Method for culturing malignant tumor cells, and kit for evaluating the efficacy of anti-cancer agents.

Abstract

The present invention aims to provide a simple and novel method for culturing malignant tumor cells, and a kit for evaluating the efficacy of anti-cancer agents. [Solution] The present invention provides a method for culturing non-adherent malignant tumor cells, comprising the step of applying non-adherent malignant tumor cells to a polyvinyl alcohol-based sponge having an average pore size of 10 μm to 100 μm and culturing them. The present invention also provides a kit for evaluating the efficacy of an anti-cancer agent, comprising a polyvinyl alcohol-based sponge having an average pore size of 10 μm to 100 μm.

Description

[Technical Field]

[0001] The technical field of the present invention relates to a method for culturing malignant tumor cells and a kit for evaluating the efficacy of anti-cancer agents. [Background technology]

[0002] Cells cultured in a planar environment often exhibit morphology, physiological function, gene expression, and drug responsiveness that differ significantly from those in vivo. Therefore, the development of three-dimensional culture technology is underway to mimic the three-dimensional tissue environment in vivo and obtain more physiologically valid results (Non-Patent Literature 1). Three-dimensional culture models have already been established and internationally recognized as an alternative to animal experiments in certain safety tests. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Jensen C, Teng Y. Front Mol Biosci. 2020 Mar 6;7:33. [Overview of the project] [Problems that the invention aims to solve]

[0004] However, there are still no established in vitro systems for screening candidate substances in drug development, including anti-cancer agents, or for drug response testing for personalized medicine. There is a need for low-cost, simple in vitro systems that can accurately evaluate candidate substances in research and clinical settings.

[0005] This invention has been made in view of these circumstances, and aims to provide a simple and novel method for culturing malignant tumor cells, and a kit for evaluating the efficacy of anti-cancer agents. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors of the present invention have found that the above problems can be solved by using a polyvinyl alcohol-based sponge with an average pore size within a predetermined range, and have completed the present invention.

[0007] The present invention provides the following: [1] A method for culturing non-adherent malignant tumor cells, comprising the step of applying non-adherent malignant tumor cells to a polyvinyl alcohol-based sponge having an average pore size of 10 μm to 100 μm and culturing them. [2] The polyvinyl alcohol-based sponge is a polyvinyl formal sponge, according to the method in [1]. [3] The polyvinyl alcohol-based sponge is hydrophilically treated, according to the method of [1] or [2]. [4] The method according to any one of [1] to [3], wherein the non-adherent malignant tumor cells are leukemia cells. [5] The method of [4], wherein the leukemia cells are chronic myeloid leukemia (CML) cells or acute myeloid leukemia (AML) cells. [6] The method according to any one of [1] to [5], further comprising the step of co-culturing with stromal cells. [7] The method according to any one of [1] to [6], wherein the leukemia cells are a chronic myeloid leukemia (CML) cell line or an acute myeloid leukemia (AML) cell line. [8] A kit for evaluating the efficacy of anti-cancer agents, comprising a polyvinyl alcohol-based sponge having an average pore size of 10 μm to 100 μm. [9] The polyvinyl alcohol-based sponge is a polyvinyl formal sponge, as described in [8].

[10] The kit according to [8] or [9], wherein the polyvinyl alcohol-based sponge is hydrophilic.

[11] The malignant tumor is non-adhesive, as described in any of [8] to

[10] .

[12] Contains interstitial cells present in a fixed and / or frozen state in a polyvinyl alcohol-based sponge, The kit described in [8] to

[11] , wherein the stromal cells are stromal cells cultured using the polyvinyl alcohol-based sponge.

[13] The kit described in [8] to

[12] , wherein the polyvinyl alcohol-based sponge has a thickness of 0.1 to 15 mm and a diameter of 1 to 37 mm and is incorporated into a multiwell plate.

[14] The polyvinyl alcohol-based sponge has a thickness of 0.5 to 3 mm and a diameter of 1 to 37 mm and is incorporated into a multiwell plate, as described in the kits of [8] to

[13] . [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a simple and novel method for culturing malignant tumor cells, and a kit for evaluating the efficacy of anti-cancer agents. [Brief explanation of the drawing]

