3D culture of primary cancer cells using tumor tissue

Abstract

The present invention addresses the problem of providing a method for producing cell masses by a three-dimensional culture method of primary cancer cells, using tumor tissue as a starting material, which method suppresses the proliferation of cells other than cancer cells such as fibroblasts while producing cell masses composed mainly of primary cancer cells with high proliferation potential, and which also has ease of handling, versatility, and high throughput. The present invention solves the problem by providing a method for producing cell masses by a three-dimensional culture method of primary cancer cells using tumor tissue, which method includes a three-dimensional culture step of culturing cells obtained from the tumor tissue on a substantially low-adhesion cell culture substrate in a medium containing 5 v / v% or less of extracellular matrix.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a cell mass by a three-dimensional culture method of primary cancer cells using tumor tissue, a cell mass, a screening method, an assessment method, and a kit. [Background technology]

[0002] Established cancer cell lines have traditionally been used to evaluate tumors in vitro. However, because cancer cell lines are nearly homogeneous cell populations adapted to the in vitro environment and have accumulated genetic mutations over time, they have been shown to lose many of the properties of the original tumor. Furthermore, it has become clear that tumors are composed of cancer cells with diverse genetic backgrounds, and because of this heterogeneity, it has been pointed out that the limited number of cancer cell lines cannot adequately explain the pathology of tumors. Therefore, to gain a more accurate understanding of tumors, culture systems using primary tumor cells have attracted attention. In addition to tumors obtained from patients, xenografts (PDXs) created by transplanting tumors into immunodeficient animals (PDXs) are also used.

[0003] The basic principle of culturing primary tumor cells is to dissociate excised tumor tissue physically or enzymatically, seed the resulting dissociated cells into a culture vessel together with culture medium, and grow them in a CO2 incubator (Non-Patent Document 1: Tissue Culture Techniques - Basics, Third Edition, Asakura Publishing Co., Ltd., 1996). To improve cancer cell growth or suppress the excessive growth of non-cancerous cells (particularly fibroblasts), cell separation by density gradient centrifugation, coating the culture vessel with an extracellular matrix, using serum-free medium, and cell separation based on differences in sensitivity to trypsin enzymes or antibiotics have been attempted. However, even with these techniques, it is difficult to achieve a high rate of cancer cell growth, and a more reliable culture method is desired.

[0004] In recent years, techniques for culturing cells in three dimensions have attracted attention as a method for culturing primary cancer cells. One such method is the organoid culture method developed by Clevers et al. at the Hubrecht Institute (Non-Patent Document 2: Sato, Toshiro, et al. "Single Lgr5 stem cells build crypt villus structures in vitro without a mesenchymal niche." Nature 459.7244 (2009): 262-265.; Non-Patent Document 3: Sato, Toshiro, et al. "Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium." Gastroenterology 141.5 (2011): 1762-1772.). Organoid culture is a culture method in which cell masses (organoids) are formed by the self-organization of adult stem cells. Specifically, adult stem cells are embedded in an extracellular matrix gel and cultured in an optimal medium. By applying this technique to primary cancer cells, they have established culture methods for colon cancer (Non-Patent Document 3), prostate cancer (Non-Patent Document 4: Gao, Dong, et al. "Organoid cultures derived from patients with advanced prostate cancer." Cell 159.1 (2014): 176-187.), and pancreatic cancer (Non-Patent Document 5: Boj, Sylvia F., et al. "Organoid models of human and mouse ductal pancreatic cancer." Cell 160.1 (2015): 324-338.). However, this method requires embedding cells in a gel at low temperatures, making it unsuitable for high throughput and insufficient for general use in drug development, etc.

[0005] The second method is the 3D-tumor growth assay (3D-TGA) developed by Molecular Response and others. Similar to the first method, cells are embedded in an extracellular matrix gel and cultured in an optimal medium. However, it differs in that a pre-culture step is performed in a culture vessel coated with extracellular matrix, and optionally, expanded cancer-associated fibroblasts (CAFs) or mesenchymal stem cells (MSCs) are embedded in the extracellular matrix gel together with cancer cells (Non-Patent Document 6: Saunders, John H., et al. "Individual patient esophageal cancer 3D models for tailored treatment." Oncotarget (2016).). Like the first method, this method requires cells to be embedded in the gel at low temperatures, making it less suitable for high throughput.

[0006] The third method, developed by Inoue et al. at the Center for Adult Diseases, is a culture method using non-dispersed cell clusters (cancer tissue-originating spheroids, CTOS) with diameters of 40 to 100 μm (Non-Patent Document 7: Kondo, Jumpei, et al. "Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer." Proceedings of the National Academy of Sciences 108.15 (2011): 6235-6240.). Specifically, the obtained CTOS are seeded on non-adhesive plates and cultured in suspension using an optimal medium. Because cancer cells can grow on CTOS without being embedded in an extracellular matrix gel, this method requires less laborious temperature control than the first and second methods. However, drug sensitivity testing requires the tedious process of selecting and rearranging CTOS of uniform size, which makes it less high-throughput. In addition, cancer cells proliferate less rapidly than with the method of embedding them in an extracellular matrix, making this method less practical.

[0007] The fourth method, developed by Nakatsukazu et al. at the National Cancer Center, uses single-cell culture plates containing dispersed cancer cells and cell culture plates treated to suppress adhesion (Patent Document 1: WO2016 / 047801). Specifically, single-cell culture plates containing dispersed cancer cells are seeded together with a medium containing 1% or more by volume of serum onto a NanoCulture Plate, a 3D culture plate manufactured by ORGANOGENIX, Inc., to culture cell clusters. ORGANOGENIX sells a culture kit (cancer organoid culture kit). This method allows cancer cells to grow without being embedded in an extracellular matrix gel, requiring less temperature-controlled manipulation than the first and second methods. Furthermore, because single-cell culture plates are used, cells can be seeded uniformly without the need for special manipulation, resulting in a higher throughput than the third method. However, only reports have been published on human lung cancer tumors and breast cancer xenograft tumors (Non-Patent Document 8: Sakamoto, Ruriko, et al. "Time-lapse imaging assay using the BioStation CT: A sensitive drug-screening method for three-dimensional cell culture." Cancer science 106.6 (2015): 757-765.), and in particular in breast cancer xenograft tumors, the proliferation of cell masses has not been clearly confirmed, and its versatility has not been sufficiently verified, raising doubts about its practicality.

