Culture medium composition

Abstract

To provide a culture medium composition for culturing an animal and plant cell and / or tissue particularly in a three-dimensional or a floating state, and a method for culturing an animal and plant cell and / or tissue using the culture medium composition.SOLUTION: There is provided a culture medium composition in which by adding nanofiber to liquid culture medium, evenly dispersing the nanofiber in the solution, and substantially holding cell and / or tissue without substantially increasing the viscosity of the solution, the composition has an effect of preventing its sedimentation. There is also provided a method for culturing a cell and / or tissue using the composition in which a cell and / or tissue is cultured in a floating state.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a medium composition for culturing animal and plant cells and / or tissues, particularly in a three-dimensional or suspended state, using nanofibers of polysaccharides or the like with enhanced dispersibility in water, and uses of the same. [Background technology]

[0002] In recent years, techniques for growing or maintaining various organs, tissues, and cells that play different roles in animals and plants outside of the body have been developed. Growing or maintaining these organs and tissues outside of the body is called organ culture and tissue culture, respectively, and growing, differentiating, or maintaining cells isolated from organs and tissues outside of the body is called cell culture. Cell culture is a technique for growing, differentiating, or maintaining isolated cells outside of the body in a medium, and is essential for analyzing the functions and structures of various organs, tissues, and cells in detail. In addition, 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., mass production of useful substances such as enzymes, cell growth factors, and antibodies, regenerative medicine to compensate for organs, tissues, and cells lost due to disease or defects, plant breeding, and creation of genetically modified crops.

[0003] Cells derived from animals are roughly divided into two types, suspension cells and adherent cells, based on their properties. Suspension cells are cells that do not require a scaffold for growth and proliferation, while adherent cells are cells that require a scaffold for growth and proliferation. The majority of cells that make up living organisms are the latter type of adherent cells. Methods for culturing adherent cells include monolayer culture, dispersion culture, embedded culture, microcarrier culture, and sphere culture.

[0004] Monolayer culture is a method of culturing target cells in a monolayer using culture vessels made of glass or synthetic polymer materials with various surface treatments, or auxiliary cells called feeder cells as a scaffold, and is the most common method. For example, culture methods using culture vessels of various shapes or properties, such as polystyrene with various surface treatments (plasma treatment, corona treatment, etc.), those coated with cell adhesion factors such as collagen, fibronectin, polylysine, or those seeded with feeder cells in advance, have been developed. However, monolayer culture has problems such as the inability to maintain the specific functions that cells have in vivo for a long period of time because its two-dimensional culture environment is completely different from the environment in vivo, the inability to reconstruct tissues similar to those in vivo, and the inability to culture large amounts of cells due to the limited number of cells per certain area (Patent Document 1). In addition, the method of culturing target cells on feeder cells may be problematic in terms of separation of feeder cells and target cells (Non-Patent Document 1).

[0005] Dispersion culture is a method in which cells are seeded in a medium, and then the culture solution is continuously stirred in a culture vessel that has been subjected to a surface treatment that inhibits cell adhesion, thereby inhibiting the adhesion of the cells to the culture vessel, and the adherent cells are cultured in a suspended state. However, the adherent cells cultured by this method cannot adhere to a scaffold, so it cannot be applied to cells that require adhesion to a scaffold for cell proliferation. In addition, since the cells are constantly crushed by shear force, they cannot exert their original cell functions, so it may not be possible to culture functional cells in large quantities (Non-Patent Document 2).

[0006] Embedding culture is a method of culturing cells by embedding and immobilizing them in a solid or semi-solid gel matrix such as agar, methylcellulose, collagen gel, gelatin, fibrin, agarose, or alginate. This method makes it possible to culture cells three-dimensionally in a state similar to that in the living body, and since the gel matrix itself may promote cell proliferation and differentiation, it is not suitable for monolayer culture or differentiation. Compared with the dispersed culture, it is possible to culture cells at a high density while maintaining the function of the cells (Patent Documents 2 and 3). Furthermore, a method has been developed in which cells are embedded in these gel base materials, microcapsules with a size of 100 to 300 μm are created, 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 continuously if the gel base material 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 collagen gel) to recover cells from the medium because the medium and microcapsules containing the gel base material have high viscosity, and the difficulty of changing the medium required for long-term culture. In recent years, a technology has been developed that enables cell recovery from a gel base material by treatment with heat or shear force, but heat and shear force can have a negative effect on cell function, and the safety of the gel base material for living organisms has not yet been clarified (Patent Documents 4 and 5, Non-Patent Documents 4, 5, 6, and 7). In addition, sol-type foods have been developed in the food industry to uniformly disperse and suspend granular foods such as finely cut fruits and vegetables and prevent them from settling or floating, but these sol-type foods do not take into consideration the recovery of the dispersed granular foods, and no consideration has been given to whether cells or tissues can be cultured in a suspended state (Patent Document 6).It is known that gellan in an aqueous solution gels under the action of calcium ions and forms a fine structure (Non-Patent Document 8).

[0007] In microcarrier culture, cells are grown in a monolayer on the surface of fine particles (hereinafter referred to as microcarriers) that are slightly heavier than water, and the fine particles are stirred in a culture vessel such as a flask to culture in a suspended state. Typically, the microcarriers used in this method have a diameter of 100 to 300 μm and a surface area of ​​3,000 to 6,000 cm. 2Microcarriers are spherical particles with a specific gravity of 1.03 to 1.05 / g and are made of materials such as dextran, gelatin, alginic acid, or polystyrene. Charged groups such as collagen, gelatin, or dimethylaminoethyl can be added to the surface of the microcarrier to facilitate cell attachment. This method is applied to mass cell culture because it makes it possible to significantly increase the culture area (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 microcarrier due to shear force during stirring and cells being damaged (Non-Patent Document 9).

[0008] Sphere culture is a method in which the target cells are allowed to form aggregates (hereinafter also referred to as spheres) consisting of tens to hundreds of cells, and then the aggregates are cultured by standing or shaking in a medium. Spheres have a high cell density, and cell-cell interactions and cell structures similar to those in the in vivo environment are reconstructed, and it is known that they can be cultured while maintaining cell functions for a long period of time compared to monolayer culture and dispersed culture methods (Non-Patent Documents 10 and 11). However, in sphere culture, if the size of the sphere is too large, it is difficult to supply nutrients and discharge waste products from inside the sphere, 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 vessel, it is difficult to increase the number of spheres per certain volume, and it is not suitable for mass culture. Furthermore, known methods for creating spheres include hanging drop culture, culture on a non-cell-adhesive surface, culture in microwells, rotary culture, culture using cell scaffolds, and aggregation using centrifugal force, ultrasound, and electric and magnetic fields. However, these methods have problems such as complicated operations, difficulty in recovering the spheres, difficulty in controlling size and mass production, unknown effects on cells, and the need for special dedicated containers and devices (Patent Document 9).

[0009] Meanwhile, plants can also be grown by culturing cells, protoplasts from which the cell walls have been removed, or organs, tissues, and callus such as plant leaves, stems, roots, meristems, seeds, embryos, and pollen in a sterile environment. Using such plant tissue and cell culture techniques, it is also possible to improve plant varieties and produce useful substances. A method of suspending plant cells and tissues in a liquid medium is known as a method for growing large quantities of plant cells and tissues in a short period of time (Non-Patent Document 12). In order to achieve good growth of these organisms, it is important to supply sufficient oxygen, maintain a uniform mixed state, and prevent cell damage. The supply of oxygen to the culture medium and the suspension of cells and tissues can be achieved either by aeration and mechanical stirring in combination or by aeration alone. The former method can lead to poor growth due to damage to cells and tissues caused by stirring, while the latter method causes less shearing of cells and tissues, but in high-density culture it can be difficult to maintain a uniform mixed state, leading to problems such as the settling of cells and tissues and reduced growth efficiency. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] JP 2001-128660 A [Patent Document 2] Japanese Patent Application Publication No. 62-171680 [Patent Document 3] Japanese Patent Application Publication No. 63-209581 [Patent Document 4] JP 2009-29967 A [Patent Document 5] JP 2005-60570 A [Patent Document 6] Japanese Patent Application Publication No. 8-23893 [Patent Document 7] JP 2004-236553 A [Patent Document 8] International Publication No. 2010 / 059775 [Patent Document 9] JP 2012-65555 A [Non-patent literature]

[0011] [Non-Patent Document 1] Klimanskaya et al., Lancet 2005,365:1636-1641 [Non-Patent Document 2] King et al., Curr Opin Chem Biol. 2007, 11:394-398. [Non-Patent Document 3] Murua et al., J. of Controlled Release 2008, 132:76-83. [Non-Patent Document 4] Mendes, Chemical Society Reviews 2008,37:2512-2529 [Non-Patent Document 5] Moon et al., Chemical Society Reviews 2012, 41:4860-4883 [Non-Patent Document 6] Pek et al., Nature Nanotechnol. 2008,3:671-675 [Non-Patent Document 7] Liu et al.,Soft Matter 2011,7:5430-5436 [Non-Patent Document 8] Perez-Campos et al., Food Hydrocolloids 2012, 28:291-300 [Non-Patent Document 9] Leung et al., Tissue Engineering 2011,17:165-172 [Non-Patent Document 10] Stahl et al.,Biochem.Biophys.Res.Comm. 2004,322:684-692 [Non-Patent Document 11] Lin et al.,Biotechnol J. 2008,3:1172-1184 [Non-Patent Document 12] Weathers et al., Appl Microbiol Biotechnol 2010,85:1339-1351 Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to solve the above-mentioned problems of the prior art, and to provide a medium composition for culturing animal and plant cells and / or tissues, particularly in a three-dimensional or suspended state, and a method for culturing animal and plant cells using the medium composition. The present invention provides a method for culturing cells and / or tissues. [Means for solving the problem]

[0013] As a result of intensive research, the present inventors have found that by mixing nanofibers made of polysaccharides such as cellulose and chitin in a liquid medium, animal and plant cells and / or tissues can be cultured in suspension in a stationary state without substantially increasing the viscosity of the liquid medium, and that the proliferation activity of cells is enhanced by culturing using this medium composition. In addition, they have found that not only water-insoluble polysaccharides such as cellulose, but also water-soluble polysaccharides such as deacylated gellan gum form fibrous structures in liquid medium, which enable animal and plant cells and / or tissues to be cultured in suspension in a stationary state without substantially increasing the viscosity of the liquid medium. They have also found that the cultured cells and / or tissues can be easily recovered from these medium compositions. Based on the above findings, the present invention has been completed through further research.

[0014] That is, the present invention is as follows:

[0015] [1] A medium composition capable of culturing cells or tissues in suspension, characterized in that it contains nanofibers. [2] The medium composition of [1], which allows replacement of the medium composition during culture and recovery of cells or tissues after the end of culture. [3] The medium composition of [1], which does not require any temperature change, chemical treatment, enzyme treatment, or shear force during cell or tissue recovery. [4] The medium composition according to [1], characterized in that the viscosity is 8 mPa·s or less. [5] The medium composition according to [1], wherein the nanofibers have an average fiber diameter of 0.001 to 1.00 μm and a ratio (L / D) of average fiber length (L) to average fiber diameter (D) of 2 to 500. [6] The medium composition according to [1], wherein the nanofibers are made of a polymer compound. [7] The medium composition according to [6], wherein the polymer compound is a polysaccharide. [8] The polysaccharide, Any water-insoluble polysaccharide selected from the group consisting of cellulose, chitin, and chitosan; or Water-soluble polysaccharides selected from the group consisting of hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid, and salts thereof. The medium composition according to [7], [9] The medium composition according to [8], wherein the polysaccharide comprises cellulose or chitin.

[10] The medium composition according to [9], characterized in that the nanofibers are obtained by pulverization.

[11] The medium composition according to any one of [1] to

[10] , which is for cell culture.

[12] The medium composition according to

[11] , wherein the cells are adherent cells or suspension cells.

[13] The medium composition according to

[12] , wherein the adherent cells are in the form of spheres.

[14] A cell or tissue culture comprising the medium composition according to any one of [1] to

[13] and a cell or tissue.

[15] A method for culturing cells or tissues, comprising culturing the cells or tissues in a medium composition according to any one of [1] to

[13] .

[16]

[14] A method for isolating cells or tissues from the culture of Methods of recovering textiles.

[17] The method according to

[16] , wherein the separation is carried out by centrifugation.

[18] A method for producing spheres, comprising culturing adherent cells in a medium composition according to any one of [1] to

[13] .

[19] A medium additive for preparing any one of the medium compositions of [1] to

[13] , characterized in that the medium additive contains the nanofiber or a water-soluble polymer compound that constitutes the nanofiber.

[20] A method for producing a medium composition, comprising mixing the medium additive according to

[19] with a medium.

[21] A method for producing a medium composition according to any one of [1] to

[13] , characterized in that the nanofiber or a water-soluble polymer compound constituting the nanofiber is mixed with a medium.

[22] A method for preserving cells or tissues, comprising preserving the cells or tissues in a medium composition according to any one of [1] to

[13] .

[23] A method for transporting cells or tissues, comprising transporting the cells or tissues in a medium composition according to any one of [1] to

[13] .

[24] A method for growing cells or tissues, comprising culturing the cells or tissues in a medium composition according to any one of [1] to

[13] .

[25] A method for subculturing adherent cells, comprising the steps of: (1) Suspension culturing of adherent cells in the medium composition according to any one of [1] to

[13] ; and (2) The cells are cultured in suspension in the step (1) without the need for detaching the cells from the culture vessel. (ii) adding a fresh medium composition described in any one of [1] to

[13] to the culture containing the adherent cells obtained, or (iii) adding all or a part of the culture containing the adherent cells obtained by the suspension culture in step (1) to a fresh medium composition described in any one of [1] to

[13] .

[26] A method for growing adherent cells, comprising suspension culturing adherent cells in a medium composition containing chitin nanofibers while the cells are attached to the chitin nanofibers.

[27] The method described in

[26] , wherein the content of chitin nanofibers in the culture medium composition is 0.0001% (weight / volume) or more and 0.1% (weight / volume) or less. Effect of the Invention

[0016] The present invention provides a medium composition containing nanofibers, particularly nanofibers made of polysaccharides. The medium composition allows cells and / or tissues to be cultured in a suspended state without shaking, rotating, or other operations that may cause damage to or loss of function of the cells or tissues. Furthermore, the medium composition allows the medium to be easily replaced during culture, and the cultured cells and / or tissues to be easily recovered. The culture method can be applied to cells and / or tissues collected from living animals or plants, and the target cells and / or tissues can be prepared in large quantities without impairing their functions. The cells and / or tissues obtained by the culture method can be used to evaluate the efficacy and toxicity of chemical substances, pharmaceuticals, etc., to mass-produce useful substances such as enzymes, cell growth factors, and antibodies, and to perform regenerative medicine to replace organs, tissues, and cells lost due to disease or defects.