[0009] [Figure 1] The experimental method for Example 1-1 is outlined below. [Figure 2] This shows the number of viable cells measured by the MTT method after three-dimensional culture using a resin sponge. N=2~3. [Figure 3] This image shows a photograph of a resin sponge stained with MTT after 7 days of three-dimensional culture. [Figure 4] This image shows an electron microscope photograph of a PVF sponge 14 days after three-dimensional culture of K562 cells. [Figure 5] The experimental methods for Examples 1-2 are outlined below. [Figure 6] The following shows the number of viable cells measured by the MTT method and the percentage of CD34-positive cells measured by flow cytometry after planar culture and three-dimensional culture of K562 and THP-1 cells using PVF sponge. [Figure 7] The experimental methods for Examples 1-3 are outlined below. [Figure 8]Shows the number of viable cells measured by the MTT method and the percentage of CD34-positive cells measured by flow cytometry after three-dimensional culture (3D) and three-dimensional co-culture (3D-C) of K562 cells and THP-1 cells using PVF sponges. [Figure 9] Shows electron micrographs of PVF sponges 14 days after three-dimensional culture and three-dimensional co-culture of THP-1 cells. [Figure 10] Shows photographs of MTT-stained PVF sponges after three-dimensional co-culture of K562 cells, U937 cells, HL60 cells, THP-1 cells, and KG-1 cells. [Figure 11] Shows photographs of MTT-stained PVF sponges after three-dimensional co-culture of MEG01 cells, CMK cells, CMY cells, UT7 cells, Mo7e cells, MOLM16 cells, MKPL1 cells, and AMKL patient cells (KK(patient)), or after three-dimensional culture of only HS-5 cells, and photographs of MTT-stained PVF sponges without culturing cells. [Figure 12] Shows photographs of MTT-stained PVF sponges after three-dimensional co-culture of MOLM16 cells, MKPL1 cells, and AMKL patient cells (KK(patient)), or after three-dimensional culture of only HS-5 cells. [Figure 13] Shows photographs (cross-sections) of MTT-stained PVF sponges after three-dimensional co-culture of MOLM16 cells, MKPL1 cells, and AMKL patient cells (KK(patient)), or after three-dimensional culture of only HS-5 cells. [Figure 14] Shows photographs of MTT-stained PVF sponges after three-dimensional co-culture when various hydrophilic treatments were performed on PVF sponges. [Figure 15] Shows the medium absorption rate of PVF sponges after each hydrophilic treatment. [Figure 16] Shows an overview of the experimental method of Example 2-2. [Figure 17] Shows the results of viable cell assays after drug addition in the three-dimensional co-culture of K562 cells, U937 cells, HL60 cells, THP-1 cells, and KG-1 cells. [Figure 18]This shows the results of a live cell assay after drug addition in a three-dimensional co-culture of CMK and CMY cells. [Figure 19] The results of a live cell assay after drug addition in a three-dimensional co-culture of UT7 and Mo7e cells are shown. [Figure 20] The results of a live cell assay after drug addition in a three-dimensional co-culture of MOLM16 and MKPL1 cells are shown. [Figure 21] This paper presents the results of live cell assays after the addition of various drug concentrations in a three-dimensional co-culture of MOLM16 and MKPL1 cells. [Figure 22] The results of a live cell assay after drug addition in a three-dimensional co-culture of MOLM16 and MKPL1 cells are shown. [Figure 23] The image shows a PVF sponge stained with MTT in a three-dimensional co-culture of RD cells, JMU-RTK2 cells, RMS-YM cells, G401 cells, and AT / RT cells. [Figure 24] The results of a live cell assay after drug addition in a three-dimensional co-culture of RD cells, JMU-RTK2 cells, RMS-YM cells, G401 cells, and AT / RT cells are shown. [Modes for carrying out the invention]

[0010] The embodiments of the present invention will be described in detail below. To avoid repetition and unnecessary complexity, similar content will be omitted from explanation as appropriate.

[0011] (1) Method for culturing malignant tumor cells One embodiment of the present invention provides a method for culturing malignant tumor cells, comprising the step of applying malignant tumor cells to a polyvinyl alcohol-based sponge and culturing them. The method of the present invention may further include the steps of sterilizing the polyvinyl alcohol-based sponge and / or hydrophilizing it. The method of the present invention may further include the step of applying stromal cells to the polyvinyl alcohol-based sponge and culturing them. The step of applying stromal cells to the polyvinyl alcohol-based sponge and culturing them may be started before the start of the step of culturing malignant tumor cells, simultaneously with the step of culturing malignant tumor cells, or after the start of the step of culturing malignant tumor cells.

[0012] The process of culturing malignant tumor cells in a polyvinyl alcohol-based sponge is not particularly limited, but may be carried out, for example, by pouring a culture medium containing suspended malignant tumor cells onto a polyvinyl alcohol-based sponge set in a culture vessel, or by immersing the polyvinyl alcohol-based sponge in a culture medium containing suspended malignant tumor cells. The culture vessel can be appropriately selected depending on the purpose, and for example, a multi-well plate, dish, or flask may be used. The culture medium can be appropriately selected depending on the cells to be cultured, and the medium may be changed as needed.

[0013] The degree of proliferation of cultured cells can be measured using methods well known to those skilled in the art. While not particularly limited, methods such as counting viable cells by collecting them and performing trypan blue staining or flow cytometry, or calculating the relative number of viable cells using their specific metabolic activity or intracellular ATP levels as indicators, can be used. Furthermore, cell proliferation can be visually observed by observing the surface or sections of a polyvinyl alcohol-based sponge after culture using an electron microscope.

[0014] (2) Kits for evaluating the efficacy of anti-cancer drugs The present invention further relates to a kit for evaluating the efficacy of anti-cancer agents, comprising a polyvinyl alcohol-based sponge. In one embodiment, the efficacy of an anti-cancer agent can be evaluated by using the kit of the present invention to culture malignant tumor cells using the method described in (1) above, adding the anti-cancer agent to the cells, and measuring the cell viability.