[0008] As described above, the culturing of primary cancer cells using tumor tissue has become more reliable through 3D culture methods, but all of these methods have problems with either the proliferation ability of cancer cells, ease of handling, high-throughput capabilities, or versatility. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2016 / 047801 [Non-patent literature]

[0010]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

Non-patent document 7

[0011] The present invention was made in consideration of the above-mentioned problems, and the inventors aim to provide a method for producing cell masses by a three-dimensional culture method of primary cancer cells, using tumor tissue as a starting material, which method suppresses the proliferation of cells other than cancer cells, such as fibroblasts, while producing cell masses composed mainly of primary cancer cells that have high proliferation potential, and which also combines ease of handling, versatility, and high throughput. [Means for solving the problem]

[0012] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that by using patient xenograft (hereinafter sometimes abbreviated as PDX) tumors as a starting material, singly dispersed cells are cultured in suspension using an optimal combination of a culture substrate and a 3D culture medium, cell masses of primary cancer cells that have high proliferation potential and are easy to handle, versatile, and capable of high throughput and that can be used in various tests can be produced by 3D culture of primary cancer cells, thereby completing the present invention.

[0013]

[0013] That is, the present invention can be exemplified as follows. [1] A method for producing cell masses by a three-dimensional culture method of primary cancer cells using tumor tissue, comprising: low Cells obtained from tumor tissue are cultured on an adhesive cell culture substrate in a medium containing 5% or less of extracellular matrix. In a floating state A method comprising a three-dimensional culture step. [2] The method according to [1], wherein the tumor tissue is a xenograft tumor. [3] The method according to [1] or [2], wherein the medium is in a sol state. [ 4 ] [1] ~[3] A cell mass of primary cancer cells obtained from tumor tissue, produced by the cell mass production method described in . [ 5 ] [1] ~[3] a step of preparing a cell mass of primary cancer cells by the method for preparing a cell mass described in the above; administering a test substance to the cell mass; and evaluating the effect of the test substance on the cell mass. A method for screening substances that have an effect on cell masses. [ 6 ] [1] ~[3] a step of preparing a cell mass of primary cancer cells by the method for preparing a cell mass described in the above; administering a test substance to the cell mass; and evaluating the effect of the test substance on the cell mass. A method for determining the effect of a substance on the cell mass. [7 A kit for producing a cell mass by a three-dimensional culture method of primary cancer cells using tumor tissue, comprising: low an adhesive cell culture substrate; for culturing cells obtained from the tumor tissue in a suspension state; A kit comprising a medium for a three-dimensional culture process containing 5 v / v% or less extracellular matrix. [Effects of the Invention]

[0014] The present invention provides a method for producing cell masses using tumor tissue as a starting material, by a three-dimensional culture method of primary cancer cells, which has high proliferation potential of cell masses composed mainly of primary cancer cells while suppressing the proliferation of cells other than cancer cells, such as fibroblasts, and which also combines ease of handling, versatility, and high throughput. [Brief explanation of the drawings]

[0015] [Figure 1] Figure (photo) showing the culture results of Example 2. *1: Pancreatic cancer (1) PDX tumor was used. The culture medium used was StemPro hESC SFM supplemented with Corning Matrigel GFR to a final concentration of 2 v / v%. *2: When spindle-shaped cells were observed on the bottom of the plate, it was determined that fibroblasts had adhered. [Figure 2] Figure (photo) showing the culture results of Example 3. *1: Pancreatic cancer (1) PDX tumor was used. PrimeSurface was used. *2: When pipetting to aspirate half of the medium in the well, it was not acceptable if the cells or cell clumps were also aspirated. [Figure 3-1] Figure (photo) showing the culture results of Example 4 (Corning Matrigel). *1: Pancreatic cancer (1) PDX tumor was used. PrimeSurface was used. *2: When pipetting to aspirate half of the medium in the well, it was not acceptable if the cells or cell clumps were also aspirated. [Figure 3-2]Figure (photo) showing the culture results of Example 4 (Cultrex BME). *1: Pancreatic cancer (1) PDX tumor was used. PrimeSurface was used. *2: When pipetting to aspirate half of the medium in the well, it was not acceptable if the cells or cell clumps were also aspirated. [Figure 3-3] Figure (photo) showing the culture results of Example 4 (Cultrex RGF BME). *1: Pancreatic cancer (1) PDX tumor was used. PrimeSurface was used. *2: When pipetting to aspirate half of the medium in the well, it was not acceptable if the cells or cell clumps were also aspirated. [Figure 3-4] Figure (photo) showing the culture results of Example 4 (Cellmatrix Type IA). *1: Pancreatic cancer (1) PDX tumor was used. PrimeSurface was used. *2: The 2v / v% well on Day 14 was observed with the naked eye because it was not in focus with the microscope. *3: When sucking up half the medium in the well by pipetting, it was not acceptable if the cells or cell clumps were sucked up along with it. [Figure 4] Figure (photo) showing the culture results of Example 5. *1: Pancreatic cancer (1) PDX tumor was used. The culture medium used was a basal medium supplemented with Corning Matrigel GFR to a final concentration of 2 v / v%. PrimeSurface culture plates were used. [Figure 5] Figure (photograph) showing the culture results of Example 6. *1: The culture medium used was StemPro hESC SFM supplemented with Corning Matrigel GFR to a final concentration of 2 v / v%. PrimeSurface culture plates were used. [Figure 6] Figure (photo) showing the culture results of Example 7. *1: The culture medium used was StemPro hESC SFM supplemented with Corning Matrigel GFR to a final concentration of 2 v / v%. PrimeSurface culture plates were used. Day 14 cell clumps were used. [Figure 7]Figure (photo) showing the culture results of Example 8. *1: The culture medium used was StemPro hESC SFM supplemented with Corning Matrigel GFR to a final concentration of 2 v / v%. PrimeSurface culture plates were used. [Figure 8] Figure (photo) showing the culture results of Example 9. *1: The culture medium used was StemPro hESC SFM supplemented with Corning Matrigel GFR to a final concentration of 2 v / v%. PrimeSurface culture plates were used. On day 7, half of the medium was replaced with culture medium supplemented with gemcitabine. On days 10 and 12, half of the medium was replaced with culture medium supplemented with gemcitabine, the concentration of which had been adjusted so that the final concentration remained unchanged. An ATP assay was performed on day 14, and the ratio of the results for each gemcitabine concentration group, with the result for a gemcitabine concentration of 0 μmol / L being considered as 100% cell viability, was calculated to create a cell viability curve. [Figure 9] FIG. 10 is a diagram (photograph) showing the culture results of Example 10. [Figure 10-1] FIG. 10 is a diagram (photograph) showing the culture results (fresh tumor) of Example 11. [Figure 10-2] FIG. 10 is a diagram (photograph) showing the culture results (frozen tumors) of Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0016] The method of the present invention for producing cell masses by three-dimensional culture of primary cancer cells using tumor tissue is characterized in that, as a three-dimensional culture step, primary cancer cells obtained from the tumor tissue are cultured on a substantially low-adhesion cell culture substrate in a medium containing 5 v / v% or less of extracellular matrix. It has generally been thought that when primary cancer cells are cultured in a suspension state in three dimensions, it is difficult to form cell clusters unless special techniques are used. Furthermore, when using a medium containing an extracellular matrix, it has been thought that cell clusters with high proliferation potential can be obtained by culturing the cells in a fixed state in a gel-like medium. However, as described in the Examples below, it was surprising that primary cancer cells could be cultured in a suspension state in a medium containing a sol-like extracellular matrix on a substantially low-adhesion cell culture substrate, while suppressing the proliferation of cells other than cancer cells, such as fibroblasts, and thus allowing the primary cancer cells to be cultured in three dimensions with high proliferation potential. This allows the production of cell masses using tumor tissue by a 3D culture method of primary cancer cells, which have high proliferation potential and can be used in various tests, and which are easy to handle, versatile, and have high throughput, especially because they are cultured in a suspended state. A suspended state does not mean that the cells are adhered to a cell culture substrate or immobile in a gel-like culture medium, but rather that they are easily movable by manipulations such as pipetting. The size of the cell masses produced by the method of the present invention is not particularly limited, but is, for example, 100 μm or more in average diameter, preferably 100 μm to 300 μm in average diameter.

