TW201619375A - Screening method of anticancer agent - Google Patents

Abstract

The present invention provides a method for culturing adhesive cancer cells, which includes suspensing and culturing the adhesive cancer cells in a culture medium containing growth factors such as transforming growth factor and the like and a structure capable of suspensing culture cells or tissues. Also, the present invention provides a method for maintaining epithelioid characters of carcinoma cells, which includes suspensing and culturing the carcinoma cells in a culture medium containing a structure capable of suspensing culture cells or tissues. Further, the present invention provides a method for inducing epithelial mesenchymal transition of a carcinoma, which includes suspensing and culturing the carcinoma cells having epithelioid character in a culture medium containing epithelial mesenchymal transit inducing factor and a structure capable of suspensing culture cells or tissues.

Description

Screening method for anticancer agent

The present invention relates to a method for culturing a cancer cell using a medium composition capable of suspending cultured cells or tissues, a screening method for an anticancer agent, and the like.

In order to study the development of an anticancer agent or to select an appropriate anticancer agent in the treatment of cancer, the cancer cells are cultured in vitro in a culture solution containing a candidate agent or an anticancer agent, and the anticancer activity of the agent for cancer cells is evaluated. . However, in the existing methods for evaluating anticancer activity, there are agents which exhibit anticancer activity in vitro, and when administered in vivo, only low effects and inability to be used are obtained, resulting in evaluation results and actual conditions in vitro. The clinical effect is not appropriate. Recently, an anticancer agent (molecular target drug) targeting a receptor for a proliferation factor such as EGF or TGF-β has been actively developed. In particular, when evaluating proliferation factors, cancer cell proliferation or angiogenesis and shape conversion (EMT: epithelial-mesenchymal transition), there is a report that the effect of the proliferation factor cannot be elicited by the cell evaluation method by the existing monolayer culture. In this case, when research and development of molecular targets targeting the proliferation factor receptor as a target, the effectiveness of the drug is determined by the previous evaluation method from the majority of candidate agents, and the actual clinical anticancer effect is inconsistent. In research and development Become a big obstacle. Moreover, when a new anticancer agent is developed, it is a major problem that a cell test by a useful proliferation factor cannot be performed.

In order to improve the problem regarding the evaluation method of the anticancer activity as described above, a method of evaluating the activity by evaluating the cell culture conditions of the in vivo environment as much as possible is developed. For example, a method in which cancer cells are cultured in an environment in which a cancer cell is inhibited from adhering to a culture container by adhering cancer cells to a carrier such as a soft agar, a collagen gel, or a hydrogel, and an anticancer agent is evaluated (Patent Document 1) Non-patent documents 1 to 6). In addition, development is carried out by coating a barrier material in which cells adhere to the surface of a culture container or by performing special processing on the surface to inhibit cell adhesion, thereby culturing the cells in a state in which the cancer cells are aggregated (sphere culture, anticancer) Method for evaluating activity (Patent Documents 2 and 3). However, these cancer cell culture methods are complicated in the production process of the culture vessel and the operation of the cell culture, and the cells are recovered from a carrier such as collagen to evaluate the anticancer activity. The operation is cumbersome, and when the carrier is a component derived from an animal, since the supply is limited, there is a case where the supply is limited, and a cell aggregate (sphere) is converged, and the size thereof becomes too large, and the cell survival rate or reproducibility is lowered. In particular, when performing a screening of a molecular target drug targeting a proliferation factor receptor or the like, a cancer cell culture method which is simple and uniform, can process a large number of samples, and has high reproducibility is required.

The present inventors have succeeded in developing a medium composition capable of culturing cells or tissues while maintaining the viscosity in the liquid medium without substantially increasing the viscosity in the liquid medium (Patent Document 4).

[Advanced technical literature] [Patent Literature]

[Patent Document 1] Japanese Patent Publication No. 2014-519813

[Patent Document 2] Japanese Patent Publication No. 2013-536689

[Patent Document 3] Japanese Patent Publication No. 2012-519281

[Patent Document 4] WO 2014/017513 A1

[Non-patent literature]

[Non-Patent Document 1] Miyazono K et al., Proc Jpn Acad Ser B Phys Biol Sci 2009, 85: 314-323

[Non-Patent Document 2] Mizushima H, et al., J Cell Sci 2009, 122: 4277-4286

[Non-Patent Document 3] Del Castillo et al., Exp Cell Res 2006, 312: 2860-2871

[Non-Patent Document 4] Wakeling AE et al., Breast Cancer Res Treat 1996, 38: 67-73

[Non-Patent Document 5] Heilmann AM et al., Cancer Res 2014, 74: 3947-3958

[Non-Patent Document 6] Sonpavde G et al., Expert Opin Investing Drugs 2014, 23: 305-315

An object of the present invention is to provide a culture technique for culturing cancer cells which can reliably reproduce clinical effects in a living body, and a screening method for an anticancer agent. Furthermore, it is an object of the present invention to provide for high-sensitivity evaluation by proliferation in vitro. A method of proliferation or epithelial-mesenchymal transition of a cancer cell of a factor.

The present inventors have found that adherent culture of adherent cancer cells is cultured in a medium composition capable of culturing cells or tissues in a suspension state without substantially increasing the viscosity in the liquid medium, and is detected by monolayer culture. The reactivity of the difficult cancer cells in the transforming growth factor, insulin-like proliferation factor and fibroblast growth factor can be detected with high sensitivity in the suspension culture, and the suspension culture technique can be used to prevent the anti-cancer of the proliferation factors. Screening of agents.

Further, the present inventors have found that when a cancer cell is cultured in a single layer, there is a tendency to switch to an interstitial type, and if it is not substantially increased in viscosity in a liquid medium, it can be cultured while maintaining a suspension state. Suspension culture of the cell or tissue culture medium can maintain the epithelial-like morphology well. In addition, if an epithelial-mesenchymal transition-inducing factor such as EGF or TGF-β is added to the suspension culture system, since it can be induced to be transformed into a mesenchymal system, the evaluation of cancer cell epithelial-mesenchymal transition can be performed by using the suspension culture system or Screening of substances that impede the transepithelial conversion of cancer cells.

The review is further based on these findings, and thus the present invention has been completed.

That is, the present invention is as follows:

[1] A method for culturing an adherent cancer cell which is reactive with any of a proliferation factor selected from the group consisting of a transforming growth factor, a tryptase growth factor, and a fibroblast growth factor, The culture method comprises: the pair is selected from the group consisting of a transforming growth factor, an insulin-like proliferation factor, and a fiber A reactive cancer cell having reactivity in any one of the proliferation factors of the group of the parent cell proliferation factor is cultured in suspension in a medium composition containing the growth factor and a structure in which the cell or tissue is suspended and cultured.

[2] The method according to [1], wherein the proliferation factor is a transgenic growth factor.

[3] The method according to [2], wherein the transformed growth factor is TGF-β 1.

[4] The method according to any one of [1] to [3] wherein the above structure contains gellan gum.

[5] The method according to any one of [1] to [4] wherein the composition of the medium has a viscosity of 8 mPa at 37 ° C. s below.

[6] A method for testing the reactivity of an adherent cancer cell to a proliferation factor, which is selected from the group consisting of a transforming growth factor, an insulin-like proliferation factor, and a fibroblast growth factor, The method comprises (1) suspending culture of an adherent cancer cell in a medium composition containing a construct capable of suspending or culturing a cell or tissue in the presence or absence of the proliferation factor; (2) measuring the cultured cancer Proliferation of cells; and (3) comparison of proliferation of cancer cells cultured in the presence of the proliferation factor and proliferation in the absence of the proliferation factor.

[7] The method according to [6], wherein the above structure contains deuterated gellan gum.

[8] The method according to [6] or [7] wherein the composition of the medium has a viscosity of 8 mPa at 37 ° C. s below.

[9] A method for screening an anticancer agent against an adherent cancer cell, wherein the adherent cancer cell is any one of a proliferation factor selected from the group consisting of a transgenic growth factor, an insulin-like growth factor, and a fibroblast growth factor Have a reaction The screening method comprises the following steps: (1) suspending and cultured the cancer cell in a medium composition containing the proliferation factor and a structure in which the cell or tissue is suspended and cultured in the presence and absence of the test substance. (2) measuring the proliferation of the cultured cancer cells; and (3) comparing the proliferation of the cancer cells cultured in the presence of the test substance with the proliferation in the absence of the test substance.

[10] The method according to [9], wherein the above structure contains deuterated gellan gum.

[11] The method according to [9] or [10] wherein the composition of the medium has a viscosity of 8 mPa at 37 ° C. s below.

[12] A method for maintaining an epithelial morphology such as a cancer cell, comprising: suspending and culturing the cancer cell in a medium composition containing a construct capable of suspending and culturing the cell or tissue.

[13] The method according to [12], wherein the above structure contains deuterated gellan gum.

[14] The method according to [12] or [13] wherein the composition of the medium has a viscosity of 8 mPa at 37 ° C. s below.

[15] A method for inducing epithelial-mesenchymal transition of the cancer cell, comprising: constituting a cancer cell having an epithelial-like morphology in a medium containing an epithelial-mesenchymal transition-inducing factor and a construct capable of suspending or culturing the cell or tissue Suspension culture.

[16] The method according to [15], wherein the above structure contains deuterated gellan gum.

[17] The method according to [15] or [16] wherein the composition of the medium has a viscosity of 8 mPa at 37 °C. s below.

[18] A method for screening a substance for inducing epithelial transition of a cancer cell, comprising: (1) in the presence or absence of a test substance, a cancer cell having an epithelial-like shape can be contained therein Suspension culture of a medium composition of cells or tissue suspensions, cultured constructs; (2) evaluation of epithelial transition of cultured cancer cells; and (3) comparison of epithelial cells of cancer cells cultured in the presence of test substances The quality conversion and the substance to be tested are not present in the epithelial-mesenchymal transition.

[19] The method according to [18], wherein the above structure contains deuterated gellan gum.

[20] The method according to [18] or [19] wherein the composition of the medium has a viscosity of 8 mPa at 37 ° C. s below.

[21] A method for screening a substance for blocking epithelial transition of a cancer cell, comprising: (1) in the presence or absence of a test substance, a cancer cell having an epithelial-like morphology is contained in the epithelium; a mass transfer inducing factor and a suspension culture medium for constructing a structure in which cells or tissues are suspended and cultured; (2) evaluating epithelial transition of cultured cancer cells; and (3) comparing in the presence of a test substance The epithelial-mesenchymal transition of the cultured cancer cells and the epithelial-mesenchymal transition in the absence of the test substance.

[22] The method according to [21], wherein the above-mentioned construct contains deuterated gellan gum.

[23] The method according to [21] or [22] wherein the composition of the medium has a viscosity of 8 mPa at 37 ° C. s below.

By the present invention, the reactivity of cancer cells to the transforming growth factor, the insulin-like proliferation factor, and the fibroblast growth factor can be detected with high sensitivity. Thus, the methods of the invention can be used to screen for anti-cancer agents that block such proliferation factors.

When the method of the present invention is used, the epithelial lineage such as cancer cells can be well maintained as compared with the monolayer culture. Further, according to the method of the present invention, since the epithelial-mesenchymal transition of cancer cells can be well reproduced in vitro, it can be used for screening of an anticancer agent which inhibits epithelial-mesenchymal transition.

(1) Medium composition used in the present invention

The present invention provides a cancer cell culture technique using a medium composition capable of culturing cells or tissues in a suspended state. The medium composition can be cultured as a cancer cell to be evaluated or a tissue containing the cancer cells while maintaining the suspension state. The medium composition can be prepared in accordance with the teachings of WO 2014/017513 A1. Hereinafter, the medium composition is referred to as a medium composition I.

The cell is the most basic unit of the animal, and the element has a cytoplasm and various cell organelles inside the cell membrane.

The cancer cells used in the present invention are mammalian cancer Cell. Examples of mammals include rodents such as mice, rats, hamsters, and woodchucks, rabbits and the like, and pigs, cows, goats, horses, sheep, and other hoofed eyes, dogs, cats, and other carnivores, humans, and monkeys. , short-tailed monkeys, cynomolgus macaques, small baboons, orangutans, chimpanzees and other primates. The mammal is preferably a rodent (mouse or the like) or a primate (human, etc.).

Examples of cancer are not limited to the following examples, and examples thereof include gastric cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, and bone marrow. Cancer, renal cell carcinoma, urinary tract cancer, liver cancer, cholangiocarcinoma, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, papillary pharyngeal cancer, laryngeal cancer, tongue cancer , fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing sarcoma, smooth muscle Sarcoma, rhabdomyosarcoma, seminoma, Wilms tumor, glioma, astrocytoma, myeloma, mesothelioma, melanoma, neuroblastoma, myeloma Cell tumors, retinal blastomas, malignant lymphomas, tissues derived from the blood of cancer patients. Examples of the cancer cell strain are not limited to the following, and include HBC-4, BSY-1, BSY-2, MCF-7, MCF-7/ADR RES, HS578T, and MDA-MB-231 as human breast cancer cell lines. , MDA-MB-435, MDA-N, BT-549, T47D, as human cervical cancer cell line HeLa, as human lung cancer cell line A549, EKVX, HOP-62, HOP-92, NCI-H 23, NCI-H 226, NCI-H 322M, NCI-H 460, NCI-H 522, DMS273, DMS114, as Caco-2 of human colorectal cancer cell line, COLO-205, HCC-2998, HCT-15, HCT-116, HT-29, KM-12, SW-620, WiDr, as human prostate cancer cell line DU-145, PC-3, LNCaP, as the human center U251, SF-295, SF-539, SF-268, SNB-75, SNB-78, and SNB-19 of neuronal cancer cell lines, as OVCAR-3, OVCAR-4, and OVCAR-5 of human ovarian cancer cell lines , OVCAR-8, SK-OV-3, IGROV-1, as human kidney cancer cell lines RXF-631L, ACHN, UO-31, SN-12 C, A498, CAKI-1, RXF-393L, 786-0 , TK-10, as a human gastric cancer cell line MKN45, MKN28, St-4, MKN-1, MKN-7, MKN-74, as a skin cancer cell line LOX-IMVI, LOX, MALME-3M, SK-MEL -2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, as a leukemia cell line CCRF-CRM, K562, MOLT-4, H L-60TB, RPMI8226, SR, UT7 /TPO, Jurkat, A431 as a human epithelial cancer cell line, A375 as a human melanocyte cell line, MNNG/HOS as a human osteosarcoma cell line, and MIAPaCa-2 as a human pancreatic cancer cell line. Examples of the cell line are not limited to the following, including HEK293 (human fetal kidney cells), MDCK, MDBK, BHK, C-33A, AE-1, 3D9, Ns0/1, NIH3T3, PC12, S2, Sf9, Sf21, High Five. (registered trademark), Vero, etc.

Suspension of cells and/or tissues in the present invention means that the cells and/or tissues are in a non-adhesive state (non-adhesive) to the culture vessel. Further, in the present invention, when the cells and/or tissues are proliferated, differentiated, or maintained, the liquid medium composition is not accompanied by external pressure, vibration, vibration in the composition, rotation operation, or the like, and the cells and/or Or the tissue is uniformly dispersed in the liquid medium composition and is in a suspended state called "suspension standing". The cultured cells and/or tissues are referred to as "suspended static culture". Further, in the "suspended suspension", the period during which the suspension can be suspended includes 5 minutes or longer, 1 hour or longer, 24 hours or longer, 48 hours or longer, 7 days or longer, etc., but the suspension state is not limited to the time.