[0015] The kit of the present invention may include, in addition to the polyvinyl alcohol-based sponge, other components necessary for cell culture. For example, these may include cells to be applied to the polyvinyl alcohol-based sponge, cell culture medium, cell culture plates or dishes, and instructions for using the kit.

[0016] In the kit of the present invention, the shape of the polyvinyl alcohol-based sponge is not particularly limited, but from the viewpoint of faster, simpler, and more accurate data measurement, it is preferable to have a shape that matches the well size of the multiwell plate, and it is more preferable that the bottom diameter be cylindrical with a diameter of 1 to 37 mm. The thickness of the polyvinyl alcohol-based sponge is preferably 0.1 to 15 mm, more preferably 0.5 to 3 mm. The polyvinyl alcohol-based sponge may be pre-assembled into the multiwell plate.

[0017] The kit of the present invention may include stromal cells present in a fixed and / or frozen state in a polyvinyl alcohol-based sponge, and the stromal cells may be stromal cells cultured in three dimensions using a polyvinyl alcohol-based sponge. Fixation may be performed using fixatives commonly used for fixing tissues and cells, such as aldehydes such as formaldehyde, glutaraldehyde, and paraformaldehyde, or organic solvents such as C1-C3 alcohols such as methanol and ethanol, or C1-C3 ketones such as acetone.

[0018] The anti-cancer agent may be one that is well known to those skilled in the art, or it may be a novel compound whose anti-cancer activity is not known. The anti-cancer agent includes, for example, drugs that have an inhibitory effect on the proliferation of malignant tumor cells or an intoxicating effect on malignant tumor cells. The efficacy of the anti-cancer agent can be evaluated, for example, by the method of measuring the degree of cell proliferation described in (1) above. The efficacy of the anti-cancer agent is not particularly limited in the screening of anti-cancer agents, but it can be evaluated quickly and easily by using a method that can be measured on a multi-well plate, specifically, for example, a method that calculates the relative number of living cells using the unique metabolic activity of living cells or the amount of intracellular ATP as indicators.

[0019] (Polyvinyl alcohol-based sponge) In one embodiment of the present invention (for example, (1) to (2) above), the polyvinyl alcohol-based sponge refers to a porous material having a fine, continuous pore structure, with polyvinyl alcohol, a synthetic resin, as the main raw material. "Having continuous pores" means that one opening on the surface of the porous material and the pores connected to it are connected internally, forming a hollow structure. Therefore, fluids such as gases and liquids that enter through one opening can pass through the internal pores and exit through (other) openings. A polyvinyl alcohol-based sponge can be obtained, for example, by mixing one or more types of polyvinyl alcohol (raw material) with an average degree of polymerization of 500 to 3000 and a degree of saponification of 80% or more to make an aqueous solution, adding a crosslinking agent, mineral acids as a catalyst, and starch as a pore-forming agent to this aqueous solution, injecting this mixture into a predetermined mold, reacting it at 40 to 80°C, removing it from the mold, and then washing it with water to remove the pore-forming agent, etc.

[0020] A polyvinyl alcohol-based sponge may be a sponge whose backbone is a resin chemically modified with polyvinyl alcohol. Preferably, the polyvinyl alcohol-based sponge may be a polyvinyl acetal-based sponge, whose backbone is a resin in which polyvinyl alcohol has been acetalized. A polyvinyl acetal-based sponge can be obtained, for example, by using aldehydes as the crosslinking agent in the above-described method for producing a polyvinyl alcohol-based sponge.

[0021] Examples of polyvinyl acetal sponges include polyvinyl formal sponges, which have a resin backbone made by acetalizing polyvinyl alcohol with aldehydes including formaldehyde. The degree of formalization can be adjusted by appropriately selecting the amount of formaldehyde, the acid concentration, the reaction temperature, or the reaction time.

[0022] Examples of commercially available polyvinyl alcohol-based sponges include the Bell Eater A series, Bell Eater D series, and Bell Eater W series, which are polyvinyl formal sponges manufactured by AION Corporation.

[0023] The polyvinyl alcohol-based sponge may be processed into a predetermined shape by cutting, punching, or other means. The shape of the polyvinyl alcohol-based sponge is not particularly limited, but can be, for example, a cube, a rectangular prism, a truncated square pyramid, a sphere, a cylinder, a truncated cone, etc., and can be appropriately selected according to the purpose. The thickness of the polyvinyl alcohol-based sponge is not particularly limited, but it is preferable that it be lower than the depth of the wells of the plate used for cell culture or the depth of the container such as the dish. The thickness of the polyvinyl alcohol-based sponge may be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15.0 mm, and may also be within the range of any two of the values ​​exemplified here.

[0024] The average pore size of the polyvinyl alcohol sponge can be appropriately selected depending on the cells to be cultured. The average pore size of the polyvinyl alcohol sponge should be large enough to accommodate the cells to be cultured, and is preferably 10 μm or larger. On the other hand, from the viewpoint of efficiently culturing cells by keeping them in the polyvinyl alcohol sponge, the average pore size of the polyvinyl alcohol sponge is preferably 100 μm or less. The average pore size of the polyvinyl alcohol sponge is preferably 10 to 100 μm, specifically, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μm, and may also be within the range of any two of the values ​​exemplified here.