[0017] Hereinafter, one embodiment of a method for producing cell aggregates by three-dimensional culture of primary cancer cells using tumor tissues using the culture method of the present invention will be described.

[0018] Substantially low-adhesion cell culture substrates that can be used in the present invention include low-adhesion culture substrates that allow for 3D suspension cell culture. Substantially low-adhesion cell culture substrates may be any cell culture substrates that are low-adhesion overall for use in 3D culture methods. Examples include cell culture substrates with hydrophilic surfaces and culture substrates whose surfaces have been treated with hydrophilic compounds. Specific examples include PrimeSurface (Sumitomo Bakelite Co., Ltd.), Corning ULA Round-bottom (Corning Incorporated), Corning ULA Flat-bottom (Corning Incorporated), and Elplasia (Kuraray Co., Ltd.). Alternatively, adhesive cell culture substrates may be treated to suppress adhesion, resulting in low-adhesion culture substrates. Examples of such treatments that can suppress adhesion include known hydrophilic and hydrophobic treatments. Low-adhesion can be determined by 3D culture of primary cancer cells using tumor tissue and confirming that cells other than cancer cells, such as fibroblasts, do not grow and proliferate in areas other than the cell masses on the substrate. Furthermore, the shape, processing, material, etc. of the substrate are not limited as long as the substrate is a cell culture substrate that is substantially low-adhesive.

[0019] The tumor tissue usable in the present invention may be any tissue fragment containing known cancer cells, such as lymphoma, myeloma, brain tumor, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colon cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, laryngeal cancer, floor of the mouth cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, paranasal sinus cancer, maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, sphenoid sinus cancer, thyroid cancer, kidney cancer, lung cancer, osteosarcoma, prostate cancer, testicular tumor, renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer, anal cancer, various other cancer cells, various stem cells, various progenitor cells, euploid progenitor cells, ES cells, iPS cells, etc. Note that the cells are not limited to a single cell, but may be a collection of multiple cell types. The origin of tumor tissue is not particularly limited, but examples include primates, including humans and monkeys; rodents, such as mice and rats; lagomorphs; felines, such as dogs and cats; ungulates, such as pigs; and perissodactyls, such as cattle and horses. In addition to tumor tissue obtained from patients as described above, xenografts (PDXs) can also be used, which are tumor tissues transplanted into immunodeficient animals. Patient-derived xenograft tumors can be generated using known techniques (Non-Patent Document 9: Cho, Sung-Yup, et al., "An integrative approach to precision cancer medicine using patient-derived xenografts," Molecules and Cells, 39.2 (2016): 77). According to the present invention, PDX tumors are particularly preferred because they can be used to produce cell masses using a 3D culture method of primary cancer cells that have the high proliferation potential of cancer cells that can be used in various tests, while also being easy to handle, versatile, and capable of high throughput. PDX tumors are particularly preferred because they enable the development of anticancer drugs with high clinical predictability using patient-derived tumors and can be widely used to evaluate therapeutic efficacy. Furthermore, tumor tissues that can be used in the present invention include fresh tumors that have been surgically removed and immersed in a tissue preservation solution (such as physiological saline or HBSS), and frozen tumors that have been immersed in a freezing solution (such as CELLBANKER 1) and frozen to keep the cells alive. Those skilled in the art can appropriately select and use a preservation method from known methods.

[0020] Unless otherwise specified, known methods can be used as three-dimensional culture methods that can be used in the present invention, and those skilled in the art can select and use them as appropriate.

[0021] In the method for producing cell masses by 3D culture of primary cancer cells using tumor tissue that can be used in the present invention, known methods can be used for the preparation of primary cancer cells. For example, methods include isolating cells from tissue fragments containing cancer cells, and using the tissue fragments as is. However, from the viewpoint of ease of handling and reproducibility of tests, the method of isolating cells is preferred. Examples of methods for isolating cells from tissue fragments containing cancer cells include separating and purifying tumor tissue fragments excised from a living body, as needed, by enzyme treatment, density gradient centrifugation, filtration, magnetic beads, flow cytometry, or other treatments. Enzyme treatment is preferred because it is a simple treatment method and allows cancer cells dispersed into single cells to be easily obtained. Note that these cell populations may be aggregates of cells derived from the same tissue but at different stages of differentiation.

