e.g., a liquid biopsy sample, a needle biopsy, a tissue biopsy) to provide isolated cells; (c) seeding the isolated cells onto the substrate; and (d) amplifying the cells by culturing in the cell culture substrate under an appropriate medium for a sufficient time to generate one or more spheroids, wherein each of the one or more spheroids is derived from a single cell.

[0090] One or more spheroids described herein are produced through single cell growth.In a preferred embodiment, one or more spheroids described herein are produced through single cell growth without cell aggregation.In some embodiments, the size of one or more spheroids is uniform.

[0091] In some embodiments, the seeding of the isolated cells in step (c) is at a concentration of between 1 cell and 10 cells / cm on the surface (i.e., the cell growth surface). 2 In some embodiments, the seeding of the isolated cells in step (c) comprises plating the cells at a density of between 10 cells / cm and 100 cells / cm on the surface. 2 In some embodiments, the seeding of the isolated cells in step (c) comprises plating the cells at a density of between 100 cells / cm and 1000 cells / cm on the surface. 2 This involves plating the cells at a density of (2).

[0092] In some embodiments, the isolated cells are cancer cells. In some embodiments, the cancer cells are isolated from human primary tumor tissue. In some embodiments, the cancer cells are isolated from a blood sample of a cancer patient.

[0093] In some embodiments, the culture 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) supplemented with B27 supplement, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

[0094] In some embodiments, the cell culture system of the present disclosure can maintain the size of single cell-derived spheroids below 200 μm in diameter to prevent cell necrosis and induce division into smaller spheroids as cells continue to grow over time. In some embodiments, the size of single cell-derived spheroids is below 150 μm in diameter. In some embodiments, the size of single cell-derived spheroids is about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 μm in diameter. Cell debris due to apoptosis / necrosis is not observed in single cell-derived spheroids after 7 days of culture, unlike platforms commonly used in the market, such as hanging drop and U-shaped ULA plates.

[0095] 5) Screening method for therapeutic agents In another aspect, the present disclosure provides a method for screening a therapeutic agent, comprising: (a) providing a single-cell-derived spheroid comprising a population of cancer cells, the single-cell-derived spheroid being prepared using a cell culture substrate disclosed herein; (b) adding a test substance to the single-cell-derived spheroid; and (c) evaluating the effect of the test substance on the single-cell-derived spheroid.

[0096] In some embodiments, the isolated cells are stem-like cells. Such stem-like cells can be identified and isolated using markers known in the art.

[0097] In some embodiments, the single cell-derived spheroids are tumor spheroids that comprise a population of cancer stem-like cells. In some embodiments, the population of cancer stem-like cells is at least about 1×10 4 In some embodiments, the population of cancer stem-like cells comprises at least about 1×10 3In some embodiments, the population of cancer stem-like cells comprises at least about 5 to 900 cancer stem-like cells, for example, 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 produced herein exhibit high stemness and epithelial-mesenchymal transition (EMT) potential.

[0098] The cancer stem-like cells described herein can be obtained from cancer cells and induced by culturing 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 cancer stem cell complete culture medium.

[0099] The population of cancer stem-like cells (CSCs) generated herein is useful for in vitro drug screening for cancer therapy and cancer immunotherapy targeting CSCs.

[0100] 6) Screening method for therapeutic agents that selectively target cancer stem-like cells In another aspect, the present disclosure provides a method for screening a therapeutic agent that selectively targets cancer stem-like cells, comprising the steps of (a) providing a single-cell-derived spheroid comprising a population of cancer stem-like cells, the single-cell-derived spheroid being prepared using a cell culture substrate having a surface as disclosed herein, (b) adding a test substance to the single-cell-derived spheroid, and (c) evaluating the effect of the test substance on the single-cell-derived spheroid.

[0101] 7) Methods for inhibiting cancer metastasis, and methods for preventing, alleviating, or treating cancer In another aspect, the disclosure provides a method for inhibiting cancer metastasis and preventing, alleviating or treating cancer, comprising administering to a cancer patient an effective amount of a therapeutic agent that selectively targets cancer stem-like cells, the therapeutic agent being screened and selected by the methods described above.