[0025] The average pore diameter of a polyvinyl alcohol-based sponge is the average value of the diameters of multiple pores present in the internal structure of the polyvinyl alcohol-based sponge. In this embodiment, the average pore diameter is defined as the average of the major axes (longitudinal distances of each pore) of a predetermined number of pores extracted from multiple pores according to predetermined criteria, and can be determined, for example, by the following measurement method.

[0026] A dry polyvinyl alcohol-based sponge is cut at a predetermined location, and the internal structure exposed on the cut surface is photographed with an electron microscope. Next, a predetermined measurement range is set on the photograph, and 20 pores with the largest major diameters are extracted from among the multiple pores present within that measurement range. Then, the major diameter of each of the 20 extracted pores is measured. Finally, the average of the 11th to 20th measurements (counting from the largest) is calculated as the average pore diameter.

[0027] The porosity of the polyvinyl alcohol-based sponge is preferably 80-95%, specifically, for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and may be within the range of any two of the values ​​exemplified here.

[0028] The porosity of a polyvinyl alcohol-based sponge is calculated using the following formula, based on the apparent volume obtained by measuring each side of a dry rectangular polyvinyl alcohol-based sponge with calipers and the true volume measured with a gas pycnometer. Porosity (%) = ((Apparent volume of sponge - True volume of sponge) / Apparent volume of sponge) × 100

[0029] Polyvinyl alcohol-based sponges may be sterilized. Sterilization methods well known to those skilled in the art can be used, such as autoclaving, UV irradiation, electron beam or gamma ray irradiation, or ethanol disinfection.

[0030] Polyvinyl alcohol-based sponges may be treated to make them hydrophilic. Examples of hydrophilic treatments include autoclaving, PBS immersion, sonication, vacuum centrifugation, and DMSO immersion. Autoclaving is preferably performed in a container such as a test tube, with the sponge immersed in a liquid that does not denature at the immersion temperature, such as water, PBS, or culture medium. PBS immersion may be performed in any liquid that does not denature at the immersion temperature, such as water or culture medium. DMSO immersion should be limited to 15 minutes or less, as prolonged immersion can cause the sponge to become transparent and significantly denatured, and it is preferable to wash the sponge with PBS or similar afterward. Although the mechanism is unknown, it is thought that such hydrophilic treatment makes it easier for the culture medium to penetrate the polyvinyl alcohol-based sponge, thus facilitating three-dimensional culture within the sponge. Hydrophilic treatment is preferable for polyvinyl alcohol-based sponges with a high degree of chemical modification to polyvinyl alcohol and higher hydrophobicity compared to regular polyvinyl alcohol-based sponges, or for polyvinyl alcohol-based sponges with small pore sizes. Furthermore, since polyvinyl alcohol-based sponges are heat-resistant, they can be autoclaved to perform sterilization and hydrophilization treatment simultaneously. From the viewpoint of enabling rapid and simple culture of malignant tumor cells and evaluation of the efficacy of anti-cancer agents, it is more preferable to perform the hydrophilization treatment by autoclaving. The effect of the hydrophilization treatment can be evaluated, for example, by measuring the culture medium absorption rate ((weight after culture medium absorption - dry weight) / weight after culture medium absorption) when the hydrophilized sponge is dried and then absorbed with culture medium. The culture medium absorption rate of the hydrophilized polyvinyl alcohol-based sponge is preferably 300% or more, and specifically, for example, it may be 300, 350, 400, 450% or more.

[0031] (Malignant tumor) In one embodiment of the present invention (for example, (1) to (2) above), malignant tumors include, for example, tumors that arise from a mutation in a gene in a normal cell. Malignant tumors can arise from any organ or tissue in the body. Malignant tumors may include, for example, solid tumors or hematological cancers. Solid tumors include, for example, one or more selected from the group consisting of lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, breast cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, uterine cancer, pancreatic cancer, giant cell tumor of bone, ovarian cancer, esophageal cancer, adrenal cancer, biliary tract cancer, small intestine cancer, ovarian cancer, ureteral cancer, renal pelvis cancer, penile cancer, testicular cancer, brain tumors (e.g., glioma), central nervous system cancers, peripheral nervous system cancers, thyroid cancer, salivary gland cancer, rhabdomyosarcoma, and carcinomas. Hematological cancers include, for example, lymphoma, leukemia, or multiple myeloma. Leukemia includes chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), and acute myeloid leukemia (AML) includes acute megakaryoblastic leukemia (AMKL). Malignant tumors may be adhesive or non-adhesive. Non-adhesive malignant tumors include, for example, hematological cancers.

[0032] Malignant tumor cells may be cells taken from a subject with a malignant tumor, or they may be cell lines. Subjects include humans and non-human mammals (e.g., one or more species such as mice, guinea pigs, hamsters, rats, rabbits, pigs, sheep, goats, cattle, horses, cats, dogs, marmosets, monkeys, or chimpanzees).