The medium composition used in the present invention may be suspended in at least one point in a temperature range (e.g., 0 to 40 ° C) at which cells or tissues can be maintained or cultured, and the cells and/or tissues may be suspended. The medium composition used in the present invention is preferably at least 1 point in the temperature range of 25 to 37 ° C, more preferably at 37 ° C, and the cells and/or tissues may be suspended and allowed to stand.

Whether it can be suspended or not can be, for example, uniformly dispersing the cells of the cultured object at a concentration of 2 × 10 ⁴ /mL in the medium composition of the evaluation object, injecting 10 mL into a 15 mL conical tube, and at least standing at 37 ° C 5 More than a minute (for example, 1 hour or more, 24 hours or more, 48 hours or more, 7 days or more) is evaluated by observing whether or not the cells are maintained in a suspended state. When more than 70% of the whole cells are in suspension, it can be concluded that the suspension state is maintained. Polystyrene beads (size 500-600 μm, manufactured by Polysciences) can also be used instead of cells for evaluation.

The medium composition used in the present invention is a composition containing a structure and a medium which can suspend and culture cells (preferably, suspension culture).

The medium composition used in the present invention is preferably a composition which can recover cells or tissues from the medium composition after the exchange treatment of the medium composition at the time of culture and after the completion of the culture, and more preferably, the cells or tissues are composed of the medium. No temperature change, chemical treatment, enzyme treatment, shearing The composition of either of the shear stresses.

The construct of the present invention is formed from a specific compound and exhibits a uniform effect of suspending cells and/or tissues. More specifically, it includes a polymer compound which forms a three-dimensional network structure by an ion collector or a polymer compound. Further, the polysaccharide is known to form a microgel by a metal ion (for example, Japanese Laid-Open Patent Publication No. 2004-129596), and the structure of the present invention also includes the microgel as an aspect.

Further, as a polymer compound, a film-like structure can be exemplified as an ion collector.

The size of the structure in the present invention is preferably such that it passes through a filter having a pore diameter of from 0.2 μm to 200 μm when it is filtered by a filter. The lower limit of the pore diameter is more preferably more than 1 μm, and it is more preferably more than 5 μm in consideration of stably suspending cells or tissues. The upper limit of the pore diameter is preferably 100 μm or less, and more preferably 70 μm or less in consideration of the size of cells or tissues.

The specific compound of the present invention is a structure which forms an amorphous shape when a specific compound is mixed with a liquid medium, and the structure is uniformly dispersed in the liquid, and has substantially no increase in viscosity of the liquid, and substantially maintains cells and/or Organize to prevent the sedimentation effect. "Substantially does not increase the viscosity of the liquid" means that the viscosity of the liquid does not exceed 8 mPa. s. At this time, the viscosity of the liquid (that is, the viscosity of the medium composition used in the present invention) is 8 mPa at 37 ° C. Below s, preferably 4mPa. s below, better for 2mPa. s below. In addition, as long as it can be shown that the structure is formed in a liquid medium, the viscosity of the liquid is not substantially increased, and the cells and/or tissues are uniformly suspended (preferably The effect of the suspension suspension is not particularly limited, and the chemical structure, molecular weight, and physical properties of the specific compound are not particularly limited.

The viscosity of the liquid containing the structure can be measured, for example, by the method described in the examples below. Specifically, an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, conical rotor: standard rotor 1°34'×R24, rotation) was used at 37 °C. It can be measured by a number of 100 rpm.

The specific compound to be used in the present invention is not particularly limited, and examples thereof include a polymer compound, and a polymer compound having an anionic functional group is preferred.

The anionic functional group may, for example, be a carboxyl group, a sulfo group, a phosphoric acid group or the like, and is preferably a carboxyl group or a salt thereof.

The polymer compound used in the present invention may be one or more selected from the group consisting of the above-mentioned anionic functional groups.

The preferred embodiment of the polymer compound used in the present invention is not particularly limited, and examples thereof include polysaccharides obtained by polymerizing ten or more monosaccharides (for example, triose, butyrate, pentose, hexose, heptose, etc.), more preferably An acidic polysaccharide having an anionic functional group can be mentioned. The acidic polysaccharide referred to herein is not particularly limited as long as it has an anionic functional group in its structure, and has, for example, a uronic acid (for example, glucuronic acid, iduronic acid, galacturonic acid, mannose). a polysaccharide of aldehyde acid, a polysaccharide having a sulfate group or a phosphate group, or a polysaccharide having the structure of the two, not only a polysaccharide obtained naturally, but also a polysaccharide or a gene produced by a microorganism. The polysaccharides produced by engineering or the use of enzymes to synthesize polysaccharides. More specifically, It can be exemplified by hyaluronic acid, lanolin, deuterated gellan (hereinafter, also referred to as DAG), rhamnose gum, scorpion gum, xanthan gum, carrageenan, sanxian gum, hexose Aldehydic acid, fucoidan, pectin, pectic acid, pectic acid, heparan sulfate, heparin, heparitin sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, sulfuric acid One or two or more of the group consisting of rhamnose and the salt thereof. The polysaccharides are preferably hyaluronic acid, DAG, eucalyptus gum, xanthan gum, carrageenan or the like. If it is considered that the cells or tissues are suspended in a low concentration and the cells or tissues are easily recovered, Better for DAG.

The salt herein may, for example, be an alkali metal salt of lithium, sodium or potassium, an alkaline earth metal salt of calcium, barium or magnesium or a salt of aluminum, zinc, copper, iron, ammonium, an organic base or an amino acid.

The weight average molecular weight of the polymer compound (polysaccharide or the like) is preferably from 10,000 to 50,000,000, more preferably from 100,000 to 20,000,000, most preferably from 1,000,000 to 10,000,000. For example, the molecular weight can be determined by gel permeation chromatography (GPC) in the form of pullulan.

In addition, DAG can use phosphorylated. This phosphorylation can be carried out by a known method.

In the present invention, a plurality of the above polysaccharides (preferably two kinds) may be used in combination. The type of the polysaccharide combination is such that the above-described structure can be formed in a liquid medium, and the viscosity of the liquid medium is not substantially increased, but the cells and/or tissues are uniformly suspended (preferably suspended and suspended), and There is no particular limitation, and the combination preferably contains at least DAG or a salt thereof. That is, a preferred polysaccharide combination comprises DAG or a salt thereof and a DAG or a salt thereof Sugars (eg, xanthan gum, alginic acid, carrageenan, eucalyptus gum, methylcellulose, locust bean gum or such salts). Specifically, the combination of polysaccharides may be DAG and rhamnose gum, DAG and sorghum gum, DAG and xanthan gum, DAG and carrageenan, DAG and Sanxian gum, DAG and locust bean gum, DAG and κ-carrageenan, DAG and sodium alginate, DAG and methyl cellulose, etc., but are not limited to these.

More specific examples of the specific compound used in the present invention include hyaluronic acid, deuterated gellan gum, eucalyptus gum, carrageenan, and xanthan gum, and the like, if it is considered that the medium composition can be lowered. The viscosity and the ease of recovery of the cells or tissues are more preferably exemplified by deuterated gellan gum or a salt thereof.

The deuterated gellanine in the present invention is a molecule of 1-3 bonded glucose, 1-4 bonded glucuronic acid, 1-4 bonded glucose and 1-4 bonded rhamnose. In the following general formula (I), R ₁ and R ₂ are each a hydrogen atom, and n is a polysaccharide having an integer of 2 or more. R ₁ may be a glyceryl group and R _{2 may} be an ethyl group. However, the content of the ethyl group and the glyceryl group is preferably 10% or less, more preferably 1% or less.

The structure in the present invention is in various forms depending on the specific compound, and if it is described for the case of de-deuterated gellan, the de-deuterated gellanine is taken back to the metal cation in the liquid medium when mixed with the liquid medium (for example, Calcium ions), by which the metal ions form an amorphous structure, suspending cells and/or tissues. The viscosity of the medium composition used in the invention prepared by deuterated gellan gum is 8 mPa. Below s, preferably at 4mPa. s below, if Considering the ease of cell or tissue recovery, it is better at 2mPa. s below.

The specific compound of the present invention can also be obtained by a chemical synthesis method. When the compound is a natural product, it is preferably obtained from various plants, various animals, and various microorganisms containing the compound by using conventional techniques, by extraction and separation and purification. . In this extraction, if water or a supercritical gas is used, the compound can be extracted more efficiently. For example, as a method for producing the gellan, the production microorganism can be cultured in a fermentation medium, and the mucosa produced in vitro can be recovered into a powder by a usual purification method, followed by drying, pulverization, and the like. Further, in the case of removing the sulphate, it is only necessary to carry out alkali treatment in the recovery of the mucosa, and the glyceryl group and the acetyl group bonded to the 1-3-bonded glucose residue may be removed and recovered. The purification method may be, for example, liquid-liquid extraction, separate precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using a Sephadex LH-20, activated carbon, silicone rubber, or the like. The purification of the impurities is removed by adsorption chromatography, adsorption/desorption treatment of the active material by thin layer chromatography, or high-speed liquid chromatography using a reverse phase column, alone or in any order. Examples of the microorganism for producing the gellan gum are not limited thereto, and examples thereof include Sphingomonas elodea and microorganisms which change the gene of the microorganism.

Therefore, commercially available products can be used for deuterated gelatin, such as KELCOGEL (CP Kelco, registered trademark) CG-LA manufactured by Sanjing Co., Ltd., Sanrongyuan F‧ F‧I (share) company manufactures "Keco Rubber (CP Kelco, registered trademark)" and so on. Also, the native type of margarine can use Sanrongyuan F. F. "Keco Gum (CP Kelco, registered trademark)" HT" manufactured by I (share) company.

The concentration of a specific compound in the medium depends on the kind of the specific compound, and the above-mentioned structure can be formed in a liquid medium in a specific compound, and the viscosity of the liquid medium is not substantially increased, and the cells and/or tissues are uniformly suspended (preferably suspended) The range of the setting is appropriately set, usually as long as 0.0005% to 1.0% (weight/capacity), preferably 0.001% to 0.4% (weight/capacity), more preferably 0.005% to 0.1% (weight/capacity), most It is preferably 0.005% to 0.05% (weight/capacity). For example, in the case of deuterated gellan gum, 0.001% to 1.0% (weight/volume), preferably 0.003% to 0.5% (weight/capacity), more preferably 0.005% to 0.1% (weight/weight) is added to the medium. The capacity) is preferably from 0.01% to 0.05% (weight/volume), preferably from 0.01% to 0.03% (weight/capacity). In the case of xanthan gum, it is preferred to add 0.001% to 5.0% (weight/volume), preferably 0.01% to 1.0% (weight/volume), more preferably 0.05% to 0.5% (weight/capacity), preferably in the medium. 0.1% to 0.2% (weight/capacity). When it is a mixture of kappa-carrageenan and locust bean gum, it is only necessary to add 0.001% to 5.0% (weight/volume), preferably 0.005% to 1.0% (weight/capacity), more preferably 0.01% to 0.1% in the medium. % (weight/capacity), preferably 0.03% to 0.05% (weight/capacity). In the case of the original type of gellan, as long as 0.05% to 1.0% (weight/capacity), preferably 0.05%, is added to the medium. 0.1% (weight / capacity) can be.

When a plurality of the above polysaccharides (preferably two kinds) are used in combination, the concentration of the polysaccharide may form the above-mentioned structure in a liquid medium in the combination of the polysaccharides, and substantially does not increase the viscosity of the liquid medium, thereby making the cells And/or the range in which the tissue is uniformly suspended (preferably suspended for standing) is appropriately set. For example, when DAG or a salt thereof is used in combination with a polysaccharide other than DAG or a salt thereof, the concentration of DAG or a salt thereof can be, for example, 0.005 to 0.02% (weight/volume), preferably 0.01 to 0.02% (weight/volume), DAG The concentration of the polysaccharide other than the salt thereof or the salt thereof may be, for example, 0.005 to 0.4 (weight/volume), preferably 0.1 to 0.4% (weight/volume).

Specific concentration range combinations are exemplified below.

DAG or its salt: 0.005 to 0.02% (preferably 0.01 to 0.02%) (weight/capacity)

Polysaccharides other than DAG

Xanthan gum: 0.1 to 0.4% (weight/capacity)

Sodium alginate: 0.1 to 0.4% (weight/capacity)

Kidney Bean Gum: 0.1 to 0.4% (weight/capacity)

Methylcellulose: 0.1 to 0.4% (weight/capacity) (preferably 0.2 to 0.4% (weight/capacity))

Carrageenan: 0.05 to 0.1% (weight/capacity)

Dathantan: 0.05 to 0.1% (weight/capacity)

Further, the concentration can be calculated by the following formula.

Concentration (%) = weight of specific compound (g) / volume of medium composition (mL) × 100

The above compound can be further converted into other derivatives according to a chemical synthesis method, and the derivative obtained therefrom can also be effectively used in the present invention. Specifically, in the case of deuterated gellanine, a hydroxyl group corresponding to R ₁ and/or R ₂ of the compound represented by the general formula (I) is substituted with a C _1-3 alkoxy group and a C _1-3 alkane. A monosaccharide residue such as a sulfonyl group, glucose or fructose, an oligosaccharide residue such as sucrose or lactose, or a derivative such as an amino acid residue such as glycine or arginine may be used in the present invention. Further, the compound can be crosslinked by using a crosslinking agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

The specific compound or a salt thereof used in the present invention may be present in any crystal form depending on the production conditions, or may be present in any hydrate, and such crystal forms or hydrates and mixtures thereof are also included in the scope of the present invention. in. Further, a solvate containing an organic solvent such as acetone, ethanol or tetrahydrofuran is also present, and such forms are included in the scope of the present invention.

The particular compounds used in the present invention may also exist as a mixture of tautomers, geometric isomers, tautomers or geometric isomers formed by intra- or extra-cyclic isomerization or mixtures thereof. The compound of the present invention, whether produced by isomerization or in the case of having an asymmetric center, may be in the form of a divided optical isomer or a mixture thereof contained in an arbitrary ratio.

The medium composition used in the present invention may have metal ions such as divalent metal ions (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), and preferably contains calcium ions. The metal ions may be, for example, calcium ions and magnesium ions, calcium ions and zinc ions, calcium ions and iron ions, Two or more types of calcium ions and copper ions are used in combination. Those skilled in the art can determine the appropriate combination. In one aspect, by containing a metal ion (for example, calcium ion) in the medium composition, the polymer compound is aggregated by the metal ion, and the polymer compound forms a three-dimensional network structure (for example, the polysaccharide is made of metal) Ions (e.g., calcium ions) form microgels to form the constructs of the present invention. The concentration of the metal ion can form the above-mentioned structure in a liquid medium in a specific compound, and substantially does not increase the viscosity of the liquid medium, and the range in which the cells and/or tissues are uniformly suspended (preferably suspended) is appropriately set. The concentration of the metal ion is from 0.1 mM to 300 mM, preferably from 0.5 mM to 100 mM, but is not limited thereto. The metal ion may also be mixed with the medium or otherwise prepared into a salt solution and added to the medium. The medium composition used in the present invention may contain an extracellular base material, a binding molecule or the like which will be described later.