[0022] A three-dimensional culture method that can be used in the present invention involves culturing primary cancer cells obtained from tumor tissue in a medium containing 5 v / v% or less of extracellular matrix on the substantially low-adhesion cell culture substrate. The extracellular matrix, medium, and primary cancer cells may be mixed in any order, but one example is to prepare a medium containing 5 v / v% or less of the extracellular matrix in a sol state, mix the primary cancer cells with this, and seed the mixture on the substantially low-adhesion culture plate. If the extracellular matrix is 5 v / v% or less, the primary cancer cells will be cultured in a suspended state. The ability to culture in a suspended state is preferable because it is easy to handle and use in various tests, and also because it allows mechanical manipulation and is easy to use in high-throughput tests.

[0023]

[0023] The extracellular matrix that can be used in the present invention includes those that can be used in known three-dimensional culture methods. For example, collagen I, collagen IV, fibronectin, lamina propionate, and the like. Nin, vitronectin, entactin, gelatin, elastin, proteoglycan, glycosaminoglycan, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, Matrigel (trademark: Corning Incorporated), Matrigel GFR (trademark: Corning Incorporated) ), Cultrex BME (trademark: TREVIGEN), Cultrex RGF BME (trademark: TREVIGEN), Cellmatrix Type IA (trademark: Nitta Gelatin Co., Ltd.) )but Examples include: The concentration of extracellular matrix that can be used in the present invention may be any concentration that allows primary cancer cells obtained from tumor tissue to be cultured three-dimensionally in a suspended state, and may be 5 v / v% or less, or may be 2.5 v / v% or less. There is no particular lower limit, but the concentration is preferably 0.1 v / v% or more, more preferably 0.2 v / v% or more, and particularly preferably 0.5 v / v% or more. However, a person skilled in the art can set the concentration appropriately depending on the type of primary cancer cells, etc.

[0024] The seeding cell density that can be used in the present invention is sufficient as long as the primary cancer cells obtained from tumor tissue can survive normally as a cell mass produced by the 3D culture method. The cell density of the primary cancer cells is usually 3 × 10 3 ~7×10 4 cells / cm 2Although the cells can be seeded at a density of 1000 kJ / ml, a preferred cell density can be set appropriately depending on the culture conditions and the culture equipment used. Known culture conditions can be used, but for example, the culture temperature is 20 to 45°C, more preferably 30 to 42°C, and particularly preferably 35 to 39°C, and the pH of the culture medium is preferably 7 to 8. The culture period can be set appropriately depending on the intended test method, but can be from 2 to 30 days, more preferably from 7 to 14 days. This method is particularly useful because it can maintain a stable, high level of activity even over a long period of time (approximately 10 days or more).

[0025] Media that can be used in the present invention include any basal cell culture medium, differentiation medium, or medium specifically for primary culture. Examples include Dulbecco's Modified Eagle's Medium (DMEM), Glasgow MEM (GMEM), RPMI 1640, Ham's F12, and serum-free media (e.g., MCDB medium). Furthermore, media supplemented with serum, various growth factors (insulin, transferrin, selenium salt, dexamethasone), or differentiation-inducing factors can also be used.

[0026] The cell masses that can be produced by the method of three-dimensional culture of primary cancer cells using tumor tissue of the present invention can be used in various test methods, including, but not limited to, a method for screening substances that act on the cell masses, a method for determining the effect of a substance on the cell masses, etc. The cell masses have high cell proliferation ability that can be used in various test methods, and because they exist in a suspended state, they have the advantages of being easy to handle, versatile, and capable of high throughput, making them suitable for use in various test methods.

[0027] Those skilled in the art can appropriately modify known methods to screen for substances that affect the cell masses of the present invention. For example, a screening method for substances that affect the cell masses according to the present invention includes the steps of preparing cell masses by a 3D culture method of primary cancer cells using tumor tissue, administering a test substance to the cell masses, and evaluating the effect of the test substance on the cell masses. Examples of substances that affect the cell masses directly or indirectly, or both directly and indirectly, include anticancer drugs, various compounds, antibodies, antibody-drug conjugates, nucleic acids, peptides, viruses, cells (NK cells, TCR-T cells, CAR-T cells, etc.), and the like. For example, since the method can screen for substances that can inhibit the proliferation of the cell masses, it can be used in the development of anticancer drugs.

[0028] Those skilled in the art can appropriately modify known methods to assess the effect of a substance on the cell mass of the present invention. For example, a method for assessing the effect of a substance on a cell mass according to the present invention includes the steps of producing a cell mass by a 3D culture method of primary cancer cells using tumor tissue, administering a test substance to the cell mass, and evaluating the effect of the test substance. Test substances for assessing the effect include, for example, anticancer drugs, various compounds, antibodies, antibody-drug conjugates, nucleic acids, peptides, viruses, and cells (NK cells, TCR-T cells, CAR-T cells, etc.). For example, by identifying an anticancer drug that can inhibit the proliferation of the cell mass from various anticancer drugs, administering that anticancer drug to the patient from whom the cell mass was derived can enhance the therapeutic effect, thereby assisting in the selection of a treatment method. Note that the combination of the cell mass and the patient from whom it was derived is not limited, and the method can also be applied to patients from whom cells of the same type as the cell mass are derived.

[0029] Known anticancer drugs can be used, and examples thereof include actinomycin D, melphalan, busulfan, carboplatin, cisplatin, cyclophosphamide, dacarbazine, oxaliplatin, procarbazine, temozolomide, ifosfamide, liposomal doxorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, bleomycin, mitoxantrone, cladribine, fluorouracil, melcaprine, pemetrexed, mettrexate, cytarabine, nelarabine, capecitabine, fludarabine, gemcitabine, pentostatin, vincristine, eribulin, paclitaxel, vinblastine, irinotecan, docetaxel, etoposide, and vinorelbine. Rubin, Nogitecan, Paclitaxel, Toretinoin, Bevacizumab, Torastuzumab, Panitumumab, Cetuximab, Ibritumomab tiuxetan, Rituximab, Gemtuzumab ozogamicin, Everolimus, Erlotinib, Lapatinib, Gefitinib, Imatinib, Dasatinib, Sunitinib, Sorafenib, Portezomib, Tamibarotene, Nimustine, Rani These include mustine, enocitabine, carumofur, cytarabine ocfosfate, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, doxifluridine, hydroxycarbamic acid, sobuzoxane, vindesine, aclarubicin, amrubicin, zinostatin stimalamer, pirarubicin, peplomycin, and nedaplatin.

