Culture medium composition and culturing method of cells or tissue using said composition

Abstract

To provide a culture medium composition for culturing animal or plant cells and / or tissue especially three dimensionally or in a floating state, and a culture method of the animal or plant cells and / or tissue using the culture medium composition.SOLUTION: The present invention provides a culture medium composition forming a structure of indeterminate form in a liquid culture medium, the structure being dispersed evenly in the solution without substantially increasing the viscosity of the solution, and by substantially holding the cells and / or tissue in the solution has the effect of preventing precipitation thereof, and provides a culturing method of cells and / or tissue using same which cultures cells and / or tissue in a floating state.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a culture medium composition containing a structure capable of suspending cells or tissues, and a method for culturing cells or tissues characterized by using the culture medium composition. The culture medium composition of the present invention and the cell culture method using the same can be suitably used particularly when culturing animal and plant cells and / or tissues in a suspended state.

Background Art

[0002] In recent years, technologies for growing or maintaining various organs, tissues, and cells that play different roles in animals and plants in vitro have been developed. Growing or maintaining these organs and tissues in vitro is called organ culture and tissue culture, respectively, and growing, differentiating, or maintaining cells separated from organs and tissues in vitro is called cell culture. Cell culture is a technique for growing, differentiating, or maintaining isolated cells in vitro, and is essential for detailed analysis of the functions and structures of various organs, tissues, and cells in vivo. In addition, the cells and / or tissues cultured by this technique are used in various fields such as the evaluation of the efficacy and toxicity of chemical substances, pharmaceuticals, etc., the mass production of useful substances such as enzymes, cell growth factors, antibodies, etc., regenerative medicine to supplement organs, tissues, and cells lost due to diseases and defects, plant variety improvement, and the creation of genetically modified crops.

[0003] Animal-derived cells are roughly divided into floating cells and adherent cells according to their properties. Floating cells are cells that do not require a substrate for growth and proliferation, and adherent cells are cells that require a substrate for growth and proliferation, but most of the cells that make up the living body are the latter adherent cells. As methods for culturing adherent cells, monolayer culture, dispersed culture, embedding culture, microcarrier culture, and sphere culture are known.

[0004] Monolayer culture is a method of culturing target cells in a monolayer using a culture vessel made of glass or synthetic polymer materials with various surface treatments, or using auxiliary cells called feeder cells as a scaffold. It is the most common and widespread method. For example, those with various surface treatments (such as plasma treatment, corona treatment, etc.) applied to polystyrene, those coated with cell adhesion factors such as collagen, fibronectin, polylysine, or those with feeder cells seeded in advance, etc., culture methods using culture vessels of various shapes or properties have been developed. However, monolayer culture has problems such as being unable to maintain the specific functions that cells have in vivo for a long time because its two-dimensional culture environment is completely different from the in vivo environment, being unable to reconstruct tissues similar to those in vivo, not being suitable for large-scale cell culture because the number of cells per unit area is limited (Patent Document 1). Also, in the method of culturing target cells on feeder cells, the separation of feeder cells and target cells may be a problem (Non-Patent Document 1).

[0005] Suspension culture is a method of culturing adherent cells in a floating state by inhibiting the adhesion of cells to the culture vessel by continuously stirring the culture solution in a culture vessel with a surface treatment that inhibits cell adhesion after seeding the cells in the medium. However, since the adherent cells cultured by this method cannot adhere to a scaffold, it cannot be applied to cells that require adhesion to a scaffold for cell proliferation. Also, since the original cell functions cannot be exerted due to being constantly disrupted by shear force, it may not be possible to culture a large number of functional cells (Non-Patent Document 2).

[0006] Embedding culture is a method of embedding and fixing cells in a solid or semi-solid gel substrate such as agar, methylcellulose, collagen, gelatin, fibrin, agarose, alginate, etc. and culturing them. This method enables three-dimensional culturing of cells in a form close to that in vivo. In some cases, the gel substrate itself can promote cell growth and differentiation. Therefore, compared to monolayer culture or dispersed culture, it is possible to culture cells at a high density while maintaining their functions (Patent Documents 2 and 3). Furthermore, a method has been developed in which microcapsules with a size of 100 to 300 μm are created with these gel substrates embedded in cells, and the cells are cultured in an aqueous medium while dispersing the microcapsules (Non-Patent Document 3). However, these methods have problems such as the inability to observe cultured cells over time when the gel substrate does not transmit visible light, the need for complicated and cell-damaging operations such as enzyme treatment (for example, collagenase treatment in the case of a collagen gel) to recover cells from the medium containing the gel substrate or microcapsules because the medium or microcapsules containing the gel substrate has a high viscosity, and difficulty in medium replacement required for long-term culture. In recent years, techniques have been developed that enable cell recovery from gel substrates by treatments such as heat and shear force. However, heat, shear force, etc. may have an adverse effect on cell functions, and the safety of the gel substrate for living organisms has not yet been clearly elucidated (Patent Documents 4 and 5, Non-Patent Documents 4, 5, 6, and 7). In addition, sol-like foods have been developed in the food field to uniformly disperse and suspend granular foods such as small-cut fruits and vegetables and prevent their precipitation and floating. However, the sol-like foods do not consider recovering the dispersed granular foods, and no examination has been made as to whether cells and tissues can be cultured in a suspended state (Patent Document 6).

[0007] Microcarrier culture involves growing cells in a monolayer on the surface of microparticles (hereinafter also referred to as microcarriers) that are slightly heavier than water, and agitating the microparticles in a culture vessel such as a flask to perform culture in a suspended state. Usually, the microcarriers used in this method have a diameter of 100 to 300 μm and a surface area of 3000 to 6000 cm 2 / g, spherical particles with a specific gravity of 1.03 to 1.05, which are composed of materials such as dextran, gelatin, alginic acid, or polystyrene. Charged groups such as collagen, gelatin, or dimethylaminoethyl can be imparted to the surface of the microcarriers to facilitate cell adhesion. Since this method can extremely increase the culture area, it is applied to the large-scale culture of cells (Patent Documents 7 and 8). However, it is difficult to attach the target cells almost uniformly to all microcarriers, and there are problems such as cells detaching from the microcarriers due to the shear force during agitation and cells being damaged (Non-Patent Document 8).

[0008] Sphere culture is a method in which target cells form aggregates (hereinafter also referred to as spheres) consisting of dozens to hundreds of cells, and then the aggregates are statically placed or shaken in a culture medium for culture. Spheres have a high cell density, and cell-cell interactions and cell structures similar to those in the in vivo environment are reconstructed. It is known that cell functions can be maintained in culture for a long time compared to monolayer culture and dispersed culture methods (Non-Patent Documents 9 and 10). However, in sphere culture, when the size of the spheres is too large, it becomes difficult to supply nutrients and discharge waste products inside the spheres, so large spheres cannot be formed. In addition, since the formed spheres need to be cultured in a dispersed state on the bottom surface of the culture container, it is difficult to increase the number of spheres per unit volume, and it is not suitable for large-scale culture. Furthermore, as methods for creating spheres, hanging drop culture, culture on a cell non-adhesive surface, culture in a microwell, rotary culture, culture using a cell scaffold, aggregation by centrifugal force, ultrasonic waves, electric fields, or magnetic fields are known. However, these methods have problems such as complicated operations, difficulty in recovering spheres, difficulty in controlling size and mass production, unclear effects on cells, and the need for special dedicated containers and devices (Patent Document 9).

[0009] On the one hand, regarding plants, cells, protoplasts with cell walls removed, or organs, tissues, calli such as leaves, stems, roots, growing points, seeds, embryos, and pollen of plants can be cultured and propagated in a sterile state. Using such plant tissue and cell culture techniques, it is also possible to improve plant varieties and produce useful substances. As a method for rapidly proliferating plant cells and tissues in large quantities in a short period, a method of suspension-culturing plant cells and tissues in a liquid medium is known (Non-Patent Document 11). To achieve their good growth, it is important to supply sufficient oxygen, maintain a uniform mixing state, and further prevent cell damage. The supply of oxygen to the culture solution and the suspension of cells and tissues can be carried out by combining aeration and mechanical stirring, or by aeration only. In the former case, growth inhibition may occur due to cell and tissue damage caused by stirring. On the other hand, in the latter case, although shear of cells and tissues is small, it may be difficult to maintain a uniform mixing state in high-density culture, resulting in problems such as sedimentation of cells and tissues and a decrease in growth efficiency.

[0010] Furthermore, for the research and development of anticancer drugs or the selection of appropriate anticancer drugs in cancer treatment, the anticancer activity of drugs against cancer cells is evaluated by culturing cancer cells in vitro in a culture solution containing candidate drugs or anticancer drugs. However, existing methods for evaluating anticancer activity have problems such as a deviation between the in vitro evaluation results and the actual clinical effects. To improve this problem, techniques for performing activity evaluation under cell culture conditions that reproduce the in vivo environment as much as possible have been developed. For example, a method has been developed in which cancer cells are embedded in a support such as soft agar, collagen gel, or hydrogel to culture the cancer cells in an environment where adhesion to the culture vessel is inhibited and evaluate the anticancer drug (Patent Document 10, Non-Patent Documents 12 and 13). In addition, methods have been developed in which the surface of the culture vessel is coated with a cell adhesion-inhibiting material or the surface is specially processed to inhibit cell adhesion and culture the cancer cells in an aggregated state (sphere culture) to evaluate the anticancer activity (Patent Documents 11 and 12).

[0011] However, these cancer cell culture methods have various problems. The process of creating the culture vessel and the operation of cell culture are complicated. The operation of recovering cells from a support such as collagen and evaluating the anti-cancer activity is complicated. When the support is an animal-derived component, it may be expensive and its supply may be restricted. There are various problems such as cell aggregates (spheres) aggregating with each other, resulting in excessive size and low cell viability and reproducibility. Moreover, when screening for anti-cancer agents, a simple, uniform, and highly reproducible cancer cell culture method that can handle a large number of samples is required.

[0012] In addition, by culturing hepatocytes in vitro in a culture solution containing a pharmaceutical candidate or a drug, various activities of the pharmaceutical candidate or the drug on hepatocytes are evaluated. However, when hepatocytes are cultured in vitro, their functions originally present in vivo may be lost. Therefore, existing hepatocyte culture methods have problems such as the inability to accurately evaluate a pharmaceutical candidate or a drug and the difficulty in evaluating a large number of samples. To overcome this problem, a technique for performing activity evaluation under cell culture conditions that reproduce the in vivo environment as much as possible has been developed. For example, methods for culturing hepatocytes on the extracellular matrix such as collagen, laminin, Matrigel (registered trademark), etc. to maintain the functions of hepatocytes have been developed (Patent Document 13, Non-Patent Documents 14, 15). Also, methods for forming cell aggregates (spheres) of hepatocytes to maintain the functions of hepatocytes have been developed by treating the surface of the culture vessel with a cell adhesion-inhibiting material, inhibiting cell adhesion by applying special processing to the container surface, vibrating the culture vessel, etc. (Patent Documents 14, 15, Non-Patent Documents 16, 17).

[0013] However, these hepatocyte culture methods have various problems. The processes of preparing the culture containers and operating cell cultures are complicated. The operations for recovering cells from supports such as collagen and evaluating hepatocyte functions are complicated. When the support is a component derived from animals, it is expensive and its supply may be restricted. There are problems such as cell aggregates (spheres) aggregating with each other and becoming overly large, resulting in low cell viability and reproducibility. Moreover, when screening drug candidates or drugs, a simple, uniform, highly reproducible hepatocyte culture method that can process a large number of samples is required.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

Patent Document 15

Non-Patent Document

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Problems to be Solved by the Invention

[0016] An object of the present invention is to solve the above problems of the prior art and provide a medium composition for culturing animal and plant cells and / or tissues, particularly in a three-dimensional or floating state, and a method for culturing animal and plant cells and / or tissues using the medium composition. Another object of the present invention is to solve the above problems of the prior art and provide a medium composition for culturing cell aggregates (spheres) of cancer cells in a three-dimensional environment and a test method for cancer cells using the medium composition. Alternatively, an object of the present invention is to solve the above problems of the prior art and provide a medium composition for culturing cell aggregates (spheres) of hepatocytes in a three-dimensional environment and a test method for cancer cells using the medium composition. Furthermore, an object of the present invention is to provide a medium additive that promotes the growth of cancer cells in the culture of cancer cells, and to provide a medium additive that suppresses the decrease in the number of hepatocytes in the culture of hepatocytes.

Means for Solving the Problems

[0017] As a result of intensive studies on the floating effect of cells and / or tissues in various compounds and liquid media containing them, the present inventors have succeeded in discovering a structure that can uniformly disperse cells and / or tissues in a floating state without substantially increasing the viscosity in the liquid medium. When using a medium composition containing at least the said structure, it was found that cells and / or tissues can grow, differentiate or be maintained while maintaining the floating state. Furthermore, it was also found that cultured cells and / or tissues can be easily recovered from the said medium composition, leading to the completion of the present invention. In addition, as a result of intensive studies on the effect of various compounds and liquid media containing them on cancer cell aggregates (spheres), the present inventors have succeeded in discovering a medium composition that can prevent the aggregation of the said spheres and disperse them uniformly. When using the said medium composition, it is possible to culture the said spheres with a high survival rate, and it was found that the activity of anticancer agents against cancer cells can be evaluated efficiently and sensitively. Furthermore, it was also found that cultured spheres can be easily recovered from the said medium composition for evaluation, leading to the completion of the present invention. Alternatively, as a result of intensive studies on the effect of various compounds and liquid media containing them on hepatocyte aggregates (spheres), the present inventors have succeeded in discovering a medium composition that can prevent the aggregation of the said spheres and disperse them uniformly. When using the said medium composition, it is possible to culture the said spheres with a high survival rate while maintaining the function as hepatocytes, and it was found that the effect of pharmaceutical candidate agents or pharmaceuticals on hepatocytes can be evaluated efficiently and sensitively. Furthermore, it was also found that cultured spheres can be easily recovered from the said medium composition for evaluation, leading to the completion of the present invention. Furthermore, the inventors of the present invention found that when deacylated gellan gum or a salt thereof was added to a medium containing cancer cells, the growth of cancer cells was significantly promoted, and thus completed the present invention. In addition, the inventors of the present invention found that when deacylated gellan gum or a salt thereof was added to a medium containing hepatocytes, the decrease in the number of hepatocytes was suppressed, and thus completed the present invention.

[0018] That is, the present invention is as follows:

[0019] (1) A culture medium composition, characterized by containing a structure capable of suspending and culturing cells or tissues. Composition. (2) The culture medium composition according to (1), which enables replacement treatment of the culture medium composition during culture and recovery of cells or tissues after completion of culture. (3) The culture medium composition according to (1), which does not require any of temperature change, chemical treatment, enzyme treatment, and shear force when recovering cells or tissues from the culture medium composition. (4) The culture medium composition according to (1), characterized in that the viscosity is 8 mPa·s or less. (5) The culture medium composition according to (1), characterized in that when the size of the structure is filtered through a filter, it passes through a filter with a pore size of 0.2 μm to 200 μm. (6) The culture medium composition according to (1), characterized in that the structure contains a polymer compound. (7) The culture medium composition according to (6), characterized in that the polymer compound contains a polymer compound having an anionic functional group. (8) The culture medium composition according to (6), characterized in that the polymer compound is a polysaccharide. (9) The culture medium composition according to (7), characterized in that at least one kind is selected from the group consisting of a carboxy group, a sulfo group, and a phosphate group as the anionic functional group. (10) The medium composition according to (8), wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectinic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratan sulfate, chondroitin sulfate, delta mannan sulfate, laminarin sulfate, and salts thereof. (11) The medium composition according to (10), wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, deacylated gellan gum, diutan gum, xanthan gum, carrageenan, and salts thereof. (12) The medium composition according to (10) or (11), wherein the polysaccharide is deacylated gellan gum or a salt thereof. (13) The medium composition according to (12), wherein the final concentration of the deacylated gellan gum or a salt thereof in the medium composition is 0.001 to 1.0% (weight / volume). (14) The medium composition according to (13), further comprising a polysaccharide other than deacylated gellan gum or a salt thereof. (15) The medium composition according to (14), wherein the polysaccharide is at least one selected from the group consisting of xanthan gum, alginic acid, carrageenan, diutan gum, and salts thereof. (16) The medium composition according to (14), wherein the polysaccharide is at least one selected from the group consisting of methylcellulose, locust bean gum, and salts thereof. (17) The medium composition according to any one of (1) to (16), further comprising a metal ion. (18) The medium composition according to (17), wherein the metal ion is a divalent metal ion. (19) The medium composition according to (18), wherein the metal ion is at least one selected from the group consisting of calcium ion, magnesium ion, zinc ion, iron ion, and copper ion. (20) The culture medium composition according to (19), wherein the metal ion is a calcium ion. (21) The culture medium composition according to (20), further comprising a metal ion other than calcium ion. (22) The culture medium composition according to (21), wherein the metal ion is at least one selected from the group consisting of magnesium ion, sodium ion, and potassium ion. (23) The culture medium composition according to any one of (1) to (22), further comprising an extracellular matrix and / or a cell adhesion molecule. (24) The culture medium composition according to (23), wherein the extracellular matrix is at least one selected from the group consisting of collagen, hyaluronic acid, and proteoglycan. (25) The culture medium composition according to (23), wherein the cell adhesion molecule is at least one selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin. (26) The culture medium composition according to any one of (1) to (25), which is for cell culture. (27) The culture medium composition according to (26), wherein the cell is an adherent cell or a floating cell. (28) The culture medium composition according to (27), wherein the adherent cell is attached to a microcarrier. (29) The culture medium composition according to (27), wherein the adherent cell is embedded in a carrier. (30) The culture medium composition according to (27), wherein the adherent cell is a sphere. (31) The culture medium composition according to (27), wherein the adherent cell is selected from the group consisting of pluripotent stem cells, cancer cells, and hepatocytes. (32) A cell or tissue culture comprising the culture medium composition according to any one of (1) to (31) and a cell or tissue. (33) A method for culturing a cell or tissue, which comprises culturing the cell or tissue in the culture medium composition according to any one of (1) to (31). (34) The culturing method according to (33), characterized in that the cells are selected from the group consisting of pluripotent stem cells, cancer cells, and hepatocytes. (35) A method for recovering cells or tissues, characterized by separating cells or tissues from the culture of (32). (36) The recovery method according to (35), characterized in that the separation is performed by filtration, centrifugation, or magnetic separation. (37) A method for producing spheroids, characterized by culturing adherent cells in the culture medium composition according to any one of (1) to (31). (38) A method for screening an anticancer agent, comprising: (a) a step of culturing cancer cells in the culture medium composition according to any one of claims 1 to 31 in the presence and absence of a test substance; and (b) a step of analyzing the change in the growth of cancer cells. (39) The method according to (38), further comprising a step of selecting, as a candidate substance, a substance that suppresses the growth of cancer cells as compared with the case in the absence of the test substance. (40) A method for screening a pharmaceutical candidate substance that acts on hepatocytes, comprising: (a) a step of culturing hepatocytes in the culture medium composition according to any one of claims 1 to 31 in the presence and absence of a test substance; and (b) a step of analyzing the change in the physiological function of hepatocytes. (41) The method according to (40), further comprising a step of selecting, as a candidate substance, a substance that suppresses or increases the physiological function of hepatocytes as compared with the case in the absence of the test substance. (42) A method for evaluating the efficacy or toxicity of a pharmaceutical candidate substance that acts on hepatocytes, comprising: (a) a step of culturing hepatocytes in the culture medium composition according to any one of claims 1 to 31 in the presence and absence of a test substance; and (b) a step of analyzing the change in the physiological function of hepatocytes. (43) A medium additive for preparing a medium composition capable of culturing cells or tissues in suspension, characterized in that a polymer compound is dissolved or dispersed in a solvent. (44) The medium additive according to (43), characterized in that it is in a sterilized state. (45) The medium additive according to (43) or (44), wherein the polymer compound is a polymer compound having an anionic functional group. (46) The medium additive according to (43) or (44), wherein the polymer compound is deacylated gellan gum or a salt thereof. (47) A method for producing a medium composition capable of culturing cells or tissues in suspension, characterized by mixing a polymer compound and a medium. (48) Mixing the medium additive according to any one of (43) to (46) with a medium. The method for producing a medium composition according to (47), characterized by the above. (49) The method for producing a medium composition according to (48), characterized in that the medium is dissolved or dispersed in a solvent. (50) The method for producing a medium composition according to (47), wherein the polymer compound is a polymer compound having an anionic functional group. (51) The method for producing a medium composition according to (50), wherein the polymer compound is deacylated gellan gum or a salt thereof. (52) The method for producing a medium composition according to (47), characterized by mixing the polymer compound and the medium with water. (53) The method for producing a medium composition according to (52), characterized by heating at 80 to 130 °C after mixing with water. (54) The method for producing a medium composition according to (53), characterized by heating at 100 to 125 °C. (55) The method for producing a medium composition according to (47), characterized by filter sterilization. (56) The method for producing a medium composition according to (55), characterized in that the filter sterilization is carried out by passing through a filter of 0.1 to 0.5 μm. (57) A culture medium additive for cancer cells containing deacylated gellan gum or a salt thereof, or diutan gum or a salt thereof. (58) The additive according to (57), which promotes the growth of cancer cells in the culture of cancer cells. (59) The additive according to (57), which is used for evaluating the anticancer activity of an anticancer agent. (60) A culture medium composition for cancer cells comprising the additive according to any one of (57) to (59). (61) A method for culturing cancer cells, comprising culturing the cancer cells in the presence of the additive according to any one of (57) to (59) or in the culture medium composition according to (60). (62) A method for evaluating the activity of an anticancer agent against cancer cells, comprising culturing the cancer cells in the presence of the additive according to any one of (57) to (59) or in the culture medium composition according to (60). (63) The method according to (61) or (62), characterized in that the cancer cells form cell aggregates in the culture medium composition for cancer cells. (64) The method according to (61) or (62), characterized in that the culture vessel for culturing the cancer cells suppresses the adhesion of cancer cells. (65) A culture medium additive for hepatocytes containing deacylated gellan gum or a salt thereof, or diutan gum or a salt thereof. (66) The additive according to (65), which suppresses the decrease in the number of hepatocytes in the culture of hepatocytes. (67) The additive according to (65), which is used for evaluating the effect of a pharmaceutical and a pharmaceutical candidate on hepatocytes. (68) A culture medium composition for hepatocytes comprising the additive according to any one of (65) to (67). (69) A method for evaluating the activity of a pharmaceutical and a pharmaceutical candidate against hepatocytes, comprising culturing the hepatocytes in the presence of the additive according to any one of (65) to (67) or in the culture medium composition according to (68). (70) The method according to (69), characterized in that the hepatocytes form cell aggregates in the culture medium composition for hepatocytes. The method according to (69), characterized in that the culture vessel for culturing the hepatocytes suppresses the adhesion of the hepatocytes.

Advantages of the Invention

[0020] The present invention provides a culture medium composition containing a specific compound (hereinafter also referred to as a specific compound), particularly a structure of a polymer compound having an anionic functional group. When using this culture medium composition, cells and / or tissues can be cultured in a floating state without operations such as shaking and rotation that may cause cell and tissue damage and loss of function. Furthermore, when using this culture medium composition, the culture medium can be easily exchanged during culture, and the cultured cells and / or tissues can be easily recovered. The present invention applies the culture method to cells and / or tissues collected from an animal organism or a plant body, and a large amount of target cells and / or tissues can be prepared without impairing their functions. The cells and / or tissues obtained by the culture method can be used for evaluating the efficacy and toxicity of chemical substances, pharmaceuticals, etc., mass-producing useful substances such as enzymes, cell growth factors, antibodies, and regenerative medicine for supplementing organs, tissues, and cells lost due to diseases and defects. In particular, the culture medium composition prepared from deacylated gellan gum is excellent and has the following characteristics. Since the concentration for expressing the performance is extremely low (about one digit lower), the influence on the culture medium components is minimized. Also, it is not easily lumped when dissolved in water, so there are few troubles even when manufacturing in large quantities. Furthermore, since the viscosity in the concentration range where the performance is expressed is low, the operability such as the recovery of cells and / or tissues is extremely good. In addition, when using the culture medium composition of the present invention, the aggregation of cancer cell aggregates (spheres) is suppressed, and since the spheres can be cultured in a dispersed state, the growth of cancer cells can be promoted. Furthermore, when using the culture medium composition, when evaluating an anticancer agent, the anticancer agent can be easily added to the culture medium, and a detection reagent for evaluating cell growth can be easily added. In addition, since the cultured cancer cells can be recovered, the functional evaluation of the recovered cells can also be easily carried out. The present invention can be suitably used when performing efficacy evaluation and screening of chemical substances, anticancer agents, etc. using the cancer cells obtained by the culture method. When cultured in the culture medium composition of the present invention, since the influence from the non-in vivo environment in two-dimensional culture is small and only cell adhesion occurs, the sensitivity of HB-EGF (heparin-binding epidermal growth factor-like growth factor), which promotes carcinogenesis, increases in cancer cells, and the sensitivity to an EGF receptor inhibitor downstream thereof can be enhanced. Furthermore, the sensitivity to an MEK inhibitor and an Akt inhibitor, which are important signal transduction pathways for cancer cell anchorage-independent growth, can also be enhanced. Alternatively, when using the culture medium composition of the present invention, the aggregation of hepatocyte aggregates (spheres) is suppressed, and since the spheres can be cultured in a dispersed state, the survival and cell functions of hepatocytes can be maintained in vitro. Furthermore, when using the culture medium composition, when evaluating a drug candidate or a drug, the drug candidate or the drug can be easily added to the culture medium, and a detection reagent for evaluating cell functions can be easily added. In addition, since the cultured hepatocytes can be recovered, the functional evaluation of the recovered cells can also be easily carried out. The present invention can be suitably used when performing efficacy and toxicity evaluation and screening of chemical substances, drug candidates or drugs, etc. using the hepatocytes obtained by the culture method. Furthermore, the medium additive containing deacylated gellan gum or a salt thereof of the present invention can greatly promote the growth of cancer cells in the culture of cancer cells. In addition, the medium additive containing the deacylated gellan gum of the present invention or a salt thereof can suppress the decrease in the number of hepatocytes in the culture of hepatocytes.

Brief Description of the Drawings

[0021]

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Mode for Carrying Out the Invention

[0022] Hereinafter, the present invention will be described in more detail. The terms used in this specification are defined as follows.