[0102] In another aspect, the present disclosure provides a composition comprising an in vitro generated single-cell-derived spheroid, wherein the single-cell-derived spheroid is prepared by the above method.

[0103] In some embodiments according to any of the above methods, the cancer cells comprise cells of a primary cell culture obtained from a tumor sample (such as a tissue or fluid sample) obtained from surgery, biopsy, or blood of a subject, such as a liquid biopsy sample, needle biopsy sample, or cervical smear sample. In some embodiments, the sample is a blood, saliva, or urine sample. In some embodiments, the sample is obtained from a cancer patient, such as a cancer patient with metastatic cancer, or a cancer patient before, during, or after 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 above methods, the cancer cells comprise cells of a cancer cell line.

[0104] In some embodiments according to any of the above methods, the test agent is a chemotherapeutic agent, such as a cytotoxic or cytostatic chemotherapeutic agent. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor, such as an immune checkpoint inhibitor. In some embodiments, the therapeutic agent is a nucleic acid medicine. In some embodiments, the therapeutic agent is a therapeutic cell composition, including, but not limited to, T cells, natural killer (NK) cells, and dendritic cells.

[0105] In some embodiments of any of the above cell culture methods, the cells are cultured for about 2 days to about 5 weeks, e.g., about 3 days to about 14 days, e.g., about 7 days. In some embodiments, the cells are cultured for 3 days and the average diameter of at least one 3D spheroid is about 40 μm to about 200 μm.

[0106] In some embodiments according to any of the above methods, the polyelectrolyte multilayer has a thickness of about 30 nm to about 30 μm (including about 100 nm to about 20 μm, such as about 500 nm).

[0107] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the foregoing summary and the following detailed description are exemplary and explanatory only and are not intended to limit the claimed subject matter. In this application, the singular form includes the plural form unless otherwise specified. It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" also include the plural form unless the context clearly dictates otherwise. In this application, when "or" is used, it means "and / or" unless otherwise specified. Furthermore, the use of the term "including" is not limiting, as are other forms such as "include", "includes", "included", etc.

[0108] As used herein, ranges and amounts may be expressed by adding "about" to a particular value or range. "About" includes the amount itself. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

[0109] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0110] 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" means that the claim may include other steps or elements only to the extent that they do not materially affect the basic and novel characteristics of the claimed method. "Consisting of" means excluding any element or step not recited in the claim. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0111] As used herein, the term "positively charged polyelectrolyte" includes monomeric units or non-polymeric molecules that contain two or more positive charges. In some instances, a positively charged polyelectrolyte also includes monomeric units or non-polymeric molecules that contain positively charged groups, neutrally charged groups, or negatively charged groups, resulting in a net positive charge.

[0112] As used herein, the term "cationic polymer" includes a plurality of monomeric units or non-polymeric molecules. In some instances, the cationic polymer is a synthetic polymer. In other instances, the cationic polymer is a natural polymer.

[0113] As used herein, the term "cationic polypeptide" refers to a polypeptide that contains two or more positive charges. In some instances, the cationic polypeptide contains positively charged amino acid residues, negatively charged residues, and polar residues, but the net charge of the polypeptide is positive. In some instances, the cationic polypeptide is 8-100 amino acids in length. In some instances, the cationic polypeptide is 8-80, 8-50, 8-40, 8-30, 8-25, 8-20, 8-15, 10-100, 10-80, 10-50, 10-40, 10-30, 10-20, 20-100, 20-80, 20-50, 20-40, 20-30, 30-100, 30-80, 30-50, 40-100, 40-80, or 50-100 amino acids in length.

[0114] As used herein, the term "negatively charged polyelectrolyte" includes monomeric units or non-polymeric molecules that contain two or more negative charges. In some instances, negatively charged polyelectrolytes also include monomeric units or non-polymeric molecules that contain positively charged groups, neutrally charged groups, or negatively charged groups, and have a net negative charge.