The construct composed of the specific compound used in the present invention exhibits an effect of suspending the cells and/or tissues in a liquid containing the specific compound structure when the cells and/or tissues are cultured in vitro (preferably suspending the cells) Set the effect). By this suspending effect, cells and/or tissues of a certain volume can be increased and cultured as compared with monolayer culture. Moreover, in the conventional suspension culture method, when the rotation or vibration operation is accompanied, there is a case where shearing stress is applied to cells and/or tissues, and the proliferation rate or recovery rate of cells and/or tissues is lowered or the function of the cells is impaired. By using the medium composition containing the specific compound structure of the present invention, cells and/or tissues can be uniformly dispersed without performing operations such as vibration, and cells and/or tissues of interest can be easily and largely obtained without loss of cell function. Also, in the past When cells and/or tissues are cultured in suspension in a medium containing a gel substrate, it is difficult to observe or recover cells and/or tissues, and the function of the cells is damaged during recovery, by using a structure containing a specific compound of the present invention. The medium composition can suspend and culture cells and/or tissues, and its function is not damaged, and can be observed and recovered. Further, the conventional medium containing the gel substrate has high viscosity and difficulty in exchanging the medium, and the medium composition containing the specific compound structure of the present invention has a low viscosity, and the medium can be easily exchanged by using a pipette or a pump.

When the cells and/or tissues are cultured using the specific compound of the present invention, the medium used for culturing the cells and/or tissues may be mixed with a specific compound to prepare a medium composition. Examples of the composition of the medium include a natural medium, a semi-synthetic medium, and a synthetic medium, or a semi-solid medium, a liquid medium, a powder medium (hereinafter also referred to as a powder medium), and the like according to the shape classification. When the cells and/or tissues are derived from animals, any medium used for culturing animal cells can be used. Examples of such a medium include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM/F12 medium, and McCoy's 5A medium. Eagles's Minimum Essential Medium (EMEM), alpha MOD medium (α MEM), MEM medium (Minimum Essential Medium), RPMI 1640 medium, Iskoff modified Dulbec medium ( Iscove's Modified Dulbecco's Medium; IMDM), MCDB131 Medium, William's Medium E (William's Medium E), IPL41 medium, Fischer's medium, StemPro34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Cambrex), X-VIVO 15 (manufactured by Cambrex), HPGM (manufactured by Cambrex), StemSpan H3000 (manufactured by Stemcell Technologies), StemSpanSFEM (manufactured by Stemcell Technologies), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biological), StemProhESCSFM (manufactured by Invitrogen), Essential 8 (registered trademark) medium (Gibco) Manufactured by the company, mTeSR1 or 2 medium (manufactured by Stemcell Technologies), Repro FF or Lipod FF2 (manufactured by Reprocell), PSGro hESC/iPSC medium (manufactured by System Biosciences) , NutriStem (registered trademark) medium (manufactured by Biological Industries), CSTI-7 medium (manufactured by Cell Science Research Institute), MesenPRO RS medium (manufactured by Gibco), MF-Medium (registered trademark) mesenchymal stem cell growth medium ( Manufactured by Toyobo Co., Ltd., Sf-900II (manufactured by Invitrogen Co., Ltd.), Opti-Pro (manufactured by Invitrogen Co., Ltd.), and the like.

The medium used for culturing cancer cells may contain a cell adhesion factor in the above medium, and examples thereof include basement membrane gel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, Fibronectin. Two or more of these cell adhesion factors may also be added in combination. In addition, for the medium used for culturing cancer cell spheres, guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, A viscous agent such as tamarind (seed gum) or methyl cellulose is mixed.

Those skilled in the art can freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, and the like to the above medium according to the purpose. In the case of animal-derived cells and/or tissue culture, one skilled in the art may add one or more other chemical components or biological components in combination according to the purpose.

The components added to the medium derived from the cells and/or tissues of the animal may include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothio Glycerin, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methyl cellulose, various cytokines, various hormones, various proliferation Factors, various extracellular matrices or various cell adhesion molecules.

Examples of the antibiotic added to the medium include a sulfa drug, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, Diclooxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbene Carbenicillin, ticarcillin, piperacillin, azlocillin, mcczlocillin, mecillinam, amdinocillin, cephalosporin Phytocin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, Ciprofloxacin (ciprofloxacin), cinoxacin, norfloxacin, perfloxacin, rosaxacin, ofloxacin, enoxacin ), pipemidic acid, sulbactam, clavulanic acid, β-Bromopenicillanic acid, β-chloropenic acid, 6-ethyl fluorenyl Formaldehyde/food light ester of acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, adenosine and sulbactam, Tazobactam, aztreonam, sulfazecin, isosulfazecin, nocardicin, m-carboxyphenol, phenylacetamide methyl phosphate, Chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, metacycline, and minocycline.

When a specific compound of the present invention is added to the above medium, the specific compound is first dissolved or dispersed in a suitable solvent (this is used as a medium additive). Thereafter, as a specific compound concentration in the medium, as described above, a concentration which allows the cells and/or tissues to be uniformly suspended (preferably suspended and suspended) without substantially increasing the viscosity of the liquid medium, for example, the medium The additive is added to the medium so as to be 0.0005% to 1.0% (weight/volume), preferably 0.001% to 0.4% (weight/volume), more preferably 0.005% to 0.1% (weight/volume), and most preferably 0.005% to 0.05% (weight / capacity) can be. For example, in the case of deuterated gellan gum, 0.001% to 1.0% (weight/volume), preferably 0.003% to 0.5% (by weight) is added to the medium. / capacity), more preferably 0.005% to 0.1% (weight / capacity), preferably 0.01% to 0.05% (weight / capacity). In the case of xanthan gum, 0.001% to 5.0% (weight/volume), preferably 0.01% to 1.0% (weight/volume), more preferably 0.05% to 0.5% (weight/capacity), more preferably 0.1, is added to the medium. % to 0.2% (weight/capacity). When it is a mixture of kappa-carrageenan and locust bean gum, 0.001% to 5.0% (weight/volume), preferably 0.005% to 1.0% (weight/volume), more preferably 0.01% to 0.1%, is added to the medium. Preferably, it is 0.03% to 0.05% (weight/capacity). In order to remove the mixed system of the deuterated gellan gum and the Daiyu Tan gum, 0.001% to 1.0% (weight/volume), preferably 0.005% to 0.01% (weight/capacity) may be added to the medium. . In order to deuterize the mixture of the blue gum and the methyl cellulose, 0.001% to 1.0% (weight/volume), preferably 0.005% to 0.2% (weight/capacity) may be added to the medium. In order to remove the mixed system of the gellan gum and the locust bean gum, 0.001% to 1.0% (weight/volume), preferably 0.01% to 0.1% (weight/volume) may be added to the medium. In order to deuterize the mixture of the blue gum and the sodium alginate, 0.001% to 1.0% (weight/volume), preferably 0.01% to 0.1% (weight/capacity) may be added to the medium. In order to remove the mixed system of the gellan gum and the xanthan gum, 0.001% to 1.0% (weight/volume), preferably 0.01% to 0.1% (weight/capacity) may be added to the medium. In order to remove the mixed system of calcined gum and kappa-carrageenan, 0.001% to 1.0% (weight/volume), preferably 0.01% to 0.1% (weight/volume) may be added to the medium. Further, the concentration can be calculated by the following formula.

Concentration (%) = weight of specific compound (g) / volume of medium composition (mL) × 100

Here, the appropriate solvent used in the medium additive Examples thereof include aqueous solvents such as water, dimethyl hydrazine (DMSO), methanol, ethanol, butanol, propanol, glycerin, propylene glycol, and butylene glycol, and the like, but are not limited thereto. At this time, the concentration of the specific compound is 0.001% to 5.0% (weight/capacity), preferably 0.01% to 1.0% (weight/capacity), more preferably 0.1% to 0.6% (weight/capacity). In this case, an additive which reduces the concentration at the time of use while improving the effect of the specific compound can be additionally added. Examples of such additives may be one or more kinds of guar gum, rosin gum, propylene glycol alginate, locust bean gum, gum arabic, karaya gum, tamarind, methyl cellulose, carboxymethyl cellulose, Polysaccharides such as agarose, tamarind gum, and pullulan are mixed. Further, when the specific compound is cultured, it can be immobilized on the surface of the carrier or carried on the inside of the carrier. The particular compound can be of any shape when provided or preserved. The specific compound can be formulated into a solid such as a tablet, a pill, a capsule, or a granule, such as a liquid in a suitable solvent and a solution or suspension dissolved in a dissolving agent or in a state of being bonded to a substrate or a monomer. Examples of the additives at the time of formulation include preservatives such as parabens; excipients such as lactose, glucose, sucrose, and mannitol; lubricants such as magnesium stearate and talc; polyvinyl alcohol and hydroxypropyl group. Adhesives such as cellulose and gelatin; surfactants such as fatty acid esters; plasticizers such as glycerin. These additives are not limited to the above, as long as the user can freely choose. Further, the specific compound in the present invention can be subjected to sterilization treatment as necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and filter sterilization. The material of the filter portion when the filter is sterilized (hereinafter, also referred to as filter sterilization) is not particularly limited, and examples thereof include glass fiber, nylon, PES (polyether oxime), and hydrophilic PVDF (polyhedral). Difluoroethylene), cellulose mixed ester, cellulose acetate, polytetrafluoroethylene, and the like. The size of the pores of the filter is not particularly limited, and is preferably from 0.1 μm to 10 μm, more preferably from 0.1 μm to 1 μm, still more preferably from 0.1 μm to 0.5 μm. In the sterilization treatment, the specific compound may be in a solid form or in a solution state.

By the above preparation, a solution or dispersion of a specific compound is added to a liquid medium, and the above-described structure is formed in a liquid medium, whereby the medium composition used in the present invention can be obtained. The medium usually has a polymer compound or a polymer compound which contains a metal ion (for example, a divalent metal ion such as calcium ion) in a sufficient concentration to form a three-dimensional network structure, and only a solution of the specific compound of the present invention is added to the liquid medium or As the dispersion, the medium composition used in the present invention can be obtained. Alternatively, the medium may be added to the medium additive (solution or dispersion of the specific compound). Further, the medium composition used in the present invention can be prepared by mixing a specific compound with a medium component in an aqueous solvent (for example, water containing ion-exchanged water or ultrapure water). Examples of the mixed state include (1) mixing a liquid medium with a medium additive (solution), (2) mixing the above polymer compound (solid such as powder) in a liquid medium, and (3) adding a powder medium to the medium additive (solution) (4) mixing the powder medium and the above polymer compound (solid such as powder) with an aqueous solvent, but is not limited thereto. In order to prevent the distribution of a specific compound in the medium composition used in the present invention, it is preferably (1) or (4), or (1) or (3).

When a specific compound is dissolved in a solvent (for example, an aqueous solvent such as water or a liquid medium) or when a specific compound and a powder medium are dissolved in a solvent, it is preferred to heat the mixture in order to promote dissolution. The temperature of heating can be enumerated, for example From 80 ° C to 130 ° C, it is preferably from 100 ° C to 125 ° C (for example, 121 ° C) which can be heat-sterilized. The solution of the specific compound obtained was cooled to room temperature after heating. The above-described structure composed of the specific compound is formed by adding the above-described metal ion (for example, a divalent metal ion such as calcium ion) to the solution (for example, adding the solution to a liquid medium). Or when a specific compound is dissolved in a solvent containing a metal ion (for example, a divalent metal ion such as calcium ion) (for example, an aqueous solvent such as water or a liquid medium), it may be heated (for example, 80 ° C to 130 ° C, preferably 100 ° C). The resulting structure can be formed from the specific compound by cooling the solution to room temperature to 125 ° C (for example, 121 ° C).

Although the method of preparing the medium composition used in the present invention is exemplified, the present invention is not limited to the examples. A specific compound is added to ion-exchanged water or ultrapure water. In this manner, the temperature at which the specific compound is dissolved (for example, 60° C. or higher, 80° C. or higher, and 90° C. or higher) is stirred and dissolved to be transparent. After dissolving, the mixture is allowed to cool while being stirred, and sterilized (for example, autoclaved at 121 ° C for 20 minutes). After returning to room temperature, the above-mentioned sterilized aqueous solution is added to the medium while stirring (for example, a homomixer or the like) in any medium used for the stationary culture, and mixed to be uniform with the medium. The mixing method of the aqueous solution and the culture medium is not particularly limited, and for example, mixing by suction or the like may be employed, and mixing using a machine such as an electromagnetic stirrer or a mechanical stirrer, a homomixer, or a homogenizer may be employed. Further, the medium composition used in the present invention may be filtered with a filter after mixing. The size of the pores of the filter used in the filtration treatment is from 5 μm to 100 μm, preferably from 5 μm to 70 μm, more preferably from 10 μm to 70 μm.

Alternatively, the powder medium and the above polymer compound (solid such as powder) are mixed with an aqueous solvent, and heated at the above temperature to prepare a medium composition used in the present invention.

For example, when deuterated gellan gum is prepared, deuterated gellan gum is added to ion-exchanged water or ultrapure water to be 0.1% to 1% (weight/capacity), preferably 0.2% to 0.5% ( Weight/capacity), more preferably from 0.3% to 0.4% (weight/capacity). Further, in another aspect, when the deuterated gellan gum is prepared, the deuterated gellan gum is added to the ion-exchanged water or the ultrapure water so as to be 0.1% to 1% (weight/capacity), preferably 0.2. % to 0.8% (weight/capacity), more preferably 0.3% to 0.6% (weight/capacity).

Thus, as long as the temperature at which the deuterated gellan gum is dissolved can be heated at any temperature, at 60 ° C or higher, preferably at 80 ° C or higher, more preferably at 90 ° C or higher (for example, 80 to 130 ° C). Dissolve until it is in a transparent state while stirring. After dissolving, the mixture was allowed to cool while stirring, and autoclaved at, for example, 121 ° C for 20 minutes. After returning to room temperature, the aqueous solution is added to the medium by stirring, for example, a DMEM/F12 medium with a homomixer or the like to achieve the desired final concentration (for example, a ratio of 0.3% aqueous solution: medium at a final concentration of 0.015%). 1:19), uniform mixing. The mixing method of the aqueous solution and the culture medium is not particularly limited, and for example, mixing by suction or the like, mixing using a machine such as an electromagnetic stirrer, a mechanical stirrer, a homomixer, or a homogenizer may be mentioned. Further, the medium composition used in the present invention may be filtered with a filter after mixing. The size of the pores of the filter used in the filtration treatment is from 5 μm to 100 μm, preferably from 5 μm to 70 μm, more preferably from 10 μm to 70 μm.

Preferred embodiments of the medium composition and the method for producing the same used in the present invention are described below.

The medium composition used in the present invention is preferably a medium composition which can suspend and culture cells or tissues, and the viscosity of the above medium composition is 8 mPa. The medium composition characterized by s below (at 37 ° C) and containing deuterated gellan gum or a salt thereof. In one aspect, the concentration of the degummed orchid or its salt in the medium composition is from 0.01 to 0.05% (weight/volume). In one aspect, the medium composition further contains a polysaccharide other than deuterated gellan gum or a salt thereof. In one aspect, the medium composition forms a structure in which cells or tissues are suspended and cultured in order to deuteriumize the gellan, and contains a divalent metal ion (for example, calcium ion) in a sufficient concentration. The concentration is, for example, 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM.

The medium composition can be produced by mixing de-deuterated gellan gum or a salt thereof with a medium. In one aspect, the medium is a liquid medium. In one aspect, the liquid medium forms a structure in which cells or tissues are suspended and cultured in order to deuteriumize the gellan, and contains a sufficient concentration of a divalent metal ion (for example, calcium ion). The concentration is, for example, 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM. In one aspect, the deuterated gellan gum or a salt thereof dissolved or dispersed in a solvent is mixed with the medium. In one aspect, the deuterated gellan gum or a salt thereof dissolved or dispersed in a solvent is in a sterilized state. In one aspect, sterilization is carried out by autoclaving, and in another aspect, sterilization is carried out by filtration sterilization. In one aspect, filter sterilization is carried out by passing through a filter of 0.1 to 0.5 μm.