[0023] The cell in the present invention is the most basic unit that constitutes an animal or a plant, and as its elements, it has cytoplasm and various organelles inside the cell membrane. At this time, the nucleus containing DNA may or may not be included inside the cell. For example, the cells derived from animals in the present invention include germ cells such as sperm and eggs, somatic cells that constitute a living body, stem cells (such as pluripotent stem cells), progenitor cells, cancer cells separated from a living body, cells that have acquired immortality ability after being separated from a living body and are stably maintained in vitro (cell lines), cells that have been artificially genetically modified after being separated from a living body, cells that have been artificially nuclear-exchanged after being separated from a living body, and the like. Examples of somatic cells that constitute a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, heart cells, esophageal cells, muscle cells (for example, smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (for example, CD34-positive cells derived from umbilical cord blood), and monocytes. The somatic cells include cells collected from any tissue such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain, or nerve tissue. A stem cell is a cell that has the ability to replicate itself and the ability to differentiate into multiple other cell lineages. Examples thereof include, but are not limited to, embryonic stem cells (ES cells), embryonal carcinoma cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and the like. Examples of pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells among the above-mentioned stem cells. A progenitor cell is a cell in an intermediate stage of differentiating from the above-mentioned stem cells into specific somatic cells or germ cells. A cancer cell is a cell derived from a somatic cell that has acquired the ability of infinite proliferation.A cell line is a cell that has acquired the ability to proliferate infinitely through artificial manipulation in vitro.

[0024] Examples of cancer tissues include, but are not limited to, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, myeloid cancer, renal cell carcinoma, ureteral cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, craniopharyngeal cancer, laryngeal cancer, tongue cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, seminoma, Wilms tumor, glioma, astrocytoma, myeloblastoma, meningioma, melanoma, neuroblastoma, medulloblastoma, retinoblastoma, malignant lymphoma, and tissues such as blood from cancer patients. Examples of cancer cell lines include, but are not limited to, HBC-4, BSY-1, BSY-2, MCF-7, MCF-7 / ADR RES, HS578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D as human breast cancer cell lines, HeLa as a human cervical cancer cell line, A549, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273, DMS114 as human lung cancer cell lines, and Caco-2, COLO-205 as human colorectal cancer cell lines , HCC-2998, HCT-15, HCT-116, HT-29, KM-12, SW-620, WiDr, human prostate cancer cell lines such as DU-145, PC-3, LNCaP, human central nervous system cancer cell lines such as U251, SF-295, SF-539, SF-268, SNB-75, SNB-78, SNB-19, human ovarian cancer cell lines such as OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, IGROV-1, human kidney cancer cell lines such as RXF-631L, ACHN, UO-31, SN-12C, A498, CAKI-1, RXF-393L, 786-0, TK-10, human gastric cancer cell lines such as MKN45, MKN28, St-4, MKN-1, MKN-7, MKN-74, skin cancer cell lines such as LOX-IMVI, LOX, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, leukemia cell lines such as CCRF-CRM, K562, MOLT-4, HL-60TB, RPMI8226, SR, UT7 / TPO, Jurkat, human epithelial-like cancer cell lines such as, A431, human melanoma cell lines such as A375, human osteosarcoma cell lines such as, MNNG / H OS, human pancreatic cancer cell lines such as MIAPaCa-2 and the like. Examples of cell lines include, but are not limited to, 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 and the like.

[0025] The hepatocytes in the present invention include, in addition to primary hepatocytes collected from liver tissue, hepatocyte cell lines established by subculturing under conditions optimized for in vitro culture, cells derived from tissues other than the liver, pluripotent stem cells such as iPS cells and ES cells, mesenchymal stem cells, peripheral blood-derived stem cells, bone marrow stem cells, adipose stem cells, hepatic stem cells, hepatocytes differentiated in vitro from hepatic progenitor cells, etc. The liver tissue is a liver collected from humans, rats, mice, guinea pigs, hamsters, rabbits, pigs, cows, horses, dogs, cats, monkeys, or the like, and may be not only a normal liver but also a cancerous liver. The primary hepatocytes can be isolated and collected from these livers by the perfusion method using collagenase, but may also be those purchased from reagent companies such as Primary Cell Co., Ltd., Becton Dickinson Japan Co., Ltd., Takara Bio Inc., Hokkaido System Science Co., Ltd., Lonza Japan Co., Ltd., Veritas Co., Ltd., Life Technologies Japan Co., Ltd., etc. The hepatocytes to be purchased may be in a frozen state or attached to a carrier such as collagen. Examples of hepatocyte cell lines include, but are not limited to, HepG2, Hep3B, HepaRG (registered trademark), JHH7, HLF, HLE, PLC / PRF / 5, WRL68, HB611, SK-HEP-1, HuH-4, HuH-7, etc.

[0026] The functions of hepatocytes in the present invention include, but are not particularly limited to, the expression of cytochrome P450 (also referred to as CYP) activities such as CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, etc., and the metabolism of pharmaceuticals by this enzyme, the conjugation of pharmaceuticals by glucuronic acid, glutathione, sulfuric acid, glycine, etc., the production of useful proteins such as albumin, apolipoprotein, thrombopoietin, etc., the secretion of bilirubin, the synthesis of urea, the synthesis of bile acids and fatty acids, the transport of pharmaceuticals by transporters, etc. In an embodiment of the present invention, the hepatocytes preferably maintain cytochrome P450 activity, albumin production, and / or the transport of pharmaceuticals by transporters (e.g., the uptake of Carboxydichlorofluorescein diacetate, Tetraethylammonium Bromide, Taurocholate, Rosvastatin, and the excretion of Carboxydichlorofluorescein) among the above functions.

[0027] The pharmaceuticals in the present invention include all substances used for medical purposes. A pharmaceutical candidate is a substance that is being searched for or developed as a candidate for a pharmaceutical, and examples include synthetic compounds, proteins, nucleic acids, saccharides, natural products, etc.

[0028] The anti-cancer agent in the present invention refers to, in addition to drugs that directly act on cancer cells to suppress the growth and function of cancer cells, drugs that do not directly act on cancer cells but suppress the growth or function of cancer cells or kill cancer cells through cooperative action with immune cells or other drugs in the living body. Examples of such anti-cancer agents include, but are not particularly limited to, alkylating agents, platinum derivatives, antimetabolites typified by 5FU-based anti-cancer agents, topoisomerase inhibitors, microtubule inhibitors, anti-cancer antibiotics typified by epirubicin, gefitinib, trastuzumab, cetuximab, erlotinib, panitumumab, lapatinib, temsirolimus, everolimus, ipilimumab, vandetanib, crizotinib, luxitinib, trametinib and other molecular target drugs. The target molecules of molecular target drugs include, but are not particularly limited to, various kinases, Her2, EGFR (epidermal growth factor receptor), PI3K (phosphatidylinositol 3-kinase), mTOR (mammalian target of rapamycin protein), Akt, CDK (cyclin-dependent kinase), VEGFR (vascular endothelial growth factor receptor), PDGFR (platelet-derived growth factor receptor), FGFR (fibroblast growth factor receptor), c-Met, Raf, p38MAPK, CTLA-4, ALK, JAK, MEK (MAPK / ERK kinase), Hsp90, histone deacetylase, etc. Furthermore, synthetic compounds, proteins, nucleic acids, saccharides, natural products, etc. that are candidates for drugs having such effects are also included in the anti-cancer agents in the present invention.

[0029] The plant-derived cells in the present invention include cells separated from each tissue of the plant body, and also include protoplasts from which the cell wall has been artificially removed from the cells.

[0030] The tissue in the present invention is a structural unit in which several types of cells with different properties and functions are assembled in a certain pattern. Examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, nerve tissue, etc. Examples of plant tissues include meristematic tissue, epidermal tissue, assimilatory tissue, mesophyll tissue, conducting tissue, mechanical tissue, parenchyma tissue, dedifferentiated cell mass (callus), etc.

[0031] When culturing cells and / or tissues by the method of the present invention, the cells and / or tissues to be cultured can be arbitrarily selected from the cells and / or tissues described above. The cells and / or tissues can be directly collected from an animal or plant body. The cells and / or tissues may be derived, grown, or transformed from an animal or plant body by a specific treatment and then collected. In this case, the treatment may be in vivo or ex vivo. Examples of animals include insects, fish, amphibians, reptiles, birds, pancrustaceans, hexapoda, and mammals. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypuses, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, rhesus monkeys, chimpanzees, and humans. There is no particular limitation on the plants, so long as the collected cells and / or tissues can be cultured in liquid. Examples of such plants include, but are not limited to, plants that produce herbal medicines (e.g., saponin, alkaloids, berberine, scopolin, plant sterols, etc.) (e.g., ginseng, periwinkle, henbane, coptis, belladonna, etc.), plants that produce pigments and polysaccharides (e.g., anthocyanins, safflower pigments, madder pigments, saffron pigments, flavones, etc.) that are used as raw materials for cosmetics and food (e.g., blueberries, safflower, madder, saffron, etc.), plants that produce pharmaceutical ingredients, and plants that are used as feed or food (rice, corn, wheat, barley, etc.).

[0032] In the present invention, the term "suspended cells and / or tissues" refers to a state in which the cells and / or tissues are not attached to a culture vessel (non-adherent). Furthermore, in the present invention, when the cells and / or tissues are proliferated, differentiated or maintained, the cells and / or tissues are allowed to remain in the liquid medium without external pressure or vibration to the liquid medium composition, or without shaking, rotating or the like in the composition. A state in which it is uniformly dispersed and floating in the culture medium is referred to as "floating and static", and culturing cells and / or tissues in this state is referred to as "floating and static culture". Also, the period during which it can be floated in "floating and static" includes at least 5 to 60 minutes, 1 hour to 24 hours, 1 day to 21 days, but is not limited to these periods as long as the floating state is maintained.

[0033] The culture medium composition of the present invention is a composition containing a structure capable of floating and culturing cells or tissues (preferably capable of floating and static culture) and a culture medium. The culture medium composition of the present invention is preferably a composition capable of exchanging the culture medium composition during culturing and recovering cells or tissues from the culture medium composition after culturing is completed. More preferably, it is a composition that does not require any of temperature change, chemical treatment, enzyme treatment, or shear force when recovering cells or tissues from the culture medium composition. The structure in the present invention is formed from a specific compound and exhibits an effect of uniformly floating cells and / or tissues. More specifically, it includes those in which polymer compounds are aggregated via ions, or those in which polymer compounds form a three-dimensional network. Also, it is known that polysaccharides form microgels via metal ions (for example, Japanese Patent Application Laid-Open No. 2004-129596), and the structure of the present invention includes such microgels as one aspect. Also, as an example of those in which polymer compounds are aggregated via ions, a film-like structure is included as one aspect. The film is illustrated in FIG. 13. The size of the structure in the present invention preferably passes through a filter with a pore size of 0.2 μm to 200 μm when filtered with a filter. As the lower limit of the pore size, more preferably, it exceeds 1 μm, and considering stably floating cells or tissues, even more preferably, it exceeds 5 μm. As the upper limit of the pore size, more preferably, it is 100 μm or less, and considering the size of cells or tissues, even more preferably, it is 70 μm or less. The specific compound in the present invention refers to a compound that forms an amorphous structure when mixed with a liquid medium, the structure is uniformly dispersed in the liquid, and can substantially retain cells and / or tissues without substantially increasing the viscosity of the liquid and prevent its sedimentation. "Not substantially increasing the viscosity of the liquid" means that the viscosity of the liquid does not exceed 8 mPa·s. The viscosity of the liquid at this time (i.e., the viscosity of the culture medium composition of the present invention) is 8 mPa·s or less, preferably 4 mPa·s or less, and more preferably 2 mPa·s or less. Furthermore, as long as the structure is formed in a liquid medium and can uniformly suspend (preferably suspend and stand still) cells and / or tissues without substantially increasing the viscosity of the liquid, the chemical structure, molecular weight, physical properties, etc. of the specific compound are not limited at all. The viscosity of the liquid containing the structure can be measured, for example, by the method described in the examples below. Specifically, it can be measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1°34´×R24, rotation speed 100 rpm) under the condition of 37°C.

[0034] Examples of the specific compound used in the present invention are not particularly limited, but include polymer compounds, preferably polymer compounds having an anionic functional group. Examples of the anionic functional group include carboxy group, sulfo group, phosphate group and their salts, and carboxy group or its salt is preferred. As the polymer compound used in the present invention, those having one or more selected from the group of the anionic functional groups can be used. Preferred specific examples of the polymer compound used in the present invention are not particularly limited, but include polysaccharides obtained by polymerizing 10 or more monosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.), and more preferably acidic polysaccharides having an anionic functional group. The acidic polysaccharide referred to here is not particularly limited as long as it has an anionic functional group in its structure. For example, uronic acid (e.g., glucuronic acid, iduronic Polysaccharides having acidic groups (e.g., glucuronic acid, galacturonic acid, mannuronic acid), polysaccharides having a sulfate group or a phosphate group in part of their structures, or polysaccharides having both structures, including not only naturally obtained polysaccharides but also polysaccharides produced by microorganisms, genetically engineered polysaccharides, or artificially synthesized polysaccharides using enzymes. More specifically, examples include hyaluronic acid, gellan gum, deacylated gellan gum (hereinafter sometimes referred to as DAG), rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthangum, hexuronic acid, fucoidan, pectin, pectinic acid, pectininic acid, heparan sulfate, heparin, heparitin sulfate, keratan sulfate, chondroitin sulfate, deltaman sulfate, laminan sulfate, and salts thereof, and those composed of one or more species selected from the group consisting of these. The polysaccharide is preferably hyaluronic acid, DAG, diutan gum, xanthan gum, carrageenan, or a salt thereof, can suspend cells or tissues at a low concentration, and considering the ease of cell or tissue recovery, most preferably DAG. The salts mentioned here include, for example, salts of alkali metals such as lithium, sodium, and potassium, salts of alkaline earth metals such as calcium, barium, and magnesium, or salts such as aluminum, zinc, copper, iron, ammonium, organic bases, and amino acids. The weight average molecular weight of these polymer compounds (such as polysaccharides) is preferably from 10,000 to 50,000,000, more preferably from 100,000 to 20,000,000, and still more preferably from 1,000,000 to 10,000,000. For example, the molecular weight can be measured by pullulan conversion using gel permeation chromatography (GPC). Furthermore, as described in the examples below, phosphorylated DAG can also be used. The phosphorylation can be carried out by known methods.

[0035] In the present invention, a plurality of (preferably two) types of the above polysaccharides can be used in combination. The types of the combination of polysaccharides are not particularly limited as long as they can form the above-described structure in a liquid medium and uniformly suspend (preferably suspend and leave still) cells and / or tissues without substantially increasing the viscosity of the liquid medium. Preferably, the combination contains at least DAG or a salt thereof. That is, a preferred combination of polysaccharides includes DAG or a salt thereof, and a polysaccharide other than DAG or a salt thereof (e.g., xanthan gum, alginic acid, carrageenan, diutan gum, methylcellulose, locust bean gum or a salt thereof). Specific combinations of polysaccharides include, but are not limited to, DAG and ramzan gum, DAG and diutan gum, DAG and xanthan gum, DAG and carrageenan, DAG and zantan gum, DAG and locust bean gum, DAG and κ-carrageenan, DAG and sodium alginate, DAG and methylcellulose, etc.

[0036] More preferred specific examples of the specific compound used in the present invention include hyaluronic acid, deacylated gellan gum, diutan gum, carrageenan, xanthan gum and salts thereof. Considering the points of being able to lower the viscosity of the medium composition and the ease of cell or tissue recovery, the most preferred example is deacylated gellan gum or a salt thereof. The deacylated gellan gum in the present invention is a linear high molecular polysaccharide having four molecules of sugar, i.e., 1-3-linked glucose, 1-4-linked glucuronic acid, 1-4-linked glucose and 1-4-linked rhamnose, as a structural unit. In the following general formula (I), it is a polysaccharide in which both R1 and R2 are hydrogen atoms and n is an integer of 2 or more. However, R1 may contain a glyceryl group and R2 may contain an acetyl group, but the contents of the acetyl group and the glyceryl group are preferably 10% or less, more preferably 1% or less. The structure in the present invention can take various forms depending on the specific compound. Taking the case of deacylated gellan gum as an example, when mixed with a liquid medium, deacylated gellan gum incorporates metal ions (such as calcium ions) in the liquid medium, forms an amorphous structure through these metal ions, and suspends cells and / or tissues. The viscosity of the medium composition of the present invention prepared from deacylated gellan gum is 8 mPa·s or less, preferably 4 mPa·s or less, and more preferably is 2 mPa·s or less considering the ease of cell or tissue recovery.

[0037] [Chemical formula]

[0038] The specific compound in the present invention can also be obtained by chemical synthesis methods. However, when the compound is a natural product, it is preferably obtained by extracting, separating, and purifying it from various plants, animals, and microorganisms containing the compound using conventional techniques. In the extraction, water or supercritical gas can be used to efficiently extract the compound. For example, as a method for producing gellan gum, a production microorganism is cultured in a fermentation medium, the mucous substance produced outside the cells is recovered by ordinary purification methods, and after processes such as drying and pulverization, it can be made into a powder. In the case of deacylated gellan gum, an alkali treatment is performed when recovering the mucous substance, and the glyceryl group and acetyl group bonded to the glucose residue bonded by 1-3 bonds are deacylated and then recovered. As purification methods, for example, liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatographies, gel filtration chromatography using Sephadex LH-20, etc., adsorption chromatography using activated carbon, silica gel, etc. or adsorption and desorption treatment of the active substance by thin layer chromatography, or high performance liquid chromatography using a reverse phase column, etc. can be used alone or in any order in combination and repeatedly to remove impurities and purify. Examples of the production microorganism of gellan gum include, but are not limited to, Sphingomonas elodea and the Examples include microorganisms with modified genes of microorganisms. In the case of deacylated gellan gum, commercially available products such as "KELCOGEL (registered trademark of CP Kelco)" manufactured by Sancrystal Co., Ltd. and "Kelcogel (registered trademark of CP Kelco)" manufactured by Saneigen FFI Co., Ltd. can be used. Also, as native gellan gum, "Kelcogel (registered trademark of CP Kelco) HT" manufactured by Saneigen FFI Co., Ltd. can be used.

[0039] The concentration of a specific compound in the medium depends on the type of the specific compound, and can be appropriately set within a range in which the specific compound forms the above-mentioned structure in the liquid medium and can uniformly suspend (preferably suspend and stand) cells and / or tissues without substantially increasing the viscosity of the liquid medium. Usually, it is 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 even more preferably 0.005% to 0.05% (weight / volume). For example, in the case of deacylated gellan gum, it may be added to the medium at 0.001% to 1.0% (weight / volume), preferably 0.003% to 0.5% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), even more preferably 0.01% to 0.05% (weight / volume), and most preferably 0.01% to 0.03% (weight / volume). In the case of xanthan gum, it may be added to the medium at 0.001% to 5.0% (weight / volume), preferably 0.01% to 1.0% (weight / volume), more preferably 0.05% to 0.5% (weight / volume), and most preferably 0.1% to 0.2% (weight / volume). In the case of a κ-carrageenan and locust bean gum mixed system, it is 0.001% to 5.0% (weight / volume), preferably 0.005% to 1.0% (weight / volume), more preferably 0 It may be added to the medium at 0.01% to 0.1% (weight / volume), most preferably 0.03% to 0.05% (weight / volume). In the case of native gellan gum, it may be added to the medium at 0.05% to 1.0% (weight / volume), preferably 0.05% to 0.1% (weight / volume).

[0040] When using a combination of a plurality of the above polysaccharides (preferably two kinds), the concentration of the polysaccharides is such that the combination of the polysaccharides forms the above-mentioned structure in the liquid medium and can uniformly suspend (preferably suspend and leave still) cells and / or tissues without substantially increasing the viscosity of the liquid medium. It can be appropriately set within a range where this is possible. For example, when using a combination of DAG or a salt thereof and a polysaccharide other than DAG or a salt thereof, the concentration of DAG or a salt thereof is exemplified as 0.005 to 0.02% (weight / volume), preferably 0.01 to 0.02% (weight / volume), and the concentration of the polysaccharide other than DAG or a salt thereof is exemplified as 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). The following are examples of specific combinations of concentration ranges. DAG or a salt thereof: 0.005 to 0.02% (preferably 0.01 to 0.02%) (weight / volume) Polysaccharide other than DAG Xanthan gum: 0.1 to 0.4% (weight / volume) Sodium alginate: 0.1 to 0.4% (weight / volume) Locust bean gum: 0.1 to 0.4% (weight / volume) Methyl cellulose: 0.1 to 0.4% (weight / volume) (preferably 0.2 to 0.4% (weight / volume)) Carrageenan: 0.05 to 0.1% (weight / volume) Daiyutan gum: 0.05 to 0.1% (weight / volume)

[0041] Note that the concentration can be calculated by the following formula. Concentration (%) = weight (g) of specific compound / volume (ml) of medium composition × 100

[0042] The above-mentioned compound can also be further converted into another derivative by chemical synthesis methods, and the derivatives thus obtained can also be effectively used in the present invention. Specifically, in the case of deacylated gellan gum, the hydroxyl groups corresponding to R1 and / or R2 of the compound represented by the general formula (I) can be substituted with a C1-3 alkoxy group, a C1-3 alkylsulfonyl group, a monosaccharide residue such as glucose or fructose, an oligosaccharide residue such as sucrose or lactose, an amino acid residue such as glycine or arginine, etc. Derivatives thus substituted can also be used in the present invention. Also, the compound can be crosslinked using a crosslinker such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0043] The specific compound or its salt used in the present invention can exist as any crystal form depending on the production conditions and can exist as any hydrate, and these crystal forms, hydrates, and their mixtures are also included in the scope of the present invention. Also, it may exist as a solvate containing an organic solvent such as acetone, ethanol, tetrahydrofuran, etc., and all these forms are included in the scope of the present invention.

[0044] The specific compound used in the present invention may exist in the form of tautomers, geometric isomers, mixtures of tautomers or geometric isomers, or mixtures thereof, generated by intra- or extra-ring isomerization. The compounds of the present invention, regardless of whether they are generated by isomerization, when having an asymmetric center, may exist in the form of resolved optical isomers or mixtures containing them in any ratio.

[0045] The culture medium composition of the present invention contains metal ions, for example, divalent metal ions (calcium ions, Magnesium ions, zinc ions, iron ions, copper ions, etc. may be present, and preferably contain calcium ions. The metal ions can be used in combination of two or more kinds, such as calcium ions and magnesium ions, calcium ions and zinc ions, calcium ions and iron ions, calcium ions and copper ions. Those skilled in the art can appropriately determine the combination. In one aspect, due to the inclusion of metal ions in the culture medium composition, the polymer compounds aggregate through the metal ions, and the polymer compounds form a three-dimensional network (for example, the polysaccharide forms a microgel through the metal ions), thereby forming the structure of the present invention. The concentration of the metal ions can be appropriately set within a range in which a specific compound forms the above-mentioned structure in the liquid culture medium and can uniformly suspend (preferably suspend and stand) cells and / or tissues without substantially increasing the viscosity of the liquid culture medium. The salt concentration is from 0.1 mM to 300 mM, preferably from 0.5 mM to 100 mM, but is not limited thereto. The metal ions may be mixed with the culture medium or a salt solution may be separately prepared and added to the culture medium. Further, the culture medium composition of the present invention may contain an extracellular matrix, an adhesion molecule, etc. described later. The present invention also includes a culture method for growing cells or tissues using the culture medium composition, a method for recovering the obtained cells or tissues by, for example, filtration, centrifugation or magnetic separation, and a method for producing spheres using the culture medium composition.

[0046] The structure composed of the specific compound used in the present invention exhibits the effect of suspending the cells and / or tissues in a liquid containing the structure of the specific compound (preferably the effect of suspending and statically culturing) when the cells and / or tissues are cultured in vitro. Due to this suspension effect, it is possible to increase and culture cells and / or tissues per unit volume compared to monolayer culture. Also, in the conventional suspension culture method when rotation or shaking operations are involved, shear force acts on the cells and / or tissues, so the growth rate and recovery rate of the cells and / or tissues may be low, or the cell function may be impaired. However, by using the culture medium composition containing the structure of the specific compound of the present invention, the cells and / or tissues can be uniformly dispersed without performing operations such as shaking, so that the target cells and / or tissues can be easily and abundantly obtained without loss of cell function. In addition, when floatingly culturing cells and / or tissues in a conventional medium containing a gel substrate, it is sometimes difficult to observe and recover the cells and / or tissues, or their function may be impaired during recovery. However, by using the culture medium composition containing the structure of the specific compound of the present invention, the cells and / or tissues can be floatingly cultured, observed, and recovered without impairing their function. Also, the conventional medium containing a gel substrate may have a high viscosity, making it difficult to exchange the medium. However, the culture medium composition containing the structure of the specific compound of the present invention has a low viscosity, so the medium can be easily exchanged using a pipette, pump, etc.

[0047] The human-derived cells and / or tissues cultured by the method of the present invention can be transplanted for therapeutic purposes to patients with diseases or disorders. At this time, the types of diseases or disorders to be treated, the pretreatment method, and the cell transplantation method are appropriately selected by the parties concerned. The engraftment of the transplanted cells into the recipient, the recovery from the disease or disorder, the presence or absence of side effects associated with the transplantation, and the therapeutic effect can be appropriately examined and judged by the general methods in transplantation therapy.

[0048] Furthermore, since cells and / or tissues are efficiently proliferated by the method of the present invention, the culture medium composition containing the specific compound and its structural body in the present invention can be used as a reagent for cell research. For example, when elucidating factors that regulate cell and tissue differentiation and proliferation, the number and type of cells, changes in cell surface differentiation markers and expressed genes when the cells are cultured in the coexistence of the cells and the target factor are analyzed. At this time, by using the culture medium composition of the present invention, not only can the number of cells to be analyzed be efficiently amplified, but also can be efficiently recovered. The culture conditions, culture apparatus, type of culture medium, type of the compound of the present invention, content of the specific compound, type of additives, content of additives, culture period, culture temperature, etc. when elucidating the target factor are appropriately selected by the parties from the ranges described in this specification. The cells that have proliferated or emerged by culturing can be observed using a standard microscope. At this time, the cultured cells may be stained with a specific antibody. The expressed genes changed by the target factor can be detected by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from the cultured cells and using Southern blotting, Northern blotting, RT-PCR method, etc. In addition, cell surface differentiation markers can be detected by ELISA or flow cytometry using specific antibodies, and the effects on differentiation and proliferation by the target factor can be observed.