[0115] As used herein, the term "anionic polymer" includes a plurality of monomeric units or non-polymeric molecules. In some instances, the anionic polymer is a synthetic polymer. In other instances, the anionic polymer is a natural polymer.

[0116] As used herein, the term "anionic polypeptide" refers to a polypeptide that contains two or more negative charges. In some instances, anionic polypeptides contain positively charged amino acid residues, negatively charged residues, and polar residues, but the net charge of the polypeptide is negative. In some cases, anionic polypeptides are 8-100 amino acids in length. In some cases, anionic polypeptides are 8-80, 8-50, 8-40, 8-30, 8-25, 8-20, 8-15, 10-100, 10-80, 10-50, 10-40, 10-30, 10-20, 20-100, 20-80, 20-50, 20-40, 20-30, 30-100, 30-80, 30-50, 40-100, 40-80, or 50-100 amino acids in length.

[0117] As used herein, the term "polymer" includes both homopolymers and copolymers, branched and unbranched polymers, and natural or synthetic polymers.

[0118] 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, including primary or metastatic cancerous growths).

[0119] 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.

[0120] The term "tumor spheroid" or "tumor cell spheroid" as used herein refers to an aggregate of tumor cells that constitutes a small mass or lump 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 between 10 μm and 500 μm. In some embodiments, the diameter of the tumor spheroid is between 40 μm and 100 μm. In some embodiments, the diameter of the tumor spheroid is between 40 μm and 70 μm.

[0121] As used herein, immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, lymphocytes (B cells and T cells). EXAMPLES

[0122] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0123] Example 1: Construction of a surface according to the invention To construct surface coatings with polyelectrolyte multilayers, plasma-treated tissue culture plates (TCP, polystyrene) were used for polycation / polyanion deposition. Plasma treatment involves exposing the surface of the TCP plate to oxygen plasma. Oxygen plasma treatment generates radical species on the surface, including silanol groups (Si-OH), alcoholic hydroxyl groups (C-OH), and carboxylic acid (COOH), which can form hydrogen bonds between the polyanion (or polyanions) and the activated TCP surface.

[0124] To construct the polyelectrolyte multilayers, both the polycation and the polyanion were dissolved in Tris-HCl buffer (pH 7.4) and rinsed with Tris-HCl buffer before layering on the TCP surface. Each layer of polycation or polyanion was layered, incubated, and then washed 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 (MW150K-300K), PLGA (MW50K-100K), PLO (0.01%) solution, PLH (MW5K-25K), and PLA (MW15K-70K) are commercially available from Sigma-Aldrich (St. Louis, MO, USA). Both polycations and polyanions are dissolved in Tris-HCl buffer (pH 7.4), rinsed with Tris-HCl buffer, and then layered on a TCP plate. Each layer of polycation or polyanion is layered and incubated for 10 minutes, then washed three times with Tris-HCl buffer for 2 minutes, 1 minute, and 1 minute. PLL / PLGA, PLO / PLGA, PLH / PLGA, and PLA / PLGA multilayer films can be fabricated by layer-by-layer deposition as follows.

[0125] In some examples, the polyelectrolyte multilayer is a PLL / PLGA multilayer, which can be constructed by sequentially layering PLL and PLGA onto a plate. Each layering step involves adding a PLL or PLGA solution to the plate surface, incubating for 10 minutes, and washing three times for 2 minutes, 1 minute, and 1 minute. In one embodiment, a substrate is constructed that is comprised of (PLGA / PLL)3. In one embodiment, a substrate coating is constructed that is comprised of (PLGA / PLL)5. In one embodiment, a substrate coating is constructed that is comprised of (PLGA / PLL)6. 10 In one embodiment, a substrate is constructed comprising (PLGA / PLA) 15 A substrate is constructed comprising:

[0126] In some examples, the polyelectrolyte multilayer is a PLO / PLGA multilayer, which can be constructed by sequentially layering PLO and PLGA onto a plate. Each layering step involves adding a PLO or PLGA solution to the plate surface, incubating for 10 minutes, and washing three times for 2 minutes, 1 minute, and 1 minute. In one embodiment, a substrate is constructed that is composed of (PLGA / PLO)3. In one embodiment, a substrate coating is constructed that is composed of (PLGA / PLO)5. In one embodiment, a substrate coating is constructed that is composed of (PLGA / PLO)6. 10 In one embodiment, a substrate is constructed that is composed of (PLGA / PLO) 15 A substrate is constructed comprising:

[0127] In some examples, the polyelectrolyte multilayer is a PLH / PLGA multilayer, which can be constructed by sequentially layering PLH and PLGA onto a plate. Each layering step involves adding a PLH or PLGA solution to the plate surface, incubating for 10 minutes, and washing three times for 2 minutes, 1 minute, and 1 minute. In one embodiment, a substrate is constructed that is comprised of (PLGA / PLH)3. In one embodiment, a substrate coating is constructed that is comprised of (PLGA / PLH)5. In one embodiment, a substrate coating is constructed that is comprised of (PLGA / PLH)6. 10 In one embodiment, a substrate is constructed comprising (PLGA / PLH) 15 A substrate is constructed comprising:

[0128] In some examples, the polyelectrolyte multilayer is a PLA / PLGA multilayer, which can be constructed by sequentially layering PLA and PLGA onto a plate. Each layering step involves adding a PLA or PLGA solution to the plate surface, incubating for 10 minutes, and washing three times for 2 minutes, 1 minute, and 1 minute. In one embodiment, a substrate is constructed that is comprised of (PLGA / PLA)3. In one embodiment, a substrate coating is constructed that is comprised of (PLGA / PLA)5. In one embodiment, a substrate coating is constructed that is comprised of (PLGA / PLA)6. 10 In one embodiment, a substrate is constructed comprising (PLGA / PLA) 15 A substrate is constructed comprising:

[0129] Example 2: Comparison of cell culture platforms for spheroid formation To establish ex vivo 3D models to facilitate clinical applications such as personalized drug testing, various culture platforms were evaluated. Ultra-low attachment surface (ULA) plates (Corning) were able to form spheroids based on cell aggregation as a platform for suspension culture, but the yield of spheroid formation was very low. In our experiments, less than 1% of cells seeded and cultured on ULA plates were able to form spheroids. For comparison, plates coated with layer-by-layer polyelectrolyte multilayers (PEMs) were evaluated for ex vivo culture of spheroids. For example, after culturing the HCT116 colon cancer cell line in DMEM complete medium for 5 to 7 days, 78.7 ± 2.7% of cells seeded and cultured on PEM-coated plates proliferated and formed cell spheroids, whereas only 0.2 ± 0.1% of cells seeded and cultured on ULA plates formed spheroids. To evaluate whether culture media influence spheroid formation on various culture platforms, we performed spheroid formation tests using serum-free medium supplemented with growth factors (SPH medium). We found that 62.2 ± 2.3% of cells seeded and cultured on PEM-coated culture plates proliferated and formed cell spheroids after 5-7 days of culture, whereas only 0.7 ± 0.1% of cells seeded and cultured on ULA plates formed spheroids. Similar to ULA plates, poly-HEMA-coated plates produced a low yield of spheroid formation. Matrigel, an embedding substrate often used for tissue-based cell culture, was also tested. The spheroids were embedded in Matrigel, which made analysis and imaging of the spheroids difficult due to blurred and out-of-focus images.