(2) Culturing cancer cells in the presence of proliferation factors (Method I) (2-1) Method for cultivating cancer cells

The present invention provides an adherent cancer cell which is reactive with any one of a proliferation factor selected from the group consisting of a transforming growth factor, an insulin-like proliferation factor, and a fibroblast growth factor, and the above-described medium composition I containing the proliferation factor A suspension culture is carried out, and a method of culturing an adherent cancer cell which is reactive from a proliferation factor selected from the group consisting of a transforming growth factor, an insulin-like proliferation factor, and a fibroblast growth factor (Method 1 of the present invention) is cultured. . In monolayer culture, the adhesion of adherent cancer cells to these proliferation factors is low, and it is difficult to evaluate. In contrast, if the suspension is cultured in the above-mentioned medium composition I, the reactivity to the proliferation factors is greatly increased. Promote proliferation.

The cancer cells used in Method I are reactive in any one selected from the group consisting of a transforming growth factor, an insulin-like growth factor, and a fibroblast growth factor. Therefore, the cancer cell exhibits a functional receptor for any of the proliferation factors selected from the group consisting of a transforming growth factor, an insulin-like growth factor, and a fibroblast growth factor.

Examples of the transforming growth factor (TGF) include TGF-α and TGF-β. It is preferably TGF-β. The β 1 to 3 subtype of TGF-β is included in the present invention. The receptor for TGF-α is ErbB1/EGFR. The receptor for TGF-β includes a type 1 receptor (TGF-β R1) and a type 2 receptor (TGF-β R2), and is included in the present invention.

Examples of insulin-like growth factor (IGF) include IGF-1 and IGF-2. It is preferably IGF-1. IGF-1 and IGF-2 are strong in addition to IGF-1 receptors In addition to activation, the binding to the insulin receptor is weak. The IGF-2 receptor binds only to IGF-2 and does not activate the intracellular signaling pathway. The IGF receptor is preferably an IGF-1 receptor.

Fibroblast growth factor (FGF) was confirmed to be FGF1 to FGF23. Since human FGF19 is an orthologue of mouse FGF15, the FGF family of humans and mice is composed of 22 members. In this specification, the FGF name is named according to the nomenclature of human FGF. FGF is preferably FGF2 (bFGF). The receptor for FGF can be enumerated as FGFR1 to 4, and is included in the present invention.

The cancer cells used in Method I are preferably reactive in any one selected from the group consisting of TGF-β, IGF-1 and FGF2, and more preferably in TGF-β. If suspended culture is carried out in the above-mentioned medium composition I, the reactivity to TGF-β is particularly enhanced, and proliferation is greatly promoted.

The cancer cells used in Methodology I are adherent cells. A cohesive cell is a scaffold protein-dependent cell that can survive and proliferate by scaffold protein binding.

Adhesive cancer cells include gastric cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, Urinary tract cancer, liver cancer, cholangiocarcinoma, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, papillary pharyngeal cancer, laryngeal cancer, tongue cancer, fibrosarcoma, mucosal sarcoma , liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, lymphangiosarcoma, Lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Evan's sarcoma, leiomyosarcoma, rhabdomyosarcoma, seminoma, Wil's tumor, glioma, astrocytoma, mesothelioma, melanin A cell such as a tumor, a neuroblastoma, a medulloblastoma, a retinal cell tumor, or the like, but is not limited thereto. Adhesive cancer cells are preferably malignant tumors derived from epithelial cells, that is, cancer cells.

In the culture method of the present invention, the adherent cancer cells reactive with the above-mentioned proliferation factor are cultured in suspension in the above-mentioned medium composition I containing the proliferation factor.

The proliferation factor used in the culture method of the present invention is usually a mammalian proliferation factor. The mammal can be exemplified by the above animals. Since the proliferation factor has cross-reactivity between most mammalian species, any mammalian proliferative factor can be used as long as it can achieve the purpose of the invention, and it is preferred to use a proliferative factor of the same mammal as the cultured cancer cell. For example, human adherent cancer cells reactive with TGF-β are preferably cultured in suspension in the above-described medium composition I containing human TGF-β. Human TGF-β refers to the amino acid sequence of TGF-β which has the natural expression of TGF-β in human organisms. In the present specification, other proteins and the like are also explained.

The proliferation factor used in the method of the present invention is preferably isolated. "Isolation" refers to the operation of removing a component other than the intended component or a cell, and is detached from the state existing in nature. Therefore, the "isolated proliferation factor X" does not contain the endogenous growth factor X produced from the cells or tissues of the cultured subject. Purity of "Isolated Protein X" (Total Protein Weight) The weight percentage of the protein X occupied therein is usually 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 99% or more, and most preferably 100%. The isolated proliferation factor contained in the medium used in the suspension culture is an exogenous addition in the medium composition I. Thus, in one aspect, the invention encompasses the step of providing a proliferation factor selected from the group consisting of a transgenic growth factor, an insulin-like growth factor, and a fibroblast growth factor. Further, in one aspect, the step of adding the proliferation factor selected from the group consisting of the isolated transforming growth factor, the insulin-like growth factor, and the fibroblast growth factor is added to the medium composition I. .

In the medium composition I, the concentration of the above-mentioned proliferation factor is a concentration that promotes proliferation of adherent cancer cells in a cultured subject. These concentrations can be appropriately set as long as they are within the technical field. The concentration of TGF-β is usually above 3 ng/mL. The concentration of IGF-1 is usually above 10 ng/mL. The concentration of FGF2 is usually above 10 ng/mL. The upper limit of the added proliferation factor is not particularly limited as long as it can promote the proliferation of the adherent cancer cells in the cultured object, and the concentration of TGF-β is preferably determined from the viewpoint of ensuring a good concentration dependence on cell proliferation. At about 30 ng/mL or less, the concentration of IGF-1 is about 100 ng/mL or less, and the concentration of FGF2 is about 100 ng/mL or less.

When the above-mentioned adhesive cancer cells are cultured by the culture method of the present invention, a tray, a flask, a plastic bag, a Teflon (registered trademark) bag, a dish, a petri dish, and a tissue culture culture which are usually used for culturing cells can be used. Dish, multi-disc, micro-disc, micro-disc, multi-disc, multi-well, cavity-type glass Culture equipment such as tablets, cell culture flasks, stirred bottles, tubes, trays, culture bags, and roller bottles are cultured. Among these culture containers, a microplate, a microplate, a multi-plate, and a porous disk are preferably used in the evaluation of a plurality of anticancer agents, evaluation of pharmaceutical candidate compounds, or pharmaceuticals. The shape of the bottom of the disk is not particularly limited, and a flat bottom, a U-shape, or a V-shape can be used, and a U-shaped type is preferably used. The material of the culture equipment is not particularly limited, and examples thereof include cellulose-based polymers such as glass, polyvinyl chloride, ethyl cellulose, and acetyl cellulose, polystyrene, polymethyl methacrylate, and polycarbonate. Ester, polyfluorene, polyurethane, polyester, polyamide, polystyrene, polypropylene, polyethylene, polybutadiene, poly(ethylene-vinyl acetate) copolymer, poly(butadiene) -styrene) copolymer, poly(butadiene-acrylonitrile) copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, polychloroprene, styrene resin , chlorosulfonated polyethylene, ethylene vinyl acetate, acrylic block copolymer and other plastic materials. These plastic materials are not only excellent in gas permeability such as oxygen or carbon dioxide, but also excellent in industrial formability, and can withstand various sterilization treatments and are preferably transparent materials in which the inside of the culture equipment can be observed. Here, the method of performing the sterilization treatment is not particularly limited, and for example, radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, or the like can be performed. Further, various surface treatments (e.g., plasma treatment, corona treatment, etc.) can be performed on the plastic materials.

The culture of the above-mentioned adhesive cancer cells can be automatically carried out under a mechanical control in a closed environment, such as cell seeding, medium exchange, cell image acquisition, culture cell recovery, and high-density culture while controlling pH, temperature, oxygen concentration, and the like. Bioreactor or automatic culture device get on. Using these devices, a new medium is supplied during the cultivation, and the required substances are not supplied to the cells and/or tissues insufficiently, and there are batch feed culture, continuous culture, and perfusion culture, and any method is used. It can be used in the culture method of the present invention. In addition, the culture container used in the bioreactor or the automatic culture device has an open culture container (for example, a culture container having a lid) which is easy to open and close, has a large contact area with the outside, and is not easily opened and closed, and has a small contact area with a small area. A container (for example, a sputum type culture container), any of the culture containers can be used in the culture method of the present invention.

In the culture method of the present invention, the form or state of the adhered cancer cells can be arbitrarily selected by those skilled in the art. The preferred embodiment is not particularly limited, and examples thereof include a state in which the adherent cancer cells are dispersed in the medium composition I in a single cell state, a state in which the adherent cancer cells adhere to the surface of the carrier, and the adherent cancer cells are embedded in The state inside the carrier, the state in which a plurality of adherent cancer cells are aggregated to form a cell mass (sphere), and the like. The adhesive cancer cells are preferably dispersed in the state of the medium composition I in a state of a single cell or a plurality of cells, and formed into a cell mass (sphere) in a suspension culture in the medium composition I (preferably suspended in suspension). to cultivate). That is, it is preferred not to use a carrier that binds or embeds cancer cells. In these states, the state in which the cell mass (sphere) is formed is to reconstitute the cell-cell interaction and the cell structure close to the living environment, and it is possible to culture under long-term maintenance of cell function and to facilitate recovery of the cell, so that it is preferable. .

The method of forming the cell agglutination block (sphere) is not particularly limited, and those skilled in the art can appropriately select it. These examples include a method of using a container having a cell non-adhesive surface, a hanging drop method, a spin culture method, and the like. A scaffold method, a centrifugation method, a method of agglutination by an electric field or a magnetic field, or the like. For example, in the case of using a container having a cell non-adhesive surface, the target cells can be cultured in a culture vessel which is subjected to a surface treatment which inhibits cell adhesion to form a sphere. When the cell non-adhesive culture container is used, the cell suspension is first prepared after taking the target cells, seeded in the culture container, and cultured. If culture is continued for about one week, the cells spontaneously form spheres. As the non-adhesive surface of the cell to be used at this time, a substance which inhibits adhesion of cells to the surface of a culture container such as a commonly used disk can be used. As such materials, agarose, agar, poly-HEMA (poly-(2-hydroxy-ethyl methacrylate) 2-methylpropenyloxyethylphosphocholine and other monomers (for example, A) may be mentioned. a copolymer of butyl acrylate or the like, poly(2-methoxymethyl acrylate), poly-N-isopropyl acrylamide, Mebiol Gel (registered trademark), etc. As long as there is no cytotoxicity, it is not limited to this.

Further, in order to obtain a cell aggregate having a uniform size for the purpose, a plurality of dents having the same diameter as the target cell aggregate can be introduced into the cell non-adhesive culture container to be used. As long as the dents are interconnected or within the diameter of the agglutination block of the target cell, the seeded cells do not form cell agglomerates between the dent and the dent when seeding the cells, indeed in the dent A cell agglutination block corresponding to its volume is formed, and a cell agglomerate group of uniform size can be obtained. The shape of the dimple at this time is preferably hemispherical or conical.

The culture composition I can be used to form a sphere. For example, by dissociating in the culture composition I to a single cell state, The target adherent cancer cells were uniformly dispersed, and allowed to stand for 3 to 10 days, and suspension culture was carried out to prepare a sphere. The spheres prepared here can be recovered by centrifugation or filtration. The size of the sphere varies depending on the cell type and the culture period, and is not particularly limited, and has a diameter of 20 μm to 1000 μm, preferably 40 μm to 500 μm, more preferably 50 μm to 300 μm, and most preferably 80 μm to 200 μm in the shape of a sphere or an elliptical sphere. . Even if the spheres continue to be cultured in the original state, the growth ability can be maintained for 10 days or longer, preferably for 13 days or longer, and more preferably for 30 days or longer, and mechanical division is performed periodically in the stationary culture, or alternatively. Single cell treatment and agglutination can maintain proliferative capacity indefinitely.

When the adherent cancer cells are cultured by the method of the present invention, the adherent cancer cells prepared by other methods may be added to the culture composition I containing the above-mentioned proliferation factor, and mixed in a manner of uniform dispersion. The mixing method at this time is not particularly limited, and for example, manual mixing such as suction, mixing using a stirrer, a vortex mixer, a microplate mixer, or a vibrating machine can be mentioned. After mixing, the obtained adherent cancer cell suspension can be statically cultured, and if necessary, the culture solution can be rotated, vibrated or stirred. The number of rotations and the frequency thereof can be appropriately set in accordance with the purpose of those skilled in the art. Further, when the medium composition must be exchanged during the stationary culture, the adherent cancer cells can be separated from the medium composition by centrifugation or filtration treatment, and the medium composition I containing the above-mentioned proliferation factor fresh can be added to the cells. Alternatively, the adherent cancer cells may be appropriately concentrated by centrifugation or filtration treatment, and then the fresh medium composition I containing the above proliferation factor may be added to the concentrate.

In a state, when suspending cultured adherent cancer cells, For example, the cancer cells are recovered from the subculture and dispersed into a single cell or to a state close to a single cell. Dispersion of cancer cells is carried out using a suitable cell dissociation solution. The cell dissociating liquid may be used alone or in combination as appropriate, for example, EDTA; trypsin, collagenase IV, proteolytic enzymes such as metalloproteinase, and the like. The dispersed adherent cancer cells are suspended in a medium composition I containing the above-mentioned proliferation factor, and suspended in culture (preferably, suspension culture). In the culture, the cancer cells are proliferated while being suspended in the medium composition I in the state of a single cell or in the state of a sphere.

The temperature at which the adherent cancer cells are cultured is usually 25 to 39 ° C, preferably 37 ° C. The CO ₂ concentration is usually 4 to 10% by volume, preferably 5% by volume, in the atmosphere of the culture. The oxygen concentration is 15 to 50% by volume, preferably 20% by volume in the culture atmosphere, and is usually 1 to 35 days in the culture period, and can be freely set for the purpose of culture.

(2-2) Method for testing the reactivity of cancer cells to proliferation factors

The present invention provides a method for testing the reactivity of a cancer cell to a proliferation factor selected from the group consisting of a transforming growth factor, an insulin-like growth factor, and a fibroblast growth factor, comprising (1) adhering cancer cells Suspension culture in the above-mentioned medium composition I in the presence and absence of the proliferation factor, (2) measurement of proliferation of cultured cancer cells, and (3) comparison of proliferation of cancer cells cultured in the presence of the proliferation factor A method of proliferation in the absence of the proliferation factor (Method 2 of the present invention).

As described above, in monolayer culture, adherent cancer cells have low responsiveness to transforming growth factor, insulin-like growth factor, and fibroblast growth factor, and, in contrast, by the above method I of the present invention, Suspension culture is carried out in the composition I, and the responsiveness to these proliferation factors is enhanced to greatly promote proliferation. Therefore, by applying this method, the reactivity of the adherent cancer cells to the above-mentioned proliferation factors can be evaluated with high sensitivity.

In the method 2 of the present invention, the definition of the terms or the test conditions (including the preferred conditions) are the same as the method 1 of the present invention unless otherwise specified.

In step (1), the adherent cancer cells to be evaluated are selected from any one selected from the group consisting of a transforming growth factor (eg, TGF-β), an insulin-like growth factor (eg, IGF-1), and a fibroblast growth factor (eg, FGF2). In the presence and absence of a group of proliferation factors, suspension culture (preferably suspension culture) is carried out in the above medium composition I. The proliferation factor is preferably TGF-β.