[0033] (stromal cells) In one embodiment of the present invention (for example, (1) to (2) above), stromal cells refer to cells that surround and support parenchymal cells that perform their functions in an organ. Stromal cells can be appropriately selected depending on the cells to be cultured, and include various types of cells such as fibroblasts, epithelial cells, reticular cells, adipocytes, vascular cells, nerve cells, and macrophages. For example, bone marrow stromal cells can be used in the culture of hematological cancer cells. Stromal cells may be cells collected from the above subject, or they may be cell lines. [Examples]

[0034] The present invention will be further explained below with reference to examples, but is not limited to these.

[0035] <Material> Polyvinyl alcohol-based sponges (Type-A3, Type-A4, Type-DA2, Type-DK, Type-DC, Type-DA, Type-DD, Type-EA, Type-EA2, Type-ED) were obtained by mixing one or more types of polyvinyl alcohol (raw material) with an average degree of polymerization of 500 to 3000 and a degree of saponification of 80% or more to make an aqueous solution. Aldehydes were added as a crosslinking agent, mineral acids as a catalyst, and starch, etc., as a porosity-forming agent to this aqueous solution. This mixture was then poured into a predetermined mold, reacted at 40 to 80°C, removed from the mold, and the porosity-forming agent, etc., was removed by washing with water. Type-DD and Type-ED were sponges with a lower degree of formalization compared to the other sponges. As controls, Type-PU, a polyurethane sponge, and Type-PE, a polyethylene sponge, were used.

[0036] The average pore diameter and porosity of each sponge were calculated using the method described below, and the results are shown in Table 1. (Average pore size) A 2mm thick, dry polyvinyl alcohol-based sponge was cut into 10mm x 10mm sections, and the internal structure exposed on the cut surface was photographed using an electron microscope (JEOL Ltd., JSM-IT300LA). A measurement range was set near the center of the photographed cut surface where the pore size distribution was minimal. From the multiple pores within this measurement range, 20 pores were extracted in descending order of their major axis. Next, the major axis of each of the 20 extracted pores was measured. Finally, the average of the 11th to 20th measurements (counting from the largest) was calculated as the average pore diameter. (Porosity) A dry sponge was cut into a rectangular prism approximately 10 x 10 x 40 mm in size, and the apparent volume was calculated by measuring the length of each side with calipers. Next, the true volume was measured using a gas pycnometer (Micromeritics AccuPyc II1340), and the porosity was calculated using the following formula. Porosity (%) = ((Apparent volume of sponge - True volume of sponge) / Apparent volume of sponge) × 100 [Table 1] JPEG0007900793000002.jpg20150

[0037] Example 1: Construction of a three-dimensional culture system using resin sponge Example 1-1 Investigation of material and pore size of the carrier Experimental method Figure 1 shows an overview of the experimental method. Type-A3 (average pore size 13.5 μm), Type-A4 (average pore size 23.2 μm), Type-DA2 (average pore size 29.4 μm), Type-EA2 (average pore size 106.0 μm) (polyvinyl formal (PVF)), Type-PU (average pore size 16.3 μm) (polyurethane (PU)), and Type-PE (average pore size 14.5 μm) (polyethylene (PE)) resin sponges were first cut into 2 mm cubes and autoclaved while immersed in PBS. Leukemia cells suspended in RPMI medium supplemented with 10% FBS were then placed in these sponges in a 3 × 10⁶ layer. 6 cells / cm 3 After seeding, the sponge containing the cells was transferred to a culture dish and cultured. Cells were harvested 1, 4, 7, and 14 days after the start of culture, and viable cells were stained and counted using the MTT method. The cultured sponge was also observed under an electron microscope. The leukemia cell lines used were K562 cells, which are chronic myeloid leukemia (CML) cells, and THP-1 cells, which are acute myeloid leukemia (AML) cells.

[0038] Experimental results Figure 2 shows the results of counting the number of viable cells. While cells could not be proliferated with PU or PE, cells could be proliferated with PVF across a wide range of stomatal diameters. However, K562 proliferated best with Type-DA2 (average stomatal diameter 29.4 μm), and THP-1 proliferated best with Type-A4 (average stomatal diameter 23.2 μm).

[0039] Figure 3 shows photographs of resin sponges stained with MTT on day 0 and day 7 of culture. The purple dye represents living cells. In K562, Type-DA2 (average pore size 29.4 μm) had a greater amount of dye than Type-DA2 (average pore size 29.4 μm), and in THP-1, Type-A4 (average pore size 23.2 μm) had a greater amount of dye than Type-DA2 (average pore size 29.4 μm). The dye adhered evenly to the carrier, indicating that the cells proliferated well within the carrier. On the other hand, in culture with PU, although cell seeding was possible, the cells died during culture, and there was almost no staining by day 7 of culture.

[0040] Figure 4 shows electron microscope images of PVF sponges after 14 days of K562 cell culture (top panel: fixed with paraformaldehyde, bottom panel: fixed with glutaraldehyde). It was observed that leukemia cells were cultured in a densely packed state within the pores.