[0030] Furthermore, after completion of the culture using the three-dimensional culture method, the cell aggregates may be used for various tests in the culture medium as is, or may be transferred to another container and used for various tests. When transferred to another container, the recovery method can be performed by a known method. Those skilled in the art can select and perform these methods as appropriate. Furthermore, since the cell aggregates are cultured in a suspended state, they have the advantage of being easily recovered.

[0031] Various test methods include known test methods, such as cell proliferation tests (MTT assay, ATP assay, etc.), live / dead cell staining analysis, phenotype screening (examination of cell morphological changes, e.g., analysis of epithelial-mesenchymal transition), histopathological analysis (HE staining, immunohistochemical staining, etc.), biochemical analysis (gene mutation analysis, mRNA expression analysis, protein expression analysis, exosome analysis, etc.), etc. Those skilled in the art can appropriately design and use test methods depending on the purpose, such as the method for screening the substance or the method for determining the effect of the substance.

[0032] According to the present invention, various test methods can be performed using a general automatic pipetting device, and procedures such as seeding cells into 96-well or 384-well plates, changing culture medium, adding drug solutions, adding assay reagents, and then acquiring electronic data using a luminescence or fluorescence measuring device, or acquiring electronic image analysis data using an automatic image analyzing device can be performed easily or with high throughput.

[0033] Those skilled in the art can easily confirm using known techniques that cell masses produced by the 3D culture method of primary cancer cells using tumor tissue of the present invention have high proliferation potential of cell masses composed mainly of primary cancer cells while suppressing the proliferation of cells other than cancer cells such as fibroblasts, and can be used for various purposes that reflect the living body. For example, whether the cell masses have functions similar to those of tumors in the living body can be confirmed by visually confirming the formation of cell masses, evaluating the proliferation of cells, evaluating the effects on the cell masses using known substances, or transplanting cell masses obtained from an animal back into another animal to evaluate their tumorigenicity.

[0034] The kit of the present invention is a kit for producing cell masses by 3D culture of primary cancer cells using tumor tissue, and includes a substantially low-adhesion cell culture substrate and a medium for the 3D culture process containing 5 v / v% or less extracellular matrix. The medium for the 3D culture process containing 5 v / v% or less extracellular matrix may be provided as a separate medium or as a mixture of the extracellular matrix and medium, so that the medium can be prepared immediately before use. The kit may also include, in an instruction manual or the like, a description of the substantially low-adhesion cell culture substrate, extracellular matrix, and medium that can be used in the cell mass production method of the present invention, so that the user of the kit can obtain them. Materials for the cell culture substrate, medium, and extracellular matrix, as well as methods for using the kit, include those that can be used in the cell mass production method of the present invention. [Example]

[0035] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0036] Example 1: Collection of tumor tissue and dispersal of cancer cells Patient-derived xenograft (PDX) tumors derived from human cancer patients were subcutaneously grown in immunodeficient mice (Super SCID mice (strain: C3H / HeJ / NOs-scid; LPS-nonresponder)) according to standard procedures. They were aseptically excised in a safety cabinet, and necrotic tumor areas were removed with surgical scissors. The tumors were immediately immersed in Japanese Pharmacopoeia saline and stored on ice. The Japanese Pharmacopoeia saline was then removed from the tumors, and the tumors were washed three times with specimen treatment solution (included in the Cancer Organoid Culture Kit, ORGANOGENIX). To prepare for 3D culture, cancer cells were dispersed as follows. After washing, tumors were placed in a 10 cm Petri dish on ice and cut into approximately 1 mm cubes with surgical scissors. Then, the tumors were collected in a 50 mL tube. Dispersion solution (included in the Cancer Organoid Culture Kit, ORGANOGENIX) was added to the tube, and the tumor pieces were enzymatically treated at 37°C for 60 min while shaking in a water bath. A sample treatment solution twice the volume of the reaction solution was added to weaken the reaction, and the cells were passed through a 100 μm cell strainer to remove undispersed residue. The tube and cell strainer were rinsed with an appropriate amount of sample treatment solution, and the cells were recovered and centrifuged at 300 × g for 5 min. After removing the supernatant, the cell pellet was resuspended in sample treatment solution and centrifuged at 300 × g for 5 min. The cell pellet was then resuspended in an appropriate amount of sample treatment solution and counted. The cells were confirmed to be single cells and used in the following experiments.

[0037] <Example 2: Seeding and culturing of cells on a 3D culture plate> First, we investigated whether 3D culture of PDX tumors was possible using a cancer organoid culture kit (ORGANOGENIX), which is said to enable 3D culture of primary cancer cells. The cancer organoid culture kit is a method of culturing cancer cells on a low-adhesion plate with an uneven scaffold structure to form cell clusters, and in a medium containing 1% or more by volume of serum. Primary cancer cells were prepared using pancreatic cancer (1) PDX tumor (obtained from the National Institute of Biomedical Innovation) according to Example 1. After dispersion, the counted cells were dispensed into a 15 mL tube in the required amount, centrifuged at 300 × g for 5 minutes to remove the supernatant, and then cultured in NanoCulture Medium P type (an accessory to the cancer organoid culture kit, ORGANOGENIX) until the cell count reached 1 × 10 5A cell suspension was prepared to give a concentration of 1 x 10 cells / mL. 150 μL of NanoCulture Medium P type was added to a 3D culture plate, NanoCulture Plate (cancer organoid culture kit accessory, ORGANOGENIX), and centrifuged at 700 × g for 5 minutes, followed by prewetting at 37°C for 10 minutes. 100 μL of the cell suspension was added to the plate, and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The number of cells seeded was 1 x 10 4 The culture medium was 250 μL / well, with the seeding date designated Day 0. Half the volume of medium was replaced as needed. As a result, no clear cell clusters were formed, and spindle-shaped fibroblasts were observed adhering to the bottom of the plate. This indicates that it is difficult to directly apply ORGANOGENIX's cancer organoid culture kit to pancreatic cancer (1) PDX tumors.