[0049] When culturing cells and / or tissues using the culturing method of the present invention, it is possible to culture them using culturing equipment such as petri dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, cell culture flasks, spinner flasks, tubes, trays, culture bags, roller bottles, etc. that are generally used for culturing cells. The material of these culturing equipment is not particularly limited, and examples include glass, polyvinyl chloride, cellulose-based polymers such as ethyl cellulose and acetyl cellulose, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, 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 copolymers, and other plastics. These plastics are not only excellent in gas permeability such as oxygen and carbon dioxide, but also excellent in industrial moldability and can withstand various sterilization treatments, and are preferably made of a transparent material that allows observation of the inside of the culturing equipment. Here, the method of performing the sterilization treatment is not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, etc. In addition, various surface treatments (for example, plasma treatment, corona treatment, etc.) may be performed on these plastics. Furthermore, these culturing equipment may be pre-coated with an extracellular matrix, cell adhesion molecules, etc.Examples of such coating materials include collagen I to XIX, gelatin, fibronectin, vitronectin, laminin-1 to 12, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulins, hyaluronic acid, superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, alginate gel, hydrogel, and their cleavage fragments. These coating materials can also be those whose amino acid sequences are artificially modified by genetic recombination technology. Further, a coating material for inhibiting adhesion to cell and / or tissue culture equipment can also be used. Examples of such coating materials include silicon, poly(2-hydroxymethyl methacrylate), poly(2-methoxymethyl acrylate), poly(2-methacryloyloxyethyl phosphorylcholine), poly-N-isopropylacrylamide, Mebiol Gel (registered trademark), etc., but are not limited thereto.

[0050] Cell and / or tissue culture can also be performed using a bioreactor or an automatic culture device that can automatically perform cell seeding, medium replacement, cell image acquisition, and recovery of cultured cells under mechanical control in a closed environment, and can perform high-density culture while controlling pH, temperature, oxygen concentration, etc. As methods for supplying a new medium during culture and supplying the required substances to cells and / or tissues without excess or deficiency using these devices, there are fed-batch culture, continuous culture, and perfusion culture, and any of these methods can be used in the culture method of the present invention. Further, as for the culture containers used in bioreactors and automatic culture devices, there are open-system culture containers (for example, culture containers having lids) and closed-system culture containers (for example, cartridge-type culture containers) that are not easily opened and closed and have a small contact area with the outside world, and any of these culture containers can be used in the culture method of the present invention.

[0051] When culturing cells and / or tissues using the specific compounds in the present invention, a medium composition can be prepared by mixing the specific compounds with the medium used for culturing cells and / or tissues. In terms of classification by the composition of such a medium, there are natural media, semi-synthetic media, synthetic media, and in terms of classification by shape, there are semi-solid media, liquid media, powder media (hereinafter sometimes referred to as powder media), and the like. When the cells and / or tissues are of animal origin, any medium used for culturing animal cells can be used. Examples of such media include Dulbecco’s Modified Eagles’s Medium (DMEM), Ham’s Nutrient Mixture F12, DMEM / F12 medium, McCoy’s 5A medium, Eagles’s Minimum Essential Medium (EMEM), alpha Modified Eagles’s Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), RPMI1640 medium, Iscove’s Modified Dulbecco’s Medium (IMDM), MCDB131 medium, William’s Medium E, IPL41 medium, Fischer‘s medium, StemPro34 (manufactured by Invitrogen ) X-VIVO 10 (manufactured by Lonza), X-VIVO 15 (manufactured by Lonza), HPGM (manufactured by Lonza), StemSpan H3000 (manufactured by STEMCELL Technologies), StemSpan SFEM (manufactured by STEMCELL Technologies), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biological), StemPro hESC SFM (manufactured by Invitrogen), Essential 8 (registered trademark) Medium (manufactured by Gibco), mTeSR1 or 2 Medium (manufactured by STEMCELL Technologies), ReproFF or ReproFF2 (manufactured by ReproCell), PSGro hESC / iPSC Medium (manufactured by System Biosciences), NutriStem (registered trademark) Medium (manufactured by Biological Industries), CSTI-7 Medium (manufactured by Institute of Cell Science), MesenPRO RS Medium (manufactured by Gibco), MF-Medium (registered trademark) Mesenchymal Stem Cell Expansion Medium (manufactured by Toyobo Co., Ltd.), Sf-900 II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), etc.

[0052] The media used for culturing cancer cells can contain cell adhesion factors in the above media. Examples thereof include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. These cell adhesion factors can also be added in combination of two or more types. Furthermore, thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose, etc. can be further mixed into the media used for culturing cancer cell spheres.

[0053] As media used for culturing hepatocytes, in addition to the above media, there are HepatoZYME-SFM (manufactured by Life Technologies), HCM (registered trademark)-Hepatocyte Culture Medium BulletKit (registered trademark, manufactured by Lonza), HBM (registered trademark)-Hepatocyte Basal Medium (manufactured by Lonza), HMM (registered trademark)-Hepatocyte Maintenance Medium (manufactured by Lonza), Modified Waymouth Medium (manufactured by Nissui Pharmaceutical Co., Ltd.), ISOM’s Medium, Hepatocyte Proliferation Medium (manufactured by Takara Bio Inc.), Hepatocyte Maintenance Medium (manufactured by Takara Bio Inc.), Hepatocyte Basal Medium (manufactured by Takara Bio Inc.), Active Maintenance Super Medium (manufactured by In Vitro ADMET Laboratories), etc. These media can contain cell adhesion factors, and examples thereof include Matrigel, collagen gel, gelatin, poly-L-lysine, poly- D-lysine, laminin, fibronectin. These cell adhesion factors can also be added in combinations of two or more. Furthermore, thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, etc. can be further mixed into the media used for culturing cancer cells or hepatocyte spheres.

[0054] When the cells and / or tissues are of plant origin, media include a basal medium such as Murashige and Skoog (MS) medium, Linsmaier and Skoog (LS) medium, White medium, Gamborg's B5 medium, Nitsch medium, Heller medium, Morel medium, etc., which are commonly used in plant tissue culture, or a modified medium in which the components of these media are modified to optimal concentrations (for example, reducing the ammonium nitrogen concentration by half, etc.), to which plant growth regulators (plant hormones) such as auxins and, if necessary, cytokinins are added at appropriate concentrations. These media can be further supplemented with, if necessary, caseinolytic enzymes, corn steep liquor, vitamins, etc. Examples of auxins include, but are not limited to, 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), etc. Auxins can be added to the medium at a concentration of, for example, about 0.1 to about 10 ppm. Examples of cytokinins include, but are not limited to, kinetin, benzyladenine (BA), zeatin, etc. Cytokinins can be added to the medium at a concentration of, for example, about 0.1 to about 10 ppm.

[0055] Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. to the above media according to the purpose. When culturing animal-derived cells and / or tissues, those skilled in the art can also add one or more combinations of other chemical components or biological components according to the purpose. Components added to the medium of animal-derived cells and / or tissues include fetal bovine serum, human serum, equine serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, and various cell adhesion molecules. Examples of cytokines added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), etc., but are not limited thereto. Hormones added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Müllerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, co Luteinizing hormone-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandin, leukotriene, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin are included, but are not limited thereto. Examples of growth factors added to the medium include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, Notch ligand (such as Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), pleiotrophin, etc., but are not limited thereto. In addition, those obtained by artificially modifying the amino acid sequences of these cytokines and growth factors by genetic recombination technology can also be added. Examples thereof include the IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor). Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, hyaluronic acid, alginate gel, various hydrogels, and further cleavage fragments thereof, etc.

[0056] Examples of antibiotics added to the medium include sulfonamides, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mezlocillin, mecillinam, anginosillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rosoxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulanic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, adinosili Examples include formaldehyde - Huudolat esters of clavulanic acid and sulbactam, tazobactam, aztreonam, sulfazecin, isosulfazecin, nocardicin, m - carboxyphenyl, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0057] When adding a specific compound in the present invention to the above medium, first dissolve or disperse the specific compound in a suitable solvent at the time of use (this is referred to as a medium additive). Then, as detailed above, as the concentration of the specific compound in the medium, a concentration that can uniformly suspend (preferably suspend and leave still) cells and / or tissues without substantially increasing the viscosity of the liquid medium, for example, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.4% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), still more preferably 0.005% to 0.05% (weight / volume). For example, in the case of deacylated gellan gum, it may be added to the medium at 0.001% to 1.0% (weight / volume), preferably 0.003% to 0.5% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), and most preferably 0.01% to 0.03% (weight / volume). Also, in another aspect, in the case of deacylated gellan gum, it may be added to the medium at 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.003% to 0.1% (weight / volume), and most preferably 0.005% to 0.03% (weight / volume). In the case of xanthan gum, it may be added to the medium at 0.001% to 5.0% (weight / volume), preferably 0.01% to 1.0% (weight / volume), more preferably 0.05% to 0.5% (weight / volume), and most preferably 0.1% to 0.2% (weight / volume). In the case of a mixed system of κ-carrageenan and locust bean gum, it may be added to the medium at 0.001% to 5.0% (weight / volume), preferably 0.005% to 1.0% (weight / volume), more preferably 0.01% to 0.1%, and most preferably 0.03% to 0.05% (weight / volume). In the case of a mixed system of deacylated gellan gum and diutan gum, it may be added to the medium at 0.001% to 1.0% (weight / volume), and most preferably 0.005% to 0.01% (weight / volume). In the case of a mixed system of deacylated gellan gum and methylcellulose, it may be added to the medium at 0.001% to 1.0% (weight / volume), and most preferably 0.005% to 0.2% (weight / volume).In the case of a mixed system of deacylated gellan gum and locust bean gum, it may be added to the medium at 0.001% to 1.0% (weight / volume), most preferably 0.01% to 0.1% (weight / volume). Deacylated... In the case of a mixed system of deacylated gellan gum and sodium alginate, it may be added to the medium at 0.001% to 1.0% (weight / volume), most preferably 0.01% to 0.1% (weight / volume). In the case of a mixed system of deacylated gellan gum and xanthan gum, it may be added to the medium at 0.001% to 1.0% (weight / volume), most preferably 0.01% to 0.1% (weight / volume). In the case of a mixed system of deacylated gellan gum and κ-carrageenan, it may be added to the medium at 0.001% to 1.0% (weight / volume), most preferably 0.01% to 0.1% (weight / volume). The concentration can be calculated by the following formula. Concentration (%) = weight of specific compound (g) / volume of medium composition (ml) × 100

[0058] Here, examples of suitable solvents for the medium additive include aqueous solvents such as water, dimethyl sulfoxide (DMSO), methanol, ethanol, butanol, propanol, glycerin, propylene glycol, butylene glycol, and various alcohols, but are not limited thereto. At this time, the concentration of the specific compound is preferably 0.001% to 5.0% (weight / volume), preferably 0.01% to 1.0% (weight / volume), more preferably 0.1% to 0.6% (weight / volume). At that time, additives that enhance the effect of the specific compound or lower the concentration during use can be further added. Examples of such additives include one or more polysaccharides such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose, carboxymethyl cellulose, agarose, tamarind seed gum, pullulan, etc. can be mixed. In addition, the specific compound can also be used by immobilizing it on the surface of the carrier or supporting it inside the carrier during culturing. The specific compound can be in any shape at the time of providing or storing. The specific compound can be tablets, pills, capsules... It can be in the form of formulated solids such as cell agents and granules, liquids such as solutions or suspensions dissolved in appropriate solvents and solubilizers, or in a state bound to a substrate or a single entity. Additives during formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose, mannitol, etc.; lubricants such as magnesium stearate, talc, etc.; binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc.; surfactants such as fatty acid esters; plasticizers such as glycerin, etc. These additives are not limited to the above, and can be freely selected as long as they are available to those skilled in the art. Also, the specific compound in the present invention may be subjected to a sterilization treatment as necessary. The sterilization method is not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, filter sterilization, etc. The material of the filter part when performing filter sterilization (hereinafter sometimes referred to as filtration sterilization) is not particularly limited, and examples include glass fiber, nylon, PES (polyethersulfone), hydrophilic PVDF (polyvinylidene fluoride), cellulose mixed ester, cellulose acetate, polytetrafluoroethylene, etc. The pore size of the filter is not particularly limited, but preferably 0.1 μm to 10 μm, more preferably 0.1 μm to 1 μm, and most preferably 0.1 μm to 0.5 μm. These sterilization treatments can be carried out whether the specific compound is in solid form or in solution state.

[0059] By adding a solution or dispersion of a specific compound to a liquid medium through the above-described preparation, the above-mentioned structure is formed in the liquid medium, and the culture medium composition of the present invention can be obtained. Usually, the culture medium contains metal ions at a concentration sufficient for the polymer compound to aggregate via ions or for the polymer compound to form a three-dimensional network. Therefore, the culture medium composition of the present invention can be obtained simply by adding a solution or dispersion of the specific compound of the present invention to the liquid medium. Alternatively, the culture medium may be added to the culture medium additive (solution or dispersion of the specific compound). Furthermore, the culture medium composition of the present invention can also be prepared by mixing the specific compound and the culture medium components in an aqueous solvent (such as water containing ion-exchanged water, ultrapure water, etc.). Examples of the mixing modes include, but are not limited to, (1) mixing the liquid medium and the culture medium additive (solution), (2) mixing the above polymer compound (solid such as powder) with the liquid medium, (3) mixing the powder culture medium with the culture medium additive (solution), and (4) mixing the powder culture medium and the above polymer compound (solid such as powder) with an aqueous solvent. In order to prevent the non-uniform distribution of the specific compound in the culture medium composition of the present invention, the modes of (1) or (4) or (1) or (3) are preferred. When dissolving the specific compound in a solvent (such as an aqueous solvent like water or a liquid medium), or when dissolving the specific compound and the powder culture medium in a solvent, it is preferable to heat the mixture to promote dissolution. Examples of the heating temperature include 80°C to 130°C, preferably 100°C to 125°C (such as 121°C) for heat sterilization. After heating, the obtained solution of the specific compound is cooled to room temperature. By adding the above-mentioned metal ions to the solution (for example, by adding the solution to a liquid medium), the above-mentioned structure composed of the specific compound is formed. Alternatively, when dissolving the specific compound in a solvent containing the above-mentioned metal ions (such as an aqueous solvent like water or a liquid medium), heating (for example, 80°C to 130°C, preferably 100°C to 125°C (such as 121°C)) and then cooling the obtained solution to room temperature can also form the above-mentioned structure composed of the specific compound.

[0060] The method for preparing the medium composition of the present invention is exemplified, but the present invention is not limited thereto. A specific compound is added to ion-exchanged water or ultrapure water. Then, while heating at a temperature at which the specific compound can be dissolved (for example, 60°C or higher, 80°C or higher, 90°C or higher), it is stirred until it becomes transparent. After dissolution, it is allowed to cool while stirring and sterilized (for example, autoclave sterilization at 121°C for 20 minutes). After returning to room temperature, while stirring any medium to be used for static culture (for example, a homomixer, etc.), the sterilized aqueous solution is added to the medium and mixed so as to be uniform with the medium. The method for mixing the aqueous solution and the medium is not particularly limited, and examples include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer. Also , after mixing, the medium composition of the present invention may be filtered through a filter. The pore size of the filter used in the filtration treatment is 5 μm to 100 μm, preferably 5 μm to 70 μm, more preferably 10 μm to 70 μm.

[0061] Alternatively, a 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 the medium composition of the present invention. For example, when preparing deacylated gellan gum, deacylated gellan gum is added to ion-exchanged water or ultrapure water so as to be 0.1% to 1% (weight / volume), preferably 0.2% to 0.5% (weight / volume), more preferably 0.3% to 0.4% (weight / volume). Also, in another aspect, when preparing deacylated gellan gum, deacylated gellan gum is added to ion-exchanged water or ultrapure water so as to be 0.1% to 1% (weight / volume), preferably 0.2% to 0.8% (weight / volume), more preferably 0.3% to 0.6% (weight / volume). And it may be heated at any temperature as long as it can dissolve the deacylated gellan gum, but it is heated with stirring at 60°C or higher, preferably 80°C or higher, more preferably 90°C or higher (for example, 80 to 130°C) until it becomes transparent. After dissolution, it is allowed to cool while stirring, and autoclave sterilization is performed at, for example, 121°C for 20 minutes. After returning to room temperature, for example, while stirring the DMEM / F12 medium with a homomixer or the like, this aqueous solution is added to the medium so as to obtain a desired final concentration (for example, when the final concentration is 0.015%, the ratio of the 0.3% aqueous solution: medium is 1:19), and uniformly mixed. The mixing method of this aqueous solution and the medium is not particularly limited, and examples include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer. Further, the culture medium composition of the present invention may be filtered through a filter after mixing. The pore size of the filter used during the filtration treatment is 5 μm to 100 μm, preferably 5 μm to 70 μm, more preferably 10 μm to 70 μm. Furthermore, after the preparation of the culture medium composition of the present invention, the structure can be sedimented by centrifugation.

[0062] The form and state of the cells and / or tissues cultured by the method of the present invention can be arbitrarily selected by those skilled in the art. Preferred specific examples thereof include, but are not particularly limited to, a state in which the cells and / or tissues are dispersed alone in the culture medium composition, a state in which the cells and / or tissues are adhered to the surface of the carrier, a state in which the cells and / or tissues are embedded inside the carrier, a state in which a plurality of cells aggregate to form cell clusters (spheres), or a state in which two or more types of cells aggregate to form cell clusters (spheres). More preferably, a state in which the cells and / or tissues are adhered to the surface of the carrier, a state in which the cells and / or tissues are embedded inside the carrier, a state in which a plurality of cells aggregate to form cell clusters (spheres), or a state in which two or more types of cells aggregate to form cell clusters (spheres). Even more preferably, a state in which the cells and / or tissues are adhered to the surface of the carrier, a state in which a plurality of cells aggregate to form cell clusters (spheres), or a state in which two or more types of cells aggregate to form cell clusters (spheres). Among these states, the state of forming cell clusters (spheres) has cell-cell interactions and cell structures reconstructed that are close to the in-vivo environment, can be cultured while maintaining cell functions in the long term, and the cells can be recovered relatively easily, so it can be cited as the most preferred state for culturing by the method of the present invention.

[0063] Examples of the carrier for supporting cells and / or tissues on the surface include microcarriers composed of various polymers, glass beads, ceramic beads, polystyrene beads, dextran beads, and the like. Examples of the polymer include vinyl resins, urethane resins, epoxy resins, polystyrene, polymethyl methacrylate polyesters, polyamides, polyimides, silicone resins, phenolic resins, melamine resins, urea resins, aniline resins, ionomer resins, polycarbonates, collagen, dextran, gelatin, cellulose, alginates, and mixtures thereof, etc. can be used. The carrier enhances cell adhesion, or Alternatively, it may be coated with a compound that enhances the release of substances from cells. Examples of such coating materials include poly(monostearoyl glyceride succinate), poly-D,L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagens I to XIX, fibronectin, vitronectin, laminins-1 to 12, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, alginate gel, various hydrogels, and further fragments thereof, etc. At this time, two or more coating materials may be combined. Furthermore, for the medium used for culturing the carrier carrying cells and / or tissues on the surface, one or more polysaccharides such as guar gum, tamarind gum, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose, carboxymethyl cellulose, agarose, tamarind seed gum, pullulan, etc. can be mixed. In addition, the carrier may contain a magnetic material, for example, ferrite. The diameter of the carrier is several tens of μm to several hundreds of μm, more preferably 100 μm to 200 μm, and its specific gravity is preferably close to 1, more preferably 0.9 to 1.2, and particularly preferably about 1.0. Examples of the carrier include, but are not limited to, Cytodex 1 (registered trademark), Cytodex 3 (registered trademark), Cytoline1 (registered trademark), Cytoline2 (registered trademark), Cytopore1 (registered trademark), Cytopore2 (registered trademark) (above, GE Healthcare Life Sciences), Biosilon (registered trademark) (NUNC), Cult ispher-G (registered trademark), Cultispher-S (registered trademark) (both from Thermo SCIENTIFIC), HILLEXCT (registered trademark), ProNectinF-COATED (registered trademark), and HILLEXII (registered trademark) (SoloHill Engineering), GEM (registered trademark) (Global Eukaryotic Microcarrier), etc. may be mentioned. The carrier may be sterilized if necessary. The sterilization method is not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and dry heat sterilization. The method of culturing animal cells using the carrier is not particularly limited, and a culturing method using a normal fluidized bed type culture tank or a packed bed type culture tank can be used. At this time, the carrier carrying cells and / or tissues on its surface can be uniformly dispersed without performing operations such as shaking by using a medium composition containing the structure of the specific compound of the present invention. Therefore, the target cells and / or tissues can be cultured without loss of cell function. The cells and / or tissues cultured by this method can be recovered by performing centrifugation or filtration treatment while still being carried on the carrier after culturing. At this time, centrifugation or filtration treatment may be performed after adding the used liquid medium. For example, the gravitational acceleration (G) during centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used during filtration treatment is 10μm to 100μm, but it is not limited thereto. Also, if a material having magnetism such as ferrite is encapsulated in the carrier, the cultured carrier can be recovered by magnetic force. The cells and / or tissues cultured by this method can be recovered by detaching them from the carrier using various chelating agents, heat treatment, or enzymes.

[0064] When embedding cells and / or tissues inside a carrier, a material composed of various polymers can be selected as the carrier. Examples of such polymers include collagen, gelatin, alginate, chitosan, agarose, polyglycolic acid, polylactic acid, fibrin adhesive, polylactic acid-polyglycolic acid copolymer, proteoglycan, glycosaminoglycan, sponges such as polyurethane foam, DseA-3D (registered trademark), poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives and copolymers thereof, polyvinyl methyl ether, polypropylene oxide, polyethylene oxide, polyvinyl Examples of temperature-responsive polymers such as partially acylated lower alcohols, polyacrylamide, polyvinyl alcohol, methyl cellulose, nitrocellulose, cellulose butyrate, polyethylene oxide, and hydrogels such as poly(2-hydroxyethylmethacrylate) / polycaprolactone can be mentioned. It is also possible to prepare a carrier for embedding cells using two or more of these polymers. Furthermore, the carrier may contain a physiologically active substance in addition to these polymers. Examples of such physiologically active substances include cell growth factors, differentiation-inducing factors, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins, or extracellular matrices, etc., and it is possible to contain a plurality of these. Examples of cell adhesion factors include poly(monostearoyl glyceride succinate), poly-D,L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagen I to XIX, gelatin, fibronectin, vitronectin, laminin-1 to 12, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, alginate gel, various hydrogels, and furthermore, cleavage fragments thereof, etc. can be mentioned. At this time, two or more cell adhesion factors may be combined. Furthermore, for the medium used for culturing the carrier embedding cells and / or tissues, one or more thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose, carboxymethyl cellulose, agarose, tamarind seed gum, pullulan, etc. can be mixed. The method of embedding cells and / or tissues in these carriers is not particularly limited. For example, a mixed solution of cells and the polymer may be aspirated into a syringe and dropped into a culture medium through an injection needle of about 25G to 19G, or dropped into a culture medium using a micropipette. The size of the bead-shaped carrier formed here is determined by the shape of the tip of the instrument used when dropping the cell and polymer mixture, preferably from several tens of μm to several thousand μm, more preferably from 100 μm to 2000 μm. The number of cells that can be cultured in the bead-shaped carrier is not particularly limited, and it may be freely selected according to this bead size. For example, in the case of a bead-shaped carrier with a diameter of about 2000 μm, up to 5 million cells can be embedded in the bead-shaped carrier of this size. Also, the cells may be dispersed one by one in the carrier, or cell aggregates formed by a plurality of cells may be formed. At this time, the carrier embedded with cells and / or tissues can be uniformly dispersed without performing operations such as stirring by using a culture medium composition containing the structure of the specific compound of the present invention. Therefore, the target cells and / or tissues can be cultured without loss of cell function. The cells and / or tissues cultured by this method can be recovered by performing centrifugation or filtration treatment in a state of being embedded in the carrier after culture. At this time, centrifugation or filtration treatment may be performed after adding the used liquid culture medium. For example, the gravitational acceleration (G) during centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used during filtration treatment is 10 μm to 100 μm, but it is not limited thereto. The cells and / or tissues cultured by this method can be dispersed and recovered by decomposing the carrier using various chelating agents, treatments such as heat and enzymes.