[0041] Examples 1-2 Comparison with planar culture

[0042] Experimental method Figure 5 shows an overview of the experimental method. In three-dimensional culture (3D), Type-A4 (average pore size 23.2 μm) PVF sponge was cut into 2 mm cubes and autoclaved while immersed in PBS. Leukemia cells were suspended in RPMI medium supplemented with 10% FBS and placed in these sponges at a rate of 1 × 10⁶ 6 cells / cm 3 (L), or 3×10 6 cells / cm 3 Seeds were seeded in (H). Cells were harvested 1, 4, 7, and 14 days after the start of culture, and viable cells were stained and counted using the MTT method, and FACS analysis was performed. In addition, in a control experiment using planar culture (2D) for comparison, 2 × 10 cells were collected. 4 cells / mL(L), or 6×10 4 The cells were suspended in culture medium at a concentration of cells / mL(H), and 2 mL of the cell suspension was added to each 35 mm dish for incubation.

[0043] Experimental results The results are shown in Figure 6. It was found that both K562 cells and THP-1 cells could be cultured at a higher density than in planar culture, regardless of the cell seeding density. Furthermore, in three-dimensional culture, the proportion of CD34-positive cells, a marker for leukemia stem cells, tended to be higher compared to planar culture, suggesting that cells could be cultured and proliferated in an environment closer to that of a living organism.

[0044] Examples 1-3: Examination of co-culture systems Experimental method Figure 7 shows an overview of the experimental method. To better mimic the in vivo microenvironment, co-culture with bone marrow stromal cells was performed. First, HS-5 stromal cells were cultured using a PVF sponge, and after 3 days, X-ray irradiation was performed to stop the proliferation of HS-5 cells. Subsequently, leukemia cells were added and co-culture was performed. The rest of the procedure was carried out under the same conditions and methods as the three-dimensional culture (3D) in Example 1-2, and viable cells were stained and counted by the MTT method, and the sponge was photographed with an electron microscope. Three-dimensional monoculture (3D) was performed as a control for three-dimensional co-culture (3D-C) of each cell type, and the two were compared.

[0045] Experimental results The results are shown in Figures 8 and 9. In THP-1 cells, the number of viable leukemia cells tended to increase with co-culture, and the proportion of CD34-positive cells, a marker for leukemia stem cells, increased compared to 3D (Figure 8). Furthermore, electron microscopy observation of the PVF sponge 14 days after THP-1 cell culture confirmed that HS-5 cells were in contact with leukemia cells in co-culture (Figure 9), suggesting that co-culture allows cells to be cultured and proliferated in an environment closer to that of a living organism compared to monoculture.

[0046] Example 2: Evaluation of the efficacy of an anti-cancer agent using a three-dimensional co-culture system Example 2-1 Investigation of pore size Experimental method Immersed in PBS and subjected to autoclaving (120 °C, 20 minutes), PVF sponges with various pore sizes (Type-A3 (average pore size 13.5 μm), Type-A4 (average pore size 23.2 μm), Type-DK (average pore size 36.8 μm), Type-DA (average pore size 73.3 μm)) (thickness 1 mm, diameter 6 mm) were placed on an adhesive cell culture plate 96-well (Sumitomo Bakelite, MS-8096F), and HS-5 cells, which are bone marrow stromal cells, were seeded at 15x10 3 cells / well. One day later, leukemia cells were seeded at 6x10 4 cells / well. Further, three days later, the living cells in the PVF sponge were stained with MTT reagent (Nacalai, MTT Cell Count Kit 23506-80), and the cell penetration into the sponge was confirmed.

[0047] <- Experimental results The results of MTT staining after three-dimensional co-culture of K562 cells, which are chronic myeloid leukemia (CML) cells, U937 cells, HL60 cells, THP-1 cells, and KG-1 cells, which are acute myeloid leukemia (AML) cells, are shown in Fig. 10. The PVF sponges used were Type-A3 (average pore size 13.5 μm), Type-A4 (average pore size 23.2 μm), Type-DK (average pore size 36.8 μm), and Type-DA (average pore size 73.3 μm). It was confirmed that for K562 cells, HL60 cells, and KG-1 cells, the cells had penetrated to the back even with a pore size of 36.8 μm (Type-DK) or more, while for U937 cells and THP-1 cells, the cells had penetrated to the back even with a pore size of 13.5 μm (Type-A3).

[0048] Figure 11 shows the results of MTT staining after three-dimensional co-culture of cells from acute megakaryoblastic leukemia (AMKL) (a subtype of acute myeloid leukemia (AML)), including MEG01 cells, CMK cells, CMY cells, UT7 cells, Mo7e cells, MOLM16 cells, MKPL1 cells, and AMKL patient cells (KK (patient)). As controls, PVF sponges cultured in three dimensions with only HS-5 cells and PVF sponges without cell culture were also stained with MTT. The PVF sponges used were Type-A3 (mean pore size 13.5 μm), Type-A4 (mean pore size 23.2 μm), Type-DK (mean pore size 36.8 μm), and Type-DA (mean pore size 73.3 μm). In AMKL cells, penetration to the back surface was confirmed from a pore size of approximately 36.8 μm (Type-DK).