[0130] Example 3: Formation of living spheroids on the culture platform of the present invention Liquid biopsy offers the possibility of obtaining cancer specimens in real time directly from blood. However, the rarity of circulating tumor cells (CTCs) hampers the whole process of ex vivo application in clinical use. Although several papers have shown the feasibility of using circulating tumor cells for screening and testing individual drugs, unbiased 3D culture models for clinical application have hardly been provided. To verify the feasibility of PEM-coated culture plates for rare cell proliferation, HCT116 cells at very low density were seeded on the surface of PEM-coated culture plates and photographed by time-lapse microscopy. Single cells showed semi-adhesion on the surface of PEM-coated culture plates and continued to proliferate to form spheroids with a diameter of about 70 μm after 7 days. Thus, single-cell-derived spheroids on PEM-coated culture plates may facilitate subsequent clinical applications by utilizing rare cell samples. Furthermore, when the density of cells seeded on the surface of PEM-coated culture plates increased, the cells continued to proliferate and formed spheroids from each single cell. However, when two spheroids are close enough to each other to form a larger spheroid, the cells undergo spheroid fusion. To establish clinical applications of rare cell proliferation technology using 3D spheroids, uniformity of spheroid size and maintenance of live spheroids are desirable to eliminate potential bias during ex vivo testing. To test the uniformity of spheroid size grown on the cell culture platform of the present invention, a total of 300 cells were seeded in wells coated with the surface of the present invention in a 96-well plate. The cells proliferated and formed spheroids. The size of the spheroids was verified with an Opera Phenix high content confocal screening system (Perkin Elmer). The average number of spheroids per well was 320 ± 54, and the average spheroid diameter in each well was 69 ± 34 μm on day 5. These results indicate that the spheroid formation ability was high and the size of the formed spheroids was uniform on the culture plate coated with PEM.Spheroids grown on either the UL platform or the cell culture platform of the present invention were isolated and stained with LIVE / DEAD assay (Invitrogen) to test cell viability. Fluorescence intensity analysis of EthD1 showed that spheroid cells cultured on the UL platform were significantly more dead than those cultured on the cell culture platform of the present invention. Furthermore, spheroids cultured on PEM-coated culture plates showed high cell viability even after freeze-thaw cycle storage, indicating high cell viability after cryobanking. Furthermore, confocal microscopy scanning revealed that the aggregated spheroids created on the ULA plate had loose cell-cell contacts, whereas the spheroids cultured on PEM-coated culture plates had tight cell-cell interactions. Thus, uniform and viable single-cell-derived spheroids may facilitate clinical applications such as personalized medicine drug testing.

[0131] Example 4: Patient-derived spheroids obtained from clinical samples To establish clinically useful patient-derived spheroids, patient-derived specimens were obtained from tissue resections, core needle biopsies, and phlebotomy blood draws and ex vivo spheroid cultures were performed. In some examples, cancer cells were obtained from tumor tissue. In some examples, cancer cells were obtained from blood samples. Live CTCs can be isolated using methods known in the art. All viable cells can be seeded onto the surface of PEM-coated culture plates, and patient-derived spheroids can be generated in 2-4 weeks. Figures 1A-C show representative images of the time course of CTC cultures generated on surfaces according to one embodiment of the present disclosure. (A) CTC cultures derived from CTCs in a blood sample from a breast cancer patient were cultured on surfaces for 7 and 14 days; (B) CTC cultures derived from CTCs in a blood sample from a head and neck squamous cell carcinoma (HNSCC) patient were cultured on surfaces for 12, 15, and 38 days; (C) CTC cultures derived from CTCs in a blood sample from a colorectal cancer (CRC) patient were cultured on surfaces for 2, 13, and 27 days. Scale bar: 50 μm.

[0132] Example 5: High concordance between ex vivo drug testing on CTC-derived spheroids and clinical responses Patient-derived CTC cultures were successfully generated ex vivo from 33 blood samples obtained from 31 cancer patients recruited in this study. Samples were obtained from patients diagnosed with breast cancer (n=13), colon cancer (n=11), head and neck cancer (n=4), urothelial cancer (n=3), lung cancer (n=1), and gastric cancer (n=1). These blood samples were collected and cultured on the surface of the cell culture platform of the present invention.

[0133] The calculated success rate of generating CTC cultures was 88% (29 of 33 samples). Four samples from breast cancer patients had insufficient sample volume to perform drug testing.

[0134] Next, the relationship between cell viability of each drug test in CTC culture and clinical response in patients was examined. A total of 167 ex vivo drug tests were produced from 29 CTC cultures on the surface of the culture platform of the present disclosure. For comparison, 42 treatment results were obtained from clinical patients who underwent paired testing. Among the 42 treatment results with known clinical responses, 35 CTC cultures that underwent paired patient treatment were classified into a clinical resistance group based on their treatment response according to the doctor's diagnosis, and the remaining 7 patient treatments were classified into a clinical sensitivity group.