The growth promoting effect of the adhesion-producing cancer cells to be evaluated by the proliferation factor can be detected in the culture period, and is not particularly limited, and is usually about 1 to 7 days.

The proliferation of the cultured cancer cells can be measured by a method known per se. For example, the number of cells of cancer cells after culture is measured. The number of cells that proliferate or appear can be counted by standard microscopes used in the field. Alternatively, the number of cells in cancer cells can be measured by colony formation method, crystal violet method, thymidine incorporation method, trypan blue staining method, ATP (adenosine 3 phosphate) assay, 3-(4,5-dimethyl group). Thiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-Dimethylthial-2-yl) -2,5-Diphenyltetrazalium Bromide (MTT) staining method, WST-1 (registered trademark) staining method, WST-8 (registered trademark) staining method, flow cytometry method, method using automatic cell number measuring device, etc. .

Proliferation of cancer cells cultured in the presence of the proliferation factor is compared to proliferation in the absence of the proliferation factor. Comparison of the proliferation of cancer cells is preferably based on statistically significant differences. Further, the degree of proliferation of the control cancer cells to which the proliferative factor is not added is measured, and the proliferation of the cancer cells exposed to the proliferative factor to be tested may be measured beforehand or may be simultaneously measured, from the precision of the experiment, From the viewpoint of reproducibility, it is preferred to simultaneously measure the person.

As a result of the comparison, it was confirmed that the cancer cells were judged to be reactive with the proliferation factor when the proliferation was promoted by the addition of the proliferation factor.

(2-3) Screening method of anticancer agent

The present invention provides a method for screening for an anticancer agent selected from any of the adherent cancer cells reactive with a proliferation factor consisting of a transforming growth factor, an insulin-like growth factor, and a fibroblast growth factor, and comprises (1) The cancer cells are cultured in suspension in the medium composition I containing the proliferation factor in the presence or absence of the test substance, (2) the proliferation of the cultured cancer cells is measured, and (3) will be examined. A method of comparing proliferation of cancer cells cultured in the presence of a substance with proliferation in the absence of a test substance (Method 3 of the present invention).

As described above, in monolayer culture, adherent cancer cells proliferate The responsiveness of the factor, the insulin-like growth factor, and the fibroblast growth factor is low. In contrast, by performing the suspension culture in the above-mentioned medium composition I by the above method I of the present invention, the responsiveness to the growth factor is improved. Since the proliferation is greatly promoted, by applying this method, substances which inhibit the proliferation of the above-mentioned proliferation factors of the adherent cancer cells can be screened with high sensitivity.

In the method 3 of the present invention, the definition of the terms or the test conditions (including the preferred conditions) are the same as the methods 1 and 2 of the present invention unless otherwise specified.

In step (1), in the presence and absence of the test substance, any one selected from the group consisting of a transforming growth factor (eg, TGF-β), an insulin-like proliferation factor (eg, IGF-1), and a fibroblast growth factor The proliferating factor in the group (for example, FGF2) is reactive and adherent cancer cells, and is cultured in suspension in the above-mentioned medium composition I containing the proliferation factor (preferably, suspension culture). The proliferation factor is preferably TGF-β.

The test substance to be supplied to the method 3 of the present invention may be any known compound or novel compound, and examples thereof include a nucleic acid, a saccharide, a lipid, a protein, a peptide, an organic low molecular compound, and a compound library produced by a combinatorial chemistry technique. (libray), random peptide library or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

The proliferation effect of the adhesion-promoting cancer cells to be evaluated by the stimulation factor can be detected in the culture period, and is not particularly limited, and is usually about 1 to 7 days.

Determining the proliferation of cultured cancer cells can be known per se Method implementation. For example, the number of cells of cancer cells after culture is measured. The number of cells that proliferate or appear can be counted by standard microscopes used in the field. The number of cells of cancer cells can be measured by colony formation method, crystal violet method, thymidine incorporation method, trypan blue staining method, ATP (adenosine 3 phosphate) assay, 3-(4,5-dimethylthiazole- 2-(4,5-Dimethylthial-2-yl-2,5-Diphenyltetrazalium Bromide (MTT) staining method, WST-1 (registered trademark) The dyeing method, the WST-8 (registered trademark) staining method, the flow cytometry method, the method using the cell number automatic measuring device, and the like are carried out.

The proliferation of cancer cells cultured in the presence of the test substance is compared with the proliferation in the absence of the test substance. Comparison of the proliferation of cancer cells is preferably based on statistically significant differences. Further, the degree of proliferation of the control cancer cells to which the test substance is not added may be measured by the measurement of the cancer cells that are in contact with the test substance, or may be simultaneously measured, and the precision and reproduction of the test may be performed. From the viewpoint of sex, it is preferred to measure at the same time.

As a result of the comparison, by adding the test substance, a substance which inhibits the proliferation of the adherent cancer cells by the proliferation factor can be selected as a candidate substance for inhibiting the proliferation of the adhesion-promoting cancer cells, or as a binding material. Candidate substances for anticancer agents of cancer cells.

Only in comparison with the above, it is not possible to negate the possibility of inhibiting the proliferation of cancer cells in the subject by evaluation irrespective of the proliferation-promoting effect of the proliferation factor added to the above-mentioned medium composition I (for example, only by cytotoxicity). Here, the proliferation factor of the evaluation target is not added to the medium composition I, and the same operations as the above steps (1) to (3) are performed, and further evaluation can be performed. The effect of the test substance on the proliferation of the cancer cell in which the proliferation factor is not dependent is estimated. Therefore, as a result of the comparison, when the test substance does not exist and does not inhibit the proliferation of the adherent cancer cell, the test substance can be selected as a substance which selectively blocks proliferation of the proliferation factor-dependent adhesion cancer cell. . Thus, a substance which selectively inhibits proliferation of a specific growth factor-dependent adherent cancer cell is superior to a candidate substance for an anticancer agent for adherent cancer cells because it non-specifically reduces the risk of damaging normal cells.

In addition, the effect of the candidate substance obtained can be confirmed in the living body. For example, the candidate substance is administered to a non-human mammal of sputum cancer (for example, a non-human mammal that recognizes an adhesion-promoting cancer cell which inhibits proliferative activity by in vitro migration), and the cancer cell is proliferated in the non-human mammal. The degree of comparison with the control non-human mammal (except for the non-administration of the candidate substance, the feeding condition is the same as that of the non-human mammal in which the candidate substance is administered), based on the comparison result, the non-human mammal can be selected. A candidate substance that inhibits the proliferation of cancer cells is a candidate substance for an anticancer agent that has been confirmed to be effective in the living body.

(3) Assessment of epithelial-mesenchymal transition in cancer cells (Methodology II) (3-1) Method for maintaining epithelial lineage of cancer cells

The present invention provides a method comprising the method of suspending culture of cancer cells in the above-described medium composition I to maintain the epithelial morphology of the cancer cells (Method 4 of the present invention). If the cancer cells are cultured in a single layer, the epithelial-mesenchymal transition is promoted, the epithelial system is lost, and the interstitial morphology is dominant. In contrast, if the cancer cells are suspended in the above-mentioned medium composition I, Raising, inhibits epithelial-mesenchymal transition, not only maintains the epithelial-like morphology, but also induces the transformation of the mesenchymal metaplasia into epithelial-like (ie, interstitial epithelial transition). The shape is dominant.

Epithelial-mesenchymal transition (EMT) refers to the process in which epithelial cells lose their cell polarity or bind to the cells of surrounding cells, can migrate, gain invasive ability, and change into mesenchymal cells. In general, cancer cells are relatively high in epithelial-mesenchymal transition from E-cadherin, and the surface epithelial-like epithelial cells are surface-to-side with N-cadherin. The high, the cells adhere to each other at a point, and the adhesion between the cells is reduced, such as the migration of interstitial lines, which is considered to be one of the causes of infiltration or metastasis to surrounding tissues.

Examples of cancer cells include gastric cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, Urethral cancer, liver cancer, cholangiocarcinoma, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, papillary pharyngeal cancer, laryngeal cancer, tongue cancer, synovial tumor, A cell such as a skin tumor, an Evan's sarcoma, a seminoma, a Wilm's tumor, a glioma, a stellate cell tumor, a myeloma, a retinal cell tumor, etc., but is not limited thereto.

The cancer cells used in Methodology II have epithelial-mesenchymal transition ability. Epithelial-mesenchymal transition ability refers to the ability to induce epithelial-mesenchymal transition by stimulation of epithelial-mesenchymal transition-inducing factors. Examples of the epithelial-mesenchymal transition-inducing factor include TGF-β, FGF (FGF1 to FGF23, preferably FGF2), and EGF receptor agonists (EGF, HB-EGF, TGF-α, β-). Animal cellulose (cellulin), amphiregulin (epiregulin), epiregulin (epiregulin), HGF, Wnt/β-catenin (Catenin), Notch, type I collagen, and the like. Usually, the epithelial stroma of cancer cells is converted to reversible, and can return to the epithelial lineage after migration to the interstitial lineage.

In the method 4 of the present invention, the cancer cells are cultured in suspension in the above medium composition I (preferably, suspension culture). By suspension culture, the epithelial-mesenchymal transition of cancer cells is inhibited, and the epithelial-like morphology is maintained. If the cancer cells are cultured in a single layer, the interstitial morphology is dominant, and the cancer cells which are dominant in the mesenchymal type are suspended in the above-mentioned medium composition I, and the interstitial epithelial transformation is induced and lost. The interstitial quality is changed to become the epithelial type. In the method 4 of the present invention, "maintaining epithelial traits such as cancer cells" includes maintaining a state in which epithelial sinusoids are dominant in cancer cells and a tumor cell having an interstitial morphological superiority to an epithelial genus. The state of advantage moves.

In the method 4 of the present invention, the culture container or the culture device used for culturing the cancer cells can be exemplified by the culture container or the culture device described in the method 1 of the present invention.

The form or state of the cancer cells cultured in the method 4 of the present invention is not particularly limited, and it is preferred that the cancer cells are dispersed in the medium composition I in a state of a single cell or a plurality of cancer cells are aggregated to form cells. The state of the block (sphere) is suspended in culture medium composition I (preferably, suspension culture). Preferably, suspension culture is carried out without using a carrier for adhering or embedding cancer cells. By binding to a carrier of cancer cells, there is a possibility of inducing epithelial-mesenchymal transition.

The method of forming the cell agglutination block (sphere) can be exemplified by the method described in the above method 1 of the present invention.

When the cancer cells are cultured by the method 4 of the present invention, cancer cells prepared by other methods may be added to the culture composition I, mixed, and uniformly dispersed. The mixing method at this time is not particularly limited, and examples thereof include manual mixing such as suction, and mixing using a stirrer, a vortex mixer, a microplate mixer, or a vibrator. After mixing, the obtained cancer cell suspension can be statically cultured, and if necessary, the culture solution can be rotated, vibrated or stirred. The number of rotations and the frequency can be appropriately set for the purpose of the manufacturer. Further, when the medium composition must be exchanged during the stationary culture, the cancer cells can be separated from the medium composition by centrifugation or filtration treatment, and the fresh medium composition I can be added to the cells. Alternatively, the cancer cells may be appropriately concentrated by centrifugation or filtration treatment, and fresh medium composition I may be added to the concentrate.

In one aspect, when the cancer cells are cultured in suspension, for example, the cancer cells are recovered from the subculture, and dispersed into a single cell or to a state close to a single cell. Dispersion of cancer cells is carried out using an appropriate cell dissociation solution. The cell dissociation liquid may be used alone or in combination as appropriate, such as EDTA, trypsin, collagenase IV, or proteolytic enzymes such as metalloproteinase. The dispersed cancer cells are suspended in the above-mentioned medium composition I, and suspended in culture (preferably, suspension culture). In the culture, the cancer cells are proliferated while being suspended in the medium composition I in the state of a single cell or in the state of a sphere.

In a preferred aspect, in method 4 of the present invention, Holding the epithelial lineage such as cancer cells and not inhibiting the interstitial epithelial transformation, the medium composition I does not contain the epithelial-mesenchymal transition-induced epithelial-mesenchymal transition-inducing factor. Examples of epithelial-mesenchymal transition-inducing factors include TGF-β, FGF (FGF1 to FGF23, preferably FGF2), and EGF receptor agonists (EGF, HB-EGF, TGF-α, β-animal cellulose, amphiregulin, Epiregulin, etc.), HGF, Wnt/β-catenin, Notch, type I collagen, and the like.

The temperature at which the cancer cells are cultured is usually 25 to 39 ° C, preferably 37 ° C. The CO ₂ concentration is usually 4 to 10% by volume, preferably 5% by volume, in the atmosphere of the culture.

In the hypoxic environment, since the epithelial-mesenchymal transition is induced, in the method 4 of the present invention, the oxygen concentration in the culture of the cancer cells is preferably higher than the partial pressure of oxygen (20% by volume) in the air.

It is not particularly limited as long as the epithelial system such as cancer cells can be maintained during the culture, and can be freely set for the purpose of culture. When the cancer cell having the superior interstitial quality is transferred to the state in which the epithelial line shape is dominant (for example, the cancer cells in the monolayer culture are recovered, and the suspension culture is carried out in the medium composition I), preferably In the medium composition I, the suspension culture is continued for 6 days or longer, preferably for 11 days or more, and the interstitial epithelial transformation is sufficiently performed.

By this operation, cancer cells can be obtained by suspension culture (preferably suspension culture) in the medium composition I, whereby cancer cells maintaining the epithelial-like morphology can be obtained. The method 4 of the present invention is also a method for modulating cancerous cells having an epithelial-like morphology.

Confirmed epithelial cells in cultured cancer cells The line shape is maintained (the epithelial line shape is superior). Method 4 of the present invention can include such confirmation steps. It was confirmed that the state in which the epithelial line shape was maintained can be performed by investigating the expression of E-Cadherin and N-Cadherin. In the state where the epithelial line shape is dominant, the expression of epithelial cadherin is relatively high, and the expression of neurocadherin is relatively low. In the state where the interstitial lineage is dominant, epithelial cadherin is the opposite. The performance is relatively low, and the performance of neurocadherin is relatively high. Therefore, as long as the expression of epithelial cadherin is relatively high and the expression of neurocadherin is relatively low, it can be judged that the cancerous cells after culture are maintained in a state in which the epithelial lineage is maintained (the epithelial lineage is superior). In addition, when the cancer cell with the superior interstitial quality is transferred to the epithelial-like state, the expression of epithelial cadherin and calcineurin in the cancer cells after culture is the same as that at the beginning of the culture. Compared with cancer cells, as long as the expression of epithelial cadherin is increased and the expression of neuro-cadherin is decreased, it can be judged that the cancer cells migrate to the epithelial-like state. The expression of epithelial cadherin and calcineurin can be performed by assessing the expression of mRNA or protein. mRNA expression can be assessed by RT-PCR, Northern blotting, nucleic acid arrays, and the like using primers or probes specific for epithelial cadherin and calcineurin. The performance of the protein can be assessed by immunohistochemical analysis (immune tissue staining, flow cytometry, etc.) using antibodies specific for epithelial cadherin and calcineurin.

(3-2) Induction method of epithelial-mesenchymal transition

The present invention provides a cancer cell comprising a epithelial genus, A method of suspension culture in the above-described medium composition I containing an epithelial-mesenchymal transition-inducing factor to induce epithelial-mesenchymal transition of the cancer cell (Method 5 of the present invention). In the method 4 of the present invention described above, the epithelial-mesenchymal transition of the cancer cells is induced by adding a sufficient amount of the epithelial-mesenchymal transition-inducing factor induced by the epithelial-mesenchymal transition in the medium composition I.