[0049] Figures 12 and 13 show the results of MTT staining after three-dimensional co-culture of MOLM16 cells, MKPL1 cells, and AMKL patient cells (KK(patient)). As a control, PVF sponges cultured in three dimensions with only HS-5 cells were also stained with MTT. Type-DA (average pore size 73.3 μm), Type-DC (average pore size 69.3 μm), Type-EA (average pore size 93.1 μm), Type-DD (average pore size 83.0 μm), and Type-ED (average pore size 107.7 μm) were used. In the case of Type-ED (average pore size 107.7 μm), the sponge was not stained, suggesting that the cells did not remain in the sponge but fell out, indicating that the three-dimensional co-culture was unsuccessful. However, in the other sponges, it was confirmed that the cells penetrated evenly throughout the sponge and were cultured successfully. In particular, Type-DD cells (average stomatal diameter 83.0 μm) were stained most intensely, and it was confirmed that these cells proliferated easily.

[0050] Example 2-2 Investigation of hydrophilic treatment Example 2-2-1 Three-dimensional co-culture using hydrophilic treated PVF sponge Experimental method Three-dimensional co-culture was performed using PVF sponges treated with various hydrophilicity-enhancing processes, and the permeability of cells to the sponges was confirmed. Each group was subjected to the following conditions. Except for autoclaving, sterilization was performed by irradiating with UV for 30 minutes. Based on the above pore size studies, Type-A3 (average pore size 13.5 μm) PVF sponges were used for THP-1 cells, and Type-DA (average pore size 73.3 μm) PVF sponges were used for MOLM16 cells and MKPL1 cells. Other conditions and methods were the same as in Example 2-1. Autoclave (TOMY LSX-500): Immerse in PBS and autoclave at 120°C for 20 minutes. PBS immersion ON / N: Immerse in PBS for 18 hours. Ultrasonic treatment: 60 minutes Vacuum centrifugation: 60 minutes Centrifugation (control): 60 minutes DMSO treatment: Type-A3 (average pore size 13.5 μm) ⇒ Immersed in DMSO and immediately washed with PBS. Type-DA (average pore size 73.3 μm) ⇒ Immerse in DMSO for 10 minutes, then wash with PBS.

[0051] Experimental results Figure 14 shows photographs of MTT-stained PVF sponges after three-dimensional co-culture, after various hydrophilic treatments were applied to the PVF sponges. In the culture of THP-1 cells using Type-A3 (average pore size 13.5 μm) PVF sponges, cell osmosis was increased in the hydrophilic-treated sponges compared to the untreated sponges. This is thought to be because the hydrophilic treatment makes it easier for the culture medium to penetrate the sponge. On the other hand, in the culture of MKPL1 cells using Type-DA (average pore size 73.3 μm) PVF sponges, cell osmosis was slightly increased in the hydrophilic-treated sponges compared to the untreated sponges in the solitary culture of HS-5 cells, but there was little difference in co-culture. This is thought to be because, with a pore size of 73.3 μm, the culture medium easily penetrates the sponge even in the untreated sponges.

[0052] Example 2-2-2 Measurement of the culture medium absorption rate of PVF sponge after hydrophilic treatment Experimental method To evaluate the effect of hydrophilic treatment, a hydrophilic-treated PVF sponge was dried and then immersed in a culture medium at 37°C for 1 minute to absorb the medium. The culture medium absorption rate was then measured. ( ((Weight after culture medium absorption - dry weight) / dry weight) ×100) The following measurements were taken. PVF sponge Type-A4 was used. The conditions for each group were as follows. Blank drying: Samples that have been dried without hydrophilic treatment. <Hydrophilic treatment> Water absorption: Soak in water for 18 hours. Ultrasonic treatment: 60 minutes Autoclave: Immerse in PBS and autoclave at 121°C for 24 minutes. PBS: Soak in PBS for 18 hours. DMSO: After immersion in DMSO, immerse in PBS for 18 hours.

[0053] Experimental results Figure 15 shows the culture medium absorption rates of PVF sponges after each hydrophilic treatment. In all hydrophilic treatment groups, the culture medium absorption rate increased significantly compared to the blank drying group. Among the hydrophilic treatment groups, the group immersed in water for 18 hours showed the highest culture medium absorption rate, but high absorption rates were also observed with shorter treatment times such as autoclaving and sonication.

[0054] Example 2-3: Evaluation of the efficacy of anti-cancer agents using a three-dimensional co-culture system. Experimental method Figure 16 shows an overview of the experimental method. A PVF sponge (1 mm thick, 6 mm in diameter) that had been immersed in PBS and autoclaved (120°C, 20 minutes) was placed in a 96-well adherent cell culture plate, and HS-5 cells, which are bone marrow stromal cells, were cultured in 15x10⁻¹⁰⁻¹ 3 Leukemia cells were seeded in a 6x10⁻¹⁶ format after 1 day. 4Cells were seeded at a rate of cells / well. After one day, a drug was added, and a cell viability assay was performed 72 hours after addition (normal, 2D: MTT method (Nakarai, MTT Cell Count Kit 23506-80), 3D: luminescent cell viability assay (Promega, CellTiter-Glo 3D Cell Viability Assay G9681)). As controls, cell cultures were performed in a group without PVF sponge and without co-culture (normal), and in a group with co-culture without PVF sponge (2D), and compared with the three-dimensional co-culture group (3D). Measurement values ​​were calculated as relative values ​​for each group, with day 0 set to 100%.