[0135] A significant differential response of cell viability to chemotherapy drugs was identified between the resistant and sensitive groups of CTC cultures (Figure 2D, calculated by Mann-Whitney t-test, p = 0.0159). With an appropriate decision threshold, CTC cultures in the sensitive group showed a much lower cell viability than those in the resistant group. A receiver operating characteristic (ROC) curve constructed from this window showed a high AUC value of 0.939 [confidence interval (CI), 0.86-1.01 (Figure 2E)]. The data demonstrated that when tested in an ex vivo drug assay with the same clinical therapeutic agent, the CTC cultures showed a drug response highly relevant to clinical treatment outcomes. Importantly, this assay method was able to be performed within 1-10 days of initial seeding. 2 Only cells were required, and spheroids for subsequent therapeutic drug screening could be generated within three weeks.

[0136] Example 6: Ex vivo drug response profiles of CTC cultures correlate with clinical treatment outcomes To validate the results of the ex vivo drug assay, therapeutic agents were tested, either alone or in combination, in CTC cultures obtained from patient blood samples. Patient A had been pretreated with oxaliplatin, docetaxel, and 5-FU and progressed after clinical treatment. Ex vivo drug assays designed with both used and potential candidate drug panels, including oxaliplatin, docetaxel, 5-FU, doxorubicin, mitomycin-c, and irinotecan, were performed in a time- and dose-dependent manner (Figure 3A). With regard to cell morphology, the diameter of CTC spheroids obtained from patient A's blood samples was approximately 30–40 μm, and the spheroid morphology was intact in the untreated Mock group. After treatment with oxaliplatin and 5-FU for 3 days, the diameter of the CTC spheroids became smaller than that of the Mock group and they were visibly leaky. When treated with doxorubicin, the CTC cultures showed obvious broken morphology, with cell debris appearing around the cells (Figure 3C). Meanwhile, patient A showed high spheroid viability of over 50% with 5-FU and docetaxel treatment, but less than 4% with doxorubicin treatment. This result is highly consistent with the clinical treatment results in resistance to clinically used drugs. Therefore, doxorubicin was a promising related candidate drug for the next line of treatment among the unused drugs (mitomycin-c and irinotecan) (Figure 3B). Furthermore, ex vivo drug testing was performed on urothelial carcinoma patient B. The size and cell viability of CTC cultures treated with cisplatin / gemcitabine combination from patient B were both smaller than those of the untreated Mock group and single-agent treated spheroids (Figures 3C and 3D). Similarly, this patient also showed a significant tumor mass shrinkage after cisplatin / gemcitabine combination therapy on CT imaging, indicating a favorable clinical response (Figure 3F). Collectively, the results of the CTC-based ex vivo drug sensitivity assay showed high concordance with clinical patient response. These results indicate the feasibility of a clinically useful drug testing platform for predicting clinical outcomes of drug treatment response.

[0137] Example 8: Co-culture of patient-derived tumor spheroids with autologous NK cells obtained from peripheral blood Peripheral blood can be used as a source of natural killer (NK) cells. Peripheral blood mononuclear cells (PBMCs) contain 10–12% of circulating NK cells. Patient-derived NK (PDNK) cells were isolated from peripheral blood of colorectal cancer patients and expanded ex vivo for 2 weeks after isolation.

[0138] Tumor spheroids derived from colon cancer patients were produced on the surface of the present invention. To examine the cytotoxicity of PDNK cells against patient-derived tumor spheroids, tumor spheroids and PDNK cells were co-cultured for 24 hours. The patient-derived tumor spheroids remained unchanged in morphology even after 24 hours of incubation, indicating that PDNK cells did not exhibit cytotoxicity against patient-derived tumor spheroids. This result indicated that autologous PDNK cells could approach patient-derived tumor spheroids on the surface of the present invention, but did not recognize the patient-derived tumor spheroids as targets for killing.