[0017] In addition, the medium composition of the present invention can maintain cells or tissues in an environment close to that in the living body, and is therefore useful for the preservation and transportation of cells and tissues.For example, when cells are cultured on a plate and transported as is, the vibration during transportation can cause the cells to peel off from the plate, etc., and the original functions of the cells can be reduced.However, the medium composition of the present invention has nanofibers that form a three-dimensional network and support the cells, allowing the cells to be held in a floating state, thereby avoiding damage to the cells caused by peeling off from the plate due to vibration during transportation, and allowing the cells to be preserved and transported while maintaining their original functions. can. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows that when HepG2 cell spheres were cultured in a medium composition, the spheres were uniformly dispersed and could be cultured in a suspended state. [Diagram 2] FIG. 1 shows that when HeLa cell spheres were cultured in the medium composition, the spheres were uniformly dispersed and could be cultured in a floating state. [Diagram 3] This figure shows that when HeLa cell spheres were cultured in a medium composition and the spheres were observed under a microscope, aggregation between the spheres was suppressed compared to existing medium. [Figure 4] FIG. 1 shows that when microcarriers to which HepG2 cells have been attached are cultured in a medium composition, the HepG2 cells can proliferate on the microcarriers. [Diagram 5] FIG. 1 shows that when HeLa cell spheres were added to a medium composition, the spheres were uniformly dispersed and in a floating state. [Figure 6] FIG. 1 shows that the medium composition induces the formation of HeLa cell spheres. [Figure 7] 1 is a diagram showing a film, which is one embodiment of a structure. The concentration of deacylated gellan gum in the medium composition is 0.02% (weight / volume). [Figure 8]FIG. 1 shows that the medium composition induces sphere formation of HepG2 cells. [Figure 9] FIG. 1 shows the floating state of laminin-coated GEMs to which HepG2 cells have been attached when cultured in a medium composition. [Figure 10] FIG. 1 shows the floating state of alginate beads in which HepG2 cells are embedded when cultured in a medium composition. [Figure 11] FIG. 2 shows the floating state of collagen gel capsules in which HepG2 cells are embedded when cultured in a medium composition. [Figure 12] FIG. 2 shows the floating state of rice-derived callus when cultured in a medium composition. [Figure 13] The viscosity of each medium composition at 25°C is shown. [Figure 14] 1 shows a scanning electron microscope photograph of the MNC-containing medium composition of Example 1. [Figure 15] 1 shows a scanning electron microscope photograph of the PNC-containing medium composition of Example 2. [Figure 16] 1 shows a scanning electron microscope photograph of the CT-containing medium composition of Example 3. [Figure 17] 1 shows a scanning electron microscope photograph of the DAG-containing medium composition of Example 4. [Figure 18] 1 shows a scanning electron microscope photograph of the Car-containing medium composition of Example 5. Dried at room temperature. [Figure 19] 1 shows a scanning electron microscope photograph of the Car-containing medium composition of Example 5. Dried at 110° C. [Figure 20] 1 shows a scanning electron microscope photograph of the Xan-containing medium composition of Comparative Example 3. [Figure 21] 1 shows a scanning electron microscope photograph of the DU-containing medium composition of Comparative Example 4. [Figure 22] 1 shows the results of observing the dispersed state of HepG2 cell spheres after 6 days of suspension culture in the MNC-containing medium composition of Example 1. From the left, the MNC concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Diagram 23]1 shows the results of observing the dispersion state of HepG2 cell spheres after 6 days of suspension culture in the PNC-containing medium composition of Example 2. From the left, the PNC concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Figure 24] 1 shows the results of observing the dispersion state of HepG2 cell spheres after 6 days of suspension culture in the CT-containing medium composition of Example 3. From the left, the CT concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Diagram 25] 1 shows the results of observing the dispersed state of HepG2 cell spheres after 6 days of suspension culture in the DAG-containing medium composition of Example 4. From the left, the DAG concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Figure 26] This shows the observation results of the dispersed state of HepG2 cell spheres after 6 days of suspension culture in the Car-containing medium composition of Example 5. From the left, the Car concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Figure 27] This shows the results of observing the dispersion state of HepG2 cell spheres after 6 days of suspension culture in the Xan-containing medium composition of Example 5. From the left, the Xan concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%. [Figure 28] 1 shows the results of observing the dispersed state of HepG2 cell spheres after 6 days of suspension culture in the DU-containing medium composition of Comparative Example 4. From the left, the DU concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Figure 29] 1 shows the observation results of the dispersed state of HepG2 cell spheres after 6 days of suspension culture in the Alg-containing medium composition of Comparative Example 5. From the left, the Alg concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%. [Diagram 30] The RLU values ​​on day 6 after initiating suspension culture of MCF7 cells in the medium compositions of Example 1' and Comparative Example 5' are shown. [Diagram 31] The RLU values ​​on day 6 after suspension culture of MCF7 cells in the medium compositions of Examples 2' and 3' are shown. [Diagram 32] The RLU values ​​on day 6 after suspension culture of MCF7 cells in the medium compositions of Example 4' and Example 5' are shown. [Diagram 33] The RLU values ​​on day 6 after suspension culture of MCF7 cells in the medium compositions of Comparative Examples 3' and 4' are shown. [Diagram 34] The RLU values ​​on day 6 after the start of suspension culture of A375 cells in the medium compositions of Example 1' and Comparative Example 5' are shown. [Diagram 35] The RLU values ​​on day 6 after suspension culture of A375 cells in the medium compositions of Examples 2' and 3' are shown. [Diagram 36] The RLU values ​​on day 6 after the start of suspension culture of A375 cells in the medium compositions of Example 4' and Example 5' are shown. [Figure 37] The RLU values ​​on day 6 after suspension culture of A375 cells in the medium compositions of Comparative Examples 3' and 4' are shown. [Figure 38] 1 shows the results of microscopic observation of the dispersed state of MCF7 cells in each medium composition on the second day after the start of suspension culture. [Figure 39] 1 shows the results of microscopic observation of the dispersed state of A375 cells in each medium composition on the second day after the start of suspension culture. [Diagram 40] 1 shows the results of microscopic observation of the dispersed state of MDCK cells in each medium composition on the fourth day after the start of suspension culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will now be described in further detail. The terms used in this specification are defined as follows.

[0020] The cell in the present invention is the most basic unit constituting an animal or a plant, and has as its elements a cytoplasm and various organelles inside the cell membrane. In this case, the nucleus containing DNA may or may not be contained inside the cell. For example, the animal-derived cells in the present invention include reproductive cells such as sperm and eggs, somatic cells constituting an organism, stem cells, progenitor cells, cancer cells isolated from an organism, cells (cell lines) isolated from an organism that have acquired immortalization ability and are stably maintained outside the body, cells isolated from an organism that have been artificially genetically modified, cells isolated from an organism that have been artificially replaced with a nucleus, etc. 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, bone cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, and muscle cells (even though they may be isolated from the body). 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, spleen, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain, or nerve tissue. Stem cells are cells that have the ability to replicate themselves and the ability to differentiate into cells of multiple lineages, and examples of stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Progenitor cells are cells that are in the middle of differentiating from the stem cells into specific somatic cells or germ cells. Cancer cells are cells derived from somatic cells and have acquired infinite proliferation ability. Cell lines are cells that have acquired infinite proliferation ability by artificial manipulation outside the body, examples of which include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human fetal kidney cell line), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), Vero, etc.

[0021] In the present invention, the plant-derived cells include cells isolated from each tissue of a plant body, and also include protoplasts obtained by artificially removing the cell wall from said cells.

[0022] In the present invention, a tissue is a structural unit in which cells having several different types of properties and functions are assembled in a certain manner, and examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, nervous tissue, etc. Examples of plant tissues include meristematic tissue, epidermal tissue, assimilation tissue, mesophyll tissue, conductive tissue, mechanical tissue, parenchyma tissue, dedifferentiated cell mass (callus), etc.

[0023] When culturing cells and / or tissues, 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 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 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 japonica, 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 foods (e.g., blueberries, safflower, madder, saffron, etc.), and plants that produce pharmaceutical ingredients.

[0024] In the present invention, the term "suspension of cells and / or tissues" refers to a state in which the cells and / or tissues are not attached to the culture vessel (non-adherent). Furthermore, in the present invention, when the cells and / or tissues are grown, differentiated or maintained, the state in which the cells and / or tissues are uniformly dispersed and suspended in the liquid medium composition without applying external pressure or vibration to the liquid medium composition or shaking or rotating operations within the composition is referred to as "suspension static culture", and culturing the cells and / or tissues in this state is referred to as "suspension static culture". In "suspension static culture", The period during which the material can be allowed to float is at least 5 minutes or more, preferably 1 hour or more, 24 hours or more, 48 hours or more, 6 days or more, or 21 days or more, but is not limited to these periods as long as the material can be kept in a floating state.

[0025] In a preferred embodiment, the medium composition of the present invention allows cells and / or tissues to be kept in suspension at least at one point in a temperature range (e.g., 0 to 40°C) that allows the maintenance and culture of cells and tissues. The medium composition of the present invention allows cells and / or tissues to be kept in suspension at least at one point in a temperature range of preferably 25 to 37°C, most preferably at 37°C.

[0026] Whether or not the cells can be left suspended can be evaluated, for example, by uniformly dispersing polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) in the medium composition to be evaluated, leaving the beads at 25°C, and observing whether the cells maintain a suspended state for at least 5 minutes (preferably 24 hours or more, 48 hours or more).

[0027] The medium composition of the present invention is a composition containing nanofibers and a medium that allows cells or tissues to be cultured in suspension (preferably in static suspension culture). The medium composition is preferably a composition that allows for a medium composition replacement process during culture and for recovery of cells or tissues after completion of culture, and more preferably a composition that does not require any of temperature change, chemical treatment, enzyme treatment, or shear force when recovering cells or tissues.

[0028] [Nanofiber] The nanofibers contained in the medium composition of the present invention exhibit the effect of uniformly suspending cells and / or tissues in a liquid medium. More specifically, examples of nanofibers contained in the medium composition of the present invention include nanofibers formed in a liquid medium by aggregation and self-organization of low molecular weight compounds or polymeric compounds via covalent bonds, ionic bonds, electrostatic interactions, hydrophobic interactions, van der Waals forces, etc., or nanofibers obtained by micronizing a relatively large fiber structure made of a polymeric compound by high pressure treatment, etc. Without being bound by theory, in the medium composition of the present invention, the nanofibers form a three-dimensional network that supports the cells and tissues, thereby maintaining the suspended state of the cells and tissues.

[0029] In this specification, nanofibers refer to fibers having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofibers used in the present invention is preferably 0.005 to 0.50 μm, more preferably 0.01 to 0.05 μm, and even more preferably 0.01 to 0.02 μm. If the average fiber diameter is less than 0.001 μm, the nanofibers may be too fine to obtain a suspending effect, and may not lead to improvement in the characteristics of the medium composition containing them.

[0030] The aspect ratio (L / D) of the nanofiber used in the present invention is obtained by average fiber length / average fiber diameter, and is usually 2 to 500, preferably 5 to 300, and more preferably 10 to 250. When the aspect ratio is less than 2, the nanofiber may lack dispersibility in the medium composition and may not have sufficient floating action. When the aspect ratio exceeds 500, it means that the fiber length becomes extremely large, and the viscosity of the composition may increase, which may cause problems in subculture operations such as medium replacement. In addition, the medium composition becomes less transparent to visible light, which leads to a decrease in transparency, making it difficult to observe cultured cells over time and may cause problems in cell evaluation using light absorption, fluorescence, luminescence, etc.

[0031] In this specification, the average fiber diameter (D) of nanofibers was determined as follows. First, a collodion support membrane manufactured by Oken Shoji Co., Ltd. was subjected to hydrophilization treatment for 3 minutes using an ion cleaner (JIC-410) manufactured by JEOL Ltd., and several drops of the nanofiber dispersion liquid to be evaluated (diluted with ultrapure water) were dropped onto it and dried at room temperature. This was observed with a transmission electron microscope (TEM, H-8000) (10,000 times) manufactured by Hitachi Ltd. at an accelerating voltage of 200 kV, and the obtained image was used to measure the fiber diameter of each of the samples (200 to 250 nanofibers), and the number average value was taken as the average fiber diameter (D).

[0032] The average fiber length (L) was determined as follows. The nanofiber dispersion to be evaluated was diluted with pure water to 100 ppm, and the nanofibers were uniformly dispersed using an ultrasonic cleaner. This nanofiber dispersion was cast onto a silicon wafer whose surface had been hydrophilized in advance using concentrated sulfuric acid, and dried at 110°C for 1 hour to obtain a sample. Using images of the obtained sample observed with a scanning electron microscope (SEM, JSM-7400F) (2,000x) manufactured by JEOL Ltd., the fiber length of each of the 150 to 250 nanofiber specimens was measured, and the number average value was determined as the average fiber length (L).

[0033] The nanofibers used in the present invention have the effect of uniformly dispersing the nanofibers in the liquid while maintaining the primary fiber diameter when mixed with a liquid medium, substantially retaining the cells and / or tissues without substantially increasing the viscosity of the liquid, and preventing their sedimentation. "Does not substantially increase the viscosity of the liquid" means that the viscosity of the liquid does not exceed 8 mPa·s. In this case, the viscosity of the liquid (i.e., the viscosity of the medium composition produced by the production method 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, there are no limitations on the chemical structure, molecular weight, physical properties, etc. of the nanofibers, so long as they exhibit the effect of uniformly suspending (preferably suspending and leaving) the cells and / or tissues without substantially increasing the viscosity of the liquid when dispersed in the liquid medium. The viscosity of the liquid containing the nanofibers can be measured by the method described in the Examples below, for example. Specifically, the viscosity can be evaluated using a tuning fork vibro viscometer (SV-1A, A&D Company Ltd.) at 25°C.

[0034] Examples of raw materials constituting the nanofibers include, but are not limited to, low molecular weight compounds and high molecular weight compounds. Preferable specific examples of low molecular weight compounds used in the present invention include, but are not limited to, amino acid derivatives such as L-isoleucine derivatives, L-valine derivatives, and L-lysine derivatives, cyclohexanediamine derivatives such as trans-1,2-diaminocyclohexanediamide derivatives, 5-aminoisophthalic acid derivatives, R-12-hydroxystearyl esters, and the like. Examples of low molecular weight gelling agents include bisphosphonic acid, 1,3,5-benzenetricarboximide, cis-1,3,5-cyclohexanetricarboxamide, 2,4-dibenzylidene-D-sorbitol, N-lauroyl-L-glutamic acid-α,γ-bis-n-butylamide, and calcium dehydroabietic acid.

[0035] Preferable specific examples of the polymer compound used in the present invention include, but are not limited to, polysaccharides and polypeptides.

[0036] The polysaccharide refers to a sugar polymer formed by polymerization of 10 or more monosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.) Polysaccharides include water-insoluble polysaccharides and water-soluble polysaccharides.