[0055] Experimental results Figure 17 shows the results of live cell assays using K562 cells (chronic myeloid leukemia (CML) cells) and U937, HL60, THP-1, and KG-1 cells (acute myeloid leukemia (AML) cells). Cytarabine (AraC) was used as the drug. Based on the results of Example 2-1, Type-A4 (mean pore size 23.2 μm) PVF sponges were used for K562, HL60, and KG-1 cells, and Type-A3 (mean pore size 13.5 μm) PVF sponges were used for U937 and THP-1 cells. In all cells, drug resistance improved in 2D compared to normal cells, and further improved in 3D.

[0056] Figures 18-21 show the results of live cell assays using CMK cells, CMY cells, UT7 cells, Mo7e cells, MOLM16 cells, and MKPL1 cells, which are cells of acute megakaryoblastic leukemia (AMKL) (a subtype of acute myeloid leukemia (AML)). The drugs used were cytarabine (AraC), ruxolitinib (Rux) and a combination of cytarabine and ruxolitinib. In addition, PVF sponges of Type-A4 (mean pore size 23.2 μm), Type-DK (mean pore size 36.8 μm), and Type-DA (mean pore size 73.3 μm) were used. Under many conditions, improved drug resistance was observed in 3D compared to normal conditions (Figures 17-19). Furthermore, in a three-dimensional co-culture of MOLM16 and MKPL1 cells using a Type-DA (average pore size 73.3 μm) PVF sponge, a synergistic effect of drug combination was observed (Figure 20).

[0057] Figure 22 shows the results of three-dimensional co-culture of MOLM16 cells and MKPL1 cells using Type-DA (mean pore size 73.3 μm) or Type-DD (mean pore size 83.0 μm) PVF sponges (1 mm or 2 mm thick). No significant differences in drug resistance were observed depending on the thickness or type of sponge.

[0058] Figures 23 and 24 show the results of MTT staining and live cell assays for solid cancer cells, specifically RD cells, JMU-RTK2 cells, RMS-YM cells, G401 cells, and AT / RT cells, respectively. Doxorubicin was used as the drug. Other than using solid tumor cells instead of leukemia cells, the conditions and methods were the same as those described in Examples 2-1 and 2-3 above. All cells had pore sizes of 36.8 μm (Type-DK) or larger, and the cells penetrated to the back of the cell. Furthermore, all cells showed improved drug resistance in 2D compared to normal cells, and further improved drug resistance in 3D.

[0059] The present invention has been described above based on examples. These examples are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible, and that such modifications also fall within the scope of the present invention.

Claims

1. A method for culturing non-adherent malignant tumor cells, comprising the step of applying non-adherent malignant tumor cells to a polyvinyl alcohol-based sponge having an average pore size of 10 μm to 100 μm and culturing them, The aforementioned polyvinyl alcohol-based sponge has been treated to make it hydrophilic. A culture method in which, if the hydrophilization treatment is performed by autoclaving, the autoclaving is performed with the sponge immersed in the liquid.

2. The method according to claim 1, wherein the polyvinyl alcohol-based sponge is a polyvinyl formal sponge.

3. The method according to claim 1 or 2, wherein the non-adherent malignant tumor cells are leukemia cells.

4. The method according to claim 3, wherein the leukemia cells are chronic myeloid leukemia (CML) cells or acute myeloid leukemia (AML) cells.

5. The method according to claim 4, further comprising the step of co-culturing with stromal cells.

6. The method according to claim 4, wherein the leukemia cells are a chronic myeloid leukemia (CML) cell line or an acute myeloid leukemia (AML) cell line.

7. The method according to claim 5, wherein the leukemia cells are a chronic myeloid leukemia (CML) cell line or an acute myeloid leukemia (AML) cell line.

8. A kit for evaluating the efficacy of an anti-cancer agent, comprising a polyvinyl alcohol-based sponge having an average pore size of 10 μm to 100 μm, The aforementioned polyvinyl alcohol-based sponge has been treated to make it hydrophilic. The aforementioned drug efficacy evaluation kit includes the step of applying malignant tumor cells to the polyvinyl alcohol-based sponge and co-culturing them with stromal cells.

9. The kit according to claim 8, wherein the polyvinyl alcohol-based sponge is a polyvinyl formal sponge.

10. The kit according to claim 8 or 9, wherein the malignant tumor is non-adhesive.

11. The polyvinyl alcohol-based sponge contains interstitial cells that are fixed and / or frozen. The kit according to claim 10, wherein the stromal cells are stromal cells cultured using the polyvinyl alcohol-based sponge.

12. The kit according to claim 11, wherein the polyvinyl alcohol-based sponge has a thickness of 0.1 to 15 mm and a diameter of 1 to 37 mm, and is incorporated into a multiwell plate.

13. The kit according to claim 12, wherein the polyvinyl alcohol-based sponge has a thickness of 0.5 to 3 mm and a diameter of 1 to 37 mm, and is incorporated into a multiwell plate.

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