[0038] To achieve culture in a suspended state, the extracellular matrix, which is usually used in gel form, was prepared in a sol-like state in the medium, and we investigated whether primary cancer cells could form cell masses without the progression and proliferation of cells other than cancer cells, such as fibroblasts. As described above, the required amount of counted cells was dispensed into a 15 mL tube and centrifuged at 300 × g for 5 min to remove the supernatant. Then, the cells were cultured in a medium containing StemPro hESC SFM (Thermo Fisher Scientific) supplemented with Corning Matrigel GFR (Corning) at a final concentration of 2 v / v% until the cell count reached 5 × 10 4A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto common low-adhesion 3D culture plates such as PrimeSurface (Sumitomo Bakelite), Corning ULA Round-bottom (Corning), Corning ULA Flat-bottom (Corning), and Elplasia (Kuraray), or onto a common adhesive planar (2D) culture plate, a 96-well microplate (Corning), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. Similarly, 1 × 10 cells were seeded onto NanoCulture Plates. 5 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 100 μL of the cell suspension was added to a plate prewetted with 150 μL of medium, and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The number of cells seeded was 1 × 10 4 cells / 200 μL / well (except NanoCulture Plate) or 1 × 10 4 The cells were cultured at 250 μL / well (NanoCulture Plate), and the day of seeding was designated Day 0. Medium replacement was performed at half the volume as appropriate. The results are shown in Figure 1. Morphology was confirmed using a phase-contrast microscope, and the presence of spindle-shaped cells on the plate bottom determined whether or not the cells were cell clusters. The presence of spindle-shaped cells on the plate bottom determined the presence of adherent fibroblasts. The results showed that fibroblasts did not adhere to the plate bottom on any of the common low-attachment 3D culture plates, such as PrimeSurface, Corning ULA Round-bottom, Corning ULA Flat-bottom, and Elplasia, and primary cancer cells formed cell clusters larger than 100 μm. On the other hand, cell clusters were formed on the common adhesive planar culture plates, such as the 96-well microplate and the low-attachment NanoCulture Plate, which has a textured surface that acts as a scaffold for cell cluster formation. However, fibroblasts adhered to the plate bottom. These results demonstrate that culture on low-attachment culture plates in medium containing a sol-like extracellular matrix can form sufficiently large primary cancer cell clusters, even in suspension culture, without the proliferation and proliferation of cells other than cancer cells, such as fibroblasts.

[0039] <Example 3: Examination of the concentration of extracellular matrix added to the medium> Primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation) according to Example 1. The required amount of cells was dispersed and counted, and then placed in a 15 mL tube. The cells were centrifuged at 300 × g for 5 minutes to remove the supernatant. Then, the cells were cultured in a medium prepared by adding Corning Matrigel GFR (Corning) to StemPro hESC SFM (Thermo Fisher Scientific) to a final concentration of 0, 0.5, 1, 2, 5, 10, 20, or 50 v / v% until the cell count reached 5 × 10 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4The seeding day was designated Day 0. No medium changes were performed, and the feasibility of half-medium changes was evaluated on Day 14. The results are shown in Figure 2. Morphology was confirmed using a phase-contrast microscope. At Corning Matrigel GFR concentrations of 0.5% v / v to 5% v / v, there was no proliferation of cells other than cancer cells, such as fibroblasts, even in a suspended state, and sufficient-sized cell clusters were formed. Furthermore, if the medium was not sufficiently sol-like when pipetting to remove half of the well, and the cells or cell clusters were also removed, a half-medium change was not possible. Half-medium changes were possible at 2% v / v or less, demonstrating ease of handling of cell clusters and their suitability for various tests.

[0040] <Example 4: Examination of the type of extracellular matrix added to the culture medium> Primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation) according to Example 1. The required amount of cells was dispersed and counted, then placed in a 15 mL tube and centrifuged at 300 × g for 5 minutes to remove the supernatant. After that, the cells were cultured in a medium prepared by adding Corning Matrigel (Corning), Cultrex BME (TREVIGEN), Cultrex RGF BME (TREVIGEN), or Cellmatrix Type IA (Nitta Gelatin) to a final concentration of 0, 0.5, 1, 2, 5, 10, or 20 v / v%. The cells were cultured until the cell count reached 5 × 10. 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4The seeding day was designated Day 0. No medium changes were performed, and the feasibility of half-medium changes was evaluated on Day 14. The results are shown in Figure 3. Morphology was confirmed using a phase-contrast microscope or the naked eye. For all extracellular matrices (Corning Matrigel, Cultrex BME, Cultrex RGF BME, or Cellmatrix Type IA), at concentrations between 0.5% and 5% v / v, there was no extensive proliferation of cells other than cancer cells, such as fibroblasts, even in a suspended state, and sufficient-sized cell clusters were formed. Furthermore, if the medium was not sufficiently sol-state when pipetting to remove half of the well, and the cells or cell clusters were also removed, a half-medium change was not possible. Half-medium changes varied depending on the type of extracellular matrix, but were feasible at concentrations between 2% and 5% v / v.

[0041] <Example 5: Examination of types of basal medium> Primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation) according to Example 1. The required amount of cells counted after dispersion was dispensed into a 15 mL tube and centrifuged at 300 × g for 5 minutes to remove the supernatant. After that, the cells were cultured in a basal medium prepared by adding Corning Matrigel GFR (Corning) to a final concentration of 2 v / v% in StemPro hESC SFM (Thermo Fisher Scientific) or StemFit AK02N (Takara Bio). The cells were cultured until the cell count reached 5 × 10 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4 The culture medium was 200 μL / well, with the seeding date designated Day 0. Half the volume of medium was replaced as needed. The results are shown in Figure 4. Morphology was confirmed using a phase-contrast microscope, and in either StemPro hESC SFM or StemFit AK02N basal medium, there was no extensive proliferation of cells other than cancer cells, such as fibroblasts, even in a suspended state, and the formation of sufficiently large cell clusters was observed.