[0037] Examples of water-insoluble polysaccharides include, but are not limited to, celluloses such as cellulose and hemicellulose; chitins such as chitin and chitosan; and the like.

[0038] Examples of water-soluble polysaccharides include acidic polysaccharides having anionic functional groups. Examples of acidic polysaccharides having anionic functional groups include, but are not limited to, polysaccharides having uronic acid in the structure (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid); polysaccharides having sulfate or phosphoric acid in the structure, or polysaccharides having both structures. More specifically, examples include those composed of one or more of the group consisting of hyaluronic acid, gellan gum, deacylated gellan gum (DAG), rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid, and salts thereof. Examples of the salts include salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; salts of aluminum, zinc, copper, iron, and the like; ammonium salts; quaternary ammonium salts such as tetraethylammonium, tetrabutylammonium, methyltributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium, and choline; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, and chloroprocaine; and salts with amino acids such as glycine, alanine, and valine.

[0039] Examples of the polypeptide include polypeptides that form fibers in living organisms, specifically, collagen, elastin, myosin, keratin, amyloid, fibroin, actin, tubulin, etc., but are not limited to these.

[0040] The raw materials constituting the nanofibers used in the present invention include not only naturally occurring substances, but also substances produced by microorganisms, substances produced by genetic engineering, or substances artificially synthesized using enzymes or chemical reactions. The raw materials constituting the nanofibers used in the present invention are preferably naturally occurring substances (i.e., substances extracted from nature) or substances obtained by modifying these substances through chemical or enzymatic reactions.

[0041] In one embodiment, the polysaccharide is a water-insoluble polysaccharide. Preferred water-insoluble polysaccharides include cellulose and chitin such as chitin and chitosan. Considering the ability to reduce the viscosity of the medium composition and the ease of recovery of cells or tissues, cellulose and chitin are most preferred.

[0042] Cellulose is a natural polymeric compound in which D-glucopyranose, a six-membered ring of glucose, is linked by β-1,4 glucoside bonds. The raw materials can be, for example, plant-derived cellulose such as wood, bamboo, hemp, jute, kenaf, cotton, agricultural crops, and food waste, or cellulose produced by microorganisms or animals such as bacterial cellulose, Cladophora, Glaucocystis, Baronia, and Hoya cellulose. Plant-derived cellulose is made up of very fine fibers called microfibrils that are further bundled together to form a higher-order structure in stages, from fibrils to lamellae to fiber cells. In addition, bacterial cellulose is made up of cellulose microfibrils secreted from bacterial cells that form a fine mesh structure with the same thickness.

[0043] In the present invention, high-purity cellulose raw materials such as cotton and bacterial cellulose can be used as they are, but it is preferable to use cellulose derived from other plants after isolating and purifying them. Cellulose that is preferably used in the present invention includes cotton cellulose, bacterial cellulose, kraft pulp cellulose, microcrystalline cellulose, etc. In particular, kraft pulp cellulose is preferably used because it has a high flotation effect. .

[0044] Chitin refers to one or more carbohydrates selected from the group consisting of chitin and chitosan. The main sugar units constituting chitin and chitosan are N-acetylglucosamine and glucosamine, respectively. In general, chitin is a sugar that has a high content of N-acetylglucosamine and is poorly soluble in an acidic aqueous solution, and chitosan is a sugar that has a high content of glucosamine and is soluble in an acidic aqueous solution. In this specification, for convenience, chitin is a sugar whose N-acetylglucosamine content in the constituent sugars is 50% or more, and chitosan is a sugar whose N-acetylglucosamine content is less than 50%. From the viewpoint of achieving a high floating effect, the higher the N-acetylglucosamine content in the sugar units constituting chitin, the more preferable it is. The N-acetylglucosamine content in the sugar units constituting chitin is preferably 80% or more, more preferably 90% or more, even more preferably 98% or more, and most preferably 100%.

[0045] As the raw material of chitin, many biological resources such as shrimp, crab, insects, shellfish, and mushrooms can be used. The chitin used in the present invention may be chitin having an α-type crystal structure such as chitin derived from crab shells or shrimp shells, or chitin having a β-type crystal structure such as chitin derived from squid shells. The outer shells of crabs and shrimps are often treated as industrial waste, and are preferable as raw materials from the viewpoint of easy availability and effective use, but a deproteinization step and a deashing step are required to remove proteins, ash, and the like contained as impurities. Therefore, in the present invention, it is preferable to use purified chitin that has already been subjected to a dematrix treatment. Purified chitin is commercially available.

[0046] In one embodiment, the polysaccharide is a water-soluble polysaccharide. Preferred water-soluble polysaccharides include deacylated gellan gum, carrageenan, etc. From the viewpoint of achieving a high flotation effect, deacylated gellan gum is the most preferred.

[0047] Deacylated gellan gum is a linear polymeric polysaccharide having four sugar molecules as constituent units: 1-3 linked glucose, 1-4 linked glucuronic acid, 1-4 linked glucose, and 1-4 linked rhamnose, and is a polysaccharide represented by the following general formula (I), in which R1 and R2 are both 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 content of the acetyl group and the glyceryl group is preferably 10% or less, more preferably 1% or less.

[0048] [ka]

[0049] A method for producing gellan gum involves culturing a producing microorganism in a fermentation medium, recovering the mucus produced outside the cells using a conventional purification method, and powdering the recovered product after drying, pulverization, etc. Deacylated gellan gum can be produced by culturing a microorganism that produces gellan gum in a fermentation medium, and recovering the mucus produced outside the cells. In this case, the mucus is subjected to an alkali treatment to deacylate the glyceryl and acetyl groups bound to the 1-3-linked glucose residues, and then the product can be recovered. The deacylated gellan gum can be purified from the recovered mucus by, for example, liquid-liquid extraction. The method can be carried out by using, alone or in any combination in any order, fractional precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20 or the like, adsorption chromatography using activated carbon, silica gel, or the like, or thin layer chromatography for adsorption and desorption of active substances, or high performance liquid chromatography using a reversed phase column, etc. Examples of microorganisms that produce gellan gum include, but are not limited to, Sphingomonas elodea and microorganisms obtained by genetically modifying said microorganisms. In the case of deacylated gellan gum, commercially available products such as "KELCOGEL (registered trademark of CP Kelco) CG-LA" manufactured by Sansho Co., Ltd. and "KELCOGEL (registered trademark of CP Kelco)" manufactured by San-Ei Gen F.F.I., Inc. can be used.

[0050] The weight-average molecular weight of the polymer compound used in the present invention is preferably 1,000 to 50,000,000, more preferably 10,000 to 20,000,000, and even more preferably 100,000 to 10,000,000. For example, the molecular weight can be estimated from the polyethylene glycol or pullulan equivalent value measured by gel permeation chromatography (GPC) or the viscosity of an aqueous solution.

[0051] In the present invention, the above polymer compounds can be used in combination of two or more kinds (preferably two kinds). The type of combination of polymer compounds is not particularly limited as long as it forms nanofibers in the medium composition or disperses as nanofibers and can suspend (preferably suspend and leave) cells and / or tissues without substantially increasing the viscosity of the liquid medium, but preferably, the combination includes at least cellulose, chitin, collagen, or deacylated gellan gum. That is, suitable combinations of polymer compounds include cellulose, chitin, collagen, or deacylated gellan gum; and other polymer compounds (e.g., xanthan gum, alginic acid, carrageenan, diutan gum, methylcellulose, locust bean gum, or salts thereof).

[0052] [Preparation of nanofibers] The medium composition of the present invention contains nanofibers prepared from the above-mentioned raw materials. The method of preparing the nanofibers differs depending on whether a water-insoluble polymeric compound (e.g., water-insoluble polysaccharides such as cellulose and chitin) is used as the raw material or a water-soluble polymeric compound (e.g., water-soluble polysaccharides such as deacylated gellan gum) is used.

[0053] When the raw material for nanofibers is a water-insoluble polymeric compound (e.g., water-insoluble polysaccharides such as cellulose and chitin), nanofibers are usually obtained by pulverizing the raw material. There are no limitations on the pulverization method, but to refine the raw material to the fiber diameter and length described below that meet the objectives of the present invention, a method that can generate strong shearing force, such as a high-pressure homogenizer, grinder (stone mill), or media stirring mill such as a bead mill, is preferred.

[0054] Among these, it is preferable to use a high-pressure homogenizer for micronization, and it is desirable to use a wet grinding method such as that disclosed in, for example, JP-A-2005-270891 and Patent No. 5232976 for micronization (grinding). Specifically, the raw materials are pulverized by ejecting a dispersion liquid in which the raw materials are dispersed from a pair of nozzles at high pressure and colliding with each other, and this can be carried out by using, for example, a Starburst System (a high-pressure grinding device manufactured by Sugino Machine Co., Ltd.) or a Nanovater (a high-pressure grinding device manufactured by Yoshida Kikai Kogyo Co., Ltd.).

[0055] When the raw material is pulverized (pulverized) using the above-mentioned high-pressure homogenizer, the degree of pulverization or homogenization depends on the pressure at which the raw material is pumped into the ultra-high-pressure chamber of the high-pressure homogenizer, the number of times the raw material is passed through the ultra-high-pressure chamber (number of treatments), and the concentration of the raw material in the aqueous dispersion. The pressure (treatment pressure) is usually 50 to 250 MPa, and preferably 150 to 245 MPa. If the pumping pressure is less than 50 MPa, the nanofibers may not be sufficiently fine, and the expected effect of the fineness may not be obtained.

[0056] In addition, the concentration of the raw material in the aqueous dispersion during the micronization treatment is 0.1% by mass to 30% by mass, preferably 1% by mass to 10% by mass. If the concentration of the raw material in the aqueous dispersion is less than 0.1% by mass, the productivity is low, and if it is more than 30% by mass, the pulverization efficiency is low, and the desired nanofibers cannot be obtained. The number of times of micronization (pulverization) treatment is not particularly limited, and depends on the concentration of the raw material in the aqueous dispersion. When the concentration of the raw material is 0.1 to 1% by mass, the number of treatments is about 10 to 100 times, but when it is 1 to 10% by mass, about 10 to 1000 times are required. In addition, when the concentration is high and exceeds 30% by mass, several thousand treatments or more are required, and the viscosity increases to a level that makes handling difficult, which is unrealistic from an industrial point of view.

[0057] When the raw material for the nanofibers is a water-soluble polymeric compound (e.g., a water-soluble polysaccharide such as deacylated gellan gum), when the substance is added to the culture medium, the substance aggregates via metal cations in the culture medium and forms nanofibers in the culture medium, which then construct a three-dimensional network, resulting in the formation of nanofibers in which cells or tissues can be cultured in suspension.

[0058] The concentration of nanofibers in the medium composition of the present invention can be appropriately set so as to enable cells and / or tissues to be suspended (preferably suspended and left stationary) without substantially increasing the viscosity of the medium, and is typically within the range of 0.0001% to 1.0% (weight / volume), for example, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (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 cellulose nanofibers, it is sufficient to add them to the culture medium at typically 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), and even more preferably 0.01% to 0.05% (weight / volume). In the case of pulp cellulose nanofibers among cellulose nanofibers, the lower limit of the concentration in the medium is preferably 0.01% (weight / volume) or more, 0.015% (weight / volume) or more, 0.02% (weight / volume) or more, 0.025% (weight / volume) or more, or 0.03% (weight / volume) or more, from the viewpoint of expressing the suspension action and enabling suspension static culture. In the case of pulp cellulose nanofibers, the upper limit of the concentration in the medium is preferably 0.1% (weight / volume) or less, or 0.04% (weight / volume) or less, from the viewpoint of not substantially increasing the viscosity of the medium. In the case of microcrystalline cellulose nanofibers, the lower limit of the concentration in the medium is preferably 0.01% (weight / volume) or more, 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more from the viewpoint of expressing the floating action. In order to enable suspension static culture, the lower limit of the microcrystalline cellulose nanofiber concentration in the medium is preferably 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more. In addition, in the case of microcrystalline cellulose nanofibers, the upper limit of the concentration in the medium is preferably 0.1% (weight / volume) or less. In the case of chitin nanofibers, they are usually added to the medium at 0.0001% to 1.0% (weight / volume), for example, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), and most preferably 0.03% to 0.07% (weight / volume). From the viewpoint of expressing the suspension action, the lower limit of the chitin nanofiber concentration in the medium is preferably 0.0001% (weight / volume) or more, 0.0003% (weight / volume) or more, 0.0005% (weight / volume) or more, or 0.001% (weight / volume) or more. From the viewpoint of enabling suspension static culture, The lower limit of the chitin nanofiber concentration in the medium is preferably 0.03% (weight / volume) or more, and the upper limit of the chitin nanofiber concentration in the medium is preferably 0.1% (weight / volume) or less. Water-insoluble nanofibers such as cellulose nanofibers and chitin nanofibers do not generally substantially increase the viscosity of the medium composition at a concentration of 0.1% (weight / volume) or less. In the case of carrageenan, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), and most preferably 0.02% to 0.1% (weight / volume) may be added to the medium. From the viewpoint of expressing the floating action and enabling the suspension static culture, the lower limit of the carrageenan concentration in the medium is preferably 0.01% or more. The upper limit of the carrageenan concentration in the medium is preferably 0.1% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the medium, it is also preferable to set the upper limit of the carrageenan to 0.04% (weight / volume) or less. In the case of deacylated gellan gum, it may be added to the medium at 0.001% to 1.0% (weight / volume), for example, 0.005% to 1.0% (weight / volume), preferably 0.003% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), even more preferably 0.01 to 0.05% (weight / volume), and most preferably 0.01% to 0.02% (weight / volume). From the viewpoint of expression of the floating action, the lower limit of the deacylated gellan gum concentration in the medium is preferably 0.005% (weight / volume) or more, or 0.01% or more. From the viewpoint of enabling floating static culture, the lower limit of the deacylated gellan gum concentration in the medium is preferably 0.01% (weight / volume) or more. From the viewpoint of not substantially increasing the viscosity of the medium, the upper limit of the deacylated gellan gum concentration in the medium is 0.05% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the medium, it is also preferable to set the upper limit of the deacylated gellan gum to 0.04 (weight / volume) % or less.