In the method 5 of the present invention, the definition of the terms or the test conditions (including the preferred conditions) are the same as the method 4 of the present invention unless otherwise specified.

In the method 5 of the present invention, a cancer cell having an epithelial-like morphology is first provided. Cancer cells having an epithelial-like morphology can be modulated by, for example, Method 4 of the present invention described above. Therefore, the method 4 of the present invention can be carried out before the method 5 of the present invention is carried out to prepare cancer cells having an epithelial-like morphology.

Next, the cancerous cells having the epithelial-like morphology are subjected to suspension culture (preferably suspension culture) in the above-described medium composition I containing the epithelial-mesenchymal transition-inducing factor. Examples of epithelial-mesenchymal transition-inducing factors include TGF-β, FGF (FGF1 to FGF23, preferably FGF2), and EGF receptor agonists (EGF, HB-EGF, TGF-α, β-animal cellulose, amphiregulin, Epiregulin, etc.), HGF, Wnt/β-catenin, Notch, type I collagen, and the like. The epithelial-mesenchymal transition inducing factor is preferably isolated. The isolated epithelial-mesenchymal transition inducing factor contained in the medium used in the suspension culture is an exogenous additive in the medium composition I. Thus, in one aspect, the invention encompasses the step of providing a single epithelial mesenchymal transition inducing factor. Also, in one aspect, it is included in the medium composition I will be separated The step of exogenous addition of epithelial-mesenchymal transition induction factor. The above medium composition I contains an epithelial-mesenchymal transition-inducing concentration epithelial-mesenchymal transition-inducing factor. The epithelial-conversion-inducing concentration of each factor can be easily set as long as it is in the technical field, for example, the TGF-β concentration is 0.1 ng/mL or more, and the HB-EGF concentration is 30 ng/mL or more.

In the method 5 of the present invention, the morphology or state of the cancer cells to be cultured is not particularly limited, and it is preferred that the cancer cells are dispersed in the medium composition I in a state of a single cell, or a plurality of cancer cells are aggregated to form cells. The state of the block (sphere) is suspended in culture medium composition I (preferably, suspension culture). Preferably, the vector in which the cancer cells are adhered or embedded is suspended culture.

In one aspect, by performing the above method 4 of the present invention, cancer cells having an epithelial-like morphology are obtained. Therefore, in the medium composition I, the epithelial-mesenchymal transition-inducing concentration of the epithelial-mesenchymal transition-inducing factor is added, and suspension culture (preferably suspension culture) is continued.

The temperature at which the cancer cells are cultured is usually 25 to 39 ° C, preferably 37 ° C. The CO ₂ concentration is usually 4 to 10% by volume, preferably 5% by volume, in the atmosphere of the culture.

In a hypoxic environment, the epithelial-mesenchymal transition is induced, and in the method 5 of the present invention, the cancer cells can be cultured in suspension in a low oxygen concentration (2 to 10% by volume, preferably 3% by volume). On the other hand, from the viewpoint of the effect of the hypoxic environment, only the effect of adding the epithelial-mesenchymal transition inducing factor is evaluated, and the oxygen concentration in the culture method of the present invention 5 is preferably made into oxygen in the air. Partial pressure (20% by volume) or more.

The culture period is not limited to the induction of epithelial-mesenchymal transition of cancer cells, and it is preferred to continue the suspension culture in the medium composition I containing the epithelial-mesenchymal transition-inducing factor, for example, for more than 6 days, preferably for more than 11 days, so that the epithelial-mesenchymal transition is sufficiently performed. .

By this operation, the epithelial-mesenchymal transition can be induced by suspending culture (preferably suspension culture) of the cancer cell having the epithelial-like morphology in the medium composition I containing the epithelial-mesenchymal transition-inducing factor. A cancerous cell with an interstitial morphology. Method 5 of the present invention is also a method of modulating cancerous cells having an interstitial morphology.

After culture, it can be confirmed that the epithelial-mesenchymal transition is induced, that is, the cancer cells have an interstitial shape (the interstitial shape is superior). Method 5 of the present invention can include such confirmation steps. It was confirmed that the induction of epithelial-mesenchymal transition can be performed by investigating the expression of epithelial cadherin and neurocadmium. The epithelial cadherin is superior in appearance, the epithelial cadherin is relatively high, and the neurocadherin is relatively low. In contrast to the interstitial morphology, the epithelial cadherin expression is opposite. Relatively low, neurocalcin expression is relatively high. Therefore, as long as the expression of epithelial cadherin is relatively low and the expression of neurocadmium is relatively high, it can be judged that the cancer cells after culture have interstitial morphology (inter-class lineage is superior). Moreover, in the cancer cells after culture, the expression of epithelial cadherin and neurocadherin is compared with the cancer cells at the beginning of the culture, as long as the expression of epithelial cadherin is decreased and the expression of neurocadmium is increased, It was judged that the epithelial-mesenchymal transition was induced. The performance of epithelial cadherin and neurocadherin can be evaluated by assessing the performance of mRNA or protein. Row. The expression of mRNA can be evaluated by RT-PCR, Northern blotting, nucleic acid arrays, and the like using primers or probes specific for epithelial cadherin and calcineurin. The performance of the protein can be assessed by immunohistochemical analysis (immune tissue staining, flow cytometry, etc.) using antibodies specific for epithelial cadherin and calcineurin.

(3-3) Screening method for substances that induce epithelial-mesenchymal transition

The present invention provides a screening method for a substance for inducing epithelial-mesenchymal transition, which comprises (1) suspending a cancer cell having an epithelial-like morphology in the above-mentioned medium composition I in the presence and absence of a test substance, (2) Evaluating the epithelial transition of cultured cancer cells and (3) comparing the epithelial transition of cancer cells cultured in the presence of the test substance with the epithelial transition in the absence of the test substance (the present invention) Method 6).

As described above, if the method 5 of the present invention is used, in vitro, the epithelial-mesenchymal transition of the cancer cells can be induced by stimulation of the epithelial-mesenchymal transition-inducing factor, and the epithelial-mesenchymal transition induction can be induced by using the test substance. Factors that screen for substances that induce epithelial-mesenchymal transition.

In the method 6 of the present invention, the definition of the terms or the test conditions (including the preferred conditions) are the same as the methods 4 and 5 of the present invention unless otherwise specified.

In the method 6 of the present invention, a cancer cell having an epithelial-like morphology is first provided. Cancer cells with epithelial-like morphology can be used as examples Method 4 is modulated as described above for the present invention. Thus, Method 4 of the present invention can be practiced prior to performing Method 6 of the present invention to modulate cancerous cells having epithelial-like morphology.

In the step (1), the cancerous cells having the epithelial-like morphology are suspended and cultured in the above-mentioned medium composition I (preferably in suspension suspension culture) in the presence or absence of the test substance.

The test substance to be supplied to the method 6 of the present invention may be any known compound and novel compound, and examples thereof include nucleic acids, saccharides, lipids, proteins, peptides, organic low molecular compounds, compound libraries prepared using combinatorial chemistry techniques, random peptide libraries or It is derived from natural ingredients such as microorganisms, animals and plants, and marine life.

In a preferred embodiment, in the step (1), in order to clarify the effect of the added test substance, the medium composition I does not contain the epithelial-mesenchymal transition-inducing concentration-derived mesenchymal transition-inducing factor. Examples of epithelial-mesenchymal transition-inducing factors include TGF-β, FGF (FGF1 to FGF23, preferably FGF2), and EGF receptor agonists (EGF, HB-EGF, TGF-α, β-animal cellulose, amphiregulin, Epiregulin, etc.), HGF, Wnt/β-catenin, Notch, type I collagen, and the like.

The culture period is not particularly limited as long as the effect of the epithelial-mesenchymal transition induction of the test substance to be added can be evaluated, and it is preferred to continue the suspension culture in the medium composition I, for example, for 6 days or more, preferably for 11 days or more, so that the epithelial space is interposed. The quality conversion is fully carried out.

The evaluation of the epithelial transition of the cultured cancer cells can be carried out by a method known in the art. For example, by investigating epithelial calcium sticky eggs This assessment can be performed for the performance of white and neurocalcin. The epithelial cadherin is superior in appearance, the epithelial cadherin is relatively high, and the neurocadherin is relatively low. In contrast to the interstitial morphology, the epithelial cadherin expression is opposite. Relatively low, neurocalcin expression is relatively high. Therefore, as long as the expression of epithelial cadherin is relatively low and the expression of neurocadherin is relatively high, it can be judged that the cancer cells after culture have a state of interstitial quality (the interstitial type is superior). Moreover, in the cancer cells after culture, the expression of epithelial cadherin and neurocadherin is compared with the cancer cells at the beginning of the culture, as long as the expression of epithelial cadherin is decreased and the expression of neurocadmium is increased, It was judged that the epithelial-mesenchymal transition was induced. The expression of epithelial cadherin and calcineurin can be performed by assessing the expression of mRNA or protein. The expression of mRNA can be evaluated by RT-PCR, Northern blotting, nucleic acid arrays, and the like using primers or probes specific for epithelial cadherin and calcineurin. The performance of the protein can be assessed by immunohistochemical analysis (immune tissue staining, flow cytometry, etc.) using antibodies specific for epithelial cadherin and calcineurin.

Therefore, the epithelial transition of the cancer cells cultured in the presence of the test substance is compared with the epithelial transition in the absence of the test substance. The comparison of epithelial-mesenchymal transitions is preferably based on statistically significant differences. Further, the degree of epithelial-mesenchymal transition of the control cancer cells to which the test substance is not added is measured, and the skin mesenchymal transition of the cancer cells contacting the test substance may be measured beforehand or may be simultaneously measured. From the viewpoint of precision and reproducibility, it is better to simultaneously measure Determiner.

As a result of the comparison, when the epithelial-mesenchymal transition is induced by the addition of the test substance, the substance can be selected as a candidate substance for inducing epithelial mesenchymal transition of the cancer.

(3-4) Screening method for substances that hinder epithelial-mesenchymal transition

The present invention provides a screening method for a substance which inhibits epithelial-mesenchymal transition of a cancer cell, comprising: (1) in the presence or absence of a test substance, the cancer cell having an epithelial-like morphology is contained in the epithelial-mesenchymal transition-inducing factor Suspension culture in medium composition I, (2) evaluation of epithelial transition of cultured cancer cells, and (3) comparison of epithelial transition of cancer cells cultured in the presence of test substance and absence of test substance Method of epithelial-mesenchymal transition (Method 7 of the present invention).

As described above, if the method 5 of the present invention is used, in vitro, the epithelial-mesenchymal transition of the cancer cells can be induced by stimulation of the epithelial-mesenchymal transition-inducing factor, by adding a test substance to the culture system. It can screen out substances that hinder the conversion of epithelial stroma.

In the method 7 of the present invention, the definition of the terms or the test conditions (including the preferred conditions) are the same as the methods 4 and 5 of the present invention unless otherwise specified.

In the method 7 of the present invention, a cancer cell having an epithelial-like morphology is first provided. Cancer cells with epithelial-like morphology can be used as examples Method 4 is modulated as described above for the present invention. Thus, the method 4 of the present invention can be carried out prior to the implementation of the method 7 of the present invention to prepare cancerous cells having an epithelial-like morphology.

In the step (1), the cancerous cells having the epithelial-like morphology are suspended and cultured in the above-mentioned medium composition I containing the epithelial-mesenchymal transition-inducing factor in the presence or absence of the test substance (preferably suspended in the suspension). Set culture).

The test substance to be supplied to the method 7 of the present invention may be any known compound and novel compound, and examples thereof include nucleic acids, saccharides, lipids, proteins, peptides, organic low molecular compounds, compound libraries prepared using combinatorial chemistry techniques, random peptide libraries or It is derived from natural ingredients such as microorganisms, animals and plants, and marine life.

Examples of epithelial-mesenchymal transition-inducing factors include TGF-β, FGF (FGF1 to FGF23, preferably FGF2), and EGF receptor agonists (EGF, HB-EGF, TGF-α, β-animal cellulose, amphiregulin, Epiregulin, etc.), HGF, Wnt/β-catenin, Notch, type I collagen, and the like. The epithelial-mesenchymal transition inducing factor is preferably isolated. The isolated epithelial-mesenchymal transition inducing factor contained in the medium used in the suspension culture is an exogenous additive in the medium composition I. Thus, in one aspect, the invention encompasses the step of providing a single epithelial mesenchymal transition inducing factor. Further, in one aspect, the step of exogenously adding the epithelial-mesenchymal transition-inducing factor to the medium composition I is included. The above medium composition I contains an epithelial-mesenchymal transition-inducing concentration epithelial-mesenchymal transition-inducing factor. The epithelial-conversion-inducing concentration of each factor is sufficient as long as it is in the technical field. For easy setting, for example, the TGF-β concentration is 0.1 ng/mL or more, and the HB-EGF concentration is 30 ng/mL or more.

In the method 7 of the present invention, the morphology or state of the cancer cells to be cultured is not particularly limited, and it is preferred that the cancer cells are dispersed in the medium composition I in a state of a single cell, or a plurality of cancer cells are aggregated to form cells. The state of the block (sphere) is suspended in culture medium composition I (preferably, suspension culture). Preferably, the vector in which the cancer cells are adhered or embedded is suspended culture.

The temperature at which the cancer cells are cultured is usually 25 to 39 ° C, preferably 37 ° C. The CO ₂ concentration is usually 4 to 10% by volume, preferably 5% by volume, in the atmosphere of the culture.

In a hypoxic environment, inducing epithelial-mesenchymal transition, in the method 7 of the present invention, cancer cells can be cultured in suspension in a hypoxic (2 to 10%, preferably 3%) environment. On the other hand, from the viewpoint of the effect of the hypoxic environment, the oxygen concentration during the culture of the method 7 of the present invention is preferably made into the air from the viewpoint of the effect of adding the epithelial-mesenchymal transition inducing factor to the test substance. Oxygen partial pressure (20%) or more.

During the culture, as long as the epithelial-mesenchymal transition-inducing factor can be evaluated, the epithelial-mesenchymal transition of the cancer cells can be performed without particular limitation, and it is preferred to continue the suspension culture, for example, for more than 6 days, preferably for more than 11 days, to effect epithelial-mesenchymal transition. Fully proceed.

The evaluation of the epithelial transition of the cultured cancer cells can be carried out by a method known in the art. This assessment can be performed, for example, by investigating the performance of epithelial cadherin and calcineurin. Epithelial lineage In the state of superiority, the expression of epithelial cadherin is relatively high, and the expression of neurocadherin is relatively low. In contrast to the state of interstitial plastids, the performance of epithelial cadherin is relatively low. The performance of neurocadherin is relatively high. Therefore, as long as the expression of epithelial cadherin is relatively low and the expression of neurocadherin is relatively high, it can be judged that the cancer cells after culture have a state of interstitial quality (the interstitial type is superior). Moreover, in the cancer cells after culture, the expression of epithelial cadherin and neurocadherin is compared with the cancer cells at the beginning of the culture, as long as the expression of epithelial cadherin is decreased and the expression of neurocadmium is increased, It was judged that the epithelial-mesenchymal transition was induced. The expression of epithelial cadherin and calcineurin can be performed by assessing the expression of mRNA or protein. The expression of mRNA can be evaluated by RT-PCR, Northern blotting, nucleic acid arrays, and the like using primers or probes specific for epithelial cadherin and calcineurin. The performance of the protein can be assessed by immunohistochemical analysis (immune tissue staining, flow cytometry, etc.) using antibodies specific for epithelial cadherin and calcineurin.