[0042] Example 6: Examination of PDX tumor types Primary cancer cells were prepared using various PDX tumors (obtained from the National Institute of Biomedical Innovation) according to Example 1. The various PDX tumors used were pancreatic cancer (1), pancreatic cancer (2), lung squamous cell carcinoma (1), lung squamous cell carcinoma (2), lung squamous cell carcinoma (3), gastric cancer (1), and colon cancer (1). After dispersion, the counted cells were aliquoted into 15 mL tubes in the required amount and centrifuged at 300 × g for 5 minutes to remove the supernatant. After that, the cells were cultured in a medium containing StemPro hESC SFM (Thermo Fisher Scientific) supplemented with Corning Matrigel GFR (Corning) to a final concentration of 2 v / v% until the cell count reached 5 × 10 cells. 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4 The seeding day was designated Day 0, with half the amount of medium replaced as needed. The results are shown in Figure 5. Morphology was confirmed using a phase-contrast microscope, and in PDX tumors derived from any cancer type or patient, there was no progression or proliferation of cells other than cancer cells, such as fibroblasts, and the formation of sufficiently large cell clusters was observed, even in a floating state.

[0043] Example 7: Histopathological analysis Primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation) according to Example 1. The required amount of cells was dispersed and counted, and then placed in a 15 mL tube. The cells were centrifuged at 300 × g for 5 minutes to remove the supernatant. After that, the cells were cultured in a medium prepared by adding Corning Matrigel GFR (Corning) to StemPro hESC SFM (Thermo Fisher Scientific) to a final concentration of 2 v / v% until the cell count reached 5 × 10 4A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4 The seeding day was designated Day 0. Half the volume of medium was replaced as needed. On Day 14, the cell clusters were collected in 1.5 mL tubes, solidified into a gel using iPGell (GenoStaff), and fixed overnight in 10% neutral buffered formalin. Paraffin-embedded specimens were prepared from the formalin-fixed cell clusters according to standard procedures and subjected to HE staining and anti-human HLA immunohistochemical staining. For comparison, formalin-fixed paraffin-embedded specimens of pancreatic cancer (1) PDX tumors were prepared and similarly subjected to HE staining and anti-human HLA immunohistochemical staining. The results are shown in Figure 6. The staining results confirmed that the cell clusters generated from pancreatic cancer (1) PDX tumors were composed of human cancer cells. Furthermore, the pancreatic cancer (1) PDX tumors and the cell clusters generated from them shared a similar structure.

[0044] Example 8: Cell proliferation study Primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation) according to Example 1. The required amount of cells was dispersed and counted, and then placed in a 15 mL tube. The cells were centrifuged at 300 × g for 5 minutes to remove the supernatant. After that, the cells were cultured in a medium prepared by adding Corning Matrigel GFR (Corning) to StemPro hESC SFM (Thermo Fisher Scientific) to a final concentration of 2 v / v% until the cell count reached 5 × 10 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.) at N = 4 (four locations per plate under the same conditions), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4The cells / 200 μL / well were seeded on Day 0. Half the medium was replaced on Days 1, 7, 10, and 12. An ATP assay was performed on Days 1, 3, 7, 10, and 14 using the CellTiter-Glo 3D Cell Viability Assay (Promega). The ratio of the results from each measurement day to the results from Day 1 was calculated, and a growth curve was created. The results are shown in Figure 7. The number of viable cells in cell aggregates prepared from pancreatic cancer (1) PDX tumors increased in a time-dependent manner, linearly increasing to approximately four-fold on Day 7 and more than seven-fold on Day 14 compared to Day 1. This demonstrated high proliferation potential.

[0045] <Example 9: Anticancer drug sensitivity test> According to Example 1, primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation). In vivo anticancer drug sensitivity tests were performed according to the following procedure, with reference to known methods. Pancreatic cancer (1) PDX tumors grown subcutaneously in immunodeficient mice [Super SCID mice (strain name: C3H / HeJ / NOs-scid; LPS-nonresponder)] were aseptically excised in a safety cabinet, and necrotic areas of the tumor were removed with surgical scissors. Subsequently, transplanted tumor pieces measuring approximately 2-3 mm square were prepared and subcutaneously transplanted into 12 or more immunodeficient mice. The tumors were then cultured until the average tumor volume was approximately 200 mm. 3 Once tumor volume reached 1000 mg / kg, the animals were divided into groups based on tumor volume and assigned to a control group or gemcitabine-treated group (N=6: 6 animals per group under the same conditions). Gemcitabine (60 mg / kg / day) was administered twice a week for 4 weeks. Tumor diameter was measured twice a week, tumor volume was calculated, and a tumor growth curve was created. The results are shown in the left panel of Figure 8.

[0046] In vitro anticancer drug sensitivity testing was performed as follows: After dispersion, the counted cells were dispensed into a 15 mL tube in the required amount and centrifuged at 300 × g for 5 minutes to remove the supernatant. After that, the cells were cultured in a medium containing StemPro hESC SFM (Thermo Fisher Scientific) supplemented with Corning Matrigel GFR (Corning) at a final concentration of 2 v / v% until the cell count reached 5 × 10 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.) at N = 4 (four locations per plate under the same conditions), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4 The cells were cultured at 200 μL / well, with the seeding date designated Day 0. A half-volume medium change was performed on Day 1. On Day 7, a half-volume medium change was performed with medium supplemented with gemcitabine to a final gemcitabine concentration of 0, 0.001, 0.01, 0.1, 1, or 10 μmol / L. On Days 10 and 12, a half-volume medium change was performed with medium adjusted to maintain the final gemcitabine concentration. On Day 14, an ATP assay was performed using the CellTiter-Glo 3D Cell Viability Assay (Promega). Cell viability curves were created by calculating the ratio of the results for each gemcitabine concentration group, with the result for a gemcitabine concentration of 0 μmol / L designated as 100% viability. The results are shown in the right panel of Figure 8. Gemcitabine inhibited the growth of pancreatic cancer (1) PDX tumors in both in vivo and in vitro sensitivity assays.