[0059] [Combination of polysaccharides] In addition to the nanofibers, multiple types (preferably two types) of polysaccharides can be used in combination. The concentration of the polysaccharide can be appropriately set within a range in which the cells and / or tissues can be uniformly suspended (preferably suspended and left stationary) without substantially increasing the viscosity of the liquid medium. For example, when a combination of nanofibers and polysaccharides is used, the concentration of the nanofibers is exemplified as 0.005 to 0.1% (weight / volume), preferably 0.01 to 0.07% (weight / volume), and the concentration of the polysaccharide is exemplified as 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). Specific examples of combinations in the concentration range are as follows. Cellulose or chitin nanofiber: 0.005 to 0.1% (preferably 0.01 to 0.07%) (weight / volume) polysaccharide Xanthan gum: 0.1-0.4% (weight / volume) Sodium alginate: 0.1 to 0.4% (weight / volume) (preferably 0.0001 to 0.4% (weight / volume)) Locust bean gum: 0.1-0.4% (weight / volume) Methylcellulose: 0.1 to 0.4% (weight / volume) (preferably 0.2 to 0.4% (weight / volume)) Carrageenan: 0.05-0.1% (weight / volume) Diutan gum: 0.05 to 0.1% (weight / volume) Native Gellan Gum: 0.0001-0.4% (weight / volume)

[0060] The concentration can be calculated by the following formula. Concentration (%) = weight of nanofibers (g) / volume of medium composition (ml) × 100

[0061] [Metal cation] In one embodiment, the medium composition of the present invention contains a metal cation, for example, a divalent metal cation (calcium ion, magnesium ion, zinc ion, iron ion, copper ion, etc.), preferably a calcium ion. In particular, when the nanofibers contained in the medium composition of the present invention are composed of a water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum), the medium composition of the present invention preferably contains the above-mentioned metal cation. By containing the metal cation, the water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum) aggregates via the metal cation to form nanofibers in the medium composition, which then construct a three-dimensional network, resulting in the formation of nanofibers that can be cultured in a suspended state for cells or tissues.

[0062] [Culture medium] Examples of media contained in the medium composition of the present invention include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Minimum Essential Medium, RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex), HPGM (Cambrex), StemSpan H3000 (Stem Cell Technology), StemSpanSFEM (Stem Cell Technology), StemlineII (Sigma-Aldrich), QBSF-60 (Quality Biologicals), StemProhESCSFM (Invitrogen), mTeSR1 or 2 medium (Stem Cell Technology), Sf-900II (Invitrogen), Opti-Pro (Invitrogen), and the like.

[0063] When the cells and / or tissues are derived from plants, examples of the medium include basic media such as Murashige-Skoog (MS) medium, Linsmeier-Skoog (LS) medium, White medium, Gamborg B5 medium, Nitsche medium, Heller medium, and Morel medium that are commonly used in plant tissue culture, or modified media in which the components of these media are modified to optimal concentrations (e.g., the ammonia nitrogen concentration is halved), to which auxins and, if necessary, plant growth regulators (plant hormones) such as cytokinins are added at appropriate concentrations. These media can be further supplemented with caseinase, corn steep liquor, vitamins, and the like, if necessary. Examples of auxins include, but are not limited to, 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D). 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.

[0064] 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-mentioned medium depending on the purpose. When culturing cells and / or tissues derived from animals, 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. depending on the purpose. Other chemical or biological components may also be added in combination with one or more of them. Components that may be added to the culture medium for cells and / or tissues of animal origin include fetal bovine serum, human serum, horse 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-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-29), interleukin-31 (IL-29), interleukin-32 (IL-29), interleukin-33 (IL-29), interleukin-34 (IL-29), interleukin-35 (IL-29), interleukin-36 (IL-29), interleukin-37 (IL-29), interleukin-38 (IL-29), interleukin-39 (IL-39), interleukin-40 (IL-39), These include, but are not limited to, 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), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), and thrombopoietin (TPO).

[0065] Hormones added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-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, and the like. Examples of hormone-releasing hormones include, but are not limited to, 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, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalins.

[0066] 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), proteases, and the like. These include, but are not limited to, protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokines, Notch ligands (Delta1, etc.), Wnt proteins, angiopoietin-like proteins 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), and pleiotrophin. It is not possible. In addition, it is also possible to add cytokines or growth factors whose amino acid sequences have been artificially altered using recombinant gene technology, such as IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

[0067] Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, hyaluronic acid, alginate gel, various hydrogels, and cleaved fragments of these.

[0068] Examples of antibiotics that may be added to the medium include sulfa preparations, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexocillin, mecillinam, anginocillin, cephalosporin, and derivatives thereof. , oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultanpicillin, adinocilicin, These include the formaldehyde fodolate esters of sulbactam and sulbactam, tazobactam, aztreonam, sulfazetine, isosulfazetine, norcadicine, m-carboxyphenyl, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0069] [Method of producing medium composition] The medium composition of the present invention can be produced by mixing the nanofibers with a medium used for culturing cells and / or tissues at a concentration that allows the cells and / or tissues to be uniformly suspended (preferably suspended and stationary) without substantially increasing the viscosity of the liquid medium. The present invention also provides a method for producing such a medium composition of the present invention.

[0070] The nanofibers may be in the form of a solid formulation such as a powder, tablet, pill, capsule, or granule, a liquid such as a dispersion in an appropriate physiological aqueous solvent, or bound to a substrate or carrier. Additives used in formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose, and mannitol; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to the above, and can be freely selected by those skilled in the art as long as they are available. Sterilization methods are not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and filter sterilization.

[0071] In a preferred embodiment, the medium composition of the present invention is prepared by mixing a dispersion of the nanofibers in a physiological aqueous solvent with a liquid medium. The dispersion may be sterilized (autoclaved, gamma ray sterilized, etc.). Alternatively, the dispersion and a powdered medium may be mixed in water. The dispersion may be mixed with a liquid medium (aqueous solution of a medium) prepared by dissolving the nanofibers, and then sterilized for use. The dispersion and the liquid medium may be sterilized separately before mixing. Examples of aqueous solvents include, but are not limited to, water and dimethyl sulfoxide (DMSO). As the aqueous solvent, water is preferred. The aqueous solvent may contain an appropriate buffer or salt. The above-mentioned nanofiber dispersion is useful as a medium additive for preparing the medium composition of the present invention. The present invention also provides such a medium additive.

[0072] The mixing ratio of nanofiber dispersion:liquid medium (aqueous medium) is usually 1:99 to 99:1, preferably 10:90 to 90:10, and more preferably 20:80 to 80:20.

[0073] In addition, when the nanofibers are composed of a water-soluble polymer compound (e.g., a water-soluble polysaccharide such as deacylated gellan gum), instead of mixing the nanofibers with the medium, the water-soluble polymer compound (e.g., a water-soluble polysaccharide such as deacylated gellan gum) may be mixed with the medium to form the nanofibers in the medium, thereby producing the medium composition of the present invention. The form of the polymer compound may be a formulated solid such as a powder, tablet, pill, capsule, or granule, a liquid such as a solution or suspension dissolved in an appropriate solvent and dissolving agent, or a state bound to a substrate or carrier. Additives used in the formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose, and mannitol; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to those mentioned above, and can be freely selected as long as they are available to those skilled in the art. The polymer compound may be sterilized as necessary by any method, including, for example, radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and filter sterilization.

[0074] In a preferred embodiment, an aqueous solution (hereinafter referred to as medium additive 2) of a water-soluble polymeric compound (e.g., a water-soluble polysaccharide such as deacylated gellan gum) is used in the production method of the present invention. The aqueous solution can be obtained by dissolving a solid (e.g., powder) of the water-soluble polymeric compound in a physiological aqueous solvent. Examples of the aqueous solvent include, but are not limited to, water and dimethyl sulfoxide (DMSO). As the aqueous solvent, water is preferred.

[0075] The aqueous solvent may contain a suitable buffer or salt. The aqueous solvent may or may not contain a divalent metal cation, but in a preferred embodiment, does not contain a divalent metal cation. When the aqueous solvent does not contain a divalent metal cation, a water-soluble polymer compound (e.g., a water-soluble polysaccharide such as deacylated gellan gum) in the aqueous solution is unlikely to form nanofibers that can suspend and culture cells or tissues, and can be stably stored in a dissolved state in water.

[0076] The medium additives may further contain additives to enhance the effect of nanofibers or to reduce the concentration when used. 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, and methylcellulose.

[0077] The following is an example of a method for producing the medium composition of the present invention, but the present invention is not limited thereto. Nanofibers are added to ion-exchanged water or ultrapure water. Then, after stirring at room temperature until the whole is uniformly dispersed, it is sterilized (for example, by sterilization at 121°C for 20 minutes). The sterilization is performed by autoclave sterilization. While stirring (for example, with a homomixer) any medium used for static culture, the sterilized nanofiber dispersion is added to the medium and mixed uniformly with the medium. There are no particular limitations on the method for mixing the aqueous solution with the medium, and examples of the method include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, mechanical stirrer, homomixer, and homogenizer.

[0078] For example, when preparing a medium composition using cellulose nanofibers, cellulose nanofibers are added to ion-exchanged water or ultrapure water to a concentration of 0.0001% to 5.0% (weight / volume), preferably 0.001% to 1.0% (weight / volume), and more preferably 0.01% to 0.6% (weight / volume). Then, the mixture is stirred at room temperature until the entire mixture is homogenized, and then sterilized (for example, autoclave sterilization at 121°C for 20 minutes). For example, while stirring a liquid medium such as DMEM medium with a homogenizer or the like, the aqueous solution is added to the medium to a desired final concentration (for example, when the final concentration is 0.03%, the ratio of 0.6% aqueous solution:medium is 1:20), and mixed uniformly. Alternatively, the liquid medium such as DMEM medium is added to the aqueous solution with a pipette to a desired final concentration (for example, when the final concentration is 0.03%, the ratio of 0.6% aqueous solution:medium is 1:20), and mixed uniformly by pipetting. The method for mixing the aqueous dispersion with the medium is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, mechanical stirrer, homomixer, or homogenizer.

[0079] [Culture method] The present invention also provides a culture method for growing cells or tissues using the medium composition of the present invention; a method for recovering the resulting cells or tissues, for example by filtration, centrifugation or magnetic separation; and a method for producing spheres using the medium composition of the present invention.

[0080] The nanofibers used in the present invention exhibit the effect of suspending (preferably suspending and leaving) the cells and / or tissues in a liquid containing the nanofibers when the cells and / or tissues are cultured in vitro. This suspension effect makes it possible to increase and culture the cells and / or tissues per given volume compared to monolayer culture. In addition, when rotation or shaking is involved in conventional suspension culture methods, shear forces are applied to the cells and / or tissues, resulting in low proliferation rates and recovery rates of the cells and / or tissues, or in cases where the functions of the cells are impaired. However, by using a medium composition containing the nanofibers of the present invention, the cells and / or tissues can be uniformly dispersed without shaking or other operations, and the desired cells and / or tissues can be obtained easily and in large quantities without loss of cell function. Furthermore, when cells and / or tissues are cultured in suspension in a medium containing a conventional gel base material, it can be difficult to observe or recover the cells and / or tissues, or their functions can be impaired during recovery, but by using a medium composition containing the nanofibers of the present invention, cells and / or tissues can be cultured in suspension and observed and recovered without impairing their functions. Furthermore, while conventional media containing a gel base material are highly viscous and may be difficult to replace, the medium composition containing the nanofibers of the present invention has a low viscosity and can be easily replaced using a pipette, pump, etc.

[0081] The human-derived cells and / or tissues cultured by the method of the present invention can be transplanted into patients with a disease or disorder for the purpose of treatment. In this case, the type of disease or disorder to be treated, the pretreatment method, and the cell transplantation method are appropriately selected by the person concerned. The engraftment of the transplanted cells into the recipient, recovery from the disease or disorder, the presence or absence of side effects associated with the transplantation, and the effectiveness of the treatment can be appropriately tested and evaluated by general methods used in transplantation therapy.

[0082] Furthermore, since cells and / or tissues can be efficiently grown in the medium composition of the present invention, the medium composition can be used as a reagent for cell research. For example, the medium composition can be used to detect factors that regulate the differentiation and growth of cells and tissues. When clarifying the target factor, the number and type of cells, changes in cell surface differentiation markers and expressed genes when cells are cultured in the presence of a target factor are analyzed. By using the medium composition of the present invention, the number of cells to be analyzed can be efficiently amplified and efficiently recovered. The culture conditions, culture device, type of medium, type of nanofiber of the present invention, content of nanofiber, type of additive, content of additive, culture period, culture temperature, etc., when clarifying the target factor, are appropriately selected by the person concerned from the ranges described in this specification. The cells that have proliferated or appeared by culture can be observed using a microscope that is standard in the field. In this case, the cultured cells may be stained with a specific antibody. The expressed genes that have changed due to the target factor can be detected by extracting RNA (ribonucleic acid) from the cultured cells and using Northern blotting, RT-PCR, etc. Furthermore, the cell surface differentiation markers can be detected by ELISA or flow cytometry using a specific antibody, and the effect of the target factor on differentiation and proliferation can be observed.

[0083] Furthermore, when the medium composition of the present invention is used, cells and / or tissues can be efficiently grown, and therefore the culture method of the present invention is excellent as a method for growing cells and / or tissues or a method for promoting the growth of cells and / or tissues. When cells and / or tissues are cultured using the medium composition of the present invention, the cells and / or tissues do not adhere to the culture vessel, are not unevenly distributed only on the bottom surface of the culture vessel, and are dispersed with a three-dimensional spread, promoting the growth. In particular, when chitin nanofibers are used as nanofibers, cells adhere to the chitin nanofibers and grow strongly using them as a scaffold, and as a result, the grown cells, cell masses (spheres, etc.) and / or tissues are connected on the nanofibers in a grape-like cluster shape. For this growth-promoting effect, it is sufficient that the medium composition contains nanofibers at a concentration sufficient to suspend the cells and / or tissues (i.e., to avoid adhesion of the cells and / or tissues to the culture vessel), and it is not essential that the cells and / or tissues can be suspended and left still (i.e., the cells and / or tissues are uniformly dispersed and suspended in the liquid medium composition without external pressure, vibration, shaking, rotation, etc.). For example, in the case of chitin nanofibers, if the concentration is 0.0001% (weight / volume) or more, which is sufficient to exhibit the floating action, the proliferation-promoting effect is exhibited even at a concentration below 0.03% (weight / volume) that enables stable floating static culture (e.g., 0.025% (weight / volume) or less, 0.02% (weight / volume) or less). In the case of microcrystalline cellulose nanofibers, if the concentration is 0.01% (weight / volume) or more, which is sufficient to exhibit the floating action, the proliferation-promoting effect is exhibited even at a concentration below 0.03% (weight / volume) that enables stable floating static culture (e.g., 0.025% (weight / volume) or less, 0.02% (weight / volume) or less). In the case of deacylated gellan gum, as long as the concentration is 0.005% (weight / volume) or more, which is sufficient to exhibit a floating effect, the proliferation-promoting effect can be achieved even at a concentration below 0.01% (weight / volume), which enables stable suspended static culture (e.g., 0.009% (weight / volume) or less, 0.008% (weight / volume) or less).

[0084] Among nanofibers, chitin nanofibers in particular have excellent cell proliferation promoting effects.