Therefore, the epithelial transition of the cancer cells cultured in the presence of the test substance is compared with the epithelial transition in the absence of the test substance. The comparison of epithelial-mesenchymal transitions is preferably based on statistically significant differences. Further, the degree of epithelial-mesenchymal transition of the control cancer cells to which the test substance is not added is measured, and the skin mesenchymal transition of the cancer cells contacting the test substance may be measured beforehand or may be simultaneously measured. From the viewpoint of precision and reproducibility, it is preferred to measure at the same time.

As a result of the comparison, by adding the test substance to block the epithelial-mesenchymal transition-inducing factor, the epithelial-mesenchymal transition of the cancer cell can be selected as a barrier to the epithelial transition of the cancer cell (preferably by the epithelium). Interstitial substances for mesenchymal transition-inducing factors, epithelial mesenchymal transition in cancer cells. Since the epithelial-mesenchymal transition contributes to the infiltration or metastasis of the cancer, the substance can be used as a medical candidate for inhibiting the invasion or metastasis of the cancer.

The contents of all publications including the patents and the patent application specification are hereby incorporated by reference in their entirety herein in their entireties in the extent of the disclosure.

[Examples]

Hereinafter, the analysis examples and test examples of the medium composition used in the present invention will be specifically described as examples, and the present invention will be described in more detail, but the present invention is not limited to the examples.

(Test Example 1: Comparison with monolayer culture method in the proliferation of SKOV3 cells stimulated with each proliferation factor)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing a deuterated gellan gum having a final concentration of 0.015% (w/v) in MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. Next, the deuterated gellane/low-adhesion culture method was sown by adding the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) to the medium composition containing the above deuterated gellan gum to make 37000. The cells/mL were dispensed into a well of a 96-well flat-bottom ultra-low-adhesive surface microplate (manufactured by Corning, Inc., #3474) to make 135 μL per well. The "low-adhesion culture method" was performed by human ovarian cancer cells. Strain SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) was sown in a medium composition containing no deuterated gellan gum, and was dispensed into a 96-well flat-bottom ultra-low-adhesive surface microplate after 37000 cells/mL (manufactured by Corning, # In the well of 3474), each well was 135 μL. The "monolayer culture method" is carried out by seeding a human ovarian cancer cell line SKOV3 in a medium composition containing no deuterated gelatin, and then dispensing into a 96-well flat-bottom microplate after 2200 cells/mL. The company manufactured, #3585), was implemented in a hole of 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, 10 times of human HB-EGF (manufactured by PEPROTECH) was added to make a final concentration of 30, 100 ng/mL, and a 10-fold concentration of human TGF-β 1 (manufactured by PEPROTECH) was used to make the final concentration to be 3, 10 ng / mL, 10 times the concentration of human PDGF-BB (manufactured by PEPROTECH) to a final concentration of 3, 10 ng / mL, 10 times the concentration of human IGF-1 (manufactured by PEPROTECH) to a final concentration of 10, 100 ng / mL and a final concentration of 0.015% (w/v) of the medium composition containing deuterated gellan gum (de-salted orchid extract group) each 15 μL. In a monolayer culture group, 15 μL of a medium composition of each proliferation factor having a concentration of only 10 times was added. Continue to train for 7 days. For day 8 of culture medium, added with ATP reagent ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corporation) so suspended, after allowed to stand at room temperature for about 10 minutes measured at FlexStation 3 (manufactured Molecular Devices Corporation) Luminescence intensity (RLU value), the number of viable cells was measured by subtracting only the luminescence value of the medium.

As a result, it was revealed that human HB-EGF and human TGF-β 1 exhibited strong pharmacological effects compared to the monolayer culture method by using the SKOV3 cell proliferation assay using the medium composition used in the present invention. Further, Table 1 shows the control values of the RLU value (ATP measurement, luminescence intensity) % on the 8th day of culture.

(Test Example 2: TGF-β inhibitor in SKOV3 cells stimulated with TGF-β Effect of proliferation)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing a deuterated gellan gum having a final concentration of 0.015% (w/v) in MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. Then, the "de-deuterated gelatin/low-adhesion culture method" is carried out by culturing a human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) into a medium composition containing the above deuterated gellan gum. After 37000 cells/mL, the wells were dispensed into a well of a 96-well flat-bottom ultra-low-adhesion surface microplate (manufactured by Corning, Inc., #3474) to make 135 μL per well. The "monolayer culture method" is carried out by seeding a human ovarian cancer cell line SKOV3 in a medium composition containing no deuterated gelatin, and then dispensing into a 96-well flat-bottom microplate after 2200 cells/mL. The company manufactured, #3585), was implemented in a hole of 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, human TGF-β 1 (manufactured by PEPROTECH) and LY364947 (manufactured by Santa Cruz Biotechnology), which are TGF-β receptor inhibitors, were added at a final concentration of 30 ng/mL and finally. A concentration of 0.015% (w/v) of the medium composition containing deuterated gellan gum (de-salted gellan addition group) was 15 μL each. In a monolayer culture group, 15 μL of a medium composition of only human TGF-β 1 and LY364947 was added at a 10-fold concentration. Continue to train for 4 days. After about 10 minutes measured at FlexStation 3 (manufactured Molecular Devices Corporation) for 5 days were cultured, ATP reagent added ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corporation) to make the suspension was allowed to stand at room temperature The luminous intensity (RLU value) was measured by subtracting the luminescence value of the medium alone.

As a result, it was confirmed that the effect of the inhibitor on cell proliferation stimulated by human TGF-β 1 can be clearly determined by using the SKOV3 cell proliferation assay using the medium composition used in the present invention. On the other hand, in the single layer culture, the effect of the inhibitor can not be judged. Table 2 shows the RLU value (ATP measurement, luminescence intensity) on the fourth day of the monolayer culture, and Table 3 shows the RLU value (ATP measurement, luminescence intensity) on the fourth day of culture using the medium composition of the present invention.

(Test Example 3: Comparison of proliferation of A431 cells stimulated with each proliferation factor and monolayer culture method)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium concentration of 0.015% (w/v) of de-deuterated gellan gum was prepared in EMEM medium (DS PHARMA BIOMEDICAL) containing 10% (v/v) fetal bovine serum or There is no unadded medium composition of deuterated gellan gum. Next, the human squamous cell carcinoma cell line A431 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) was sown in the medium composition to which the above deuterated gellan gum was added, and was dispensed into a 96-well flat-bottom ultra-low-adhesive surface after being 37,000 cells/mL. The wells of a microplate (manufactured by Corning, #3474) were 135 μL per well. The monolayer culture method was carried out by seeding human squamous cell carcinoma cell line A431 in a medium composition containing no deuterated gellan gum, and then was dispensed into a 96-well flat-bottom microplate after being prepared at 2200 cells/mL (manufactured by Corning Co., Ltd.). , # 3585), the hole was made to have 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, 10 times the concentration of human HB-EGF (manufactured by PEPROTECH) was added to a final concentration of 100 ng/mL, and 10 times the concentration of human EGF (manufactured by PEPROTECH) to a final concentration of 10, 30 ng/mL. 10 times the concentration of human bFGF (manufactured by PEPROTECH Co., Ltd.) to a final concentration of 30, 100 ng/mL, 10 times the concentration of human TGF-β 1 (manufactured by PEPROTECH) to a final concentration of 10, 30 ng/mL, 10 times concentration Human PDGF-BB (manufactured by PEPROTECH) has a final concentration of 1, 10 ng/mL, and a 10-fold concentration of human IGF-1 (manufactured by PEPROTECH) to a final concentration of 10, 100 ng/mL and a final concentration of 0.015% (w/v). The medium composition containing the deuterated gellan gum (de-salted orchid extract group) was each 15 μL. In a monolayer culture group, 15 μL of a medium composition of each proliferation factor having a concentration of only 10 times was added. Continue to train for 7 days. For day 8 of culture medium, added with ATP reagent ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corporation) so suspended, after allowed to stand at room temperature for about 10 minutes measured at FlexStation 3 (manufactured Molecular Devices Corporation) Luminescence intensity (RLU value), the number of viable cells was measured by subtracting only the luminescence value of the medium.

As a result, it was revealed that human HB-EGF, human EGF, human bFGF, human TGF-β1, and human IGF1 were strong in comparison with the monolayer culture method by using the A431 cell proliferation assay using the medium composition used in the present invention. Drug effect. Further, Table 4 shows the control values of the RLU value (ATP measurement, luminescence intensity) % on the 8th day of the static culture.

(Test Example 4: Comparison of EMT marker expression of SKOV3 cells stimulated with human HB-EGF and monolayer culture method)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing a deuterated gellan gum having a final concentration of 0.015% (w/v) in MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. Then, the deuterated gellanine/low-binding culture method was sown by adding the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) to the medium composition to which the deuterated gellan gum was added, thereby making it 37,000 cells. After /mL, the mixture was dispensed into a well of a 96-well flat-bottom ultra-low-adhesion surface microplate (manufactured by Corning, Inc., #3474) to carry out 135 μL per well. The monolayer culture method was carried out by seeding a human ovarian cancer cell line SKOV3 in a medium composition containing no deuterated gellan gum, and then was dispensed into a 96-well flat-bottom microplate (manufactured by Corning, 7400 cells/mL, #3585) The holes were made with 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, 10 times of human HB-EGF (manufactured by PEPROTECH Co., Ltd.) was added to make a final concentration of 30, 100 ng/mL, and a final concentration of 0.015% (w/v) containing deuterated gellan gum. The culture medium composition (de-salted gelatin-added group) and the culture medium composition (monolayer culture group) of human HB-EGF at a 10-fold concentration were each 15 μL, and then culture was continued for 11 days. The culture solution containing the cancer cells was collected on the 12th day, and the cells were collected by centrifugation (400 g, 3 minutes). Total RNA was extracted from the cells using an RNeasy Mini Kit (manufactured by QIAGEN). Total RNA and PrimeScript ^TM RT Master Mix (manufactured TAKARA BIO Inc.) using GeneAmp PCR System 9700 (Applied Biosystems, Inc.) reverse transcription reaction synthesize cDNA. Each cDNA sample used in the PCR reaction was fractionated and diluted to 1/10 with sterilized water. Further, the sample used in the calibration curve was set using a fractionation so that the mixed cDNA was set in a quantitative range of from 1/3 to 1/43 in a ratio of 3 times. For the PCR reaction, each cDNA sample, a sample, a Premix Ex Taq ^TM (manufactured by TAKARA BIO Co., Ltd.), and various Taqman probes (manufactured by Applied Biosystems) were used, and a 7500 Instant Polymerase Chain Reaction System (Real Time PCR System) was used. Applied by Applied Biosystems). The mRNA of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was specifically used as an intrinsic control, and the expression of E-cadherin and N-cadherin mRNA was corrected by the value of GAPDH. Each probe used (Applied Biosystems, Inc.) is shown below.

GAPDH: HS99999905

E-cadherin: HS01023894

N-Cadherin: HS00983056

As a result, it was revealed that the SKOV3 cells using the medium composition used in the present invention have a higher expression amount of E-cadherin and a lower expression amount of N-cadherin than the monolayer culture method, and are considered to be associated with monolayer culture. Than, the shape is superior to the epithelium. Further, compared with the monolayer culture, it was revealed that the human HB-EGF showed a strong effect of lowering the expression of E-cadherin and promoting the expression of N-cadherin. Table 5 shows the expression values of E-cadherin mRNA on the 12th day of static culture, and Table 6 shows the expression values of N-Cadherin mRNA.

(Test Example 5: Effect of TGF-β receptor inhibitor on E-cadherin expression of SKOV3 cells stimulated with human TGF-β 1 )

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing a deuterated gellan gum having a final concentration of 0.015% (w/v) in MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. The deuterated gellanine/low-adhesion culture method was sown by adding the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) to the medium composition containing the above deuterated gellan gum to make 37000 cells/mL. Thereafter, the mixture was dispensed into a well of a 96-well flat-bottom ultra-low-adhesive surface microplate (manufactured by Corning, Inc., #3474) to carry out 135 μL per well. Each tray was kept in a static state in an incubator (37 ° C, 5% CO ₂ ) in such a manner that the final concentration of CO ₂ was 30 ng/mL. On the first day of culture, human TGF-β 1 (manufactured by PEPROTECH) and LY364947 (manufactured by Santa Cruz Biotechnology), which are TGF-β receptor inhibitors, and a final concentration of 0.015% (w/v) were added. The medium composition containing the deuterated gellan gum (de-salted orchid extract group) was each 15 μL, and the culture was continued for 11 days. The culture solution containing the cancer cells was collected on the 12th day, and the cells were collected by centrifugation (400 g, 3 minutes). Total RNA was extracted from the cells using an RNeasy Mini Kit (manufactured by QIAGEN). Total RNA and PrimeScript ^TM RT Master Mix (manufactured TAKARA BIO Inc.) using GeneAmp PCR System 9700 (Applied Biosystems, Inc.) reverse transcription reaction synthesize cDNA. Each cDNA sample used in the PCR reaction was fractionated and diluted to 1/10 with sterilized water. Further, the sample used in the calibration curve was set using a fractionation so that the mixed cDNA was set in a quantitative range of from 1/3 to 1/43 in a ratio of 3 times. PCR reactions using each cDNA sample, a calibration sample, Premix Ex Taq ^TM (TAKARA BIO Inc.) and various Taqman probes (manufactured by Applied Biosystems), using real time polymerase chain reaction system 7500 (manufactured by Applied Biosystems) embodiment . The mRNA of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was specifically used as an intrinsic control, and the expression of E-cadherin mRNA was corrected by the value of GAPDH. The following shows each probe used (Applied Biosystems)

GAPDH: HS99999905

E-cadherin: HS01023894

As a result, it was revealed that SKOV3 cells using the medium composition used in the present invention were stimulated with human TGF-β 1 and the expression amount of E-cadherin was lowered. In addition, LY364947 showed a concentration-dependent inhibition by a decrease in the expression of human TGF-β1. Table 7 shows the expression values of E-cadherin mRNA on the 12th day of static culture.

(Test Example 6: Effect of selective inhibitor on Panc02, 03 cell proliferation stimulated by human HB-EGF)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. The solution was used to prepare a medium composition of de-salted gellan gum having a final concentration of 0.015% (w/v) in DMEM medium (manufactured by WAKO Co., Ltd.) containing 10% (v/v) fetal bovine serum. The medium composition of the deuterated gellan gum was added. The deuterated gellanine/low-binding culture method was sown by adding the human pancreatic cancer cell line Panc02, 03 (manufactured by ATCC) to the medium composition containing the above deuterated gellan gum to make 37000 cells/ After the mL was dispensed into a well of a 96-well flat-bottom ultra-low-adhesive surface microplate (manufactured by Corning, Inc., #3474), each well was 135 μL. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, 10 times the concentration of human HB-EGF (manufactured by PEPROTECH), Gefitinib which is an EGF receptor inhibitor, and TGF- were added at a final concentration of 100 ng/mL. LY364947, a β-receptor inhibitor, Masitinib, which is a c-kit/FGF receptor-3 inhibitor (all manufactured by Santa Cruz Biotechnology), and a final concentration of 0.015% (w/v) 15 μL of each of the culture medium of the sulphurized gelatin (de-salted orchid extract group) was continued for 10 days. For 11 days culture fluid, ATP reagent added ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corporation) so suspended, after allowed to stand at room temperature for about 10 minutes measured at FlexStation 3 (manufactured Molecular Devices Corporation) Luminescence intensity (RLU value), the number of viable cells was measured by subtracting only the luminescence value of the medium.