[0065] The method for forming cell aggregates (spheres) is not particularly limited and can be appropriately selected by those skilled in the art. Examples thereof include a method using a container having a cell non-adhesive surface, a hanging drop method, a rotary culture method, a three-dimensional scaffold method, a centrifugation method, a method using aggregation by an electric field or a magnetic field, etc. For example, regarding the method using a container having a cell non-adhesive surface, the target cells can be cultured in a culture container subjected to a surface treatment that inhibits cell adhesion to form spheres. When using this cell non-adhesive culture container, first, the target cells are collected, and then a cell suspension is prepared, seeded in the culture container, and cultured. When culturing is continued for about a week, the cells spontaneously form spheres. As the cell non-adhesive surface used at this time, a surface of a generally used culture container such as a petri dish coated with a substance that inhibits cell adhesion can be used. Such substances include agarose, agar, poly-HEMA (poly-(2-hydroxy-ethyl methacrylate)), a copolymer of 2-methacryloyloxyethyl phosphorylcholine and other monomers (for example, butyl methacrylate, etc.), poly(2-methoxymethyl acrylate), poly-N-isopropylacrylamide, mebiol gel (registered trademark), etc. However, as long as there is no cytotoxicity, it is not limited to these. In addition, as a method for forming cell aggregates (spheres), the methods described in NATURE BIOTECHNOLOGY, VOL.28, NO.4, APRIL 2010, 361-366, NATURE PROTOCOLS, VOL.6, NO.5, 2011, 689-700, NATURE PROTOCOLS, VOL.6, NO.5, 2011, 572-579, Stem Cell Research, 7, 2011, 97-111, Stem Cell Rev and Rep, 6, 2010, 248-259, etc. can also be used. In addition, the culture medium used for culturing to form spheres can also contain components that accelerate the formation of spheres or promote their maintenance. Examples of components having such effects include dimethyl sulfoxide, superoxide dismutase, ceruloplasmin, catalase, peroxidase, L-ascorbic acid, L-ascorbic acid phosphate ester, tocopherol, flavonoid, uric acid, bilirubin, selenium-containing compounds, transferrin, unsaturated fatty acids, albumin, theophylline, forskolin, glucagon, dibutyryl cyclic AMP, etc. Examples of selenium-containing compounds include sodium selenite, sodium selenate, dimethyl selenide, hydrogen selenide, selenomethionine, Se-methylselenocysteine, selenocystathionine, selenocysteine, selenohomocysteine, adenosine-5'-phosphoselenate, Se-adenosylselenomethionine, ROCK inhibitors such as Y27632, Fasudil (HA1077), H-1152, Wf-536, etc. In addition, in order to obtain cell aggregates of a uniform size as intended, a plurality of depressions having the same diameter as the intended cell aggregates can be introduced onto the cell non-adhesive culture vessel used. If these depressions are in contact with each other or within the diameter range of the intended cell aggregates, when the cells are seeded, the seeded cells will surely form cell aggregates of a size corresponding to the volume in the depressions without forming cell aggregates between the depressions, and a population of cell aggregates of a uniform size can be obtained. The shape of the depressions at this time is preferably hemispherical or conical. Alternatively, spheres can also be formed based on a support having cell adhesiveness. Examples of such supports include collagen, polyrotaxane, polylactic acid (PLA), poly(lactic-co-glycolic acid) copolymer (PLGA), hydrogel, etc. In addition, spheres can also be formed by co-culturing with feeder cells. As feeder cells for promoting sphere formation, any adherent cells can be used, but preferably feeder cells corresponding to various cells are desirable. Although not limited, for example, when forming spheres of cells derived from the liver or cartilage, examples of such feeder cells include COS-1 cells and vascular endothelial cells as suitable cell types. Furthermore, spheres can also be formed using a culture composition containing the structure of the specific compound of the present invention. At this time, the concentration of the specific compound is, as detailed above, a concentration that can uniformly suspend (preferably suspend and leave still) cells and / or tissues without substantially increasing the viscosity of the liquid medium, for example, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.3% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), and even more preferably 0.01% to 0.05% (weight / volume). Then, the specific compound may be added to the medium used for sphere formation. Also In another aspect, the concentration of the specific compound is, as detailed above, a concentration that can uniformly suspend (preferably suspend and leave still) cells and / or tissues without substantially increasing the viscosity of the liquid medium, for example, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.3% (weight / volume), more preferably 0.003% to 0.1% (weight / volume), and even more preferably 0.005% to 0.05% (weight / volume). Then, the specific compound may be added to the medium used for sphere formation. The spheroids are prepared by uniformly dispersing the target cells in a medium containing the structure of the specific compound and allowing them to stand and culture for 3 to 10 days. The spheroids prepared here can be recovered by centrifugation or filtration. For example, the gravitational acceleration (G) during centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used for filtration is 10 μm to 100 μm, but it is not limited thereto. In addition, the cultured spheroids can be recovered by magnetic force using magnetic microparticles coated with an antibody that specifically binds to the target cells on the surface. Examples of such magnetic microparticles include Dynabeads (manufactured by Invitrogen), MACS MicroBeads (manufactured by Miltenyi Biotec), BioMag (manufactured by Technichemical), etc. The size of the spheroids varies depending on the cell type and the culture period and is not particularly limited. However, when they are spherical or ellipsoidal, they have 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. Such spheroids can maintain their proliferative ability for a period of 10 days or more, preferably 13 days or more, and more preferably 30 days or more by continuing static culture as it is. However, by performing mechanical division regularly during static culture, or by further performing single-cell treatment and aggregation, they can maintain their proliferative ability substantially indefinitely. The culture vessel used for culturing the spheroids is not particularly limited as long as it can generally culture animal cells. For example, flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, chamber slides, cell culture flasks, spinner flasks, petri dishes, tubes, trays, culture bags, roller bottles, EZ SPHERE( (manufactured by Asahi Glass Co., Ltd.), Sumilon Celltight Plate (manufactured by Sumitomo Bakelite Co., Ltd.), etc. Among these culture vessels, when evaluating a large number of anticancer agents, or evaluating pharmaceutical candidate compounds or pharmaceuticals, microplates, microwell plates, multiplates, and multiwell plates are preferably used. The well bottom shape of these plates is not particularly limited, but flat-bottomed, U-shaped, and V-shaped ones can be used, and preferably U-shaped ones are used. The material of these culture devices is not particularly limited, and examples thereof include plastics such as glass, polyvinyl chloride, cellulose-based polymers, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, and polypropylene.

[0066] The medium used for static culture of spheroids can contain cell adhesion factors, and examples thereof include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Two or more of these cell adhesion factors can be added in combination. Furthermore, thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose, carboxymethyl cellulose, agarose, tamarind seed gum, and pullulan can be further mixed into the medium used for culturing spheroids. By using the medium composition containing the structure of the specific compound of the present invention, it can be uniformly dispersed in the culture solution without performing operations such as shaking, so that the target cells and / or tissues can be cultured as spheroids without loss of cell function. Cultured by static culture according to this method The formed spheres can be recovered by performing centrifugation or filtration after culturing. At this time, after adding the used liquid medium, centrifugation or filtration may be performed. For example, the gravitational acceleration (G) during centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used during filtration is 10 μm to 100 μm, but it is not limited thereto. Further, the cultured spheres can be recovered by magnetic force using magnetic microparticles coated with an antibody that specifically binds to the target cells on the surface. Examples of such magnetic microparticles include Dynabeads (manufactured by Invitrogen), MACS MicroBeads (manufactured by Miltenyi Biotec), BioMag (manufactured by Technichemical), etc. The recovered spheres can be dispersed as single cells by dissociating them using various chelating agents, heat, filters, enzymes, etc. The recovery of cells and the exchange of the medium composition can also be achieved by performing centrifugation, filtration, or magnetic recovery using a bioreactor or an automatic culture device that can be operated in a closed environment under mechanical control.

[0067] As a method for statically culturing plant-derived cells and / or tissues, callus, which is an undifferentiated mass of plant cells, can be cultured. The induction of callus can be performed by methods known for each plant species used. For example, after sterilizing the surface of a part of the tissue of a differentiated plant body (e.g., sections of roots, stems, leaves, seeds, meristems, embryos, pollen, etc.) using 70% alcohol, 1% sodium hypochlorite solution, etc. as necessary, a tissue piece of an appropriate size (e.g., a root section of about 1 to about 5 mm square) is cut out using a scalpel, etc. as necessary, and the tissue piece is seeded on a pre-sterilized callus induction medium by aseptic operation using a clean bench, etc. and aseptically cultured under appropriate conditions. The callus induced here may be immediately subjected to liquid culture for mass propagation, or it can also be maintained as a stock strain by subculturing in a subculture medium. The subculture can be performed using either a liquid medium or a solid medium. When starting static culture using the medium composition of the present invention, the amount of the plant cell mass inoculated varies depending on the growth rate of the target cells, the culture mode (batch culture, fed-batch culture, continuous culture, etc.), the culture period, etc. For example, when culturing a plant cell mass such as callus, the wet weight of the cell mass with respect to the medium composition of the present invention is 4 to 8 (weight / volume (w / v))%, preferably 5 to 7 (w / v)%, and it is inoculated into the medium composition of the present invention. The particle size of the plant cell mass during culture is 1 mm to 40 mm, preferably 3 mm to 20 mm, more preferably 5 mm to 15 mm. Here, the "particle size" means, for example, the diameter when the plant cell mass is spherical, the major axis when it is ellipsoidal, and the maximum length that can be similarly obtained in other shapes.

[0068] When culturing cells and / or tissues, the temperature is usually 25 to 39°C, preferably 33 to 39°C for animal cells. The CO2 concentration is usually 4 to 10% by volume in the culture atmosphere, and 4 to 6% by volume is preferred. The culture period is usually 3 to 35 days, but it may be freely set according to the purpose of the culture. The culture temperature of plant cells is usually 20 to 30°C, and if light is required, it may be cultured under an illuminance condition of 2000 to 8000 lux. The culture period is usually 3 to 70 days, but it may be freely set according to the purpose of the culture.

[0069] When culturing cells and / or tissues by the method of the present invention, cells and / or tissues separately prepared with respect to the culture composition of the present invention may be added and mixed so as to be uniformly dispersed. The mixing method at that time is not particularly limited, and examples include manual mixing such as pipetting, and mixing using devices such as a stirrer, a vortex mixer, a microplate mixer, and a shaker. After mixing, the culture solution may be left in a static state, or the culture solution may be rotated, shaken, or stirred as necessary. The rotation speed and frequency may be appropriately set according to the purpose of those skilled in the art. Also, when it becomes necessary to replace the medium composition during the static culture period, after separating the cells and / or tissues and the medium composition by centrifugation or filtration treatment, a new medium composition The substance may be added to cells and / or tissues. Alternatively, after appropriately concentrating the cells and / or tissues by centrifugation or filtration, a new culture medium composition may be added to this concentrated solution. For example, the gravitational acceleration (G) during centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used during filtration is 10 μm to 100 μm, but it is not limited thereto. In addition, cells and / or tissues cultured can be separated by magnetic force using magnetic microparticles coated with an antibody that specifically binds to the target cells on the surface. Examples of such magnetic microparticles include Dynabeads (manufactured by Invitrogen), MACS MicroBeads (manufactured by Miltenyi Biotec), BioMag (manufactured by Technichemical), magnetic microspheres (manufactured by Polysciences Inc.), and the like. The exchange of these culture medium compositions can also be performed by a bioreactor or an automatic culture device that can be executed in a closed environment under mechanical control.

[0070] Since cancer cells are efficiently proliferated by the method of the present invention, the culture medium composition containing the specific compound in the present invention can be used for the evaluation of anticancer agents against cancer cells. For example, when evaluating an anticancer agent that inhibits the growth of cancer cells, the number and type of cells, changes in cell surface differentiation markers and expressed genes when the cancer cells and the anticancer agent are co-cultured are analyzed. At this time, by using the culture medium composition of the present invention, not only can the number of cells to be analyzed be efficiently amplified, but also can be efficiently recovered. In the present invention, in particular, a culture medium additive for cancer cells containing deacylated gellan gum or a salt thereof, and a culture medium composition for cancer cells containing the additive can be used for the evaluation of cancer cell growth or anticancer activity, etc. The concentration of the deacylated gellan gum or a salt thereof at that time is as described above. And even when using diutan gum, cancer cells are proliferated, but considering the particularly excellent growth effect on cancer cells, the fact that bubbles are less likely to occur in the culture solution because it can be used at a low concentration (the aforementioned preferred concentration), and the fact that cancer cells are easily recovered, deacylated gellan gum is more preferable.

[0071] As a more specific method for screening anticancer agents, there may be mentioned a method including (a) a step of culturing cancer cells in the culture medium composition of the present invention in the presence and absence of a test substance, and (b) a step of analyzing changes in the growth of cancer cells. The method may further include a step of selecting a substance that suppresses the growth of cancer cells and / or a step of recovering cancer cells, as compared with the case in the absence of the test substance. "Change" means that the growth of cancer cells increases or decreases. The analysis can be performed by the above method, but is not limited thereto.

[0072] When evaluating the activity of an anticancer agent, the culture conditions, culture utensils, culture apparatus, type of medium, type of specific compound, content of specific compound, type of additive, content of additive, culture period, culture temperature, type of anticancer agent, content of anticancer agent, etc. can be appropriately determined by the parties within the ranges described in this specification. Cells that have grown or appeared by culturing can be observed using a standard microscope in the art. When measuring the number of cells, the colony formation method, crystal violet method, thymidine incorporation method, trypan blue staining method, ATP (adenosine triphosphate) measurement method, 3-(4,5-Dimethylthial-2-yl)-2,5-Diphenylt etrazalium Bromide (MTT) staining method, WST-1 (registered trademark) staining method, WST-8 (registered trademark) staining method, flow cytometry method, method using a cell number automatic measurement device, etc. can be used. Among these, the WST-8 (registered trademark) staining method can be most preferably used. Also, when evaluating the cytotoxicity to cells, the lactate dehydrogenase (LDH) activity measurement method, CytoTox-ONE (registered trademark) method, etc. can be used. Alternatively, after staining the cultured cells with a specific antibody, the cell surface differentiation marker can be detected by ELISA or flow cytometry, and the effects on growth and apoptosis by the anticancer agent can be observed. Furthermore, genes whose expression has changed due to the anticancer agent can be extracted from the cultured cells as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) It can be detected by methods such as extraction, Southern blotting, Northern blotting, RT-PCR method, etc.

[0073] Since the survival and function of hepatocytes are maintained by the method of the present invention, the medium composition containing the specific compound in the present invention can be used when evaluating various effects of pharmaceuticals or pharmaceutical candidate substances on hepatocytes. For example, when evaluating the toxic effect of a pharmaceutical candidate substance on hepatocytes, the number and type of cells, the changes in cell surface differentiation markers and expressed genes when culturing hepatocytes in coexistence with the test substance to be evaluated are analyzed. At this time, by using the medium composition of the present invention, not only the survival and function of the hepatocytes to be analyzed can be maintained, but also the hepatocytes can be efficiently recovered.

[0074] Examples of the method for screening a pharmaceutical candidate substance acting on hepatocytes include a method comprising: (a) a step of culturing hepatocytes in the medium composition of the present invention in the presence and absence of a test substance; and (b) a step of analyzing changes in the physiological functions of hepatocytes. Examples of the method for evaluating the efficacy or toxicity of a pharmaceutical candidate substance acting on hepatocytes include a method comprising: (a) a step of culturing hepatocytes in the medium composition of the present invention in the presence and absence of a test substance; and (b) a step of analyzing changes in the physiological functions of hepatocytes. These methods may further include a step of selecting a substance that suppresses or increases the physiological functions of hepatocytes as compared with the case in the absence of the test substance, and / or a step of recovering hepatocytes. The said change means that the physiological functions of hepatocytes (for example, hepatocyte proliferation, enzyme activity of cytochrome P450, etc.) increase or decrease. And when the physiological function of hepatocytes increases, evaluations such as low efficacy or toxicity, and when the physiological function of hepatocytes decreases, high efficacy or toxicity, etc. can be made.

[0075] When evaluating the activity of the pharmaceutical candidate substance, the culture conditions, culture vessels, culture apparatus, type of culture medium, type of specific compound, content of specific compound, type of additive, content of additive, culture period, culture temperature, type of pharmaceutical or pharmaceutical candidate substance or its content, etc. are appropriately selected by the parties from the ranges described in this specification. The cells maintained or emerged by culturing can be observed using a standard microscope in the art. When measuring the cell count, methods such as colony formation method, crystal violet method, thymidine incorporation method, trypan blue staining method, ATP (adenosine triphosphate) measurement method, 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 an automatic cell counter, etc. can be used. Among these, the WST-8 (registered trademark) staining method can be most preferably used. When evaluating the cytotoxicity to cells, methods such as lactate dehydrogenase (LDH) activity measurement method, CytoTox-ONE (registered trademark) method, etc. can be used. Also, after staining the cultured cells with a specific antibody, cell surface differentiation markers can be detected by ELISA (Enzyme-linked immu nosorbent assay) or flow cytometry, and the effects of the pharmaceutical or pharmaceutical candidate substance on cell growth and apoptosis can be observed. Genes whose expression has changed due to a pharmaceutical or pharmaceutical candidate substance can be detected by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from the cultured cells and using methods such as Southern blotting, Northern blotting, RT-PCR method, etc. Also, proteins whose expression has changed due to a pharmaceutical or pharmaceutical candidate substance can be detected by ELISA, Western blotting, flow cytometry method, etc. Furthermore, the enzyme activity of cytochrome P450 can be detected by using methods such as radioisotope method, high performance liquid chromatography method, luminescence method, colorimetric method, etc. to detect the structural conversion activity of the substrate by the enzyme.

Example

[0076] The present invention will be described in more detail by specifically presenting analysis examples and test examples of the culture medium composition of the present invention as examples, but the present invention is not limited thereto. invention will be further described in detail, but the present invention is not limited by these.

[0077] (Analysis Example 1: Viscosity Measurement and Cell Suspension Test of a Medium Containing Deacylated Gellan Gum) Preparation and Viscosity Measurement of a Medium Containing Deacylated Gellan Gum Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in pure water to a concentration of 0.4% (w / v), and then heated and stirred at 90 °C until dissolved. While stirring this aqueous solution, it was allowed to cool to room temperature and autoclaved at 121 °C for 20 minutes. 50 mL of 2-fold concentrated DMEM / F-12 medium (manufactured by Aldrich) and 47.5 mL of sterilized water were placed in a 300 mL tall beaker, and 2.5 mL of the deacylated gellan gum aqueous solution was added while stirring with a homomixer (3000 rpm) at room temperature, and stirring was continued for 1 minute to prepare a medium composition with a final concentration of deacylated gellan gum of 0.01%. Similarly, medium compositions were prepared by adding deacylated gellan gum aqueous solutions so that the final concentrations were 0.02, 0.03, and 0.05% (w / v). The viscosity of this medium composition was measured at 37 °C using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., Viscometer TVE-22L, standard rotor 1°34´×R24) at a rotational speed of 100 rpm for 5 minutes. Cell Suspension Test of a Medium Containing Deacylated Gellan Gum The human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was suspended in EMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum at a density of 250,000 cells / mL. 10 mL of this suspension was seeded into an EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and then cultured in a CO2 incubator (5% CO2) for 3 days. 10 mL of the suspension of the obtained spheres (diameter 100 - 200 μm) was centrifuged (200G, 5 minutes) to precipitate the spheres, and 1.0 mL of the sphere suspension was prepared by removing the supernatant. Subsequently, 1.0 mL of the deacylated gellan gum-containing medium prepared above was placed in 1.5 mL Eppendorf tubes, and 10 μL of the HeLa cell sphere suspension was further added. By tapping the cell mass was dispersed, incubated at 37 °C, and the dispersion state of the cells after 1 hour was visually observed.

[0078] (Comparative Example) Preparation of Methylcellulose and Collagen-Containing Medium Preparation of Methylcellulose-Containing Medium 100 mL of DMEM / F-12 medium (manufactured by Aldrich) was placed in a 200 mL eggplant flask, and 0.1 g of methylcellulose (M0387, manufactured by Aldrich) was added. While cooling in an ice bath, the mixture was stirred to dissolve the methylcellulose. Using this solution, medium compositions were prepared by adding aqueous methylcellulose solutions to achieve final concentrations of 0.1, 0.3, 0.6, and 1.0% (w / v). Preparation of Collagen-Containing Medium 1 mL of 10-fold concentrated DMEM / F-12 medium (manufactured by Aldrich), 1 mL of reconstitution buffer (manufactured by Nitta Gelatin Inc.), and 1.5 mL of pure water were added to 6.5 mL of 0.3% Cellmatrix type I-A (manufactured by Nitta Gelatin Inc.), and a 0.2% collagen-containing medium was prepared while stirring in ice. Similarly, medium compositions were prepared by adding collagen to achieve final concentrations of 0.01, 0.05, 0.1, and 0.2% (w / v). For the medium compositions prepared above, suspension tests and viscosity measurements of HeLa cell spheres were also carried out in the same manner as for the deacylated gellan gum-containing medium. However, the viscosity of 1.0% (w / v) methylcellulose was measured at 50 rpm because it was outside the measurement range of the device.

[0079] [Table 1]

[0080] [Table 2]

[0081] [Table 3]

[0082] [Test Example] Next, the usefulness of the medium composition of the present invention in cell culture will be specifically described in the following test examples, but the present invention is not limited thereto. The concentration (%) of CO2 in the CO2 incubator was expressed as the volume % of CO2 in the atmosphere. Also, PBS means phosphate buffered saline (manufactured by Sigma-Aldrich Japan), and FBS means fetal bovine serum (manufactured by Biological Industries). Also, (w / v) represents the weight per unit volume.

[0083] (Test Example 1: Cell Proliferation Test When Single Cells are Dispersed) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.015% (w / v) to IMDM medium (manufactured by Gibco) containing 10% (v / v) fetal bovine serum and 10 ng / mL of thrombopoietin (manufactured by Wako). Subsequently, the human leukemia cell line UT7 / TPO was seeded into the above medium composition containing desacylated gellan gum at a density of 20,000 cells / mL, and then dispensed into the wells of a 6-well flat-bottom microplate (manufactured by Corning) at 5 mL per well. Similarly, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into a medium composition containing 0.015% (w / v) desacylated gellan gum in EMEM medium (manufactured by Wako) containing 10% (v / v) fetal bovine serum at a density of 20,000 cells / mL, and then dispensed into the wells of a 6-well flat-bottom microplate (manufactured by Corning) at 5 mL per well. These cell suspensions were cultured in a CO2 incubator (5% CO2) in a static state for 3 days. Then, a part of the culture solution was collected, the same amount of trypan blue staining solution (manufactured by Invitrogen) was added, and the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.). After seeding into the medium composition added with desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) and then dispensed into the wells of a 6-well flat-bottom microplate (manufactured by Corning) at 5 mL per well. These cell suspensions were cultured in a CO2 incubator (5% CO2) in a static state for 3 days. Then, a part of the culture solution was collected, the same amount of trypan blue staining solution (manufactured by Invitrogen) was added, and the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.).

[0084] As a result, it was confirmed that UT7 / TPO cells and HeLa cells could be uniformly cultured in a floating state by using the medium composition of the present invention and could grow in the said medium composition. Table 4 shows the number of UT7 / TPO cells and HeLa cells after 3 days of floating static culture.

[0085]

Table 4

[0086] (Test Example 2: Cell growth test when culturing cell line-derived spheres) The human hepatoma cell line HepG2 (manufactured by DS Pharma Biomedical Co., Ltd.) was suspended in DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum to a concentration of 250,000 cells / mL. Then, 10 mL of this suspension was seeded into EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and cultured in a CO2 incubator (5% CO2) for 7 days. Similarly, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was suspended in EMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum to a concentration of 250,000 cells / mL. Then, 10 mL of this suspension was seeded into EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and cultured in a CO2 incubator (5% CO2) for 7 days. 2.5 mL of the suspension of the spheres (diameter 100 - 200 μm) of each cell line obtained here was centrifuged (200 G, 5 minutes) to precipitate the spheres and remove the supernatant. Subsequently, 10 mL of the above medium was added to and suspended in this sphere (about 800 pieces), and then transferred to a flat-bottom tube (manufactured by BM Instruments Co., Ltd.). Similarly, a suspension of spheres was prepared using a medium composition in which 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was added to the above medium, and transferred to a flat-bottom tube (manufactured by BM Instruments Co., Ltd.). The medium composition containing 0.015% (w / v) of deacylated gellan gum was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolving it by stirring while heating at 90°C, autoclaving this aqueous solution at 121°C for 20 minutes, and then adding it to DMEM medium containing 10% (v / v) fetal bovine serum at a 1 / 20 dilution.

[0087] The above-mentioned sphere suspension was statically cultured in a CO2 incubator (5% CO2) at 37°C for 3 days, and then 2 volumes of the medium were added, followed by centrifugation (200G, 5 minutes) to precipitate the spheres, and the supernatant was removed. Here, a part of the spheres was collected, and their shape was observed with an optical microscope (CK30-F100, manufactured by OLMPUS). Subsequently, the collected spheres were washed once with 10 mL of PBS, and then 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 5 minutes. After adding 9 mL of the above medium, the cells were collected by centrifugation (200G, 5 minutes). To a part of 2 mL of the cell suspension obtained here, an equal volume of trypan blue staining solution (manufactured by Invitrogen) was added, and the number of live and dead cells was measured using a hemocytometer (manufactured by ERMA Sales Co., Ltd.).

[0088] As a result, the spheres of HepG2 cells and HeLa cells can be cultured in a floating state by using the medium composition of the present invention and it was confirmed that the cells proliferated efficiently in the medium composition. Moreover, it was confirmed that the medium composition of the present invention has a lower proportion of dead cells when the cells are proliferated compared to the existing medium, and has an excellent effect of promoting cell proliferation. At this time, the spheres cultured in the existing medium had settled on the bottom surface of the culture vessel. Furthermore, when the shape of the cultured spheres was observed with an optical microscope, no aggregation of spheres was observed in the medium composition of the present invention, whereas aggregation of spheres was observed in the existing medium. Table 5 shows the relative cell numbers of HepG2 cells and HeLa cells when the number of cells cultured in a medium not containing deacylated gellan gum was set to 1. Table 6 shows the relative dead cell rates when the dead cell rate (number of dead cells / number of live cells) when cultured in a medium not containing deacylated gellan gum was set to 1. Figures 1 and 2 respectively show the floating states of the spheres of HepG2 cells and HeLa cells cultured in the medium composition of the present invention. Furthermore, Figure 3 shows the shape of the spheres of the cultured HeLa cells.

[0089]

Table 5

[0090] [Table 6]

[0091] (Test Example 3: Cell Proliferation Test in Adherent Culture of Human Pluripotent Stem Cells) Human pluripotent stem cells (hPSCs) are usually maintained and proliferated under planar culture conditions where they are adhered onto a feeder or a culture dish coated with Matrigel. To evaluate the toxicity of deacylated gellan gum on hPSCs, deacylated gellan gum was added to mTeSR medium (manufactured by STEM CELL Technologies) at a concentration of 0.000% to 0.020% (w / v) under planar culture conditions using Matrigel (manufactured by BD Biosciences), and the effect on the proliferation of hPSCs was examined. At this time, the Kyoto University 253G1 strain was cultured as human iPS cells, and the Kyoto University KhES-1 strain was cultured as human ES cell line. The culture medium composition added with deacylated gellan gum at the above concentration was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolving it by stirring while heating at 90°C, autoclaving this aqueous solution at 121°C for 20 minutes, and then adding it to the mTeSR medium to reach the predetermined concentration. As a result, for both human iPS cells and human ES cells, the same number of cells as in the normal mTeSR medium could be obtained even in the culture medium added with deacylated gellan gum, and no toxicity due to deacylated gellan gum was observed. The results are shown in Figure 4. The number of cells after culture in Figure 4 is shown as a relative value with the number of cells obtained by seeding hPSCs on a Matrigel-coated culture dish and culturing them in mTeSR medium containing deacylated gellan gum for 5 days, with the number of cells in mTeSR medium not containing deacylated gellan gum set as 1.