[0085] In the culture method of the present invention, both suspension cells and adherent cells can be used. Adherent cells are cells that require a scaffold for growth and proliferation. Suspension cells are cells that do not require a scaffold for growth and proliferation. In the culture method of the present invention, adherent cells are preferably used. When adherent cells are used in the method of the present invention, the adherent cells do not adhere to the bottom surface of the culture vessel, and are not unevenly distributed only on the bottom surface of the culture vessel, but are dispersed with a three-dimensional spread and proliferate in a state attached to nanofibers or in a state of spheres. In particular, when chitin nanofibers are used as the nanofibers, the cells adhere to the chitin nanofibers and proliferate strongly using them as a scaffold, and as a result, the proliferated cells and cell masses (spheres, etc.) are connected on the nanofibers in a grape-like cluster shape. This makes it possible to culture the adherent cells in suspension. As a result, the proliferation of the adherent cells is promoted more than when they are cultured in a state attached to the bottom surface of the culture vessel. In addition, the proliferation rate is higher than when they are cultured in a state attached to the bottom surface of the culture vessel. Adherent cells can be cultured at any density.

[0086] In the culture method of the present invention, since the adherent cells can be cultured in suspension by the culture method of the present invention, it is possible to subculture the adherent cells by simply adding a fresh medium composition of the present invention to the cultured product after the culture, or by adding all or a part of the cultured product to a fresh medium composition of the present invention, without the need for a cell detachment operation from the culture vessel. The present invention also provides such a subculture method for adherent cells. Therefore, by using the subculture method of the present invention, the adherent cells can be subcultured without the cell detachment operation from the culture vessel. In addition, by using the subculture method of the present invention, the culture scale of the adherent cells can be expanded without the cell detachment operation from the culture vessel. The detachment operation of the cells from the culture vessel can be performed by treatment with a chelating agent (e.g., EDTA) and / or a proteolytic enzyme (e.g., trypsin, collagenase). The subculture method of the present invention is advantageous for subculture of adherent cells that are highly sensitive to the cell detachment operation from the culture vessel (e.g., adherent cells whose viability decreases due to the detachment operation, and adherent cells whose characteristics are easily changed due to the detachment operation). Examples of adherent cells that are highly sensitive to cell detachment from the culture vessel include, but are not limited to, human pluripotent stem cells; human progenitor cells; primary cells prepared from tissues such as hepatic cells, renal cells, chondrocytes, vascular cells, and adipocytes; and cells that produce biopharmaceuticals (pharmaceutical proteins), such as MDCK cells, HEK293 cells, and CHO cells.

[0087] The medium composition of the present invention allows adherent cells to be cultured at a high density and allows cells and / or tissues to grow efficiently, so that the culture method of the present invention is useful for producing useful substances by in vitro cell culture. Cells producing a useful substance are subjected to suspension culture in the medium composition of the present invention, and the useful substance is isolated from the culture, thereby obtaining the useful substance. Examples of useful substances include, but are not limited to, antibodies, enzymes (urokinase, etc.), hormones (insulin, etc.), cytokines (interferon, interleukin, tumor necrosis factor, colony stimulating factor, growth factor, etc.), vaccine antigens, and other physiologically active substances (proteins, peptides, etc.). Cells producing useful substances include non-transformed cells such as skin cells, chondrocytes, liver cells, pancreatic cells, and kidney cells, and transformed cells into which a gene encoding a useful substance or a gene involved in the biosynthesis of a useful substance has been introduced. The cells producing a useful substance may be adherent cells or suspension cells, but are preferably adherent cells. The cells producing a useful substance are preferably cells that secrete the useful substance outside the cells. Specific examples of cells that produce useful substances include, but are not limited to, HEK293, CHO-K1, BHK-21, MDCK, Vero, HepG2, MCF-7, and the like, which are introduced with a gene encoding a useful substance or a gene involved in the biosynthesis of a useful substance. Cells used for producing useful substances such as recombinant proteins are well known to those skilled in the art, and these cells can be used in the method of the present invention. When expanding the culture scale, the above-mentioned subculture method of the present invention may be used to add a fresh medium composition of the present invention to the culture after culture without detaching the cells from the culture vessel, or all or a part of the culture after culture may be added to a fresh medium composition of the present invention. When isolating a useful substance from a culture, it is necessary to remove the cells from the culture, but since the viscosity of the medium composition of the present invention is not substantially increased by the addition of nanofibers and the cells are suspended in the medium composition, the cells can be removed by a simple method such as centrifugation or filtration. In addition, the nanofibers in the medium composition can also be removed by a simple method such as centrifugation or filtration.Methods for isolating useful substances from cultures are well known to those skilled in the art, and biochemical methods for separating and purifying physiologically active substances, such as chromatography (e.g., ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and other chromatography), can be applied.

[0088] When culturing cells and / or tissues using the culture method of the present invention, a method generally used for culturing cells is Culture can be performed using culture equipment such as petri dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, tubes, trays, culture bags, roller bottles, etc. The materials of these culture equipment are 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. In addition, these plastics may be subjected to various surface treatments (e.g., plasma treatment, corona treatment, etc.). Furthermore, these culture equipment may be coated in advance with an extracellular matrix, cell adhesion molecules, etc. Examples of such coating materials include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin, hyaluronic acid, superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, alginic acid gel, hydrogel, and further cleavage fragments thereof. These coating materials may also be used in which the amino acid sequence has been artificially modified by gene recombination technology. Coating materials for inhibiting adhesion of cells and / or tissues to cultureware may also be used. Such coating materials include, but are not limited to, silicone, poly(2-hydroxymethyl methacrylate), poly(2-methacryloyloxyethyl phosphorylcholine), and the like.

[0089] The culture of cells and / or tissues can also be performed using a bioreactor or an automatic culture device that can perform cell seeding, medium replacement, cell image acquisition, and cultured cell recovery automatically in a closed environment under mechanical control and that can culture at high density while controlling pH, temperature, oxygen concentration, etc. Methods for replenishing new medium during culture using these devices and supplying required substances to cells and / or tissues without excess or deficiency include fed-batch culture, continuous culture, and perfusion culture, and any of these methods can be used in the culture method of the present invention.

[0090] 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 examples thereof include, but are not limited to, a state in which the cells and / or tissues are dispersed alone in a medium composition, a state in which the cells and / or tissues are attached to the surface of a carrier, a state in which the cells and / or tissues are embedded inside a carrier, a state in which a plurality of cells are assembled to form a cell cluster (sphere), or a state in which two or more types of cells are assembled to form a cell cluster (sphere), and more preferably a state in which the cells and / or tissues are attached to the surface of a carrier, a state in which the cells and / or tissues are embedded inside a carrier, a state in which a plurality of cells are assembled to form a cell cluster (sphere), or a state in which two or more types of cells are assembled to form a cell cluster (sphere), and even more preferably a state in which the cells and / or tissues are attached to the surface of a carrier, a state in which a plurality of cells are assembled to form a cell cluster (sphere), or a state in which two or more types of cells are assembled to form a cell cluster (sphere). Of these states, the state in which cell masses (spheres) are formed is the most preferable state for culturing using the method of the present invention, since cell-cell interactions and cell structures similar to those in the in vivo environment are reconstructed, allowing cells to be cultured while maintaining their functions over the long term, and cells can be recovered relatively easily.

[0091] Examples of carriers for supporting cells and / or tissues on the surface include microcarriers made of various polymers, glass beads, ceramic beads, etc. Examples of such polymers include vinyl resins, urethane resins, epoxy resins, polystyrene, polymethyl methacrylate polyesters, polyamides, polyimides, silicone resins, phenolic resins, meracyl resins, etc. Examples of the material that can be used include amine resin, urea resin, aniline resin, ionomer resin, polycarbonate, collagen, dextran, gelatin, cellulose, alginate, and mixtures thereof. The carrier may be coated with a compound that enhances cell adhesion or enhances the release of substances from cells. Examples of such coating materials include poly(monostearoylglyceride succinic acid), poly-D,L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, alginic acid gel, various hydrogels, and further cleaved fragments thereof. In this case, two or more coating materials may be combined. Furthermore, one or more polysaccharides such as guar gum, tamarind gum, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, etc., can be mixed with the medium used for culturing the carrier having cells and / or tissues on its surface. The carrier may also 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, Cytodex1 (registered trademark), Cytodex 3 (registered trademark), Cytoline1 (registered trademark), C Cytoline2 (registered trademark), Cytopore1 (registered trademark), Cytopore2 (registered trademark), (GE Healthcare Life Sciences), Examples include iosilon (registered trademark) (NUNC), Cultispher-G (registered trademark), Cultispher-S (registered trademark) (Thermo SCIENTIFIC), HILLEXCT (registered trademark), ProNectinF-COATED (registered trademark), and HILLEXII (registered trademark) (SoloHill Engineering). The carrier may be sterilized as necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and dry heat sterilization. The method for culturing animal cells using the carrier is not particularly limited, and a culture method using a normal fluidized bed culture tank or a packed bed culture tank can be used. In this case, the carrier having cells and / or tissues supported on its surface can be uniformly dispersed without shaking or other operations by using the medium composition containing the nanofibers of the present invention, so that the target cells and / or tissues can be cultured without losing cell function. The cells and / or tissues cultured by this method can be recovered by performing centrifugation or filtration while still supported on the carrier after the culture. In this case, centrifugation or filtration may be performed after adding the liquid medium used. For example, the gravitational acceleration (G) during centrifugation is 100 to 400 G, and the pore size of the filter used during filtration is 10 μm to 100 μm, but is not limited thereto. In addition, if a magnetic material such as ferrite is incorporated 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.

[0092] When cells and / or tissues are embedded inside a carrier, materials 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 their copolymers, polyvinyl methyl ether, polypropylene oxide, polyethylene oxide, polyvinyl chloride ... Examples of suitable bioactive materials include temperature-sensitive polymers such as partial acetylated vinyl alcohol, and hydrogels such as polyacrylamide, polyvinyl alcohol, methylcellulose, nitrocellulose, cellulose butyrate, polyethylene oxide, and poly(2-hydroxyethylmethacrylate) / polycaprolactone. In addition, it is also possible to prepare a carrier for embedding cells by using two or more of these polymers. Furthermore, the carrier may have a physiologically active substance other than these polymers. Examples of such physiologically active substances include cell growth factors, differentiation-inducing factors, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins, and extracellular matrices, and it is also possible to contain a plurality of these. Furthermore, one or more thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, and methylcellulose can be mixed with the medium used for culturing the carrier in which cells and / or tissues are embedded.

[0093] The method of embedding cells and / or tissues in these carriers is not particularly limited, but for example, a mixture of cells and the polymer may be sucked into a syringe and dropped into a medium through a needle of about 25G to 19G, or a micropipette may be used. The size of the beaded carrier formed here is determined by the shape of the tip of the instrument used to drop the mixture of cells and the polymer, and is preferably several tens of μm to several thousand μm, more preferably 100 μm to 2000 μm. The number of cells that can be cultured on the beaded carrier is not particularly limited, but may be freely selected according to the bead size. For example, in the case of a beaded carrier with a diameter of about 2000 μm, up to 5 million cells can be embedded in the beaded carrier of this size. In addition, the cells may be dispersed individually in the carrier, or a cell mass formed by a collection of multiple cells may be formed. In this case, the carrier in which the cells and / or tissues are embedded can be uniformly dispersed without stirring or other operations by using the medium composition containing the nanofibers of the present invention, so that 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 in a state in which they are embedded in the carrier after the culture. In this case, centrifugation or filtration may be performed after adding the liquid medium used. For example, the gravitational acceleration (G) during centrifugation is 100 to 400 G, and the pore size of the filter used in the filtration is 10 μm to 100 μm, but 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, heat, enzymes, etc.

[0094] The method for forming cell aggregates (spheres) is not particularly limited, and can be appropriately selected by those skilled in the art. Examples include a method using a vessel with a cell non-adhesive surface, the hanging drop method, the rotation culture method, the three-dimensional scaffold method, the centrifugation method, and a method using aggregation by an electric field or a magnetic field. For example, in the method using a vessel with a cell non-adhesive surface, the target cells can be cultured in a culture vessel that has been subjected to a surface treatment that inhibits cell adhesion, and spheres can be formed. When using this cell non-adhesive culture vessel, first, the target cells are collected, and then a cell suspension is prepared, and the cells are seeded in the culture vessel and cultured. After culturing for about a week, the cells spontaneously form spheres. The cell non-adhesive surface used at this time can be a surface of a culture vessel such as a petri dish that is commonly used and coated with a substance that inhibits cell adhesion. Such materials include, but are not limited to, agarose, agar, poly-HEMA (poly-(2-hydroxyethyl methacrylate)), copolymers of 2-methacryloyloxyethyl phosphorylcholine and other monomers (e.g., butyl methacrylate, etc.), and the like, as long as they are not cytotoxic.

[0095] In addition, as a method for forming cell aggregates (spheres), NATURE BIOTECHNOLOGY, VOL. 28, NO. 4, APRIL 2010, 361-366, N Methods described in ATURE 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, a component that accelerates the formation of spheres or promotes their maintenance can be added to the medium used for culturing to form spheres. Examples of components having such effects include dimethyl sulfoxide, superoxide dismutase, ceruloplasmin, catalase, peroxidase, L-ascorbic acid, L-ascorbic acid phosphate, tocopherol, flavonoids, uric acid, bilirubin, selenium-containing compounds, transferrin, unsaturated fatty acids, albumin, theophylline, forskolin, glucagon, dibutyluryl cAMP, ROCK inhibitors such as Y27632, Fasudil (HA1077), H-1152, and Wf-536. Examples of selenium-containing compounds include sodium selenite, sodium selenate, dimethyl selenide, hydrogen selenide, selenomethionine, Se-methylselenocysteine, selenocystathionine, and selenocysteine. , selenohomocysteine, adenosine-5'-phosphoselenate, and Se-adenosylselenomethionine. In order to obtain a uniform cell aggregate of the desired size, a plurality of depressions of the same diameter as the desired cell aggregate can be introduced on the non-cell-adherent culture vessel used. If these depressions are in contact with each other or are within the range of the diameter of the desired cell aggregate, when cells are seeded, the seeded cells will reliably form cell aggregates of a size corresponding to the volume in the depressions without forming cell aggregates between the depressions, and a cell aggregate population of uniform size can be obtained. In this case, the shape of the depression is preferably hemispherical or conical.

[0096] Alternatively, spheres can be formed on a support having cell adhesive properties, such as collagen, polyrotaxane, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), and hydrogel. In addition, spheres can be formed by co-culturing with feeder cells. Any adhesive cells can be used as feeder cells for promoting sphere formation, but feeder cells suitable for various cells are preferable. For example, when forming spheres of cells derived from liver or cartilage, examples of suitable cell types for the feeder cells include, but are not limited to, COS-1 cells and vascular endothelial cells. Furthermore, spheres can be formed using a culture composition containing the nanofibers of the present invention. In this case, the nanofibers can be added to a medium used for sphere formation so that the concentration of the nanofibers is a concentration that allows suspension culture of cells (preferably suspension static culture). For example, the nanofibers can be added to a medium used for sphere formation so that the concentration of the nanofibers is usually 0.0001% to 1.0% (weight / volume), 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). The spheres are prepared by uniformly dispersing the target cells in a medium containing the nanofibers and culturing them for 3 to 10 days. The spheres prepared here can be collected by centrifugation or filtration. For example, the gravitational acceleration (G) during centrifugation is 100 to 400 G, and the pore size of the filter used for filtration is 10 μm to 100 μm, but is not limited to these. In addition, the cultured spheres can be collected by magnetic force using magnetic microparticles whose surface is coated with an antibody that specifically binds to the target cells. Examples of such magnetic microparticles include Dynabeads (manufactured by Veritas), MACS Microbeads (manufactured by Miltenyi Biotec), and BioMag (manufactured by Techno Chemical).