[0139] For comparison, the NK-92MI cell line was employed as a parallel control group for PDNK cells. Patient-derived tumor spheroids were co-cultured with NK-92MI cells. As a result, it was observed that the patient-derived tumor spheroids were recognized and rapidly surrounded by NK-92MI cells within 2 hours. The patient-derived tumor spheroids were then further destroyed by NK-92MI cells, and debris of the patient-derived tumor spheroids was observed on the surface of the present invention. Analysis showed that the cytotoxicity of NK-92MI cells against patient-derived tumor spheroids was dramatically higher than that of autologous PDNK cells. Analysis was performed by cell-based assays based on luminescence or biochemical assays based on luminescence or fluorescence.

[0140] [Methods and Materials] Immunofluorescence staining and microscopy. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde in PBS buffer for 30 min, permeabilized with 0.1% Triton X-100 in PBS buffer for another 30 min, and blocked with 5% BSA for 1 h. Cells were then stained with rabbit anti-human pan-cytokeratin (panCK, Abcam, Cambridge, UK), a colon-specific marker, followed by nuclear staining with goat anti-rabbit 647 secondary antibody and DAPI (In VitroGen). Both unstained and immunostained cells were photographed with a Nikon Ti Eclipse inverted fluorescence microscope.

[0141] Capture and release of circulating tumor cells (CTCs) from clinical patients. Peripheral blood and tissue samples were obtained from patients with breast cancer, colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), and urothelial cancer at Chang Gung Memorial Hospital (Linkou, Taoyuan County), Taipei Veterans General Hospital (Taipei, Taiwan), and National Taiwan University Hospital (Taipei, Taiwan). Tissues obtained by surgical resection or needle biopsy were stored in ice-cold DMEM medium and transplanted immediately after surgical resection. A total of 2 mL of whole blood sample was collected from each patient in ethylenediaminetetraacetic acid (EDTA) Vacutainer® tubes (BD Biosciences) and used for capture and release of CTCs on the CTC capture platform.

[0142] Spheroid formation on the culture platform of the present invention. Cell samples to be used for culture on the culture platform of the present invention are collected in suspension medium. Cell suspension medium is added to each well. Cells can be cultured in a humidified 37°C incubator with 5% CO2 atmosphere. Half of the medium is replaced once or twice a week. Cultured spheroids (tumor masses) can be maintained on the culture platform of the present invention for more than two weeks, depending on the cell density and spheroid size requirements.

[0143] Statistical analysis. All statistical analyses were performed using GraphPad Prism software (version 6.0c, La Jolla, CA). Statistical analysis was performed using Student's t-test, and a p value of 0.05 was used to determine statistical significance between two groups. All results shown are the mean ± SEM of at least three replicate experiments.

Claims

1. 1. A method for preparing a cell culture, comprising: (a) providing a cell culture article having a surface coated with a polyelectrolyte multilayer and optionally an absorbable polymer; (b) seeding a plurality of cancer cells onto the surface, the plurality of cancer cells being obtained from a body fluid sample of a cancer patient; and (c) culturing the cancer cells in an appropriate medium for a sufficient time to generate a cell culture, wherein the cell culture comprises a three-dimensional (3D) cell culture comprising a plurality of tumor spheroids attached to the coated surface; A method for preparing a cell culture, comprising:

2. A method for evaluating a cancer therapeutic agent, comprising: (a) preparing a cell culture according to the method of claim 1; (b) optionally incubating the cell culture with a plurality of immune cells; (c) contacting the cell culture with a therapeutic agent; (d) assessing the effect of the therapeutic agent on the cell culture; and (e) determining that the cancer patient is responsive to the therapeutic agent if the therapeutic agent is effective on the cell culture, and determining that the cancer patient is not responsive to the therapeutic agent if the therapeutic agent is not effective on the cancer cell culture; A method for evaluating a cancer therapeutic agent, comprising:

3. The method according to claim 1 or 2, wherein the suitable medium contains a Rho-associated protein kinase (ROCK) inhibitor, and the ROCK inhibitor has a structural formula having an isoquinoline, 4-amidopyridine or 4-amidopyrrolopyridine skeleton.