[0092] (Test Example 4: Sedimentation Inhibition Test of Deacylated Gellan Gum in Spheroid Culture of Human Pluripotent Stem Cells) hPSCs form spheres in low-adhesion culture dishes such as Petri dishes. For example, the spheres can be formed using any of the methods described in NATURE BIOTECHNOLOGY, VOL.28, NO.4, APRIL 2010, 361-366, NATURE PROTOCOLS, VOL.6, NO.5, 2011, 689-700, NATURE PROTOCOLS, VOL.6, NO.5, 2011, 572-579, Stem Cell Research, 7, 2011, 97-111, Stem Cell Rev and Rep, 6, 2010, 248-259. hPSCs (Kyoto University 253G1 strain or Kyoto University KhES-1 strain) maintained on feeder cells (mouse embryonic fibroblasts) were collected. After removing the feeder cells by natural sedimentation, the hPSCs were resuspended in mTeSR medium supplemented with the Rho kinase inhibitor Y-27632 (10 μM). Subsequently, hPSCs colonies of a certain size were seeded onto a Petri dish (manufactured by BD Falcon), and spheres were formed by culturing in a CO2 incubator (5% CO2) at 37°C. Medium exchange was performed on the 1st and 3rd days after passage with mTeSR medium without Y-27632, and passage was performed every 5 days with mTeSR medium containing Y-27632. The spheres of hPSCs (4th day of culture) prepared in this way were suspended in a medium composition (prepared by the same method as in Test Example 3) obtained by adding 0.000% to 0.020% (w / v) of deacylated gellan gum to mTeSR medium, and then transferred to a cuvette. This cuvette was left overnight in a CO2 incubator (5% CO2) at 37°C to examine the effect of deacylated gellan gum on suppressing the sedimentation of the spheres. The results are shown in Fig. 5. As shown in Fig. 5, the addition of deacylated gellan gum enabled the spheres to be maintained in a three-dimensional floating state in the medium in all concentration ranges. On the other hand, it was found that the spheres sedimented to the bottom of the culture vessel and could not be in a floating state in the existing medium without the addition of deacylated gellan gum. Also, the effect of deacylated gellan gum was common to human iPS cells and human ES cells. From the above results, it was shown that deacylated gellan gum can keep the spheres of hPSCs in a floating state. PRIL 2010, 361-366, NATURE PROTOCOLS, VOL.6, NO.5, 2011, 689-700, NATURE PROTOCOLS, VOL.6, NO.5, 2011, 572-579, Stem Cell Research, 7, 2011, 97-111, Stem Cell Rev and Rep, 6, 2010, 248-259, any method can be used to form them. hPSCs (Kyoto University 253G1 strain or Kyoto University KhES-1 strain) maintained on feeder cells (mouse embryonic fibroblasts) were collected. After removing the feeder cells by natural sedimentation, the hPSCs were resuspended in mTeSR medium supplemented with the Rho kinase inhibitor Y-27632 (10 μM). Subsequently, hPSCs colonies of a certain size were seeded onto a Petri dish (manufactured by BD Falcon), and spheres were formed by culturing in a CO2 incubator (5% CO2) at 37°C. Medium exchange was performed on the 1st and 3rd days after passage with mTeSR medium without Y-27632, and passage was performed every 5 days with mTeSR medium containing Y-27632. The spheres of hPSCs (4th day of culture) prepared in this way were suspended in a medium composition (prepared by the same method as in Test Example 3) obtained by adding 0.000% to 0.020% (w / v) of deacylated gellan gum to mTeSR medium, and then transferred to a cuvette. This cuvette was left overnight in a CO2 incubator (5% CO2) at 37°C to examine the effect of deacylated gellan gum on suppressing the sedimentation of the spheres. The results are shown in Fig. 5. As shown in Fig. 5, the addition of deacylated gellan gum enabled the spheres to be maintained in a three-dimensional floating state in the medium in all concentration ranges. On the other hand, it was found that the spheres sedimented to the bottom of the culture vessel and could not be in a floating state in the existing medium without the addition of deacylated gellan gum. Also, the effect of deacylated gellan gum was common to human iPS cells and human ES cells. From the above results, it was shown that deacylated gellan gum can keep the spheres of hPSCs in a floating state.

[0093] (Test Example 5: Cell Proliferation Test in Sphere Culture of Human Pluripotent Stem Cells) It was examined whether hPSCs can be cultured in a tube in a three-dimensional floating state. hPSC spheres (600 to 800 cells / 3 mL) prepared and passaged in the same manner as in Test Example 4 were seeded into 5 mL polystyrene tubes (manufactured by BD Falcon) in mTeSR medium containing 0.000%, 0.015% or 0.020% (w / v) deacylated gellan gum so that the number of spheres was the same, and cultured in a CO2 incubator (5% CO2) at 37 °C for 5 days. For medium replacement, 3 times the volume of DMEM / F-12 medium (manufactured by Sigma) was added to the culture solution, and then the spheres were sedimented by centrifugation (100 G, 3 minutes), and new medium was added to the spheres. This was done on the 1st and 3rd days after passage. On the 5th day, after adding an equal volume of D MEM / F-12 medium (manufactured by Sigma), all the spheres were collected by centrifugation (100 G, 3 minutes), dissociated into single cells using trypsin-EDTA solution (manufactured by Invitrogen), and the cell number was measured using a nucleocounter (manufactured by Chemometec). As a result, in the medium without deacylated gellan gum, the spheres sank to the bottom of the tube and formed large cell masses without showing growth. However, in the medium containing 0.015% or 0.020% (w / v) deacylated gellan gum, the size of the spheres increased in a three-dimensional floating state. When the number of seeded cells was set to 1, about 10 times the number of cells was obtained on the 5th day, and cell growth was observed. The results are shown in Fig. 6. Fig. 6 relatively shows the number of cells on the 5th day when the number of seeded cells is set to 1. Also, in human ES cells, actually 3,000,000 cells per 3 mL could be obtained on the 5th day of culture in a polystyrene tube (corresponding to about 1,000,000,000 cells in the case of 1000 mL of medium).

[0094] (Test Example 6: Confirmation Test for Maintenance of Undifferentiated State in Sphere Culture of Human Pluripotent Stem Cells) ​Regarding hPSCs sphere cells cultured in suspension and statically in mTeSR medium containing 0.015% or 0.020% (w / v) deacylated gellan gum, the maintenance of their undifferentiated ability was examined by flow cytometry analysis. In a polystyrene tube, cells of human ES cells (KhES-1) passaged 3 times and human iPS cells (253G1) passaged 4 times in a spherical state were collected. Then, they were stained with antibodies against SSEA4 (#MAB4304, manufactured by Millipore), a surface marker indicating the undifferentiated state of hPSCs, and TRA-1-60 (#MAB4360, manufactured by Millipore), and the positive rate of antibody staining of the cells was evaluated using FACSCantoII (manufactured by Becton Dickinson). The results are shown in Figure 7. As shown in Figure 7, for both A: human iPS cells (253G1) and B: human ES cells (KhES-1), more than 90% of the cells cultured in suspension and statically in the supplemented medium containing deacylated gellan gum expressed pluripotent stem cell markers, similar to the cells maintained on Matrigel. Note that only the secondary antibody staining was performed as a negative control. As described above, it was found that hPSCs spheres cultured in suspension and statically in the supplemented medium containing deacylated gellan gum maintained undifferentiated state for both human iPS cells and human ES cells. After that, for human iPS cells (253G1), cells passaged 4 times were collected. Then, they were stained with antibodies against SSEA4 (#MAB4304, manufactured by Millipore), a surface marker indicating the undifferentiated state of hPSCs, and TRA-1-60 (#MAB4360, manufactured by Millipore), and the positive rate of antibody staining of the cells was evaluated using FACSCantoII (manufactured by Becton Dickinson). The results are shown in Figure 7. As shown in Figure 7, for both A: human iPS cells (253G1) and B: human ES cells (KhES-1), more than 90% of the cells cultured in suspension and statically in the supplemented medium containing deacylated gellan gum expressed pluripotent stem cell markers, similar to the cells maintained on Matrigel. Note that only the secondary antibody staining was performed as a negative control. As described above, it was found that hPSCs spheres cultured in suspension and statically in the supplemented medium containing deacylated gellan gum maintained undifferentiated state for both human iPS cells and human ES cells.

[0095] (Test Example 7: Characterization Analysis of Spherically Cultured Human Pluripotent Stem Cells 1) Using mTeSR medium (STEMCELL Technologies, Inc.) containing 0.020% (w / v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) prepared by the same method as in Test Example 3, human iPS cells (253G1) or human ES cells (KhES-1) spheres were subcultured a total of 9 times by the same method as in Test Example 4. For each cell after culture, the spheres on the first day of passage were placed on mouse embryonic fibroblasts. After that, for human iPS cells (253G1), cells passaged 4 times were collected. Then, they were stained with antibodies against SSEA4 (#MAB4304, manufactured by Millipore), a surface marker indicating the undifferentiated state of hPSCs, and TRA-1-60 (#MAB4360, manufactured by Millipore), and the positive rate of antibody staining of the cells was evaluated using FACSCantoII (manufactured by Becton Dickinson). The results are shown in Figure 7. As shown in Figure 7, for both A: human iPS cells (253G1) and B: human ES cells (KhES-1), more than 90% of the cells cultured in suspension and statically in the supplemented medium containing deacylated gellan gum expressed pluripotent stem cell markers, similar to the cells maintained on Matrigel. Note that only the secondary antibody staining was performed as a negative control. As described above, it was found that hPSCs spheres cultured in suspension and statically in the supplemented medium containing deacylated gellan gum maintained undifferentiated state for both human iPS cells and human ES cells. The following day, the cells were treated with 300 μg / mL thymidine (Sigma-Aldrich) overnight. Subsequently, the cells were treated with 100 ng / mL colcemid (Nacalai Tesque), dissociated into single cells with trypsin-EDTA solution, and hypotonic treatment was performed with 0.075 M KCl. The cells were then fixed with Carnoy's fixative (methanol:acetic acid = 3:1). The karyotype of the fixed cells was analyzed by the Q-banding method (a test outsourced to Chromosome Science Lab, Ltd.). As a result, it was found that both human iPS cells and human ES cells cultured in suspension in the medium composition of the present invention maintained a normal karyotype. The results are shown in Figure 8.

[0096] (Test Example 8: Characterization of sphere-cultured human pluripotent stem cells 2) The mTeSR medium (STEM C) containing 0.020% (w / v) deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) prepared in the same manner as in Test Example 3 was used. Using a ELISA kit (manufactured by ELL Technologies), human iP was prepared in the same manner as in Test Example 4. S cell (253G1) spheres were subcultured a total of 21 to 22 times every 5 days. The spheres were then fixed in 4% (w / v) paraformaldehyde (Nacalai Tesque, Inc.). After soaking in PBS containing 20% sucrose (w / v), they were embedded in a freezing embedding medium (OCT compound, Sakura Finetech Japan, Inc.). Sections 12 μm thick were prepared using a cryostat (Thermo Scientific, Inc.) to reveal the undifferentiated portions of hPSCs. The cells were stained with antibodies against NANOG (#4903, Cell Signaling), which shows mitochondrial activity, OCT3 / 4 (#sc-5279, Santa Cruz), and SSEA4 (#MAB4304, Millipore). As a result, it was revealed that the cells cultured in suspension in a medium composition containing deacylated gellan gum expressed the undifferentiated markers of pluripotent stem cells. As described above, it was found that the spheres of human iPS cells cultured in suspension in a medium composition containing deacylated gellan gum for more than 100 days maintained their undifferentiated state. The results are shown in Figure 9.

[0097] (Test Example 9: Cell Growth Test When Culturing Cell Lines Attached to Microcarriers) Microcarrier Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) was suspended in PBS at a concentration of 0.02 g / mL and allowed to stand overnight Then, the supernatant was discarded, and the microcarriers were washed twice with fresh PBS. Subsequently, they were suspended again in PBS at a concentration of 0.02 g / mL and autoclaved at 121°C for 20 minutes. Subsequently, the microcarriers were washed twice with 70% ethanol and three times with PBS, and then suspended in DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum at a concentration of 0.02 g / mL. Using this microcarrier suspension, 20 mL of DMEM medium containing 120 mg of Cyt odex (registered trademark) 1 and 40,000,000 HepG2 cells (containing 10% (v / v) fetal bovine serum) was prepared, and this cell suspension was cultured in a beaker pre-treated with a silicone coating agent (manufactured by Asahi Tec Glass) at 37°C for 6 hours with stirring (100 rpm) using a stirrer. Here, it was confirmed by microscopy that the HepG2 cells had adhered to the microcarriers. Subsequently, the microcarriers with adhered cells were washed twice with DMEM medium containing 10% (v / v) fetal bovine serum and suspended in 3 mL of the same medium.

[0098] To 20 mL of DMEM medium containing 10% (v / v) fetal bovine serum or a medium composition obtained by adding 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) to this medium, 300 μL of the above microcarrier suspension was added respectively, and the mixture was cultured at 37°C for 3 days. At this time, the culture solution without deacylated gellan gum was cultured while stirring with a stirrer (100 rpm). After the culture, the attachment state of the cells on the microcarriers was confirmed under a microscope, and then the microcarriers were sedimented by centrifugation (200G, 5 minutes). After washing the microcarriers with 10 mL of PBS, 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was incubated at 37°C for 5 minutes. Further, after adding 9 mL of DMEM medium containing 10% (v / v) fetal bovine serum, the microcarriers were removed using a cell strainer with a mesh size of 70 μm (manufactured by BD Falcon). Cells were recovered from the filtrate obtained here by centrifugation (200G, 5 minutes). The cells were suspended in 500 μL of the medium, and an equal amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the suspension, and then the number of viable cells was measured using a hemocytometer (manufactured by ELMA Sales Co., Ltd.). As a result, the culture solution without deacylated gellan gum contained 123,000 cells, while the culture solution containing deacylated gellan gum contained 1,320,000 cells. As described above, it was confirmed that the medium composition containing the structure of the specific compound of the present invention has an excellent cell growth promoting effect compared to the existing medium even when cell culture is carried out using microcarriers. Fig. 10 shows the attachment state of HepG2 cells when microcarrier culture was carried out for 3 days using the medium composition containing the structure of the specific compound of the present invention.

[0099] (Test Example 10: Cell suspension test using cell line-derived spheres) Xanthan gum (KELTROL CG, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 1% (w / v), and then dissolved by stirring while heating at 90°C. Using this aqueous solution, DMEM / F-12 medium compositions with final concentrations of 0.1, 0.15, and 0.2% (w / v) of xanthan gum were prepared. Also, an aqueous solution containing 0.2% (w / v) of κ-carrageenan (GENUGEL WR-80-J, manufactured by Sankyo Co., Ltd.) and 0.2% (w / v) of locust bean gum (GENUGUM RL-200-J, manufactured by Sankyo Co., Ltd.) was prepared by heating at 90°C, and using this aqueous solution, DMEM / F-12 medium (manufactured by Sigma) compositions containing 0.03, 0.04, and 0.05% (w / v) of κ-carrageenan and locust bean gum were prepared.

[0100] Using the same method as in Test Example 2, HeLa cell spheres were created, and several tens of spheres were added to 1 mL of each of the media prepared above. After that, they were left standing at 37°C for 1 hour, and the floating state of the sphere cells was visually observed. As a result, it was confirmed that the HeLa cell spheres were maintained in a floating state in all of the above medium compositions. Furthermore, after adding an equal amount of medium to this cell suspension, it was confirmed that the HeLa cell spheres could be sedimented and recovered by centrifugation (300 to 400 G, 5 minutes). The floating states when culturing the HeLa cell spheres in the medium composition of the present invention are shown in FIG. 11 respectively. Also, the viscosities measured by the same method as in Analysis Example 1 are shown in Tables 7 and 8.

[0101]

Table 7

[0102]

Table 8

[0103] (Test Example 11: Cell suspension test using a filtered medium composition) A DMEM / F-12 medium composition containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was prepared using the same method as in Test Example 2. Subsequently, 1 mL of this medium composition was filtered through filters with pore sizes of 70 μm, 40 μm (BD Falcon), 30 μm, 20 μm (AS ONE), 10 μm (Partec), 5 μm, 1.2 μm, 0.45 μm, and 0.2 μm (Sartorius Stedim Japan), respectively. Approximately several tens of HepG2 cell spheres prepared using the same method as in Test Example 2 were added to the above filtrate, and then allowed to stand at 37°C for 1 hour. The floating state of the sphere cells was observed visually. As a result, it was confirmed that the HepG2 cell spheres were maintained in a floating state in the medium composition that passed through filters with a pore size of 10 μm or more, but precipitated in the medium composition that passed through filters with a pore size of 5 μm or less. Furthermore, it was confirmed that the HepG2 cell spheres in the floating state could be sedimented and recovered by centrifugation at 300 G for 5 minutes at room temperature or by centrifugation at 200 G for 5 minutes at room temperature after adding an equal volume of medium.

[0104] (Test Example 12: Sphere Formation Test) Using the same method as in Test Example 2, a composition of EMEM medium (manufactured by WAKO) containing 0.01% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared. Subsequently, HeLa cells were added to a concentration of 1000 cells / mL and then dispensed into a 24-well plate (manufactured by Corning). The plate was cultured in suspension at 37°C for 9 days, and then the formation of spheres was confirmed under a microscope. Furthermore, the sphere cells were sedimented by centrifugation at 300G for 5 minutes, washed once with 5 mL of PBS, and then 100 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added and incubated at 37°C for 5 minutes. To 100 μL of the cell suspension obtained here, 100 μL of EMEM medium containing 10% (v / v) fetal bovine serum was added, and an equal amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the cell suspension. Then, the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.). As a result, it was confirmed that the number of HeLa cells increased up to 170,000 cells / mL. The sphere of HeLa cells formed with the medium composition of the present invention is shown in FIG. 12.

[0105] (Test Example 13: Optical Microscopic Observation of Structures) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in pure water to a concentration of 0.4 %(w / v), and then heated and stirred at 90°C to dissolve. 95 mL of 2-fold concentrated DMEM / F-12 medium (manufactured by Aldrich) was placed in a 300 mL beaker, and 5 mL of the deacylated gellan gum aqueous solution was added while stirring with a magnetic stirrer at room temperature. Stirring was continued for 5 minutes to prepare a medium composition with a final concentration of deacylated gellan gum of 0.02%. Furthermore, the prepared medium composition was stirred for 5 minutes with a homomixer (3000 rpm). The prepared medium composition was observed with an optical microscope (KEYENCE, BIOREVO BZ-9000). The observed structure is shown in FIG. 13.

[0106] (Test Example 14: Preparation by Mixing and Overheating of Powder Medium and DAG) 20 mg of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) and 1.58 g of DMEM / F-12 medium (manufactured by Life Technologies) were placed in a 200 mL Erlenmeyer flask, and 100 mL of pure water was poured in. Autoclaved at 121 °C for 20 minutes to prepare a DMEM / F-12 medium composition with a deacylated gellan gum concentration of 0.02%. Dextran beads Cytodex1 (Size 200 μm, manufactured by GE Healthcare Life Sciences) were added to the prepared medium, and the dispersion state of the beads was visually confirmed. The floating dispersion state was evaluated as ○, the partially sedimented / dispersed state as △, and the sedimented state as ×. The results are shown in Table 9.

[0107]

Table 9

[0108] (Test Example 15: Preparation of a medium composition mixed with polysaccharides) Xanthan gum (KELTROL CG, manufactured by Sankyo Co., Ltd.) was suspended in pure water to a concentration of 0.5% (w / v), and then dissolved by stirring while heating at 90 °C. Similarly, sodium alginate (Duck Alginate NSPM, manufactured by Hood Chemifa), locust bean gum (GENUGUM RL-200-J, manufactured by Sankyo Co., Ltd.), κ-carrageenan (GENUGEL WR-80-J, manufactured by Sankyo Co., Ltd.), and diutan gum (KELCO CRETE DG-F, manufactured by Sankyo Co., Ltd.) were made into 0.5% (w / v) aqueous solutions. This aqueous solution was mixed with a 0.2 or 0.1% (w / v) deacylated gellan gum solution and a 10-fold concentrated DMEM / F-12 medium, and heated at 80 °C for 30 minutes. After cooling to room temperature, an aqueous solution of 7.5% sodium bicarbonate was added to prepare a DMEM / F-12 medium composition containing deacylated gellan gum at a final concentration of 0.01, 0.02% (w / v) and other polysaccharides at final concentrations of 0.1, 0.2, 0.3, 0.4% (w / v). Also, after preparing a medium containing deacylated gellan gum in the same manner as above, powder of methylcellulose (cP400, manufactured by Wako Pure Chemical Industries, Ltd.) was added. It was stirred in an ice bath to dissolve the methylcellulose, and a DMEM / F-12 medium composition containing deacylated gellan gum at final concentrations of 0.01, 0.02% (w / v) and other methylcellulose at final concentrations of 0.1, 0.2, 0.3, 0.4% (w / v) was prepared.

[0109] Polystyrene beads (Size 500 - 600 μm, manufactured by Polysciences Inc.) were added to the medium prepared above, and the dispersion state of the beads was visually confirmed. The floating dispersion state was evaluated as ○, the partially sedimented / dispersed state as △, and the sedimented state as ×. The results are shown in Table 10.

[0110]

Table 10

[0111] (Test Example 16: Viscosity measurement of medium composition mixed with polysaccharides) In the same manner as in the polysaccharide mixture system of Test Example 15, a DMEM / F-12 medium containing deacylated gellan gum at final concentrations of 0.005, 0.01% (w / v) and other polysaccharides was prepared. The polysaccharides were prepared so that the final concentrations were 0.1% (w / v) for xanthan gum, sodium alginate, and locust bean gum, 0.2% (w / v) for methylcellulose, and 0.05% (w / v) for κ-carrageenan and diutan gum. The states of the respective medium compositions and the viscosities measured in the same manner as in Analysis Example 1 are shown in Tables 11 - 16.

[0112]

Table 11

[0113]

Table 12

[0114]

Table 13

[0115]

Table 14

[0116]

Table 15

[0117]

Table 16

[0118] (Test Example 17: Preparation of a Medium Composition with Altered Divalent Metal Ion Concentrations) DMEM / F-12 (D9785, manufactured by Aldrich) containing no calcium chloride, magnesium sulfate, or magnesium chloride was used, and a DMEM / F-12 medium composition containing 0.02% (w / v) deacylated gellan gum was prepared in the same manner as in Test Example 14. Also, a DMEM / F-12 medium composition with calcium chloride or magnesium sulfate and magnesium chloride added was prepared so that the final concentrations would be the specified amounts in the DMEM / F-12 medium. From the specified composition of the DMEM / F-12 medium, the respective final concentrations were 0.116 g / L of calcium chloride, 0.049 g / L of magnesium sulfate, and 0.061 g / L of magnesium chloride. Dextran beads Cytodex1 (GE Healthca re Life Sciences product) was added, and after 2 days, the dispersion of the beads was visually confirmed. The floating dispersion state was evaluated as ○, the partially sedimented / dispersed state as △, and the sedimented state as ×. The results are shown in Table 17.

[0119]

Table 17

[0120] (Test Example 18: Preparation of a Medium Composition with Post-Added Divalent Metal Ions) A 0.1% (w / v) deacylated gellan gum solution and a 5-fold concentrated DMEM / F-12 medium (calcium chloride, magnesium sulfate, magnesium chloride free, D9785, manufactured by Aldrich), 1167 mg of calcium chloride, 489 mg of magnesium sulfate, and 287 mg of magnesium chloride were dissolved in 300 mL of pure water to prepare a salt solution. An aqueous deacylated gellan gum solution and pure water were placed in a 200 mL tall beaker, and the solution was stirred at 200 rpm using an Ikarian type stirring blade. Solution A, which was a mixture of the medium solution and water, was added and stirred for 10 minutes as it was. Then, the salt solution was added, and further 1.6 mL of a 7.5% aqueous sodium bicarbonate solution was added to prepare a DMEM / F-12 medium composition containing 0.02% deacylated gellan gum at the final concentration. The mixing amounts of each solution are shown in the table. Four hours after the preparation, the dispersion evaluation of polystyrene beads and Cytodex 1 was performed on six medium compositions. The results are shown in Tables 18 and 19.

[0121]

Table 18

[0122]

Table 19

[0123] (Test Example 19: Preparation of Various Medium Compositions) A 0.1% (w / v) deacylated gellan gum solution and high-concentration culture media were prepared. The high-concentration culture media were 10-fold concentrated MEM (M0268, manufactured by Aldrich), RPMI-1640 (R6504, manufactured by Aldrich), and 5-fold concentrated DMEM (autoclave-compatible medium, manufactured by Nissui). A 0.1% (w / v) deacylated gellan gum solution was mixed with each high-concentration culture medium and pure water for concentration adjustment, and heated at 80 °C for 30 minutes. After cooling to room temperature, an aqueous solution of 7.5% sodium bicarbonate was added to prepare culture medium compositions containing deacylated gellan gum at final concentrations of 0.01, 0.02, and 0.03% (w / v), respectively. Regarding the nine prepared culture medium compositions, the suspension and dispersion states of polystyrene beads and dextran beads Cytodex1 were evaluated as ○ for the floating and dispersed state, △ for the partially sedimented / dispersed state, and × for the sedimented state. The results are shown in Tables 20 and 21.

[0124] [Table 20]

[0125] [Table 21]

[0126] (Test Example 20: Measurement of particle size distribution of culture medium composition containing deacylated gellan gum) Following Analytical Example 1, a DMEM / F-12 culture medium composition containing 0.038% (w / v) deacylated gellan gum was prepared. The medium was prepared by stirring at 3000 rpm and 6000 rpm for 1 minute using a homomixer. Regarding the particle size distribution of this culture medium composition, it was measured using a Multisizer 4 (precision particle size distribution measuring device based on the Coulter principle) manufactured by Beckman Coulter, Inc., and the median diameter (d50) of the volume-based particle size distribution was determined. The results are shown in Table 22.

[0127] [Table 22]

[0128] (Test Example 21: Phosphorylation of deacylated gellan gum) 1 g of deacylated gellan gum and 40 mL of pure water were weighed into a 100 mL glass test tube, heated at 100 °C for 30 minutes to prepare a suspension. 1 g of phosphoric acid aqueous solution (85%) was added to this suspension, and the mixture was heated under reflux for 5 hours. Then, while stirring for 12 hours, it was allowed to cool to room temperature The resulting white suspension was poured into 99% ethanol (500 mL). The resulting cottony white solid was collected by filtration and dried to obtain a light brown solid (0.4 g) as the phosphate of deacylated gellan gum. The introduction of the phosphate group was confirmed by Fourier transform infrared spectroscopy (IR-Prestage21, manufactured by Shimadzu Corporation) (1700 cm-1; P-OH, 1296 cm-1, 1265 cm-1; P=O). After decomposing the light brown solid with a microwave heating decomposition apparatus (ETHOS TC, manufactured by Milestone General), the content of phosphorus atoms was measured by an inductively coupled plasma optical emission spectrometer (ICP-OES) (SPS 5520, manufactured by SII Nanotechnology Inc) The result was 3.5 wt% (n = 2).