[0047] <Example 10: Study using frozen tumor tissue> Pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation) were excised according to Example 1. The tumors were immersed in CELLBANKER 1 (Takara Bio Inc.) and frozen in a programmable deep freezer (Neppa Gene Co., Ltd.) to prepare frozen tumors. The frozen tumors were thawed in a 37°C warm bath and washed with HBSS (Thermo Fisher Scientific). Primary cancer cells were then prepared according to Example 1. The required amount of cells was counted after dispersion and dispensed into a 15 mL tube. After centrifugation at 300 × g for 5 minutes to remove the supernatant, the cells were cultured in a medium containing StemPro hESC SFM (Thermo Fisher Scientific Inc.) supplemented with Corning Matrigel GFR (Corning Inc.) to a final concentration of 2 v / v% until the cell count reached 5 × 10 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL. 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.) at N = 4 (four locations per plate under the same conditions), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4 The seeding day was designated Day 0. On Day 3, 50 μL of medium supplemented with gemcitabine was added to each well (250 μL / well) to achieve final gemcitabine concentrations of 0, 0.001, 0.01, 0.1, 1, or 10 μmol / L. ATP assays were performed on Days 0, 3, and 7 using the CellTiter-Glo 3D Cell Viability Assay (Promega). The ratio of the results from each measurement day to the Day 0 result was calculated to create a growth curve. The cell viability curve was created by calculating the ratio of the results for each gemcitabine concentration group, with the result for a gemcitabine concentration of 0 μmol / L being considered 100% cell viability. These results are shown in Figure 9. The number of viable cells in cell clusters prepared from once-frozen pancreatic cancer (1) PDX tumors increased in a time-dependent manner, increasing approximately fourfold on Day 7 compared to Day 0. As in Example 9, gemcitabine inhibited the proliferation of cell clusters derived from pancreatic cancer (1) PDX tumors, even when once-frozen tumor tissue was used.

[0048] <Example 11: Examination of subculture> Fresh tumor-derived cell clusters were obtained using pancreatic cancer (1) PDX tumors (obtained from the National Institute of Biomedical Innovation, Japan) according to Example 1, and frozen tumor-derived cell clusters were obtained according to Example 10. The cell clusters were collected in 50 mL tubes and centrifuged at 300 × g for 5 minutes. The supernatant was removed, and the cells were suspended in TrypLE (Thermo Fisher Scientific) and enzymatically treated in a 37 °C warm bath. A 10-fold volume of HBSS (Thermo Fisher Scientific) was added to the reaction solution to weaken the reaction, and the cells were passed through a 100 μm cell strainer to remove undispersed debris. The tube and cell strainer were rinsed with an appropriate amount of HBSS, and the cells were collected and centrifuged at 300 × g for 5 minutes. After removing the supernatant, the cell pellet was resuspended in HBSS and centrifuged at 300 × g for 5 minutes. The cell pellet was then resuspended in an appropriate amount of HBSS and counted. The cells were confirmed to be single cells and used in the following experiments. The required amount of cells was dispensed into a 15 mL tube and centrifuged at 300 × g for 5 min to remove the supernatant. Then, the cells were cultured in a medium containing StemPro hESC SFM (Thermo Fisher Scientific) supplemented with Corning Matrigel GFR (Corning) at a final concentration of 2 v / v% until the cell count reached 5 × 10 4 cells / mL (from fresh tumor) or 2 × 10 4 A cell suspension was prepared to give a concentration of 1 × 10 cells / mL (derived from frozen tumor). 200 μL of the suspension was seeded onto PrimeSurface (Sumitomo Bakelite Co., Ltd.) at N = 4 (four locations per cell under the same conditions), and static culture was initiated in a CO2 incubator set at 37°C and 5% CO2. The seeded cell number was 1 × 10 4 cells / 200 μL / well (fresh tumor derived) or 4 × 10 3The cells were seeded at 200 μL / well (derived from frozen tumors), and the day of seeding was designated Day 0. On Day 3, 50 μL of medium supplemented with gemcitabine was added to each well (250 μL / well) to achieve final gemcitabine concentrations of 0, 0.001, 0.01, 0.1, 1, or 10 μmol / L. ATP assays were performed on Days 0, 3, and 7 using the CellTiter-Glo 3D Cell Viability Assay (Promega). The ratio of the results from each measurement day to the Day 0 result was calculated to create a growth curve. The cell viability curve was created by calculating the ratio of the results for each gemcitabine concentration group, with the result for a gemcitabine concentration of 0 μmol / L being considered 100% cell viability. These results are shown in Figure 10. In cell clusters (also called subcultures) reconstructed from cells of cell clusters derived from pancreatic cancer (1) PDX tumors, regardless of whether they were derived from fresh or frozen tumors, the number of viable cells increased in a time-dependent manner, increasing approximately two-fold on Day 7 compared to Day 0. As in Examples 9 and 10, gemcitabine inhibited the growth of reconstructed cell clusters derived from pancreatic cancer (1) PDX tumors. [Industrial Applicability]

[0049] The present invention provides a method for producing cell masses using human tumor tissue as a starting material, by a 3D culture method for primary cancer cells, which allows for the high proliferation potential of cell masses composed mainly of primary cancer cells while suppressing the proliferation of cells other than cancer cells, such as fibroblasts, and also combines ease of handling, versatility, and high throughput. This makes it possible to produce cell masses in in vivo tissues simply and inexpensively, which can contribute to drug screening, drug efficacy evaluation, drug safety evaluation, regenerative medicine, etc.

Claims

1. A method for producing a cell mass by a three-dimensional culture method of primary cancer cells using tumor tissue, comprising: A method comprising a three-dimensional culture step of culturing cells obtained from the tumor tissue in a suspension state on a low-adhesion cell culture substrate in a medium containing 0.1 v / v% or more and 5 v / v% or less of extracellular matrix.

2. The method of claim 1 , wherein the tumor tissue is a xenograft tumor.

3. The method according to claim 1 or 2, wherein the culture medium is in a sol state.

4. A step of preparing a cell mass of primary cancer cells by the method for preparing a cell mass according to any one of claims 1 to 3; administering a test substance to the cell mass; and evaluating the effect of the test substance on the cell mass. A method for screening substances that have an effect on cell masses.

5. A step of preparing a cell mass of primary cancer cells by the method for preparing a cell mass according to any one of claims 1 to 3; administering a test substance to the cell mass; and evaluating the effect of the test substance on the cell mass. A method for determining the effect of a substance on the cell mass.

6. A kit for producing a cell mass by a three-dimensional culture method of primary cancer cells using tumor tissue, comprising: A low-adhesion cell culture substrate; A kit comprising a culture medium for a three-dimensional culture process containing 0.1 v / v% or more and 5 v / v% or less of extracellular matrix for culturing cells obtained from the tumor tissue in a suspended state.

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