4. 3. The method of claim 1 or 2, wherein the surface is coated with a polyelectrolyte multilayer and an absorbable polymer, and the absorbable 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) (PDLA), polyethyleneimine (PEI), poly-L-lactide (PLLA), poly-D-lactide (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (p-HEMA), and derivatives thereof.

5. 3. The method of claim 2, wherein the therapeutic agent is selected from the group consisting of one or more chemotherapeutic agents, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, and combinations thereof.

6. 6. The method of claim 5, wherein the therapeutic agent comprises an immune checkpoint inhibitor and the cell culture is further incubated with a plurality of immune cells.

7. 7. The method of claim 6, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CLTA-4 inhibitor.

8. 8. 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.

9. The method of claim 5 , wherein the therapeutic agent comprises a therapeutic cell composition.

10. 10. The method of claim 9, wherein the therapeutic cell composition comprises T cells, natural killer (NK) cells, or dendritic cells.

11. 10. The method of claim 9, wherein the therapeutic cell composition comprises chimeric antigen receptor T (CAR-T) cells or chimeric antigen receptor-natural killer (CAR-NK) cells.

12. 6. The method of claim 5, wherein the therapeutic agent comprises one or more chemotherapeutic agents.

13. 13. The method of claim 12, wherein the one or more chemotherapeutic agents are cytotoxic or cytostatic chemotherapeutic agents.

14. The method of claim 5, wherein the therapeutic agent comprises a nucleic acid drug.

15. 3. The method of claim 1, wherein the polyelectrolyte multilayer is formed by a layer-by-layer method.

16. 3. The method of claim 1 or 2, wherein the cell culture is in direct contact with the outermost layer of the polyelectrolyte multilayer.

17. 17. The method of claim 16, wherein the outermost layer is a polycation or a polyanion.

18. 18. 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.

19. 18. The method of claim 17, wherein the polyanion is poly(L-glutamic acid) (PLGA) or poly(L-aspartic acid) (PLAA).

20. 3. The method of claim 1, wherein the polyelectrolyte multilayer comprises n pairs of bilayers of polycations and polyanions, where n is an integer from 1 to 30.

21. 3. The method of claim 1, wherein the polyelectrolyte multilayer comprises n bilayers of polycation and polyanion, and an additional layer of polyanion, where n is an integer in the range of 1 to 30.

22. 3. The method of claim 1, wherein the polyelectrolyte multilayer comprises n bilayers of polycation and polyanion, and an additional layer of polycation, where n is an integer in the range of 1 to 30.

23. 3. The method of claim 1 or 2, wherein the body fluid sample comprises serum, plasma, whole blood, urine or ascites.

24. 3. The method of claim 1 or 2, wherein the body fluid sample comprises a blood sample.

25. 3. The method of claim 1 or 2, wherein the plurality of cancer cells comprises circulating tumor cells (CTCs).

26. 3. The method of claim 1 or 2, wherein each of the tumor spheroids is generated via single cell proliferation.

27. 3. The method of claim 1 or 2, wherein each of the tumor spheroids has an average diameter of about 50 μm to about 150 μm.

28. The seeding in step (b) is performed at 1000 cells / cm on the substrate. 2 10. The method of claim 1, comprising plating the cells at a density of less than 1000 ng / ml.

29. 3. The method of claim 2, wherein the effect of the therapeutic agent on the cell culture is determined by performing cell-based and / or biochemical assays to determine the size, morphology, physical properties, biological properties, and / or kinetic properties of the cell culture.

30. The method of claim 1 or 2, wherein the thickness of the polyelectrolyte multilayer as a thin film ranges from about 30 nm to about 30 μm.

31. A therapeutic agent evaluated by the method of claim 2 for use in treating cancer.

32. The therapeutic agent described in claim 31, selected from the group consisting of one or more chemotherapeutic agents, immune checkpoint inhibitors, nucleic acid drugs, therapeutic cell compositions, and combinations thereof.

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