[0129] (Test Example 22: Preparation of a medium composition containing phosphorylated deacylated gellan gum) Any amount of phosphorylated deacylated gellan gum (30 mg) and 1.56 g of DMEM / F-12 medium (manufactured by Life Technologies) were placed in a 200 mL Erlenmeyer flask, and 100 mL of pure water was poured in. Autoclaved at 121 °C for 20 minutes to prepare a DMEM / F-12 medium composition with a deacylated gellan gum concentration of 0.03%. Dextran beads Cytodex1 (manufactured by GE Healthcare Biosciences) were added to the prepared medium, and the dispersion state of the beads was visually confirmed. At a phosphorylated deacylated gellan gum concentration of 0.03% (w / v), the dispersion state of the beads was observed.

[0130] (Test Example 23: Preparation of a medium composition containing deacylated gellan gum) The deacylated gellan gum aqueous solution and the medium solution were mixed by adding them at the ratios shown in the table below to prepare a DMEM / F-12 medium composition with a deacylated gellan gum concentration of 0.02%. The dispersion state of polystyrene beads (Size 500-600μm, manufactured by Polyscien ces Inc.) was evaluated. The results are shown in Tables 23 and 24. By allowing it to stand for 1 day or more, the styrene beads were dispersed under all conditions.

[0131]

Table 23

[0132]

Table 24

[0133] (Test Example 24: Preparation of Medium Composition Using Filter) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a final concentration of 0.02 or 0.04% (w / v), and then dissolved by heating at 90 °C for 30 minutes or at 121 °C for 20 minutes. Further, 100 mL of this aqueous solution was filtered through a polyethersulfone membrane filter with a pore size of 0.22 μm (manufactured by Corning). Subsequently, the filtrate was mixed with DMEM / F-12 medium (manufactured by Sigma-Aldrich) at 2 to 4 times the concentration, and then shaken for 1 hour using a mild mixer (SI-24, manufactured by Taitec) to prepare medium compositions containing desacylated gellan gum at a final concentration of 0.01 or 0.015% (w / v), respectively (for example, 25 mL each of a 0.02% (w / v) desacylated gellan gum aqueous solution and a 2-fold concentrated DMEM / F-12 medium were mixed to prepare 50 mL of a 0.01% (w / v) desacylated gellan gum medium composition). Spheres of HepG2 cells were prepared using the same method as in Test Example 2. After adding several tens of spheres to 1 mL of each of the media prepared above, they were allowed to stand at 37 °C, and the floating state of the sphere cells after 1 hour and 1 night was visually observed. As a result, it was confirmed that the spheres of HepG2 cells were maintained in a floating state in all of the above medium compositions. Furthermore, after adding 2 volumes of medium, it was confirmed in all medium compositions that the spheres of HepG2 cells sedimented by centrifuging the cell suspension (500 G, 5 minutes) and the cells could be recovered. When visually confirming the dispersion state of the spheres after 1 night, the results of evaluation with a floating dispersion state as ○, a partially sedimented / dispersed state as △, and a sedimented state as × are shown in Table 25.

[0134]

Table 25

[0135] (Test Example 25: Cell proliferation test when culturing cell line-derived spheres) The human fetal kidney cell line HEK293 (manufactured by DS Pharma Biomedical Co., Ltd.) was suspended in EMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum to a concentration of 250,000 cells / mL. 10 mL of this suspension was seeded into EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and then cultured in a CO2 incubator (5% CO2) for 2 days. 10 mL of the suspension of HEK293 cell spheres (diameter 100 - 200 μm) obtained here was centrifuged (200G, 5 minutes) to sediment the spheres and remove the supernatant, and then suspended in 1 mL. Subsequently, 10 mL of the above medium was added to 200 μL of this sphere suspension (the number of cells was approximately 200,000) and suspended, and then transferred to a flat-bottom tube (manufactured by BM Instruments Co., Ltd.). Similarly, a suspension of spheres was prepared using a medium composition in which 0.015% (w / v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was added to the above medium, and transferred to a flat-bottom tube (manufactured by BM Instruments Co., Ltd.). The medium composition containing 0.015% (w / v) deacylated gellan gum was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolving it by stirring while heating at 90°C, autoclaving this aqueous solution at 121°C for 20 minutes, and then adding it to EMEM medium containing 10% (v / v) fetal bovine serum at a 1 / 20 dilution.

[0136] The above sphere suspension was statically cultured in a CO2 incubator (5% CO2) at 37°C for 5 days, then 2 volumes of medium were added and centrifuged (500G, 5 minutes) to sediment the spheres and remove the supernatant. Subsequently, the recovered spheres were washed once with 10 mL of PBS, then 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and incubated at 37°C for 5 minutes. After adding 9 mL of the above medium, the cells were recovered by centrifugation (500G, 5 minutes). To a part of 2 mL of the cell suspension obtained here, an equal volume of trypan blue staining solution (manufactured by Invitrogen) was added, and then the number of live and dead cells was measured using a hemocytometer (manufactured by ELMA Sales Co., Ltd.). As a control, a medium composition without deacylated gellan gum was prepared and the same experiment was conducted.

[0137] As a result, HEK293 cell spheres were suspended by using the medium composition of the present invention. It was confirmed that the medium composition of the present invention can be cultured in a state where cells can grow efficiently, and that the medium composition of the present invention has a lower rate of dead cells when cells are grown compared to a medium composition that does not contain deacylated gellan gum, and is therefore superior in promoting cell growth. In this case, spheres cultured in the existing medium settled to the bottom of the culture vessel. Table 26 shows the relative cell numbers for HEK293 cells when the cell number was cultured in a medium not containing deacylated gellan gum, which was set to 1. Table 27 also shows the relative cell death rates when the cell death rate (number of dead cells / number of live cells) was cultured in a medium not containing deacylated gellan gum, which was set to 1.

[0138] [Table 26]

[0139] [Table 27]

[0140] (Test Example 26: Cell proliferation test when insect cells are cultured) Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. The resulting solution was sterilized in an autoclave at 121°C for 20 minutes. This solution was used to add Sf-900 (registered trademark) IIISFM medium (Gibco) to a final concentration of 0.015. A medium composition supplemented with %(w / v) deacylated gellan gum was prepared. Subsequently, Sf9 cells (manufactured by Gibco) derived from Spodoptera frugiperda were seeded into the above-prepared medium composition supplemented with deacylated gellan gum to a density of 100,000 cells / mL, and then dispensed into wells of a 24-well flat-bottom microplate (manufactured by Corning) at 1 mL per well. These cell suspensions were statically cultured at 25°C in an incubator for 5 days. Thereafter, a portion of the culture broth was collected, an equal volume of trypan blue staining solution (manufactured by Invitrogen) was added, and the number of viable cells was measured using a hemocytometer (manufactured by ELMA Sales Co., Ltd.). As a control, a medium composition without deacylated gellan gum was prepared, and the same experiment was conducted.

[0141] As a result, it was confirmed that Sf9 cells can be uniformly cultured in a floating state by using the medium composition of the present invention and can grow in the said medium composition. Furthermore, it was confirmed that the medium composition of the present invention has an excellent cell growth promoting effect when cells are grown as compared with a medium composition without deacylated gellan gum. Table 28 shows the number of Sf9 cells after 5 days of floating static culture.

[0142]

Table 28

[0143] (Test Example 27: Cell Growth Test When Culturing CD34-Positive Cells) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.015% (w / v), thrombopoietin (manufactured by WAKO) at 20 ng / mL, and stem cell factor (SCF, manufactured by WAKO) at 100 ng / mL to StemSpan SFEM medium (manufactured by StemCell Technologies). Subsequently, CD34-positive cells derived from human umbilical cord blood (manufactured by Lonza) were seeded into the above-mentioned medium composition supplemented with desacylated gellan gum to a density of 10,000 cells / mL, and then dispensed into the wells of a 24-well flat-bottom microplate (manufactured by Corning) at 1 mL per well. These cell suspensions were statically cultured in a CO2 incubator (5% CO2) at 37°C for 7 days. Thereafter, a part of the culture solution was collected, an equal volume of trypan blue staining solution (manufactured by Invitrogen) was added, and the number of viable cells was measured using a hemocytometer (manufactured by ELMA Sales Co., Ltd.). In addition, three volumes of medium were added to the remaining culture solution, and centrifugation (500 G, 5 minutes) was performed to precipitate all the cells. As a control, a medium composition without desacylated gellan gum was prepared, and the same experiment was conducted.

[0144] As a result, it was confirmed that CD34-positive cells can be uniformly cultured in a floating state by using the medium composition of the present invention and can proliferate in the said medium composition. Furthermore, it was confirmed that the medium composition of the present invention has a cell growth promoting effect equal to or better than that of existing media without desacylated gellan gum. Also, it was confirmed that the cells sedimented by centrifugation and could be recovered. Table 29 shows the relative cell numbers of the cells proliferated from CD34-positive cells after 7 days of floating static culture when the number of cells cultured in a medium without desacylated gellan gum was set as 1.

[0145]

Table 29

[0146] (Test Example 28: Sphere Formation Test) Using the same method as in Test Example 2, a composition of DMEM medium (manufactured by Wako) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared. Subsequently, HepG2 cells were added to a cell concentration of 15,000 cells / mL, and then 1 mL was dispensed into a 24-well plate (manufactured by Corning). After culturing this plate in suspension at 37°C for 7 days, the formation of spheres was confirmed under a microscope. Furthermore, the sphere cells were sedimented by centrifugation at 400 G for 5 minutes, washed once with 5 mL of PBS, then 100 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako) was added, and it was incubated at 37°C for 5 minutes. To 100 μL of the cell suspension obtained here, 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the cell suspension. Then, the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.). As a result, it was confirmed that HepG2 cells formed spheres in the medium composition of the present invention, and the number of cells also increased up to 80,800 cells / mL. The spheres of HepG2 cells formed in the medium composition of the present invention are shown in Fig. 14.

[0147] (Test Example 29: Cell Suspension Test Using Spheres Derived from Cell Lines) Dyutangum (KELKO-CRETE DG, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then heated at 90°C and dissolved by stirring while heating. Using this aqueous solution, a DMEM / F-12 medium composition with a final concentration of 0.1% (w / v) for Dyutangum was prepared. Also, an aqueous solution containing 0.5% (w / v) native gellan gum (Kelcogel HT, manufactured by San-Ei Gen F.F.I., Inc.) was prepared by heating at 90°C, and using this aqueous solution, DMEM / F-12 medium (manufactured by Sigma) compositions containing 0.05% and 0.1% (w / v) native gellan gum were prepared.

[0148] Using the same method as in Test Example 2, HeLa cell spheres were prepared. After adding several tens of spheres to 1 mL of the medium prepared above, they were allowed to stand at 37°C for 1 hour, and the floating state of the sphere cells was visually observed. As a result, it was confirmed that the HeLa cell spheres were maintained in a floating state in all of the above medium compositions. Furthermore, it was confirmed that the HeLa cell spheres sedimented by centrifuging (200G, 5 minutes) the cell suspension containing 0.1% (w / v) of Diyuetan gum, and the cells could be recovered.

[0149] (Test Example 30: Cell suspension test 1 using magnetic beads having cell adhesion ability) GEM (registered trademark, Global Eukaryotic Microcarrier, manufactured by GLS Science Inc.) coated with laminin or fibronectin ) The suspension solution was dispensed in 500 μL aliquots into 1.5 mL capacity microtest tubes (manufactured by Eppendorf). Using a magnet stand (TA4899N12, manufactured by Tamagawa Seiki Co., Ltd.), GEM was accumulated from the above GEM suspension solution to remove the solvent. Furthermore, after washing the GEM twice with 500 μL of DMEM medium (manufactured by Wako) containing 10% (v / v) fetal bovine serum, it was suspended in 500 μL of the same medium. This suspension was dispensed at 50 μL per well into a Sumilon Celltight Plate 24F (manufactured by Sumitomo Bakelite Co., Ltd.), which is a cell low-adhesion plate. Subsequently, separately prepared HepG2 cells were added to a concentration of 250,000 cells / mL, and the final volume was adjusted to 500 μL / well with the same medium. After manually stirring this cell suspension, the plate was allowed to stand overnight in a CO2 incubator (5% CO2). After confirming cell adhesion on the GEM under a microscope, the cell suspension was transferred to a 1.5 mL capacity microtest tube (manufactured by Eppendorf), and the cell-attached GEM was accumulated using the above magnet stand to remove the supernatant.

[0150] Using the same method as in Test Example 2, a composition of DMEM medium (manufactured by Wako) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared. 1 mL of each of this medium composition or the same medium without deacylated gellan gum prepared above was added to the HepG2 cell-adherent GEM (laminin or fibronectin coating) prepared above, suspended, and then transferred to a Sumilon Celltight Plate 24F. Subsequently, the plate was allowed to stand in a CO2 incubator (5% CO2) for 6 days, after which the cell culture medium was transferred to a 1.5 mL capacity microtest tube (manufactured by Eppendorf), and the cell-adherent GEM was aggregated while gently pipetting on the above magnetic stand to remove the supernatant. The GEM was washed once with 1 mL of PBS, 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako) was added, and the mixture was incubated at 37°C for 10 minutes. To 200 μL of the cell suspension obtained here, 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and an equal amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the cell suspension. Then, the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.).

[0151] As a result, it was confirmed that the GEM with adhered HepG2 cells can be cultured in a floating state by using the medium composition of the present invention, and the cells proliferated efficiently in the said medium composition. Moreover, it was confirmed that the medium composition of the present invention has an excellent effect of promoting cell proliferation compared with the existing medium without deacylated gellan gum. Also, it was possible to aggregate the HepG2 cell-adherent GEM from the medium composition of the present invention by using magnetic force, and it was further confirmed that HepG2 cells can be recovered from the GEM. Table 30 shows the number of cells when HepG2 cells were cultured on GEM for 6 days in a medium containing or not containing deacylated gellan gum. Also, Fig. 14 shows the floating state when the laminin-coated GEM with adhered HepG2 cells was cultured in the medium composition of the present invention.

[0152] [Table 30]

[0153] (Test Example 31: Cell suspension test 2 using magnetic beads with cell adhesion ability) As in Test Example 30, a fibronectin-coated GEM (registered trademark, Global Eukaryotic Microcarrier, GL Sciences Co., Ltd.) was used. The cells were suspended in MF-Medium (registered trademark) mesenchymal stem cell proliferation medium (manufactured by Toyobo Co., Ltd.). This suspension was dispensed at 50 μL per well onto a low cell adhesion plate, Sumilon Celltite Plate 24F (manufactured by Sumitomo Bakelite Co., Ltd.). Subsequently, separately prepared human bone marrow-derived mesenchymal stem cells (manufactured by Cell Applications) were added to a concentration of 250,000 cells / mL, and the plate was left to stand overnight in a CO2 incubator (5% CO2) in the same manner as in Test Example 30 to prepare GEMs to which mesenchymal stem cells were attached.

[0154] Using the same method as in Test Example 2, a composition of MF-Medium (registered trademark) mesenchymal stem cell growth medium (manufactured by Toyobo Co., Ltd.) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansei Co., Ltd.) was prepared. 1 mL each of this medium composition or the same medium without deacylated gellan gum was added to the mesenchymal stem cell-adherent GEM (fibronectin-coated) prepared above, suspended, and then transferred to a Sumilon Celltight Plate 24F. Subsequently, the plate was left standing in a CO2 incubator (5% CO2) for 4 days, after which the cell culture medium was transferred to a 1.5 mL micro test tube (manufactured by Eppendorf), and the cell-adherent GEM was accumulated while gently pipetting on the above magnetic stand to remove the supernatant. This GEM was washed once with 1 mL of PBS, 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako) was added, and it was incubated at 37°C for 10 minutes. To 200 μL of the cell suspension obtained here, 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and to a part of the cell suspension, an equal amount of trypan blue staining solution (manufactured by Invitrogen) was added, and then the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.).

[0155] As a result, it was confirmed that the GEM to which mesenchymal stem cells were adhered could be cultured in a floating state by using the medium composition of the present invention, and the cells proliferated efficiently in the said medium composition. Moreover, it was confirmed that the medium composition of the present invention has an excellent effect of promoting cell proliferation as compared with existing media not containing deacylated gellan gum. Also, it was confirmed that it is possible to accumulate the mesenchymal stem cell-adherent GEM from the medium composition of the present invention by using magnetic force, and further that mesenchymal stem cells can be recovered from this GEM. Table 31 shows the number of cells when mesenchymal stem cells were cultured on GEM for 4 days in a medium containing or not containing deacylated gellan gum.

[0156]

Table 31

[0157] (Test Example 32: Cell suspension test using alginate beads) The following test was carried out according to the method of the alginate three-dimensional culture kit manufactured by PG Research Co., Ltd. Separately prepared HepG2 cells were added to 2.5 mL of a sodium alginate solution (manufactured by PG Research Co., Ltd.) so as to be 400,000 cells / mL, and further human recombinant laminin 511 (manufactured by Veritas Co., Ltd.) was added so as to be 5 μg / mL to prepare a cell suspension. After collecting this cell suspension with a 5 mL syringe (manufactured by Terumo Corporation) equipped with a sonde, a 22G injection needle (manufactured by Terumo Corporation) was attached to this syringe. Subsequently, 10 drops of this cell suspension were added to each well of a 24-well flat-bottom microplate (manufactured by PG Research Co., Ltd.) to which 2 mL of an aqueous calcium chloride solution (manufactured by PG Research Co., Ltd.) had been added. After allowing to stand at room temperature for 10 minutes and then confirming the formation of alginate beads, the calcium chloride solution was removed, 2 mL of PBS was added, and the mixture was allowed to stand at room temperature for 15 minutes. Further, after removing the PBS, 2 mL of DMEM medium (manufactured by Wako) containing 10% (v / v) fetal bovine serum was added, and the mixture was allowed to stand at room temperature for 15 minutes. After removing the medium, 1 mL of a medium composition of DMEM medium (manufactured by Wako) containing 0.03% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) and 10% (v / v) fetal bovine serum or the same medium without deacylated gellan gum prepared in the same manner as in Test Example 2 was added to each well, and static culture was carried out in a CO2 incubator (5% CO2) for 8 days. The medium was changed on the 4th day of culture.

[0158] The cultured alginate beads were transferred to a 1.5 mL micro test tube (manufactured by Eppendorf) using a 1 mL chip, and then 1 mL of sodium citrate solution (manufactured by PG Research Co., Ltd.) was added to each tube and stirred at room temperature for 15 minutes to dissolve the alginate beads. Subsequently, the cells were sedimented by centrifugation at 300 G for 3 minutes, and the supernatant was removed. To these cells, 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added and incubated at 37 °C for 5 minutes. To 200 μL of the cell suspension obtained here, 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and to a part of the cell suspension, an equal amount of trypan blue staining solution (manufactured by Invitrogen) was added, and then the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.).

[0159] As a result, it was confirmed that the alginate beads embedding HepG2 cells can be cultured in a floating state by using the culture medium composition of the present invention, and the cells can proliferate efficiently in the culture medium composition. Moreover, it was confirmed that the culture medium composition of the present invention has an excellent effect of promoting cell proliferation as compared with existing culture media not containing deacylated gellan gum. Table 32 shows the number of cells when HepG2 cells were cultured in alginate beads for 8 days in a culture medium containing or not containing deacylated gellan gum. Also, Fig. 16 shows the floating state when alginate beads embedding HepG2 cells were cultured with the culture medium composition of the present invention.

[0160]

Table 32

[0161] (Test Example 33: Cell suspension test using collagen gel capsules) A: Tissue culture collagen Cellmatrix (registered trademark) Type I-A (Cellmatrix, manufactured by Nitta Gelatin Inc.), B: 10-fold concentrated DMEM / F-12 medium (A (manufactured by Aldrich), C: Reconstitution buffer (2.2 g of sodium hydrogen carbonate and 4.77 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were added to 100 mL of 0.05 N sodium hydroxide solution), filtered and sterilized) were each cooled in ice and mixed so that A:B:C = 8:1:1. Furthermore, human recombinant laminin 511 (manufactured by Veritas Co., Ltd.) was added to a concentration of 5 μg / mL to prepare 500 μL of a collagen mixed solution. HepG2 cells prepared separately were added to this mixed solution to a concentration of 200,000 cells / mL, and the entire volume was collected using a 1.5 mL syringe (manufactured by Terumo Corporation) equipped with a 25G injection needle (manufactured by Terumo Corporation). Subsequently, the cell suspension was dropped one drop at a time using the above syringe into a flat-bottomed tube (manufactured by BM Instruments Co., Ltd.) containing 10 mL of DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) pre-warmed at 37 °C and containing 10% (v / v) fetal bovine serum. After incubating in a 37 °C water bath for 10 minutes to confirm the formation of amorphous collagen gel capsules with a diameter of approximately 2 mm, deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was added to a final concentration of 0.04% in the same manner as in Test Example 2, and the capsules were gently stirred and suspended. Subsequently, the tube was statically cultured in a CO2 incubator (5% CO2) for 5 days. (While cooling each of them in ice, they were mixed so that A:B:C = 8:1:1. Further, human recombinant laminin 511 (manufactured by Veritas Co., Ltd.) was added to a concentration of 5 μg / mL to prepare 500 μL of a collagen mixed solution. HepG2 cells prepared separately were added to this mixed solution to a concentration of 200,000 cells / mL, and the entire volume was collected using a 1.5 mL syringe (manufactured by Terumo Corporation) equipped with a 25G injection needle (manufactured by Terumo Corporation). Subsequently, the cell suspension was dropped one drop at a time using the above syringe into a flat-bottomed tube (manufactured by BM Instruments Co., Ltd.) containing 10 mL of DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) pre-warmed at 37 °C and containing 10% (v / v) fetal bovine serum. After incubating in a 37 °C water bath for 10 minutes to confirm the formation of amorphous collagen gel capsules with a diameter of approximately 2 mm, deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was added to a final concentration of 0.04% in the same manner as in Test Example 2, and the capsules were gently stirred and suspended. Subsequently, the tube was statically cultured in a CO2 incubator (5% CO2) for 5 days.)

[0162] 25 mL of PBS was added to the culture solution containing collagen gel capsules, and the collagen gel capsules were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. Again, 25 mL of PBS was added and centrifuged, and the supernatant was removed so that the remaining volume was 5 mL. After adding 20 μL of 1% (W / V) collagenase L (manufactured by Nitta Gelatin Inc.) to this solution, it was shaken at 37 °C for 2 hours. After confirming the dissolution of the collagen gel, 10 mL of PBS was added, and the cells were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to these cells and incubated at 37 °C for 5 minutes. To the cell suspension obtained here, 4 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and the cells were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. The obtained cells were suspended in 2 mL of the same medium as above, and an equal amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of it, and then the number of viable cells was measured using a hemocytometer (manufactured by Erma Sales Co., Ltd.).

[0163] As a result, it was confirmed that the collagen gel capsules embedding HepG2 cells can be cultured in a floating state by using the culture medium composition of the present invention, and the cells proliferate efficiently in the culture medium composition. Moreover, it was confirmed that the culture medium composition of the present invention has an excellent effect of promoting cell proliferation compared with existing media that do not contain deacylated gellan gum. Table 33 shows the number of cells when HepG2 cells were cultured in a collagen gel capsule for 5 days in a medium containing or not containing deacylated gellan gum. Also, Fig. 17 shows the floating state when the collagen gel capsules embedding HepG2 cells were cultured in the culture medium composition of the present invention.

[0164] [Table 33]

[0165] (Test Example 34: Recovery test of spheres using a filter) Using the same method as in Test Example 2, a composition of 0.015% deacylated gellan gum (KELCOG EL CG-LA, manufactured by Sankyo Co., Ltd.) and DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum was prepared. Also, as a control, the same medium without deacylated gellan gum was prepared. Using the same method as in Test Example 2, HepG2 cell spheres were created, and the spheres were added to 1 mL of the medium prepared above so that the number of cells was 86,000 each, and then left standing at 37°C for 1 hour, and the floating state of the sphere cells was visually observed. Furthermore, the cell suspension was added onto a cell strainer (manufactured by Becton Dickinson) with a mesh size of 40 μm, and the spheres were captured on the filter. Subsequently, 10 mL of PBS was poured in from the back of the filter to collect the spheres into a 15 mL tube, and the spheres were sedimented by centrifugation at 300 G for 5 minutes. After removing the supernatant, 500 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the spheres and incubated at 37°C for 5 minutes. To the cell suspension obtained here, 1 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of it, and then the number of viable cells was measured using a hemocytometer (manufactured by ERMA Sales Co., Ltd.). As a result, it was confirmed that the HepG2 cell spheres were maintained in a floating state in the above medium composition. Furthermore, by filtering the sphere suspension containing 0.015% deacylated gellan gum, it was confirmed that the HepG2 cell spheres could be recovered at a recovery rate equivalent to that of the medium without deacylated gellan gum. Table 34 shows the relative number of cells recovered from the medium containing deacylated gellan gum when the number of HepG2 cells recovered by filtration using the medium without deacylated gellan gum was set to 1.

[0166]

Table 34

[0167] (Test Example 35: Cell suspension test of spheres using a mixture of various polysaccharides) Using the same method as in Test Example 15, a DMEM / F-12 medium composition was prepared by combining xanthan gum (KELTROL CG, manufactured by Sankyo Co., Ltd.), sodium alginate (Duck Alginate NSPM, manufactured by Hood Chemifa), locust bean gum (GENUGUM RL-200-J, manufactured by Sankyo Co., Ltd.), methylcellulose (cP400, manufactured by Wako Pure Chemical Industries, Ltd.), κ-carrageenan (GENUGEL WR-80-J, manufactured by Sankyo Co., Ltd.), pectin (GENU pectin LM-102AS, manufactured by Sankyo Co., Ltd.), or diutan gum (KELCO CRETE DG-F, manufactured by Sankyo Co., Ltd.) with deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.). Spheres of HepG2 cells were prepared using the same method as in Test Example 2. After adding several tens of spheres to 1 mL of the medium prepared above, they were allowed to stand at 37°C, and the floating state of the sphere cells after 1 hour and overnight was visually observed. As a result, it was confirmed that the spheres of HepG2 cells were maintained in a floating state in all of the above medium compositions. Furthermore, after adding a two-fold volume of medium, it was confirmed in all medium compositions that the spheres of HepG2 cells sedimented by centrifuging the cell suspension (500G, 5 minutes) and the cells could be recovered. When visually confirming the dispersion state of the spheres after overnight, the results of evaluation with the floating dispersion state as ○, the partially sedimented / dispersed state as △, and the sedimented state as × are shown in Tables 35 and 36. Note that - in the table indicates not implemented.