[0097] The size of the sphere varies depending on the cell type and culture period and is not particularly limited, but if it is spherical or ellipsoidal in shape, it has a diameter of 20 μm to 1000 μm, preferably 40 μm to 500 μm, and more preferably 50 μm to 300 μm. Such spheres can retain their proliferation ability for a period of 10 days or more, preferably 13 days or more, and more preferably 30 days or more when left in static culture, but can also retain their proliferation ability for an essentially indefinite period by periodically mechanically dividing the spheres during static culture or by further subjecting the spheres to single-cell disaggregation and aggregation. The culture vessel used for culturing spheres is not particularly limited as long as it is generally capable of culturing animal cells, and examples include flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc. The medium used for static culture of spheres can contain cell adhesion factors, examples of which include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Two or more of these cell adhesion factors can also be added in combination. Furthermore, the medium used for culture of spheres can be further mixed with a thickener such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, or methylcellulose. By using the medium composition containing the nanofibers of the present invention, the nanofibers can be uniformly dispersed in the culture solution without shaking or other operations, so that the target cells and / or tissues can be cultured as spheres without loss of cell function. The spheres cultured in a stationary state by this method can be collected by centrifugation or filtration after the culture. In this case, centrifugation or filtration may be performed after adding the liquid medium used. For example, the gravitational acceleration (G) during centrifugation is 100 to 400 G, and the pore size of the filter used in the filtration process is 10 μm to 100 μm, but is not limited thereto. In addition, the cultured spheres can be collected by magnetic force using magnetic fine particles whose surface is coated with an antibody that specifically binds to the target cells. Examples of such magnetic fine particles include Dynabeads (manufactured by Veritas), MACS microbeads (manufactured by Miltenyi Biotec), and BioMag (manufactured by Techno Chemical). The collected spheres can be further dissolved using various chelating agents, heat, filters, enzymes, etc., to disperse them into single cells.

[0098] As a method for statically culturing cells and / or tissues derived from plants, callus, which is a mass of undifferentiated plant cells, can be cultured. Callus induction can be performed by a method known for each plant species used. For example, the surface of a portion of tissue (e.g., roots, stems, leaf segments, seeds, growing points, embryos, pollen, etc.) of a differentiated plant body is sterilized with 70% alcohol or 1% sodium hypochlorite solution, etc., as necessary, and then a tissue piece of appropriate size (e.g., a root segment of about 1 to about 5 mm square) is cut out with a scalpel or the like, and the tissue piece is seeded on a callus induction medium that has been sterilized in advance by aseptic manipulation using a clean bench or the like, and aseptically cultured under appropriate conditions. The callus induced here may be immediately subjected to liquid culture for mass proliferation, or it may be maintained as a seed strain by subculturing it in a subculture medium. Subculture may be performed using either a liquid medium or a solid medium. The amount of plant cell masses inoculated when starting static culture using the medium composition of the present invention 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., but for example, when culturing plant cell masses such as callus, they are inoculated into the medium composition of the present invention so that the wet weight of the cell masses relative to the medium composition of the present invention is 4 to 8 (weight / volume (w / v))%, preferably 5 to 7 (w / v)%. The particle size of the plant cell masses during culture is 3 mm to 40 mm, preferably 3 mm to 20 mm, more preferably 5 mm to 15 mm. Here, the term "particle size" refers to, for example, the diameter of a plant cell mass when it is spherical, and refers to the major axis of a plant cell mass when it is ellipsoidal, and similarly refers to the maximum length that the plant cell mass can assume for other shapes.

[0099] The temperature for culturing cells and / or tissues is usually 25 to 39°C, preferably 33 to 39°C, for animal cells. The CO2 concentration in the culture atmosphere is usually 4 to 10% by volume, preferably 4 to 6% by volume. The culture period is usually 3 to 35 days, but can be freely set according to the purpose of the culture. The culture temperature for plant cells is usually 20 to 30°C, and if light is required, the cells can be cultured under an illumination condition of 2000 to 8000 lux. The culture period is usually 3 to 70 days, but can be freely set according to the purpose of the culture.

[0100] When culturing cells and / or tissues by the method of the present invention, the cells and / or tissues prepared separately may be added to the culture composition of the present invention and mixed so as to be uniformly dispersed. The mixing method is not particularly limited, and examples include manual mixing such as pipetting, and mixing using equipment such as a stirrer, a vortex mixer, a microplate mixer, and a shaker. After mixing, the culture solution may be left in a stationary state, or the culture solution may be rotated, shaken, or stirred as necessary. The number of rotations and frequency may be appropriately set according to the purpose of the person skilled in the art. In addition, when it becomes necessary to replace the medium composition during the stationary culture period, the cells and / or tissues and the medium composition may be separated by centrifugation or filtration, and then new medium composition may be added to the cells and / or tissues. Alternatively, the cells and / or tissues may be appropriately concentrated by centrifugation or filtration, and then new medium composition may be added to this concentrated solution. For example, the gravitational acceleration (G) during centrifugation is 100 to 400 G, and the pore size of the filter used for filtration is 10 μm to 100 μm, but is not limited thereto. In addition, the cultured cells and / or tissues can be separated by magnetic force using magnetic microparticles whose surface is coated with an antibody that specifically binds to the target cells. Examples of such magnetic microparticles include Dynabeads (manufactured by Veritas), MACS Microbeads (manufactured by Miltenyi Biotec), and BioMag (manufactured by Techno Chemical). The exchange of these medium compositions can also be performed by a bioreactor or an automatic culture device that can be performed in a closed environment under mechanical control.

[0101] [Method of preserving or transporting cells or tissues] The present invention also provides a preservation method and transportation method for preserving cells or tissues using the medium composition of the present invention. In the preservation or transportation method of the present invention, the medium composition of the present invention is used, so that cells or tissues can be preserved or transported in a floating state (preferably in a floating stationary state).

[0102] Cells and tissues to be preserved or transported include those mentioned above as cells and tissues that can be used for culture using the medium composition of the present invention.

[0103] In addition to the above-mentioned components, the medium composition of the present invention used for storage or transportation may contain various components that have a cell life-prolonging effect when cells or tissues are stored in an unfrozen state. Such components include sugars (excluding polysaccharides) (e.g., monosaccharides, disaccharides), antioxidants (e.g., SOD, vitamin E, or glutathione), hydrophilic polymers (e.g., polyvinylpyrrolidone), and chiral substances. Examples of such additives include sugar alcohols (eg, mannitol, sorbitol), and glycerol.

[0104] In the preservation or transportation method of the present invention, the desired cells or tissues are dispersed in the medium composition of the present invention and then placed in a sealable container. Examples of the container include, but are not limited to, flasks, plastic bags, Teflon (registered trademark) bags, tubes, culture bags, etc. During preservation or transportation, there is a risk of leakage of the contents or contamination by bacteria and the like from the outside world. To avoid contamination, containers containing dispersions of cells or tissues in the media compositions of the present invention are preferably sealed.

[0105] The temperature during storage or transportation is not particularly limited as long as the survival of the cells or tissues is maintained, but is usually 37°C or lower. A lower temperature can avoid a decrease in survival of the cells or tissues during storage or transportation, but the medium composition of the present invention is usually stored or transported at a temperature above the melting point so that the cells or tissues do not freeze. Therefore, the temperature during storage or transportation is usually maintained at -5 to 42°C, preferably 1 to 37°C, more preferably 4 to 32°C, and even more preferably 18 to 30°C.

[0106] In order to enable storage or transportation of cells or tissues in a suspended state, the temperature during storage or transportation is preferably a temperature at which the medium composition of the present invention enables cells or tissues to be suspended in a suspended state. The temperature at which cells or tissues can be suspended in a suspended state can be appropriately set depending on the type of raw material constituting the nanofiber.

[0107] In one embodiment, carrageenan (preferably κ-carrageenan) is used as a raw material for constituting the nanofibers contained in the medium composition of the present invention used in the storage or transportation method of the present invention. The medium composition of the present invention containing nanofibers composed of carrageenan has a floating effect at 25° C. or less, but loses the floating effect at 37° C. Therefore, the desired cells or tissues can be stored or transported in a floating, stationary state at 25° C. or less (preferably 0 to 25° C.), and after completion of storage or transportation, the temperature is raised to 37° C. or more (e.g., 37 to 40° C., preferably 37° C.) to cause the floating cells or tissues to settle, thereby making it possible to easily recover the cells or tissues.

[0108] The storage or transportation period is not particularly limited as long as the cells or tissues can be maintained in a viable state in the medium composition of the present invention, but is usually at least 1 hour and no more than 10 days, preferably 1 to 8 days, and more preferably 1 to 3 days. During the storage or transportation period, the cells or tissues are preferably maintained in a suspended, stationary state in the medium composition of the present invention.

[0109] By using the preservation or transportation method of the present invention, cells or tissues can be maintained in a floating state, thereby avoiding damage to the cells or tissues due to detachment from the plate due to vibration during transportation or aggregation of cells or tissues that come into contact with each other due to sedimentation, and allowing the cells or tissues to be preserved and transported while maintaining their original functions. EXAMPLES

[0110] The present invention will be explained in more detail below by specifically describing examples of the medium composition of the present invention. The materials, amounts used, ratios, treatment contents and treatment procedures shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited to the specific examples shown below.

[0111] Reference Example 1 Viscosity measurement and cell suspension test of medium containing high-temperature heat-treated deacylated gellan gum Preparation of medium containing deacylated gellan gum and viscosity measurement Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was suspended in pure water to a concentration of 0.4% (w / v), and then heated and stirred at 90°C to dissolve. The aqueous solution was allowed to cool to room temperature while stirring, and sterilized in an autoclave at 121°C for 20 minutes. 50 mL of double-concentration DMEM / F-12 medium (Aldrich) and 47.5 mL of sterile water were placed in a 300 mL tall beaker, and 2.5 mL of 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, a medium composition with a final concentration of 0.02% was prepared. A medium composition was prepared by adding an aqueous solution of deacylated gellan gum to give a concentration of 0.01, 0.03, or 0.05% (w / v). The viscosity of this medium composition was measured at 37°C for 5 minutes at 100 rpm using an E-type viscometer (Toki Sangyo Co., Ltd., Viscometer TVE-22L, standard rotor 1°34'×R24).

[0112] Cell suspension test in medium containing deacylated gellan gum Human cervical cancer cell line HeLa (DS Pharma Biomedical) was suspended in EMEM medium (WAKO) containing 10% (v / v) fetal bovine serum at 250,000 cells / mL, and 10 mL of this suspension was seeded on EZ SPHERE (Asahi Glass Co., Ltd.) and cultured for 3 days in a CO2 incubator (5% CO2). 10 mL of the suspension of spheres (diameter 100-200 μm) obtained here was centrifuged (200 G, 5 minutes) to settle the spheres, and 1.0 mL of 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 HeLa cell sphere suspension was added. The cell mass was dispersed by tapping, incubated at 37 °C, and the dispersed state of the cells was visually observed after 1 hour.

[0113] [Table 1]

[0114] Comparative Example Preparation of methylcellulose and collagen-containing medium Preparation of methylcellulose-containing medium 100 mL of DMEM / F-12 medium (Aldrich) was placed in a 200 mL eggplant flask, and 0.1 g of methylcellulose (M0387, Aldrich) was added. The mixture was stirred while cooling in an ice bath to dissolve the methylcellulose. This solution was used to prepare medium compositions with methylcellulose aqueous solutions added to final concentrations of 0.1, 0.3, 0.6, and 1.0% (w / v). Preparation of collagen-containing medium 6.5 mL of 0.3% Cell Matrix Type IA (Nitta Gelatin) was mixed with 1 mL of 10x DMEM / F-12 medium (Aldrich), 1 mL of reconstitution buffer (Nitta Gelatin), and 1.5 mL of pure water, and the mixture was stirred on ice to prepare a 0.2% collagen-containing medium. Similarly, medium compositions were prepared to which collagen was added to give final concentrations of 0.01, 0.05, 0.1, and 0.2% (w / v). The HeLa cell sphere suspension test and viscosity measurement were also performed for the medium composition prepared above, in the same manner as for the medium containing deacylated gellan gum, except that the viscosity of 1.0% (w / v) methylcellulose was measured at 50 rpm, which was within the measurement range of the device.

[0115] [Table 2]

[0116] [Table 3]

[0117] Reference test example In the following reference test examples, the CO2 concentration (%) in the CO2 incubator is shown as the volume % of CO2 in the atmosphere. PBS means phosphate buffered saline (Sigma-Aldrich Japan), and FBS means fetal bovine serum (Biological Industries). (w / v) means weight per volume.

[0118] Reference test example 1: Cell proliferation test when single cells are dispersed Deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolved by stirring while heating at 90°C, and the resulting aqueous solution was sterilized by autoclaving at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w / v) to IMDM medium (Gibco Co., Ltd.) containing 10% (v / v) fetal bovine serum and 10 ng / mL thrombopoietin (WAKO Co., Ltd.). Subsequently, human leukemia cell line UT7 / TPO was seeded in the medium composition containing the above-mentioned deacylated gellan gum to a concentration of 20,000 cells / mL, and then dispensed into the wells of a 6-well flat-bottom microplate (Corning Co., Ltd.) at 5 mL per well. Similarly, human cervical cancer cell line HeLa (DS Pharma Biomedical) was seeded in a medium composition containing 10% (v / v) fetal bovine serum (WAKO) and 0.015% (w / v) deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.) to give a cell density of 20,000 cells / mL, and then dispensed into wells of a 6-well flat-bottom microplate (Corning) at 5 mL per well. These cell suspensions were cultured in a stationary state in a CO2 incubator (5% CO2) for 3 days. After that, a portion of the culture medium was collected, and the same amount of trypan blue staining solution (Invitrogen) was added, and the number of live cells was measured using a hemocytometer (Elma Sales Co., Ltd.).

[0119] As a result, it was confirmed that UT7 / TPO cells and HeLa cells can be uniformly cultured in a suspension state by using the above medium composition, and that they proliferate in the medium composition. The cell counts of UT7 / TPO cells and HeLa cells after 3 days of static suspension culture are shown in Table 4.