As a result, it was confirmed that the culture medium composition used in the present invention was stimulated by human HB-EGF which is a living ligand of the EGF receptor, and the proliferation of Panc02, 03 cells was confirmed. Further, it is understood that this hyperproliferative action is inhibited by gefitinib which is an EGF receptor inhibitor. Table 8 shows On the eleventh day of the stationary culture, the RLU value (ATP measurement, luminescence intensity) of the conditions of addition to HB-EGF was added.

(Test Example 7: EGF receptor inhibitor for selective confirmation of SKOV3 cell proliferation and E-cadherin expression stimulated by human HB-EGF)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing a deuterated gellan gum having a final concentration of 0.015% (w/v) in MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. Then, the deuterated gellanine/low-binding culture method was sown by adding the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) to the medium composition to which the deuterated gellan gum was added, thereby making it 37,000 cells. After /mL, the mixture was dispensed into a well of a 96-well flat-bottom ultra-low-adhesion surface microplate (manufactured by Corning, Inc., #3474) to carry out 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, 10 times concentration of human HB-EGF (manufactured by PEPROTECH), gefitinib which is an EGF receptor inhibitor, and TGF-β receptor were added at a final concentration of 100 ng/mL. LY364947, an inhibitor, massetinib (both manufactured by Santa Cruz Biotechnology), a c-kit/FGF receptor-3 inhibitor, and a final concentration of 0.015% (w/v) of a medium containing deuterated gellan Each of the compositions (de-salted gellan addition group) was 15 μL, and cultivation was continued for 11 days. After about 10 minutes measured at FlexStation 3 (manufactured Molecular Devices Corporation) for 5 days were cultured, ATP reagent added ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corporation) to make the suspension was allowed to stand at room temperature Luminescence intensity (RLU value), the number of viable cells was measured by subtracting only the luminescence value of the medium. Further, the culture solution containing the cancer cells was collected on the 12th day, and the cells were collected by centrifugation (400 g, 3 minutes). Total RNA was extracted from the cells using an RNeasy Mini Kit (manufactured by QIAGEN). Total RNA and PrimeScript ^TM RT Master Mix (manufactured TAKARA BIO Inc.) using GeneAmp PCR System 9700 (Applied Biosystems, Inc.) reverse transcription reaction synthesize cDNA. Each cDNA sample used in the PCR reaction was fractionated and diluted to 1/10 with sterilized water. Further, the sample used in the calibration curve was set using a fractionation so that the mixed cDNA was set in a quantitative range of from 1/3 to 1/43 in a ratio of 3 times. PCR reactions using each cDNA sample, a calibration sample, Premix Ex Taq ^TM (TAKARA BIO Inc.) and various Taqman probes (manufactured by Applied Biosystems), using real time polymerase chain reaction system 7500 (manufactured by Applied Biosystems) embodiment . The mRNA of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was specifically used as an intrinsic control, and the expression of E-cadherin mRNA was corrected by the value of GAPDH. Each probe used (Applied Biosystems, Inc.) is shown below.

GAPDH: HS99999905

E-cadherin: HS01023894

As a result, it was confirmed that the medium composition used in the present invention was stimulated by human HB-EGF which is a bioligand of the EGF receptor, and the proliferative effect of SKOV3 cells was confirmed. This hyperproliferation acts to selectively inhibit gefitinib of the EGF receptor inhibitor. In addition, the expression of E-cadherin was reduced by stimulation with human HB-EGF. Although gefitinib which is an EGF receptor inhibitor was shown to have an inhibitory effect by the decrease in the expression of human HB-EGF, the inhibitory effect was not confirmed for other receptor inhibitors. Table 9 shows the RLU value (ATP measurement, luminescence intensity) under the conditions of HB-EGF addition on the fifth day of static culture. Further, Table 10 shows the expression values of E-cadherin mRNA on the 12th day of the stationary culture.

(Test Example 8: Selective confirmation test of TGF-β 1 receptor inhibitor for E-cadherin expression of SKOV3 cells stimulated with human TGF-β 1 )

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing 0.01% (w/v) of de-salted gelatin in a MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. Then, the deuterated gellanine/low-binding culture method was sown by adding the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) to the medium composition to which the deuterated gellan gum was added, thereby making it 37,000 cells. After /mL, the mixture was dispensed into a well of a 96-well flat-bottom ultra-low-adhesion surface microplate (manufactured by Corning, Inc., #3474) to carry out 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, human TGF-β 1 (manufactured by PEPROTECH), gefitinib which is an EGF receptor inhibitor, and TGF-β were added to a final concentration of 100 ng/mL. LY364947, a c-kit/FGF receptor-3 inhibitor, which is a C-kit/FGF receptor-3 inhibitor (all manufactured by Santa Cruz Biotechnology), and a final concentration of 0.015% (w/v) containing deuterated gellan The medium composition (de-deuterated orchid extract group) was each 15 μL, and cultivation was continued for 11 days. The culture solution containing the cancer cells was collected on the 12th day, and the cells were collected by centrifugation (400 g, 3 minutes). Total RNA was extracted from the cells using an RNeasy Mini Kit (manufactured by QIAGEN). Total RNA and PrimeScript ^TM RT Master Mix (manufactured TAKARA BIO Inc.) using GeneAmp PCR System 9700 (Applied Biosystems, Inc.) reverse transcription reaction synthesize cDNA. Each cDNA sample used in the PCR reaction was fractionated and diluted to 1/10 with sterilized water. Further, the sample used in the calibration curve was set using a fractionation so that the mixed cDNA was set in a quantitative range of from 1/3 to 1/43 in a ratio of 3 times. PCR reactions using each cDNA sample, a calibration sample, Premix Ex Taq ^TM (TAKARA BIO Inc.) and various Taqman probes (manufactured by Applied Biosystems), using real time polymerase chain reaction system 7500 (manufactured by Applied Biosystems) embodiment . The mRNA of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was specifically used as an intrinsic control, and the expression of E-cadherin mRNA was corrected by the value of GAPDH. Each probe used (Applied Biosystems, Inc.) is shown below.

GAPDH: HS99999905

E-cadherin: HS01023894

As a result, it was revealed that the medium composition used in the present invention was stimulated by human TGF-β 1 and the expression amount of E-cadherin was lowered. Further, although gefitinib which is a TGF-β 1 receptor inhibitor is selectively inhibited, the inhibitory effect of the inhibitor is not observed for other receptors. Table 11 shows the E-cadherin mRNA expression values on the 12th day of static culture.

(Test Example 9: Effect of molecular target drugs on SKOV3 cell proliferation and E-cadherin expression stimulated by human HB-EGF)

The deuterated gellan gum (KELCOGEL CG-LA, manufactured by Sanken Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to obtain 0.3% (w/v), and then stirred while heating at 90 °C. After dissolving, the aqueous solution was autoclaved at 121 ° C for 20 minutes. Using this solution, a medium containing a deuterated gellan gum having a final concentration of 0.015% (w/v) in MaCoy's 5a medium (DS PHARMA BIOMEDICAL) containing 15% (v/v) fetal bovine serum was prepared. Or no added media composition without deuterated gellan gum. Then, the deuterated gellanine/low-binding culture method was sown by adding the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL Co., Ltd.) to the medium composition to which the deuterated gellan gum was added, thereby making it 37,000 cells. After /mL, the mixture was dispensed into a well of a 96-well flat-bottom ultra-low-adhesion surface microplate (manufactured by Corning, Inc., #3474) to carry out 135 μL per well. Each tray was kept in a static state in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the first day of culture, a 10-fold concentration of human HB-EGF (manufactured by PEPROTECH), gefitinib as an EGF receptor inhibitor, and a MEK inhibitor were added to each other at a final concentration of 100 ng/mL. Trametinib, MK-2206 which is an Akt inhibitor, Cyt387 which is a JAK inhibitor (all manufactured by Santa Cruz Biotechnology), and final denaturized gelatin containing 0.015% (w/v) The medium composition (de-deuterated orchid extract group) was each 15 μL, and cultivation was continued for 11 days. For 7 days culture fluid, ATP reagent added ^{150μL (CellTiter-Glo TM Luminescent Cell} Viability Assay, Promega Corporation) so suspended, after allowed to stand at room temperature for about 10 minutes measured at FlexStation 3 (manufactured Molecular Devices Corporation) Luminescence intensity (RLU value), the number of viable cells was measured by subtracting only the luminescence value of the medium. Further, the culture solution containing the cancer cells was collected on the 12th day, and the cells were collected by centrifugation (400 g, 3 minutes). Total RNA was extracted from the cells using an RNeasy Mini Kit (manufactured by QIAGEN). Total RNA and PrimeScript ^TM RT Master Mix (manufactured TAKARA BIO Inc.) using GeneAmp PCR System 9700 (Applied Biosystems, Inc.) reverse transcription reaction synthesize cDNA. The synthesized cDNA was dispensed and diluted to 1/10 with sterile water and used in PCR. The synthesized cDNA was dispensed, mixed, and used in the calibration curve preparation. It is set at a quantitative ratio ranging from 1/3 to 1/43 in a ratio of 3 times. Using each cDNA sample, a calibration sample, Premix Ex Taq ^TM (TAKARA BIO Inc.) and various Taqman probes (manufactured by Applied Biosystems), using real time polymerase chain reaction system 7500 (manufactured by Applied Biosystems) embodiment PCR. The mRNA of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was specifically used as an intrinsic control, and the expression of E-cadherin mRNA was corrected by the value of GAPDH. Each probe used (Applied Biosystems, Inc.) is shown below.

GAPDH: HS99999905

E-cadherin: HS01023894

As a result, it was confirmed that the medium composition of the present invention was stimulated by human HB-EGF which is a living ligand of the EGF receptor, and the proliferative effect of SKOV3 cells was confirmed. Further, it was revealed that MK-2206, which is an Akt inhibitor, selectively inhibits the hypertrophy. On the other hand, trimetinib showed a weak inhibitory effect, and Cyt387 showed no inhibitory effect. In addition, it is clear that the expression of E-cadherin is reduced by the stimulation of human HB-EGF. However, with respect to human HB-EGF, the expression of E-cadherin was lowered, and no inhibitory effect was observed by the three inhibitors. Table 12 shows the RLU value (ATP measurement, luminescence intensity) under the conditions of HB-EGF addition on the 7th day of static culture. Further, Table 13 shows the expression values of E-cadherin mRNA on the 12th day of the stationary culture.

[Possibility of industrial use]

By the present invention, the reactivity of cancer cells in the transforming growth factor, the insulin-like proliferation factor, and the fibroblast growth factor can be detected with high sensitivity. Thus, the methods of the invention are useful in screening for anticancer agents that block such proliferation factors.

When the method of the present invention is used, the epithelial morphology such as cancer cells can be well maintained as compared with the monolayer culture. Moreover, if the method of the present invention is used, since the epithelial mesenchymal transition of the cancer cells can be reproduced in vitro, the resistance is good. Screening of anticancer agents that impede epithelial transition is useful.

The contents described herein are included in all publications including patents, patent application specifications, and scientific publications, which are hereby incorporated by reference in its entirety to the extent the extent

This patent application is based on Japanese Patent Application No. 2014-195814 (filed on Sep. 2, 2014), the entire contents of which are incorporated herein by reference.

Claims (23)

A method for culturing an adherent cancer cell line, wherein the adherent cancer cell line is reactive with any one of a proliferation factor selected from the group consisting of a transforming growth factor, an insulin-like growth factor, and a fibroblast growth factor, the culture method The invention comprises: an adhesive cancer cell which is reactive with any one of the proliferation factors selected from the group consisting of a transforming growth factor, an insulin-like proliferation factor and a fibroblast growth factor, and the cell or tissue can be contained in the proliferation factor The culture medium of the suspended and cultured construct is subjected to suspension culture. The culture method according to claim 1, wherein the proliferation factor is a transgenic growth factor. The culture method according to claim 2, wherein the transforming growth factor is TGF-β 1 . The culture method according to any one of claims 1 to 3, wherein the structure contains gellan gum. The culture method according to any one of claims 1 to 4, wherein the composition of the medium has a viscosity at 37 ° C of 8 mPa. s below. A method for testing the reactivity of an adherent cancer cell to a proliferation factor, the proliferation factor being selected from the group consisting of a transforming growth factor, an insulin-like proliferation factor, and a fibroblast growth factor, the method comprising: 1) culturing an adherent cancer cell in the presence or absence of the proliferation factor, and containing a construct capable of suspending and culturing the cell or tissue Suspension culture in the base composition; (2) measurement of proliferation of cultured cancer cells; and (3) comparison of proliferation of cancer cells cultured in the presence of the proliferation factor and proliferation in the absence of the proliferation factor. The method of claim 6, wherein the structure is a degummed gellan. The method of claim 6 or 7, wherein the composition of the medium has a viscosity at 37 ° C of 8 mPa. s below. An anticancer agent screening method for adherent cancer cells, which is responsive to any one of a plurality of proliferation factors selected from the group consisting of a transforming growth factor, an insulin-like growth factor, and a fibroblast growth factor. The screening method comprises (1) suspending and cultured the cancer cells in a medium composition containing the proliferation factor and a structure in which the cells or tissues are suspended and cultured in the presence and absence of the test substance; (2) The growth of the cultured cancer cells is measured; and (3) the proliferation of the cancer cells cultured in the presence of the test substance and the proliferation in the absence of the test substance are compared. The screening method according to claim 9, wherein the structure comprises deuterated gellan gum. The screening method according to claim 9 or claim 10, wherein the composition of the medium has a viscosity at 37 ° C of 8 mPa. s below. A method for maintaining an epithelial morphology such as a cancer cell, comprising: constituting the cancer cell in a structure containing a cell or tissue suspension and culture The medium composition is suspended in culture. The maintenance method according to claim 12, wherein the structure contains deuterated gellan gum. The maintenance method according to claim 12 or claim 13, wherein the composition of the medium has a viscosity at 37 ° C of 8 mPa. s below. A method for inducing epithelial transition of a cancer cell comprises: suspending a cancer cell having an epithelial-like morphology in a culture medium containing an epithelial-mesenchymal transition-inducing factor and a construct capable of suspending or culturing the cell or tissue . The method of inducing the invention of claim 15, wherein the structure comprises deuterated gellan gum. The method according to claim 15 or 16, wherein the composition of the medium has a viscosity at 37 ° C of 8 mPa. s below. A screening method for screening a substance for transepithelial transition of a cancer cell, comprising: (1) in the presence and absence of a test substance, a cancer cell having an epithelial-like shape may contain a cell or Suspension culture of tissue composition of tissue suspension and culture; (2) evaluation of epithelial transition of cultured cancer cells; and (3) comparison of epithelial transition of cancer cells cultured in the presence of test substance The epithelial transition is performed in the absence of the substance to be tested. The screening method according to claim 18, wherein the structure comprises deuterated gellan gum. The screening method described in claim 18 or 19 of the patent application, The viscosity of the medium composition at 37 ° C is 8 mPa. s below. A screening method for screening substances for blocking epithelial transition of cancer cells, comprising: (1) transforming epithelial mesenchymal cells with epithelial stroma in the presence and absence of a test substance Inducing factor and suspension culture in a medium composition of a construct which can suspend or culture cells or tissues; (2) evaluating epithelial transition of cultured cancer cells; and (3) comparing culture in the presence of a test substance The epithelial-mesenchymal transition of the cancer cells and the absence of the test substance are in the epithelial-mesenchymal transition. The screening method according to claim 21, wherein the structure comprises deuterated gellan gum. The screening method according to claim 21 or 22, wherein the composition of the medium has a viscosity at 37 ° C of 8 mPa. s below.