[0168]

Table 35

[0169]

Table 36

[0170] Comparison of dispersibility between beads and cells 1 For the deacylated gellan gum-containing medium and methylcellulose-containing medium prepared in the above (comparative example), the dispersion states of dextran beads Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) and HeLa cell spheres were compared. The results are shown in a table. Since the dispersion states of Cytodex1 and HeLa cell spheres are well correlated, Cytodex1 can be used as a cell sphere model.

[0171]

Table 37

[0172]

Table 38

[0173] Comparison of the dispersibility of beads and cells 2 For the polysaccharide and deacylated gellan gum-containing medium prepared in Test Example 15, the dispersion states of polystyrene beads (Size 500 - 600 μm, manufactured by Polysciences Inc.) and HepG2 cell spheres were compared. The floating dispersion state was evaluated as ○, the partially sedimented / dispersed state as △, and the sedimented state as ×. The results are shown in a table. Since the dispersion states of polystyrene beads and HepG2 cell spheres are well correlated, polystyrene beads can be used as a cell sphere model.

[0174]

Table 39

[0175] (Test Example 36: Suspension culture test of rice-derived plant callus) Fifty mature seeds of rice Nipponbare (purchased from Kotohigashi Agricultural Cooperative) selected by saline selection were transferred to a 50 mL polystyrene tube (manufactured by BD Falcon) and washed with 50 mL of sterilized water. Thereafter, it was stirred in 30 mL of 70% ethanol water for 1 minute. After removing the ethanol water, 30 mL of Kitchen Highter (manufactured by Kao Corporation) was added and stirred for 1 hour. After removing the Kitchen Highter, it was washed 4 times with 50 mL of sterilized water. The seeds sterilized here were placed on 1.5 mL / well (24-well flat-bottom microplate (manufactured by Corning)) of Murashige and Skoog basal medium (M9274, manufactured by Sigma-Aldrich) containing 2 μg / mL of 2,4-dichlorophenoxyacetic acid (manufactured by Sigma-Aldrich) and agar. It was cultured for 3 weeks under the conditions of 30 °C, 16 hours in the dark / 8 hours in the dark, and the cream-colored callus (1-2 mm) grown on the scutellum of the seeds was collected.

[0176] Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved and sterilized at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.03% (w / v) to Murashige and Skoog basal medium (M9274, manufactured by Sigma-Aldrich) containing 2 μg / mL of 2,4-dichlorophenoxyacetic acid (manufactured by Sigma-Aldrich). 15 calli prepared above were added to 10 mL / flat-bottom tube (manufactured by BM Instruments) of this medium composition and cultured with shaking at 25 °C for 7 days. As a result, it was confirmed that rice-derived calli can be cultured in a suspended state by using the medium composition of the present invention, and the calli are maintained in the medium composition. The suspended state when rice-derived calli are cultured in the medium composition of the present invention is shown in Fig. 18.

[0177] (Test Example 37: Cell proliferation test when HeLa cells are dispersed) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.015% (w / v) or 0.030% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded at 50,000 cells / mL into the above-mentioned medium composition supplemented with desacylated gellan gum, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 200 μL per well. As a negative control, HeLa cells suspended in the same medium without desacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 8 days. After adding 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) to the culture broth after 3 and 8 days of culture, it was incubated at 37 °C for 100 minutes, and the absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECT RA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone. The cell density was measured under a microscope after mixing 20 μL of the stirred solution obtained by pipetting the culture broth containing the cells after 8 days of culture with 20 μL of 0.4% Trypan Blue stain (manufactured by Invitrogen).

[0178] As a result, it was confirmed that HeLa cells can be cultured in a uniformly dispersed state without excessive size of cell aggregates by using the medium composition of the present invention, and can proliferate efficiently in the said medium composition. The results of microscopic observation of the cell aggregates of HeLa cells after 8 days of culture are shown in Fig. 19. Also, the absorbance at 450 nm (corresponding to the number of HeLa cells) after 3 and 8 days of static culture is shown in Table 40. The cell density of HeLa cells after 8 days of culture is shown in Table 41.

[0179]

Table 40

[0180]

Table 41

[0181] (Test Example 38: Cell Proliferation Test When A549 Cells and HCT116 Cells are Dispersed) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.015% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum or McCoy's 5a medium (manufactured by DS Pharma Biomedical Co., Ltd.). Subsequently, the human lung cancer cell line A549 (manufactured by DS Pharma Biomedical Co., Ltd.) or the human colorectal cancer cell line HCT116 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition containing deacylated gellan gum to a density of 50,000 cells / mL, and then dispensed into wells of a 96-well flat-bottomed ultra-low attachment surface microplate (manufactured by Corning, #3474) at 200 μL per well. As a negative control, A549 cells and HCT116 cells suspended in the same medium without deacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 7 days. After culturing for 3, 5, and 7 days, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added to the culture medium, and then incubated at 37°C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0182] As a result, A549 cells and HCT116 cells can be cultured in a uniformly dispersed state without excessive size of cell aggregates by using the culture medium composition of the present invention, and it was confirmed that they grow efficiently in the culture medium composition. After culturing for 5 days The results of microscopic observation of aggregates of A549 cells and HCT116 cells are shown in Fig. 20. Also, the absorbance at 450 nm (corresponding to the number of A549 cells) after static culture for 3, 5, and 7 days is shown in Table 42, and the absorbance at 450 nm (corresponding to the number of HCT116 cells) is shown in Table 43.

[0183] [Table 42]

[0184] [Table 43]

[0185] (Test Example 39: Cell Growth Test Using a U-bottom Low-Adhesion Surface Plate) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.015% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded at 50,000 cells / mL in the above medium composition supplemented with desacylated gellan gum, and then dispensed into wells of a 96-well U-bottom low-adhesion surface microplate (manufactured by Sumitomo Bakelite, #MS-9096U) at 200 μL per well. As a negative control, HeLa cells suspended in the same medium without desacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 7 days. To the culture supernatants after 2, 5, and 7 days of culture, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0186] As a result, it was confirmed that by using the medium composition of the present invention, efficient growth was achieved in the same medium composition even on other low-adhesion plates. Table 44 shows the absorbance at 450 nm (corresponding to the number of HeLa cells) after 2, 5, and 7 days of static culture.

[0187]

Table 44

[0188] (Test Example 40: Cell proliferation test using low-adhesion surface plates of other companies) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then heated at 90 °C and dissolved by stirring while heating. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at final concentrations of 0.005% and 0.030% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded at 50,000 cells / mL in the above medium composition to which desacylated gellan gum had been added, and then dispensed into wells of a 96-well flat-bottomed low-adhesion surface microplate (manufactured by IWAKI, #Ez-BindShut) at 200 μL per well. As a negative control, HeLa cells suspended in the same medium without desacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 7 days. To the culture solution after 3 days of culture, 20 μL of a WST-8 solution (manufactured by Dojindo Laboratories) was added, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0189] As a result, it was confirmed that by using the medium composition of the present invention, efficient growth can be achieved with this medium composition even on other low-adhesion plates. Table 45 shows the absorbance at 450 nm (corresponding to the number of HeLa cells) after 3 days of static culture.

[0190]

Table 45

[0191] (Test Example 41: Cell Growth Comparison Test with Happy Cell ASM Medium) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.015% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Happy Cell ASM medium (manufactured by biocroi) was prepared with DMEM medium (manufactured by WAKO) to a specified concentration (mixed 1:1) in advance. Subsequently, human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) or human lung cancer cell line A549 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded at 50,000 cells / mL into the above-mentioned medium composition with added deacylated gellan gum or Happy Cell ASM medium composition, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 200 μL per well. As a negative control, suspensions of HeLa cells and A549 cells in the same medium without deacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 5 days. To the culture medium after 3 and 5 days of culture, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added, and then incubated at 37°C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0192] As a result, by using the medium composition of the present invention, it was confirmed that growth was more efficient in this medium composition compared to Happy Cell ASM. The absorbance at 450 nm (corresponding to the number of HeLa cells) after 3 and 5 days of static culture is shown in Table 46, and the absorbance at 450 nm (corresponding to the number of A549 cells) is shown in Table 47. The absorbance at 450 nm (corresponding to the number of HeLa cells) is shown in Table 46, and the absorbance at 450 nm (corresponding to the number of A549 cells) is shown in Table 47.

[0193]

Table 46

[0194]

Table 47

[0195] (Test Example 42: Cell Proliferation Test Using Other Polysaccharides) Diyu Tangum (KELCO CRETE DG-F, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 1.5% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, medium compositions were prepared by adding Diyu Tangum at final concentrations of 0.2% and 0.3% (w / v) to DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% (v / v) fetal bovine serum. Subsequently, human lung cancer cell line A549 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium compositions supplemented with Diyu Tangum to a concentration of 50,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 200 μL per well. As a negative control, A549 cells suspended in the same medium without Diyu Tangum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days. To the culture solution after 3 days of culture, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added, and then incubated at 37°C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0196] As a result, it was confirmed that by using the medium composition of the present invention, it can grow efficiently in the medium composition containing other polysaccharides. The absorbance at 450 nm (corresponding to the number of A549 cells) after 3 days of static culture is shown in Table 48.

[0197]

Table 48

[0198] (Test Example 43: Cell Proliferation Test Using Various Anticancer Agents) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3 %(w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.030% (w / v) and various anticancer agents at final concentrations of 0.001, 0.01, 0.1, and 1 μM to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Adriamycin (manufactured by WAKO), Paclitaxel (manufactured by WAKO), or Mitomycin C (manufactured by WAKO) was used as the anticancer agent. Subsequently, human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition supplemented with deacylated gellan gum to a concentration of 50,000 cells / mL, and then dispensed into wells of a 96-well flat-bottomed ultra-low attachment surface microplate (manufactured by Corning, #3474) at 200 μL per well. Note that, as a non-additive control, HeLa cells suspended in the same medium containing only deacylated gellan gum at a final concentration of 0.030% (w / v) were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 7 days. After culturing for 3, 5, and 7 days, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added to the culture medium, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone. The cell density was measured by pipetting and stirring the culture medium containing cells after 5 days of culture, mixing 20 μL of the stirred solution with 20 μL of Trypan Blue stain 0.4% (manufactured by Invitrogen), and counting under a microscope.

[0199] As a result, it was confirmed that the anti-cancer agent can be efficiently evaluated by the cell proliferation test method using the culture medium composition of the present invention. In addition, Table 49 shows the absorbance at 450 nm (corresponding to the number of HeLa cells) after 3, 5, and 7 days of static culture. Table 50 shows the cell density of HeLa cells after 5 days.

[0200] [Table 49]

[0201] [Table 50]

[0202] (Test Example 44: Maintenance and Function Test of Human Primary Hepatocytes) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.015% or 0.030% (w / v) to HBM medium (manufactured by Lonza Japan Co., Ltd.) supplemented with additives (HCMsingleQuots (registered trademark), BSA-Fatty acid free, EGF, Ascorbic acid, Transferrin, Insulin, GA-1000, Hydrocortisone 21 hemisuccinate; manufactured by Lonza Japan Co., Ltd.). Subsequently, frozen human primary hepatocytes (manufactured by Xenotech) were seeded into the above medium composition containing desacylated gellan gum at a density of 250,000 cells / mL, and then dispensed into wells of a 96-well U-bottom ultra-low attachment surface microplate (manufactured by Sumitomo Bakelite Co., Ltd., PrimeSurface, MS-9096U) at 200 μL per well. As a negative control, human primary hepatocytes suspended in the same medium without desacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 3 days.

[0203] 1. Measurement of viable cell count After adding 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) to the culture medium after 4 hours, 8 hours, and 1 day of culture, it was incubated at 37 °C for 100 minutes, and the absorbance at 450 nm was measured with an absorbance meter (SPECTRA MAX 190, manufactured by Molecular Devices) to measure the number of viable cells.

[0204] 2. Analysis of albumin secretion After 3 days of culture, the culture medium containing hepatocytes was collected and centrifuged (400 g, 3 minutes) to collect the culture supernatant. The human albumin concentration in the medium was determined by Albumin ELISA It was measured using a quantification kit (manufactured by Bethyl Laboratories).

[0205] 3. Expression analysis of mRNA by real-time PCR method After culturing for 8 hours, the culture solution containing hepatocytes was collected and centrifuged (400 g, 3 minutes) to collect the cells. Total RNA was extracted from the cells using the RNeasy Mini kit (manufactured by QIAGEN). Using the total RNA and PrimeScript (registered trademark) RT Master Mix (manufactured by Takara Bio Inc.), a reverse transcription reaction was performed using the GeneAmp PCR System 9700 (manufactured by Applied Biosystems) to synthesize cDNA. Each cDNA sample used in the PCR reaction was a diluted one-tenth with sterilized water by dispensing. Also, the sample used for the calibration curve was set in the quantification range from 1 / 3 to 1 / 243 with a three-fold common ratio using the dispensed and mixed cDNA NA. The PCR reaction was carried out using each cDNA sample, calibration sample, Premix Ex Taq (registered trademark) (manufactured by Takara Bio Inc.) and various Taqman probes (manufactured by Applied Biosystems) using the 7500 Real Time PCR System (manufactured by Applied Biosystems). Specificity was calculated with the mRNA of GAPDH as an endogenous control, and the expression of each mRNA was corrected with the value of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), with the negative control set as 100%.

[0206] Each probe used (manufactured by Applied Biosystems) is shown below. GAPDH:HS99999905 Albumin:HS99999922 Cyp3A4:HS00604506 Cyp2C9:HS02383631 PXR (Pregnane X receptor):HS01114267 ApoA1(Apolipoprotein A1):HS00163641

[0207] As a result, it was confirmed that the medium composition of the present invention has the effect of suppressing the decrease in the number of living cells by keeping and protecting human primary hepatocytes in a dispersed state. In addition, it was confirmed that the albumin-producing ability and the mRNA group expression ability related to pharmacokinetics of the medium composition were higher than those of the negative control. Table 51 shows the absorbance at 450 nm (corresponding to the number of human primary hepatocytes) after 4 hours, 8 hours, and 1 day of static culture. Table 52 shows the albumin value in the culture supernatant after 3 days of static culture. Table 53 shows each mRNA expression value when the negative control after 8 hours of static culture was set to 100%. Figure 21 shows the state of the cells when human primary hepatocytes were cultured for 4 hours.

[0208]

Table 51

[0209]

Table 52

[0210]

Table 53

[0211] (Test Example 45: Maintenance and Function Test of Cynomolgus Monkey Primary Hepatocytes) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. This solution A medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.015% or 0.030% (w / v) to HBM medium (manufactured by Lonza Japan Co., Ltd.) supplemented with additives (HCMsingleQuots (registered trademark), BSA-Fatty acid free, EGF, Ascorbic acid, Transferrin, Insulin, GA-1000, Hydrocortisone 21 hemisuccinate; manufactured by Lonza Japan Co., Ltd.) using a liquid. Subsequently, cryopreserved cynomolgus monkey primary hepatocytes (manufactured by Inal Research Co., Ltd.) were seeded into the above medium composition added with the deacylated gellan gum to a density of 250,000 cells / mL, and then dispensed into wells of a 96-well U-bottom ultra-low attachment surface microplate (manufactured by Sumitomo Bakelite Co., Ltd., PrimeSurface, MS-9096U) at 200 μL per well. As a negative control, cynomolgus monkey primary hepatocytes suspended in the same medium without deacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 3 days.

[0212] 1. Measurement of viable cell count After culturing for 4 hours, 8 hours, 1 day, and 3 days, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added to the culture medium, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (SPECTRA MAX 190, manufactured by Molecular Devices) to measure the number of viable cells.

[0213] 2. Analysis of albumin secretion amount After culturing for 3 days, the culture medium containing hepatocytes was collected and centrifuged (400 g, 3 minutes) to collect the culture supernatant. The human albumin concentration in the medium was measured using an Albumin ELISA Quantitation kit (manufactured by Bethyl Laboratories).

[0214] 3. Analysis of mRNA expression by real-time PCR method After culturing for 1, 2, and 3 days, the culture medium containing hepatocytes was collected and centrifuged (400 g, 3 minutes) to recover the cells. Total RNA was extracted from the cells using the RNeasy Mini kit (manufactured by QIAGEN). Using the total RNA and PrimeScript (registered trademark) RT Master Mix (manufactured by Takara Bio Inc.), reverse transcription reaction was performed using the GeneAmp PCR System 9700 (manufactured by Applied Biosystems) to synthesize cDNA. Each cDNA sample used in the PCR reaction was a dispensed and diluted 1 / 10 with sterile water. Also, the sample used for the calibration curve was the dispensed and mixed cDNA, and it was set in the quantification range from 1 / 3 to 1 / 243 with a three-fold common ratio. The PCR reaction was carried out using each cDNA sample, calibration sample, Premix Ex Taq (registered trademark) (manufactured by Takara Bio Inc.) and various Taqman probes (manufactured by Applied Biosystems) with the 7500 Real Time PCR System (manufactured by Applied Biosystems). Specificity was calculated with the mRNA of GAPDH as the endogenous control, and the expression of each mRNA was corrected with the value of GAPDH. The various probes (manufactured by Applied Biosystems) used are shown below.

[0215] The various probes (manufactured by Applied Biosystems) used are shown below. GAPDH:Rh02621745 Albumin:Rh02789672 ApoA1 (Apolipoprotein A1):Rh02794272

[0216] As a result, it was confirmed that the culture medium composition of the present invention has the effect of suppressing the decrease in the number of viable cells by protecting cynomolgus primary hepatocytes. Also, the hepatocytes cultured in the said culture medium composition have the ability to produce albumin and the ability to express the mRNA group of Albumin and ApoA1, which are negative controls. It was confirmed to be higher compared to the control. Table 54 shows the absorbance at 450 nm (corresponding to the number of cynomolgus monkey primary hepatocytes) after static culture for 4 hours, 8 hours, 1 day, and 3 days. Table 55 shows the albumin value in the culture supernatant after 3 days of static culture. Also, Table 56 shows the mRNA expression value of Albumin and Table 57 shows the mRNA expression value of ApoA1 when the negative control was set to 100% after 2 and 3 days of static culture. Figure 22 shows the cell state when cynomolgus monkey primary hepatocytes were cultured for 4 hours.

[0217]

Table 54

[0218]

Table 55

[0219]

Table 56

[0220]

Table 57

[0221] (Test Example 46: Maintenance and Function Test of Hepatocytes in Collagen-Coated Microplates) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved and sterilized at 121°C for 20 minutes. Using this solution, additives (HCMsingleQuots (registered trademark), BSA-Fatty acid free, EGF, Ascorbic acid, Transferrin, A medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.015% or 0.030% (w / v) to an HBM medium (manufactured by Lonza Japan Co., Ltd.) supplemented with insulin, GA-1000, and hydrocortisone 21 hemisuccinate (manufactured by Lonza Japan Co., Ltd.). Subsequently, cryopreserved cynomolgus monkey primary hepatocytes (manufactured by Inal Research Co., Ltd.) were seeded into the above medium composition containing deacylated gellan gum at a density of 100,000 cells / mL, and then dispensed into wells of a 96-well collagen-coated microplate (manufactured by IWAKI, 4860-010) at 200 μL per well. As a negative control, cynomolgus monkey primary hepatocytes suspended in the same medium without deacylated gellan gum were dispensed. Subsequently, the plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days.

[0222] 1. Measurement of viable cell count After culturing for 1 day, 20 μL of a WST-8 solution (manufactured by Dojindo Laboratories) was added to the culture medium, and then incubated at 37°C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (SPECTRA MAX 190, manufactured by Molecular Devices) to measure the number of viable cells.

[0223] 2. Analysis of albumin secretion After culturing for 3 days, the culture medium containing hepatocytes was collected and centrifuged (400 g, 3 minutes) to collect the culture supernatant. The human albumin concentration in the medium was measured using an Albumin ELISA Quantitation kit (manufactured by Bethyl Laboratories).

[0224] As a result, by using the culture medium composition of the present invention, it was confirmed that even when using a plate coated with collagen, the decrease in the number of living cells was suppressed by protecting primary hepatocytes with the culture medium composition. It was also confirmed that the ability to produce albumin was higher in the culture medium composition than in the negative control. Table 58 shows the absorbance at 450 nm (corresponding to the number of cells of cynomolgus monkey primary hepatocytes) after 1 day of static culture. Table 59 shows the albumin value in the culture supernatant after 3 days of static culture.

[0225]

Table 58

[0226]

Table 59

[0227] (Test Example 47: Comparative Test with Happy Cell ASM Medium) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, additives (HCMsingleQuots (registered trademark), BSA-Fatty acid free, EGF, Ascorbic acid, Transferrin, Insulin, GA-1000, Hydrocortisone 21 hemisuccinate; manufactured by Lonza Japan Co., Ltd.) were added to HBM medium (manufactured by Lonza Japan Co., Ltd.) and mixed with DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) at a ratio of 1:1 to prepare a medium composition with a final concentration of 0.015% (w / v) of desacylated gellan gum. Happy Cell ASM medium (manufactured by biocroi) was prepared with DMEM medium in advance to a specified concentration (mixed 1:1). Subsequently, frozen primary human hepatocytes (manufactured by Xenotech) were seeded in the above medium composition with desacylated gellan gum added to a density of 250,000 cells / mL or the Happy Cell ASM medium composition, and then dispensed into the wells of a 96-well U-bottom ultra-low attachment surface microplate (manufactured by Sumitomo Bakelite Co., Ltd.) at 200 μL per well. As a negative control, a suspension of primary human hepatocytes in the same medium without desacylated gellan gum was dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 6 days.

[0228] 1. Measurement of viable cell count After culturing for 2 hours, 4 hours, 8 hours, 1 day, 4 days, and 6 days, 20 μL of WST-8 solution (manufactured by Dojindo Laboratories) was added to the culture medium, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (SPECTRA MAX 190, manufactured by Molecular Devices) to measure the number of viable cells.

[0229] As a result, by using the culture medium composition of the present invention, it was confirmed that it is excellent in suppressing the decrease in the number of living cells by protecting primary hepatocytes as compared with Happy Cell ASM. Table 60 shows the absorbance at 450 nm (corresponding to the number of human primary hepatocytes) after 2 hours, 4 hours, 8 hours, 1 day, 4 days, and 6 days of static culture.

[0230]

Table 60

[0231] (Test Example 48: Toxicity Test of Compounds on Hepatocytes) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. On the other hand, HepG2 cells were mixed with DMEM medium (manufactured by WAKO) to a concentration of 100,000 cells / mL, and the cell suspension was dispensed into the wells of a 96-well flat-bottomed ultra-low attachment surface microplate (manufactured by Corning, #3474) at 100 μL per well. Troglitazone (manufactured by WAKO, #71750) at each concentration was added to the above deacylated gellan gum aqueous solution, and 10 μ L of this solution was added to 100 μL of the above cell suspension. By the above treatment, a cell suspension with a DMSO concentration of 0.18% (v / v), a Troglitazone concentration of 20.0, 40.0, 60.0, 100 (μmol / L), and a deacylated gellan gum concentration of 0.015% (w / v) was prepared. Subsequently, this plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day.

[0232] 1. Measurement of the number of living cells 50 μL of the culture solution after 1-day culture was dispensed into a 96-well titer plate (manufactured by Corning). After adding 50 μL of CellTiter-Glo (registered trademark) reagent (manufactured by Promega) to this culture solution, it was incubated at room temperature for 10 minutes, and the number of viable cells was measured by measuring the luminescence intensity with a multiplate reader (manufactured by Molecular Devices, FlexStation3). 2. Measurement of lactate dehydrogenase (LDH) activity To 100 μL of the culture solution after 1-day culture, 100 μL of DMEM medium (manufactured by WAKO) was added, and the plate was centrifuged at 440G for 15 minutes. 100 μL of the supernatant was dispensed into a 96-well titer plate (manufactured by Corning), 100 μL of the reaction mixture of the cytotoxicity detection kit (manufactured by Roche Applied Science) was added, and it was allowed to stand at room temperature in the dark for 30 minutes. Subsequently, according to the protocol of the above kit, the absorbance at 490 nm (reference; 600 nm) was measured with an absorptiometer (manufactured by Molecular Devices, SPECTRA MAX 190) to measure the proportion of damaged cells, that is, the cytotoxicity rate (%).

[0233] As a result, it was confirmed that Troglitazone has cytotoxicity to hepatocytes using the medium composition of the present invention. Table 61 shows the relative cell number and cytotoxicity rate (%) when the condition without addition after 1-day culture was set to 1.

[0234]

Table 61

[0235] (Test Example 49: Cell proliferation test by ATP quantification method using A549 cells) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.005% (w / v), 0.015% (w / v), or 0.030% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, human lung cancer cell line A549 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded at a density of 100,000 cells / mL into the above medium composition supplemented with desacylated gellan gum, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 100 μL per well. As a negative control, A549 cells suspended in the same medium without desacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 5 days. For the culture supernatants after 1, 3, and 5 days of culture, 100 μL of an ATP reagent (CellTiter-Glo (registered trademark ) Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for approximately 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices). The number of viable cells was measured by subtracting the luminescence value of the medium alone. For the WST-8 measurement, 10 μL of a WST-8 solution (manufactured by Dojindo Laboratories) was added to the cells after 3 days of culture, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0236] As a result, it was confirmed by the ATP measurement method that A549 cells grow efficiently by using the culture medium composition of the present invention. Table 62 shows the RLU values (ATP measurement, luminescence intensity) after 1, 3, and 5 days of static culture. Table 63 shows the absorbance at 450 nm (WST-8) and the RLU value (ATP measurement, luminescence intensity) after 3 days of culture.