[0120] [Table 4]

[0121] Reference test example 2: Cell proliferation test when culturing cell line-derived spheres Human hepatoma cell line HepG2 (DS Pharma Biomedical) was suspended in DMEM medium (WAKO) containing 10% (v / v) fetal bovine serum at 250,000 cells / mL, 10 mL of this suspension was seeded on EZ SPHERE (Asahi Glass Co., Ltd.) and cultured for 7 days in a CO2 incubator (5% CO2). Similarly, human cervical cancer cell line HeLa (DS Pharma Biomedical) was suspended in EMEM medium (WAKO) containing 10% (v / v) fetal bovine serum at 250,000 cells / mL, 10 mL of this suspension was seeded on EZ SPHERE (Asahi Glass Co., Ltd.) and cultured for 7 days in a CO2 incubator (5% CO2). 2.5 mL of the suspension of spheres (diameter 100 to 200 μm) of each cell line obtained here was centrifuged (200 G, 5 minutes) to settle the spheres and remove the supernatant. Subsequently, 10 mL of the above medium was added to the spheres (about 800 pieces) to suspend them, and then transferred to a flat-bottom tube (manufactured by BM Equipment 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 Sansho Co., Ltd.) was added to the above medium, and transferred to a flat-bottom tube (manufactured by BM Equipment Co., Ltd.). The medium composition containing 0.015% (w / v) deacylated gellan gum was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolving it by stirring while heating at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes and then added at a 1 / 20 dilution to DMEM medium containing 10% (v / v) fetal bovine serum.

[0122] After the sphere suspension was statically cultured in a CO2 incubator (5% CO2) at 37°C for 3 days, a double volume of medium was added and centrifuged (200G, 5 minutes) to precipitate the spheres, and the supernatant was removed. A portion of the spheres was then taken and their shape was observed under an optical microscope (OLYMPUS, CK30-F100). The collected spheres were then washed once with 10mL of PBS, after which 1mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (WAKO) was added and incubated at 37°C for 5 minutes. After adding 9mL of the medium, the cells were collected by centrifugation (200G, 5 minutes). The same amount of trypan blue staining solution (Invitrogen) was added to a portion of the 2mL cell suspension obtained here, and the number of live and dead cells was measured using a hemocytometer (Elma Sales Co., Ltd.).

[0123] As a result, it was confirmed that the spheres of HepG2 cells and HeLa cells can be cultured in a suspended state by using the above medium composition, and that the cells grow efficiently in the medium composition. Moreover, it was confirmed that the medium composition has a lower rate of dead cells when the cells are grown compared to the existing medium, and has an excellent effect of promoting cell growth. At this time, the spheres cultured in the existing medium settled on the bottom of the culture vessel. Furthermore, when the shape of the cultured spheres was observed with an optical microscope, no association between the spheres was observed in the medium composition, whereas association between the spheres was observed in the existing medium. Table 5 shows the relative cell numbers of HepG2 cells and HeLa cells when the cell number when cultured in a medium not containing deacylated gellan gum is set to 1. Table 6 shows the relative cell death rate when the cell death rate (number of dead cells / number of live cells) when cultured in a medium not containing deacylated gellan gum is set to 1. Figures 1 and 2 show the floating state of HepG2 cell and HeLa cell spheres when cultured in the medium composition. Figure 3 shows the shape of the cultured HeLa cell spheres.

[0124] [Table 5]

[0125] [Table 6]

[0126] Reference Test Example 3: Cell proliferation test when culturing cell lines attached to microcarriers Microcarrier Cytodex® 1 (GE Healthcare Li (manufactured by Fe Sciences) was suspended in PBS at 0.02 g / mL and left to stand overnight. After that, the supernatant was discarded and the microcarrier was washed twice with fresh PBS. Then, the microcarrier was suspended again in PBS at 0.02 g / mL and sterilized by autoclaving at 121°C for 20 minutes. The microcarrier was then 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 0.02 g / mL. Using the microcarrier suspension, 20 mL of DMEM medium (containing 10% (v / v) fetal bovine serum) containing 120 mg of Cytodex (registered trademark) 1 and 4,000,000 HepG2 cells was prepared, and the cell suspension was cultured in a beaker previously treated with a silicon coating agent (manufactured by Asahi Techno Glass Co., Ltd.) at 37°C for 6 hours while stirring with a stirrer (100 rpm). Here, it was confirmed with a microscope that the HepG2 cells were attached to the microcarrier. Subsequently, the microcarriers to which the cells had been attached were washed twice with DMEM medium containing 10% (v / v) fetal bovine serum, and suspended in 3 mL of the same medium.

[0127] 300 μL of the above microcarrier suspension was added to 20 mL of DMEM medium containing 10% (v / v) fetal bovine serum or to a medium composition prepared by adding 0.015% (w / v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) to this medium, and cultured at 37°C for 3 days. At this time, the culture medium not containing deacylated gellan gum was cultured while stirring (100 rpm) with a stirrer. After the culture, the adhesion state of the cells on the microcarrier was confirmed under a microscope, and the microcarrier was precipitated by centrifugation (200G, 5 minutes). After washing the microcarrier with 10 mL of PBS, 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO Co., Ltd.) was added and incubated at 37°C for 5 minutes. Further, 9 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added, and then the microcarriers were removed using a cell strainer with a mesh size of 70 μm (manufactured by BD Falcon). The cells were collected from the filtrate obtained here by centrifugation (200 G, 5 minutes). The cells were suspended in 500 μL of medium, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the suspension, and the number of live cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.). As a result, the culture medium containing no deacylated gellan gum contained 123,000 cells, while the culture medium 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 has a superior effect of promoting cell proliferation compared to existing medium, even when cell culture is performed using a microcarrier. The adhesion state of HepG2 cells when microcarrier culture is performed using the medium composition containing the structure of the specific compound for 3 days is shown in FIG. 4.

[0128] Reference Test Example 4: Cell suspension test using cell line-derived spheres Xanthan gum (KELTROL CG, Sansho 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) for xanthan gum were prepared. In addition, an aqueous solution containing 0.2% (w / v) κ-carrageenan (GENUGEL WR-80-J, Sansho Co., Ltd.) and 0.2% (w / v) locust bean gum (GENUGUM RL-200-J, Sansho Co., Ltd.) was heated at 90°C to prepare DMEM / F-12 medium (Sigma) compositions containing 0.03, 0.04, and 0.05% (w / v) κ-carrageenan and locust bean gum.

[0129] HeLa cell spheres were prepared using the same method as in Reference Test Example 2, and several tens of spheres were added to 1 mL of the medium prepared above, and the medium was left 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 could be precipitated and collected by adding an equal amount of medium to the cell suspension and then centrifuging (300 to 400G, 5 minutes). The floating state of the HeLa cell spheres when cultured in the medium compositions is shown in Figure 5. The viscosity measured in the same manner as in Analysis Example 1 is shown in Tables 7 and 8.

[0130] [Table 7]

[0131] [Table 8]

[0132] Reference Test Example 5: Cell suspension test using a filtered medium composition A DMEM / F-12 medium composition containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.) was prepared using the same method as in Reference Test Example 2. Subsequently, 1 mL of this medium composition was filtered using 70 μm and 40 μm filters (BD Falcon), 30 μm and 20 μm filters (AS ONE), 10 μm filters (Partec), 5 μm, 1.2 μm, 0.45 μm, and 0.2 μm filters (Sartorius Stedim Japan). About several tens of HepG2 cell spheres prepared using the same method as in Reference Test Example 2 were added to the above filtrate, and the mixture was left to stand at 37° C. for 1 hour, and the floating state of the sphere cells was visually observed. As a result, the HepG2 cell spheres were maintained in a floating state in the medium composition that had passed through a filter of 10 μm or more, but in the medium composition that had passed through a filter of 5 μm or less. Furthermore, it was confirmed that the HepG2 cell spheres in the suspended state could be precipitated and collected by centrifugation at 300 G for 5 minutes at room temperature, or by adding an equal volume of medium and then centrifuging at 200 G for 5 minutes at room temperature.

[0133] Reference Test Example 6: Sphere Formation Test A composition of EMEM medium (manufactured by WAKO) containing 0.01% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared using the same method as in Reference Test Example 2. HeLa cells were then added to a concentration of 1000 cells / mL, and then dispensed into a 24-well plate (manufactured by Corning Co., Ltd.). After suspension culture on this plate at 37°C for 9 days, the formation of spheres was confirmed under a microscope. Furthermore, the sphere cells were precipitated 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 Co., Ltd.) was added and incubated at 37°C for 5 minutes. 100μL of EMEM medium containing 10% (v / v) fetal bovine serum was added to 100μL of the cell suspension obtained here, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the cell suspension, and the number of live cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.). As a result, it was confirmed that the HeLa cells had increased to 170,000 cells / mL. The HeLa cell sphere formed in the medium composition is shown in Figure 6.

[0134] Reference test example 7: Optical microscope observation of structure Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) 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 double-concentration DMEM / F-12 medium (Aldrich Corporation) was placed in a 300 mL tall beaker, and 5 mL of deacylated gellan gum aqueous solution was added while stirring with a magnetic stirrer at room temperature. The mixture was stirred for 5 minutes to prepare a medium composition with a final concentration of deacylated gellan gum of 0.02%. The medium composition was further stirred for 5 minutes with a homomixer (3000 rpm). The prepared medium composition was observed with an optical microscope (KEYENCE Corporation, BIOREVO BZ-9000). The observed structure is shown in FIG. 7.

[0135] Reference Test Example 8: Preparation by mixing and heating powder medium and DAG 20 mg of deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) and 1.58 g of DMEM / F-12 medium (Life Technologies) were placed in a 200 mL Erlenmeyer flask, and 100 mL of pure water was poured in. The flask was sterilized in an autoclave 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, GE Healthcare Life Sciences) were added to the prepared medium, and beads were then added. The dispersion state of the seeds was visually confirmed. A floating dispersion state was evaluated as ◯, a partial settling / dispersion state as △, and a settling state as ×. The results are shown in Table 9.

[0136] [Table 9]

[0137] Reference Test Example 9: Preparation of medium composition containing polysaccharides Xanthan gum (KELTROL CG, Sansho Co., Ltd.) was added at a concentration of 0.5% (w / v). After suspending in pure water to a concentration of 100%, the mixture was dissolved by stirring while heating at 90° C. Similarly, 0.5% (w / v) aqueous solutions were prepared for sodium alginate (Duck Alginate NSPM, Food Chemifa), locust bean gum (GENUGUM RL-200-J, Sansho Co., Ltd.), κ-carrageenan (GENUGEL WR-80-J, Sansho Co., Ltd.), and diutan gum (KELCO CRETE DG-F, Sansho Co., Ltd.). This solution was mixed with 0.2 or 0.1% (w / v) deacylated gellan gum solution and 10x DMEM / F-12 medium, and heated at 80°C for 30 minutes. After cooling to room temperature, 7.5% sodium bicarbonate solution was added to prepare DMEM / F-12 medium compositions containing deacylated gellan gum at final concentrations of 0.01 and 0.02% (w / v) and other polysaccharides at final concentrations of 0.1, 0.2, 0.3, and 0.4% (w / v). In addition, a medium containing deacylated gellan gum was prepared in the same manner as above, and then methylcellulose (cP400, manufactured by WAKO Co., Ltd.) powder was added. The methylcellulose was dissolved by stirring in an ice bath to prepare DMEM / F-12 medium compositions containing deacylated gellan gum at final concentrations of 0.01 and 0.02% (w / v) and other methylcelluloses at final concentrations of 0.1, 0.2, 0.3, and 0.4% (w / v).

[0138] 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 state was evaluated as ○ for floating dispersion, △ for partial sedimentation / dispersion, and × for sedimentation. The results are shown in Table 10.

[0139] [Table 10]

[0140] Reference Test Example 10: Viscosity measurement of medium composition containing polysaccharides DMEM / F-12 media containing deacylated gellan gum and other polysaccharides at final concentrations of 0.005 and 0.01% (w / v) were prepared in the same manner as in the polysaccharide mixture system of Reference Test Example 9. 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 state of each medium composition and the viscosity measured in the same manner as in Analysis Example 1 are shown in Tables 11 to 16.

[0141] [Table 11]

[0142] [Table 12]

[0143] [Table 13]

[0144] [Table 14]

[0145] [Table 15]

[0146] [Table 16]

[0147] Reference Test Example 11: Preparation of medium composition with different divalent metal cation concentrations Using DMEM / F-12 (D9785, manufactured by Aldrich) that does not contain calcium chloride, magnesium sulfate, or magnesium chloride, a DMEM / F-12 medium composition containing 0.02% (w / v) deacylated gellan gum was prepared in the same manner as in Reference Test Example 8. In addition, a DMEM / F-12 medium composition was prepared to which calcium chloride, magnesium sulfate, or magnesium chloride was added so that the final concentration was the specified amount of DMEM / F-12 medium. Based on the specified composition of DMEM / F-12 medium, the final concentrations were 0.116 g / L calcium chloride, 0.049 g / L magnesium sulfate, and 0.061 g / L magnesium chloride, respectively. The medium composition was prepared by adding dextran beads Cytodex1 (GE Healthcare Re Life Sciences) was added, and after 2 days, the dispersion of the beads was visually confirmed. The state of floating dispersion was evaluated as ○, the state of partial sedimentation / dispersion as △, and the state of sedimentation as ×. The results were Shown in Table 17.

[0148] [Table 17]

[0149] Reference Test Example 12: Preparation of medium composition with post-addition of divalent metal cations A salt solution was prepared by dissolving 0.1% (w / v) deacylated gellan gum solution, 5x concentration DMEM / F-12 medium (not containing calcium chloride, magnesium sulfate, or magnesium chloride, D9785, manufactured by Aldrich), 1167 mg calcium chloride, 489 mg magnesium sulfate, and 287 mg magnesium chloride in 300 mL of pure water. The deacylated gellan gum aqueous solution and pure water were placed in a 200 mL tall beaker, and the solution was stirred at 200 rpm using an anchor-type stirring blade. Liquid A, which was a mixture of medium liquid and water, was added and stirred for 10 minutes. Next, the salt solution was added, and 1.6 mL of 7.5% sodium bicarbonate aqueous solution was added to prepare a DMEM / F-12 medium composition containing deacylated gellan gum at a final concentration of 0.02%. The amount of each liquid mixed is shown in the table. Four hours after preparation, the dispersion evaluation of polystyrene beads and Cytodex1 was performed for six medium compositions. The results are shown in Tables 18 and 19.

[0150] [Table 18]

[0151] [Table 19]

[0152] Reference Test Example 13: Preparation of various medium compositions A 0.1% (w / v) deacylated gellan gum solution and a high-concentration medium solution were prepared. The high-concentration medium solutions were 10x MEM (M0268, Aldrich), RPMI-1640 (R6504, Aldrich), and 5x DMEM (medium compatible with high-pressure sterilization, Nissui). The 0.1% (w / v) deacylated gellan gum solution, each high-concentration medium, and pure water for adjusting the concentration were mixed and heated at 80°C for 30 minutes. After cooling to room temperature, a 7.5% aqueous sodium bicarbonate solution was added to prepare medium compositions containing deacylated gellan gum at final concentrations of 0.01, 0.02, and 0.03% (w / v). The six medium compositions thus prepared were evaluated based on whether the polystyrene beads and dextran beads, Cytodex1, were suspended or dispersed, whether they were partially sedimented or dispersed, and whether they were completely sedimented. The results are shown in Tables 20 and 21.