[0237]

Table 62

[0238]

Table 63

[0239] (Test Example 50: Comparison with monolayer culture method in cell growth test using anticancer agent) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition with a final concentration of 0.015% (w / v) of deacylated gellan gum added to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum or an unadded medium composition without deacylated gellan gum was prepared. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded in the above medium composition with deacylated gellan gum added to a density of 37,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. In the monolayer culture method, the human cervical cancer cell line HeLa was seeded in the above medium composition without deacylated gellan gum to a density of 37,000 cells / mL Thereafter, it was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured statically in a CO2 incubator (37 °C, 5% CO2). On the first day of culture, a medium composition containing various anticancer agents at 10-fold concentration and deacylated gellan gum at a final concentration of 0.015% (w / v) to a final concentration of 0.001 to 1 μM (deacylated gellan gum addition group) and a medium composition containing only various anticancer agents at 10-fold concentration (monolayer culture group) were each added at 15 μL, and the culture was continued for another 3 days. Adriamycin (manufactured by WAKO), Paclitaxel (manufactured by WAKO), or Mitomycin C (manufactured by WAKO) was used as the anticancer agent. To the culture solution on the fourth day, 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone. For the WST-8 measurement, after adding 15 μL of the WST-8 solution (manufactured by Dojindo Laboratories), it was incubated at 37 °C for 100 minutes, and the absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0240] As a result, it was found that the efficacy of Mitomycin C was stronger by the cell growth test method using the medium composition of the present invention than by the monolayer culture method. Table 64 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the fourth day of static culture. Table 65 shows the %Control values of the absorbance at 450 nm (WST-8 measurement) on the fourth day of static culture.

[0241]

Table 64

[0242]

Table 65

[0243] (Test Example 51: Comparison with monolayer culture method in cell proliferation test using apoptosis inducer) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition with 0.015% (w / v) deacylated gellan gum added or an unadded medium composition without deacylated gellan gum was prepared in DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded at 37,000 cells / mL in the above medium composition with deacylated gellan gum added, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. For the monolayer culture method, the human cervical cancer cell line HeLa was seeded at 37,000 cells / mL in the above medium composition without deacylated gellan gum, and then dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured in a static state in a CO2 incubator (37°C, 5% CO2). On the first day of culture, the final concentration was from 0.2 to 10 A medium composition containing various apoptosis inducers at 10-fold concentration and deacylated gellan gum at a final concentration of 0.015% (w / v) (deacylated gellan gum addition group) and a medium composition containing only various apoptosis inducers at 10-fold concentration (monolayer culture group) were each added in an amount of 15 μL, and then the culture was continued for 3 days. As the apoptosis inducer, Apoptosis Inducer set (manufactured by Merck Millipore, APT800: Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide) was used. To 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture solution on the 4th day and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the luminescence value of the medium alone. For the WST-8 measurement, after adding 15 μL of a WST-8 solution (manufactured by Dojindo Laboratories), it was incubated at 37 °C for 100 minutes, and the absorbance at 450 nm was measured using a spectrophotometer (SPECTRA MAX 190, manufactured by Molecular Devices), and the number of living cells was measured by subtracting the absorbance of the medium alone.

[0244] As a result, it was found that by the cell growth test method using the medium composition of the present invention, the drug effects of Camptothecin and Etoposide were shown to be stronger than those of the monolayer culture method. The %Control values of the RLU values (ATP measurement, luminescence intensity) on the 4th day of static culture are shown in Table 66. The %Control values of the absorbance at 450 nm (WST-8 measurement) on the 4th day of static culture are shown in Table 67.

[0245]

Table 66

[0246]

Table 67

[0247] (Test Example 52: Comparison with monolayer culture method in HeLa cell proliferation test using Trametinib and MK-2206) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved and sterilized at 121°C for 20 minutes. Using this solution, a medium composition with 0.015% (w / v) deacylated gellan gum added or an unadded medium composition without deacylated gellan gum was prepared in DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition with deacylated gellan gum added to a density of 37,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. For the monolayer culture method, the human cervical cancer cell line HeLa was seeded into the above medium composition without deacylated gellan gum to a density of 7,400 cells / mL, and then dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured in a static state in a CO2 incubator (37°C, 5% CO2). On the first day of culture, from a final concentration of 0.001 to 3 To make it 0 μM, 15 μL each of a medium composition containing each anticancer agent at 10-fold concentration and deacylated gellan gum at a final concentration of 0.015% (w / v) (deacylated gellan gum addition group) and a medium composition containing only each anticancer agent at 10-fold concentration (monolayer culture group) were added, and then the culture was continued for 5 days. As the anticancer agents, Trametinib (manufactured by Santa Cruz, MEK inhibitor) and MK-2206 (manufactured by Santa Cruz, Akt inhibitor) were used. To 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium on the 6th day and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone.

[0248] As a result, it was found that by the cell growth test method using the medium composition of the present invention, the drug effects of MK-2206 and Trametinib were shown to be stronger than those of the monolayer culture method. Table 68 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 4th day of static culture.

[0249]

Table 68

[0250] (Test Example 53: A549 cell growth using Trametinib and MK-2206 Comparison with the monolayer culture method in the test) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition with a final concentration of 0.015% (w / v) desacylated gellan gum added to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum or an unadded medium composition without desacylated gellan gum was prepared. Subsequently, the human lung cancer cell line A549 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition with desacylated gellan gum added to a density of 14,800 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. For the monolayer culture method, the human lung cancer cell line A549 was seeded into the above medium composition without desacylated gellan gum to a density of 14,800 cells / mL, and then dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state. On the first day of culture, 15 μL each of a medium composition containing a 10-fold concentration of each anticancer agent and a final concentration of 0.015% (w / v) desacylated gellan gum (desacylated gellan gum addition group) and a medium composition with only a 10-fold concentration of each anticancer agent (monolayer culture group) were added to reach a final concentration of 0.001 to 30 μM, and then the culture was continued for 5 days. As anticancer agents, Trametinib (manufactured by Santa Cruz, MEK inhibitor) and MK-2206 (manufactured by Santa Cruz, Akt inhibitor) were used. To the culture solution on the sixth day, 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone.

[0251] As a result, it was found that the cell growth test method using the culture medium composition of the present invention showed a stronger drug effect of MK-2206 compared to the monolayer culture method. Table 69 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 4th day of static culture.

[0252]

Table 69

[0253] (Test Example 54: Comparison with the monolayer culture method in the proliferation effect of HeLa cells stimulated with human HB-EGF) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved and sterilized at 121°C for 20 minutes. Using this solution, a medium composition with a final concentration of 0.015% (w / v) of deacylated gellan gum added to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum or an unadded medium composition without deacylated gellan gum was prepared. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical) was seeded into the above medium composition with deacylated gellan gum added to a concentration of 37000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. For the monolayer culture method, the human cervical cancer cell line HeLa was seeded into the above medium composition without deacylated gellan gum to a concentration of 37000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured in a static state in a CO2 incubator (37°C, 5% CO2). On the first day of culture, final concentrations of 10, 30, 1 A medium composition containing 10-fold concentrated human HB-EGF (heparin-binding epidermal growth factor-like growth factor, manufactured by PeproTech) and deacylated gellan gum at a final concentration of 0.015% (w / v) (deacylated gellan gum-added group) and a medium composition containing only 10-fold concentrated human HB-EGF (monolayer culture group) were each added in an amount of 15 μL, and the culture was subsequently continued for 7 days. To the culture solutions on the 6th and 8th days, 150 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone.

[0254] As a result, it was found that the cell growth promoting effect of human HB-EGF was stronger by the HeLa cell growth test method using the medium composition of the present invention than by the monolayer culture method. The %Control values of the RLU values (ATP measurement, luminescence intensity) on the 6th day of static culture are shown in Table 70. The %Control values of the RLU values (ATP measurement, luminescence intensity) on the 8th day of static culture are shown in Table 71.

[0255]

Table 70

[0256]

Table 71

[0257] (Test Example 55: Comparison with the monolayer culture method in the growth effect of A549 cells stimulated with human HB-EGF) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then heated at 90°C It was dissolved by stirring while heating, and this aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition containing 10% (v / v) fetal bovine serum (manufactured by Wako) with 0.015% (w / v) deacylated gellan gum added or an unadded medium composition without deacylated gellan gum was prepared. Subsequently, the human lung cancer cell line A549 (manufactured by DS Pharma Biomedical) was seeded into the above medium composition with deacylated gellan gum added to a density of 14,800 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. For the monolayer culture method, the human lung cancer cell line A549 was seeded into the above medium composition without deacylated gellan gum to a density of 14,800 cells / mL, and then dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured in a static state in a CO2 incubator (37°C, 5% CO2). On the first day of culture, 15 μL each of a medium composition containing 10-fold concentrated human HB-EGF (manufactured by PeproTech) at final concentrations of 10, 30, 100 ng / mL and 0.015% (w / v) deacylated gellan gum (deacylated gellan gum addition group) and a medium composition with only 10-fold concentrated human HB-EGF (monolayer culture group) were added, and then the culture was continued for 7 days. To the culture solutions on the 6th and 8th days, 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the luminescence value of the medium alone.

[0258] As a result, it was found that the cell growth promoting effect of human HB-EGF was stronger by the A549 cell growth test method using the culture medium composition of the present invention than by the monolayer culture method. Table 72 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 6th day of static culture. Table 73 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 8th day of static culture.

[0259] [Table 72]

[0260] [Table 73]

[0261] (Test Example 56: Comparison with the monolayer culture method in the growth effect of A431 cells stimulated with human HB-EGF) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition with a final concentration of 0.015% (w / v) desacylated gellan gum added to EMEM medium (manufactured by DS Pharma Biomedical Co., Ltd.) containing 10% (v / v) fetal bovine serum or an unadded medium composition without desacylated gellan gum was prepared. Subsequently, the human squamous cell carcinoma cell line A431 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition with desacylated gellan gum added to a density of 37,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. For the monolayer culture method, the human squamous cell carcinoma cell line A431 was seeded into the above medium composition without desacylated gellan gum to a density of 37,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured in a static state in a CO2 incubator (37 °C, 5% CO2). On the first day of culture, 15 μL each of a medium composition containing 10-fold concentrated human HB-EGF (manufactured by PEPROTECH) at a final concentration of 10, 30, 100 ng / mL and desacylated gellan gum at a final concentration of 0.015% (w / v) (desacylated gellan gum addition group) and a medium composition containing only 10-fold concentrated human HB-EGF (monolayer culture group) were added, and then the culture was continued for 7 days. To the culture supernatants on the 6th and 8th days, 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone.

[0262] As a result, it was found that the cell growth promoting effect of human HB-EGF was stronger by the A431 cell growth test method using the culture medium composition of the present invention than by the monolayer culture method. Table 74 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 6th day of static culture. Table 75 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 8th day of static culture.

[0263] [Table 74]

[0264] [Table 75]

[0265] (Test Example 57: Comparison with monolayer culture method in the growth action of SKOV3 cells stimulated with human HB-EGF) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition with 0.015% (w / v) desacylated gellan gum added to McCoy's 5a medium (manufactured by DS Pharma Biomedical Co., Ltd.) containing 15% (v / v) fetal bovine serum or an unadded medium composition without desacylated gellan gum was prepared. Subsequently, the human ovarian cancer cell line SKOV3 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition with desacylated gellan gum added to a density of 37,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. The monolayer culture method involved seeding the human ovarian cancer cell line SKOV3 into the above medium composition without desacylated gellan gum to a density of 37,000 cells / mL, and then dispensing it into the wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured statically in a CO2 incubator (37 °C, 5% CO2). On the first day of culture, 15 μL each of a medium composition containing 10-fold concentrated human HB-EGF (manufactured by PEPROTECH) and 0.015% (w / v) desacylated gellan gum at final concentrations of 10, 30, and 100 ng / ml (desacylated gellan gum addition group) and a medium composition containing only 10-fold concentrated human HB-EGF (monolayer culture group) were added, and then the culture was continued for 8 days. To the culture medium on the 6th and 9th days, 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended. After standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone.

[0266] As a result, it was found that the cell growth promoting effect of human HB-EGF was stronger by the SKOV3 cell growth test method using the culture medium composition of the present invention than by the monolayer culture method. Table 76 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 6th day of static culture. Table 77 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 9th day of static culture.

[0267] [Table 76]

[0268] [Table 77]

[0269] (Test Example 58: Comparison with the monolayer culture method in the mRNA expression of VEGF in HeLa cells stimulated with human HB-EGF) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition with 0.015% (w / v) deacylated gellan gum added to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum or an unadded medium composition without deacylated gellan gum was prepared. Subsequently, the human cervical cancer cell line HeLa (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above-mentioned medium composition supplemented with deacylated gellan gum to a density of 37,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. The monolayer culture method involved seeding the human cervical cancer cell line HeLa into the above-mentioned medium composition without deacylated gellan gum to a density of 37,000 cells / mL, and then dispensing it into wells of a 96-well flat-bottom microplate (manufactured by Corning, #3585) at 135 μL per well. Each plate was cultured statically in a CO2 incubator (37 °C, 5% CO2). On the first day of culture, a medium composition containing 10-fold concentrated human HB-EGF (manufactured by PeproTech) at final concentrations of 10, 30, 100 ng / mL and deacylated gellan gum at a final concentration of 0.015% (w / v) (deacylated gellan gum-added group) and a medium composition containing only 10-fold concentrated human HB-EGF (monolayer culture group) were each added at 15 μL, and the culture was continued for 6 days. On the seventh day, the culture solution containing cancer cells was collected, and the cells were recovered by centrifugation (400 g, 3 minutes). Total RNA was extracted from the cells using the RNeasy Mini kit (manufactured by QIAGEN). Using the total RNA and PrimeScript (registered trademark) RT Master Mix (manufactured by Takara Bio Inc.), reverse transcription reaction was performed using the GeneAmp PCR System 9700 (manufactured by Applied Biosystems) to synthesize cDNA. Each cDNA sample used in the PCR reaction was dispensed and diluted 1 / 10 with sterilized water. was used. The samples used for the calibration curve were the dispensed and mixed cDNA, and were set within a quantification range from 1 / 3 to 1 / 243 with a three-fold common ratio. The PCR reaction was carried out using each cDNA sample, calibration sample, Premix Ex Taq (registered trademark) (manufactured by Takara Bio Inc.) and various Taqman probes (manufactured by Applied Biosystems) using the 7500 Real Time PCR System (manufactured by Applied Biosystems). Specificity was determined using GAPDH (Glyceraldehyde The mRNA of 3-phosphate dehydrogenase was used as an endogenous control, and the expression of VEGF (Vascular endothelial growth factor) mRNA was corrected with the value of GAPDH and calculated with the negative control set at 100%. Each probe used (manufactured by Applied Biosystems) is shown below. GAPDH:HS99999905 VEGF: HS00173626

[0270] As a result, it was found that HeLa cells cultured using the culture medium composition of the present invention showed a stronger promoting effect on the mRNA expression of VEGF by human HB-EGF as compared with the monolayer culture method. Table 78 shows the VEGF mRNA expression values when the negative control on the 7th day of static culture was set at 100%.

[0271]

Table 78

[0272] (Test Example 59: Effect of Gefinitib on the proliferation of A549 cells stimulated with human HB-EGF) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was autoclaved at 121°C for 20 minutes. Using this solution, a medium composition with 0.015% (w / v) desacylated gellan gum added or an unadded medium composition without desacylated gellan gum was prepared in DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human lung cancer cell line A549 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded in the above medium composition with desacylated gellan gum added to a density of 14,800 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 135 μL per well. Each plate was cultured statically in a CO2 incubator (37°C, 5% CO2). On the first day of culture, each anticancer agent was added to a final concentration of 0.1 to 30 μM, human HB-EGF was added to a final concentration of 0 ng / ml or 100 ng / ml, and 15 μL each of a medium composition containing each anticancer agent and human HB-EGF (manufactured by PEPROTECH) at 10-fold concentration and desacylated gellan gum at a final concentration of 0.015% (w / v) (desacylated gellan gum addition group) was added, and then the culture was continued for 5 days. Gefitinib (manufactured by Santa Cruz, an EGF receptor inhibitor) was used as the anticancer agent. To the culture medium on the sixth day, 150 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the luminescence value of the medium alone.

[0273] As a result, in the A549 cell proliferation test method using the medium composition of the present invention and human HB-EGF, the inhibitory effect of Gefitinib was stronger under the culture conditions with HB-EGF added. Table 79 shows the %Control values of the RLU values (ATP measurement, luminescence intensity) on the sixth day of static culture.

[0274]

Table 79

[0275] (Test Example 60: Effects of Gefinitib and Elrotinib on the Proliferation of A431 Cells Stimulated with Human HB-EGF) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sanjing Co., Ltd.) at 0.3 After suspending in ultrapure water (Milli-Q water) to a concentration of %(w / v), it was dissolved by stirring while heating at 90 °C, and this aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition with 0.015% (w / v) deacylated gellan gum added or an unadded medium composition without deacylated gellan gum was prepared in EMEM medium (manufactured by DS Pharma Biomedical Co., Ltd.) containing 10% (v / v) fetal bovine serum. Subsequently, the human squamous cell carcinoma cell line A431 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the medium composition with the above-mentioned deacylated gellan gum added to a concentration of 37,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottomed ultra-low attachment surface microplate (manufactured by Corning, #3474) to a volume of 135 μL per well. Each plate was cultured in a static state in a CO2 incubator (37 °C, 5% CO2). On the first day of culture, for each anticancer agent to a final concentration of 0.1 to 30 μM, and for human HB-EGF to a final concentration of 0 ng / ml or 100 ng / ml, 15 μL each of a medium composition containing each anticancer agent and human HB-EGF (manufactured by PEPROTECH) at a 10-fold concentration and 0.015% (w / v) deacylated gellan gum (deacylated gellan gum-added group) was added, and then the culture was continued for 7 days. As anticancer agents, Gefitinib (manufactured by Santa Cruz, an EGF receptor inhibitor) and Elrotinib (manufactured by Santa Cruz, an EGF receptor inhibitor) were used. To the culture supernatants on the 4th, 6th, and 8th days, 150 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone.

[0276] As a result, cell growth of A431 cells under low-adhesion culture conditions was observed in the culture method combining the culture medium composition of the present invention and human HB-EGF. Furthermore, the inhibitory effects of Gefitinib and Elrotinib on the HB-EGF growth-promoting action were determined by the A431 cell growth test method combining the culture medium composition of the present invention and human HB-EGF. Regarding the growth-promoting action of human HB-EGF, the RLU values (ATP measurement, luminescence intensity) on the 4th, 6th, and 8th days of static culture are shown in Table 80. Furthermore, regarding the action of each anticancer agent on the human HB-EGF growth-promoting action, the %Control values of the RLU values (ATP measurement, luminescence intensity) on the 4th and 8th days of static culture are shown in Table 81.

[0277]

Table 80

[0278]

Table 81

[0279] (Test Example 61: Cell Growth Test when MCF-7 Cells are Dispersed) Desacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding desacylated gellan gum at a final concentration of 0.005% (w / v) or 0.015% (w / v) to EMEM medium (manufactured by DS Pharma Biomedical Co., Ltd.) containing 10% (v / v) fetal bovine serum. Subsequently, the human breast cancer cell line MCF-7 (manufactured by DS Pharma Biomedical Co., Ltd.) was seeded into the above medium composition supplemented with desacylated gellan gum to a density of 50,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 100 μL per well. As a negative control, MCF-7 cells suspended in the same medium without desacylated gellan gum were dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 5 days. For the culture broth after 2 and 5 days of culture, 100 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for approximately 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices). The number of viable cells was measured by subtracting the luminescence value of the medium alone. For the WST-8 measurement, 10 μL of a WST-8 solution (manufactured by Dojindo Laboratories) was added to the cells after 2 and 5 days of culture, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0280] As a result, MCF-7 cells proliferated efficiently by using the medium composition of the present invention. This was confirmed by the ATP measurement method and the WST-8 measurement method. Table 82 shows the RLU values (ATP measurement, luminescence intensity) after 2 and 5 days of static culture. Table 83 shows the absorbance at 450 nm (WST-8) after 2 and 5 days of culture. The results of microscopic observation of the aggregates of MCF-7 cells after 5 days of culture are shown in Figure 23.

[0281]

Table 82

[0282]

Table 83

[0283] (Test Example 62: Cell Proliferation Test When A375 Cells and MNNG / HOS Cells are Dispersed ) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved and sterilized at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.005% (w / v) or 0.015% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum or EMEM medium (manufactured by DS Pharma Biomedical). Subsequently, the human melanoma cell line A375 (manufactured by ATCC) or the human osteosarcoma cell line MNNG / HOS (manufactured by ATCC) was seeded into the above medium composition containing deacylated gellan gum at a density of 50,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 100 μL per well. As a negative control, A375 cells and MNNG / HOS cells suspended in the same medium without deacylated gellan gum were dispensed. Subsequently, this plate was placed in an incubator at C After seeding in the medium composition with added gellan gum, it was dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 100 μL per well. As a negative control, A375 cells and MNNG / HOS cells suspended in the same medium without deacylated gellan gum were dispensed. Subsequently, this plate was placed in an incubator at C It was cultured in an O2 incubator (37 °C, 5% CO2) in a static state for 4 days. To the culture solution after 4 days of culture, 100 μL of an ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured with a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone. After adding 10 μL of a WST-8 solution (manufactured by Dojindo Laboratories) to the culture solution after 4 days of culture, it was incubated at 37 °C for 100 minutes, and the absorbance at 450 nm was measured with an absorbance meter (SPECTRA MAX 190, manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the absorbance of the medium alone.

[0284] As a result, A375 cells and MNNG / HOS cells can be cultured in a uniformly dispersed state without excessive size of cell aggregates by using the culture medium composition of the present invention, and it was confirmed that they grow efficiently with the said culture medium composition. The results of microscopic observation of the aggregates of A375 cells and MNNG / HOS cells after 4 days of culture are shown in Fig. 24. Also, in A375 cells, the absorbance at 450 nm (WST-8) and the RLU value (ATP measurement, luminescence intensity) after 4 days of static culture are shown in Table 84. In MNNG / HOS cells , the absorbance at 450 nm (WST-8) and the RLU value (ATP measurement, luminescence intensity) after 4 days of static culture are shown in Table 85.

[0285]

Table 84

[0286]

Table 85

[0287] (Test Example 63: Cell Proliferation Test When Dispensing MIAPaCa-2 Cells) Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), and then dissolved by stirring while heating at 90 °C. This aqueous solution was autoclaved at 121 °C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.005% (w / v) or 0.015% (w / v) to DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum. Subsequently, the human pancreatic cancer cell line MIAPaCa-2 (manufactured by ATCC) was seeded in the above medium composition with added deacylated gellan gum to a density of 50,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (manufactured by Corning, #3474) at 100 μL per well. As a negative control, a suspension of MIAPaCa-2 cells in the same medium without deacylated gellan gum was dispensed. Subsequently, this plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 6 days. To the culture solution after 6 days of culture, 100 μL of an ATP reagent (CellTiter-Glo® Luminescent Cell Viability Assay, manufactured by Promega) was added and suspended, and after standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the medium alone. After 6 days of culture, 10 μL of a WST-8 solution (manufactured by Dojindo Laboratories) was added to the culture solution, and then incubated at 37 °C for 100 minutes. The absorbance at 450 nm was measured using an absorbance meter (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the medium alone .

[0288] As a result, MIAPaCa-2 cells can be cultured in a uniformly dispersed state without excessive size of cell aggregates by using the culture medium composition of the present invention, and it was confirmed that they grow efficiently in the culture medium composition. The results of microscopic observation of cell aggregates of MIAPaCa-2 cells after culturing for 6 days are shown in Fig. 25. Also, in MIAPaCa-2 cells, the absorbance at 450 nm (WST-8) and the RLU value (ATP measurement, luminescence intensity) after 4 days of static culture are shown in Table 86.

[0289] [Table 86]

[0290] (Test Example 64: Concentration and Dilution of Medium Containing Deacylated Gellan Gum) DMEM medium (manufactured by Wako) containing 0.015% (w / v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by Samchang Co., Ltd.) prepared by the same method as in Test Example 2 was dispensed into 15 mL centrifuge tubes (manufactured by VIOLAMO) at 10 mL each, and centrifuged (700 G, 5 minutes) with a swinging rotor LC-200 (manufactured by Tomy Seiko Co., Ltd.) to precipitate deacylated gellan gum. Then, 8 mL of the supernatant was removed with an aspirator, thereby concentrating the deacylated gellan gum-containing medium. Further, DMEM medium (manufactured by Wako) not containing deacylated gellan gum was added to the concentrated medium and mixed by pipetting to prepare media with arbitrary concentration ratios. On one hand, human hepatoma cells HepG2 (manufactured by DS Pharma Biomedical Co., Ltd.) were suspended in DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum to a concentration of 500,000 cells / mL. 10 mL of this suspension was seeded into EZ SPHERE (manufactured by Asahi Glass Co., Ltd.), and then cultured in a 37°C, CO₂ incubator (5% CO₂) for 7 days. 10 mL of the suspension of the obtained spheres (diameter 100 - 200 μm) was sedimented by centrifugation (200G, 5 minutes), and 1.0 mL of the sphere suspension was prepared by removing the supernatant. 100 μL of this sphere suspension was added to the medium prepared at the above arbitrary concentration, and the spheres were dispersed by pipetting. After incubating at 37°C, the dispersion state of the spheres after 1 hour was visually observed. The results are shown in Table 87.

[0291] As shown in Table 87, deacylated gellan gum can be concentrated and diluted to an arbitrary concentration after being prepared as a medium composition. It was confirmed that the medium composition concentrated and diluted in this way has a floating effect on the spheres.

[0292]

Table 87

[0293] (Test Example 65: Preparation of DMEM / Ham’s F12 Medium Containing Deacylated Gellan Gum) 120 mg of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) was suspended in 72 mL of pure water and dissolved by heating and stirring at 90°C. Pure water was added thereto to prepare 720 mL of a solution containing 0.017% (w / v) of deacylated gellan gum, and then sterilized using a sterilizing filter (pore size: 0.22 μm). On the other hand, a powder medium (Life Technologies) obtained by equally mixing DMEM / Ham's F12 and sodium hydrogen carbonate was added with pure water in an amount corresponding to one-tenth of the recommended value at the time of medium preparation to prepare an 80 mL aqueous solution at a 10-fold concentration, and then sterilized using a sterilizing filter (pore size: 0.22 μm). These were mixed with stirring at 25°C under sterile conditions to prepare 800 mL of a target medium having a deacylated gellan gum concentration of 0...

Claims

【Claim 1】 The invention described in the specification or the drawings.

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