[0153] [Table 20]

[0154] [Table 21]

[0155] Reference Test Example 14: Measurement of particle size distribution of medium composition containing deacylated gellan gum A DMEM / F-12 medium composition containing 0.038% (w / v) deacylated gellan gum was prepared in the same manner as in Reference Example 1. The medium was mixed at 3000 rpm and 6000 rpm using a homogenizer. The medium composition was prepared by stirring at 1000 rpm for 1 minute. The particle size distribution of the medium composition was measured using a Multisizer 4 (a precision particle size distribution measuring device based on the Coulter principle) manufactured by Beckman Coulter, Inc., and the median diameter (d50) of the volumetric particle size distribution was calculated. The results are shown in Table 22.

[0156] [Table 22]

[0157] Reference Test Example 15: 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 and 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 heated under reflux for 5 hours. After that, it was allowed to cool to room temperature while stirring for 12 hours. The white suspension obtained by this was poured into 99% ethanol (500 mL). The resulting flocculent white solid was filtered and dried to obtain a light brown solid (0.4 g) as the phosphoric acid oxide of deacylated gellan gum. The introduction of phosphate groups was confirmed by Fourier transform infrared spectroscopy (IR-Prestage21, Shimadzu Corporation) (1700 cm-1; P-OH, 1296 cm-1, 1265 cm-1; P=O). The light brown solid was decomposed using a microwave heating decomposition apparatus (ETHOS TC, Milestone General) and then analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES) (SPS 5520, SII Nano Technology). Therefore, the phosphorus atom content was measured and found to be 3.5 wt% (n=2).

[0158] Reference Test Example 16: Preparation of medium composition containing phosphorylated deacylated gellan gum An arbitrary amount of phosphorylated deacylated gellan gum (30 mg) and 1.56 g of DMEM / F-12 medium (Life Technologies) were placed in a 200 mL Erlenmeyer flask, and 100 mL of pure water was poured in. The flask was sterilized by autoclaving 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 (GE Healthcare Biosciences) were added to the prepared medium, and the dispersion state of the beads was visually confirmed. The dispersion state of the beads was observed at a phosphorylated deacylated gellan gum concentration of 0.03% (w / v).

[0159] Reference Test Example 17: Preparation of medium composition containing deacylated gellan gum The deacylated gellan gum aqueous solution and the medium solution were mixed 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%, and the dispersion state of polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) was evaluated. The results are shown in Tables 23 and 24. By leaving the mixture to stand for one day or more, the styrene beads were dispersed under all conditions.

[0160] [Table 23]

[0161] [Table 24]

[0162] Reference Test Example 18: Preparation of medium composition using a filter Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) 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. Furthermore, 100 mL of this aqueous solution was filtered through a polyethersulfone membrane filter (Corning) with a pore size of 0.22 μm. Subsequently, the filtrate was mixed with 2- to 4-fold concentration DMEM / F-12 medium (Sigma-Aldrich), and then shaken for 1 hour in a mild mixer (SI-24, Taitec) to prepare medium compositions containing deacylated gellan gum at a final concentration of 0.01 or 0.015% (w / v). (For example, 25 mL of 0.02% (w / v) deacylated gellan gum aqueous solution and 2-fold concentration DMEM / F-12 medium were mixed to prepare 50 mL of 0.01% (w / v) deacylated gellan gum medium composition.) HepG2 cell spheres were prepared using the same method as in Reference Test Example 2, and several tens of spheres were added to 1 mL of the medium prepared above, and the medium was left to stand at 37°C, and the floating state of the sphere cells was visually observed after 1 hour and overnight. As a result, it was confirmed that the HepG2 cell spheres were maintained in a floating state in all of the above medium compositions. After adding twice the volume of medium, the cell suspension was centrifuged (500G It was confirmed that HepG2 cell spheres settled and the cells could be recovered by incubation for 10 min (5 min) in all medium compositions. The dispersion state of the spheres after one night was visually confirmed and rated as ○ for floating dispersion, △ for partial settlement / dispersion, and × for settlement. The results are shown in Table 25.

[0163] [Table 25]

[0164] Reference Test Example 19: Cell proliferation test when culturing cell line-derived spheres Human fetal kidney cell line HEK293 (DS Pharma Biomedical) was suspended in EMEM medium (WAKO) containing 10% (v / v) fetal bovine serum at 250,000 cells / mL, and 10 mL of this suspension was seeded on EZ SPHERE (Asahi Glass Co., Ltd.) and cultured for 2 days in a CO2 incubator (5% CO2). 10 mL of the suspension of HEK293 cell spheres (diameter 100-200 μm) obtained here was centrifuged (200 G, 5 minutes) to settle the spheres, the supernatant was removed, and the spheres were suspended in 1 mL. Subsequently, 10 mL of the above medium was added to 200 μL of this sphere suspension (cell number approximately 200,000 cells) and then transferred to a flat-bottom tube (BM Equipment 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, Sansho Co., Ltd.) was added to the above medium, and transferred to a flat-bottom tube (BM Instruments Co., Ltd.). The medium composition to which 0.015% (w / v) deacylated gellan gum was added was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.) in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolving the gellan gum by stirring while heating at 90°C, sterilizing the aqueous solution in an autoclave at 121°C for 20 minutes, and then adding the solution at a 1 / 20 dilution to an EMEM medium containing 10% (v / v) fetal bovine serum.

[0165] The above-mentioned sphere suspension was cultured in a CO2 incubator (5% CO2) at 37°C for 5 days, and then a double volume of medium was added and centrifuged (500G, 5 minutes) to precipitate the spheres, and the supernatant was removed. The collected spheres were then washed once with 10mL of PBS, after which 1mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added and incubated at 37°C for 5 minutes. After adding 9mL of the above-mentioned medium, cells were collected by centrifugation (500G, 5 minutes). After adding the same amount of trypan blue staining solution (manufactured by Invitrogen) to a portion of the 2mL cell suspension obtained here, the number of live cells and dead cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.). As a control, a medium composition not containing deacylated gellan gum was prepared and the same experiment was performed.

[0166] As a result, it was confirmed that HEK293 cell spheres can be cultured in a suspended state by using the medium composition, and that the cells grow efficiently in the medium composition. Moreover, it was confirmed that the medium composition had a lower rate of dead cells during cell proliferation than a medium composition not containing deacylated gellan gum, and was therefore superior in promoting cell proliferation. At this time, 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.

[0167] [Table 26]

[0168] [Table 27]

[0169] Reference Test Example 20: Cell proliferation test when culturing insect cells 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. % (w / v) of deacylated gellan gum was added to prepare a medium composition. Sf9 cells (Gibco) derived from Spodoptera frugiperda were then seeded in the medium composition containing the deacylated gellan gum to give a concentration of 100,000 cells / mL, and then dispensed into wells of a 24-well flat-bottom microplate (Corning) at 1 mL per well. These cell suspensions were statically cultured in an incubator at 25°C for 5 days. After that, a portion of the culture solution was collected, and the same amount of trypan blue staining solution (Invitrogen) was added, after which the number of live cells was measured using a hemocytometer (Elma Sales Co., Ltd.). As a control, a medium composition not containing deacylated gellan gum was prepared, and the same experiment was performed.

[0170] As a result, it was confirmed that the Sf9 cells can be uniformly cultured in a suspended state by using the medium composition, and that the cells grow in the medium composition. Furthermore, it was confirmed that the medium composition has a superior effect of promoting cell growth when the cells are grown, compared to a medium composition that does not contain deacylated gellan gum. The cell count of Sf9 cells after 5 days of suspension static culture is shown in Table 28.

[0171] [Table 28]

[0172] Reference Test Example 21: Cell proliferation test when culturing CD34 positive cells Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolved by stirring while heating at 90°C, and the resulting aqueous solution was sterilized by autoclaving at 121°C for 20 minutes. A medium composition was prepared by adding deacylated gellan gum to StemSpan SFEM medium (StemCell Technologies) at a final concentration of 0.015% (w / v), thrombopoietin (WAKO) at 20 ng / mL, and stem cell factor (SCF, WAKO) at 100 ng / mL using this solution. Subsequently, CD34 positive cells (Lonza) derived from human umbilical cord blood were seeded in the medium composition to which the deacylated gellan gum was added to a concentration of 10,000 cells / mL, and then dispensed into the wells of a 24-well flat-bottom microplate (Corning Corporation) at 1 mL per well. These cell suspensions were statically cultured in a CO2 incubator (5% CO2) at 37°C for 7 days. After that, a portion of the culture medium was collected, and the same amount of trypan blue staining solution (Invitrogen) was added, and the number of live cells was measured using a hemocytometer (Elma Sales Co., Ltd.). In addition, three times the volume of medium was added to the remaining culture medium, and all the cells were precipitated by centrifugation (500G, 5 minutes). As a control, a medium composition not containing deacylated gellan gum was prepared, and a similar experiment was performed.

[0173] As a result, it was confirmed that CD34 positive cells can be uniformly cultured in a suspended state by using the medium composition, and that they proliferate in the medium composition. Furthermore, it was confirmed that the medium composition has a cell proliferation promoting effect equal to or greater than that of existing media that do not contain deacylated gellan gum. It was also confirmed that the cells can be recovered by settling by centrifugation. The relative cell numbers of cells proliferated from CD34 positive cells after 7 days of suspension static culture, when the number of cells cultured in a medium that does not contain deacylated gellan gum is set to 1, are shown in Table 29.

[0174] [Table 29]

[0175] Reference Test Example 22: Sphere formation test A composition of DMEM medium (manufactured by WAKO) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared using the same method as in Reference Test Example 2. HepG2 cells were then added to the medium so that the cell concentration was 15,000 cells / mL, and 1 mL of the medium was dispensed into a 24-well plate (manufactured by Corning Co., Ltd.). After the plate was subjected to suspension static culture at 37°C for 7 days, the formation of spheres was confirmed under a microscope. Furthermore, the sphere cells were precipitated by centrifugation at 400G for 5 minutes, washed once with 5 mL of PBS, and then 100 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO Co., Ltd.) was added and the mixture was incubated at 37°C for 5 minutes. 100μL of DMEM medium containing 10% (v / v) fetal bovine serum was added to 100μL of the cell suspension obtained here, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of the cell suspension, and the number of live cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.). As a result, it was confirmed that HepG2 cells formed spheres in the medium composition, and the cell number increased to 80800 cells / mL. The HepG2 cell spheres formed in the medium composition are shown in Figure 8.

[0176] Reference Test Example 23: Cell suspension test using cell line-derived spheres Diutan gum (KELKO-CRETE DG, Sansho 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. Using this aqueous solution, a DMEM / F-12 medium composition with a final diutan gum concentration of 0.1% (w / v) was prepared. In addition, an aqueous solution containing 0.5% (w / v) native gellan gum (Kelkogel HT, San-Ei Gen F.F.I. Co., Ltd.) was heated at 90°C to prepare a DMEM / F-12 medium (Sigma) composition containing 0.05 and 0.1% (w / v) native gellan gum.

[0177] HeLa cell spheres were prepared using the same method as in Reference Test Example 2, and several tens of spheres were added to 1 mL of the medium prepared above, and the medium was left 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 were precipitated by centrifuging (200G, 5 minutes) this cell suspension containing 0.1% (w / v) diutan gum, and the cells could be recovered.

[0178] Reference Test Example 24: Cell suspension test 1 using magnetic beads with cell adhesion ability GEM (Global Eukaryotic Microcarrier, registered trademark, GL Sciences, Inc.) coated with laminin or fibronectin 500μL of the suspension was dispensed into 1.5mL micro test tubes (Eppendorf) and the GEMs were collected from the suspension using a magnetic stand (TA4899N12, Tamagawa Seiki Co., Ltd.) to remove the solvent. The GEMs were washed twice with 500μL of DMEM medium (WAKO) containing 10% (v / v) fetal bovine serum, and then suspended in 500μL of the same medium. 50μL of this suspension was dispensed per well into a low cell adhesion plate, Sumilon Celltite Plate 24F (Sumitomo Bakelite Co., Ltd.). Then, HepG2 cells prepared separately were added to a concentration of 250,000 cells / mL, and the final volume was adjusted to 500μL / well with the same medium. After manually stirring the cell suspension, the plate was left to stand overnight in a CO2 incubator (5% CO2). After confirming cell adhesion on the GEMs under a microscope, the cell suspension was transferred to a 1.5 mL microtest tube (Eppendorf), and the cell-attached GEMs were accumulated using the magnetic stand described above, followed by removal of the supernatant.

[0179] A composition of DMEM medium (manufactured by WAKO) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared using the same method as in Reference Test Example 2. 1 mL of this medium composition or the above medium without deacylated gellan gum was added to the HepG2 cell-attached GEM (laminin or fibronectin coating) prepared above, suspended, and then transferred to a Sumilon Celltite Plate 24F. The plate was then left to stand in a CO2 incubator (5% CO2) for 6 days, after which the cell culture solution was transferred to a 1.5 mL micro test tube (manufactured by Eppendorf Co., Ltd.), and the cell-attached GEM was accumulated by gently pipetting on the magnetic stand, and the supernatant was removed. 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 for 10 minutes at 37° C. 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added to 200 μL of the cell suspension obtained here, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a portion of the cell suspension, after which the number of live cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.).

[0180] As a result, it was confirmed that the GEM with HepG2 cells attached thereto can be cultured in a suspended state by using the medium composition, and that the cells grow efficiently in the medium composition. Moreover, the medium composition has a higher growth rate than existing media that do not contain deacylated gellan gum. It was confirmed that the medium composition has an excellent effect of promoting cell proliferation. It was also confirmed that the HepG2 cell-attached GEMs can be accumulated from the medium composition by using magnetic force, and that the HepG2 cells can be recovered from the GEMs. The number of cells when HepG2 cells were cultured on GEM for 6 days in a medium containing or not containing deacylated gellan gum is shown in Table 30. In addition, the floating state when laminin-coated GEM to which HepG2 cells were attached and cultured in the medium composition is shown in Figure 9.

[0181] [Table 30]

[0182] Reference Test Example 25: Cell suspension test 2 using magnetic beads with cell adhesion ability As in Reference Test Example 24, a fibronectin-coated GEM (registered trademark, Global Eukaryotic Microcarrier, GL Sciences) 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 Reference Test Example 24 to prepare GEMs to which mesenchymal stem cells were attached.

[0183] Using the same method as in Reference Test Example 2, a composition of MF-Medium (registered trademark) mesenchymal stem cell proliferation medium (manufactured by Toyobo Co., Ltd.) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) was prepared. 1 mL of this medium composition or the above medium not containing deacylated gellan gum was added to the mesenchymal stem cell-attached GEM (fibronectin coating) prepared above, suspended, and then transferred to a Sumilon Celltite Plate 24F. Subsequently, this plate was left to stand in a CO2 incubator (5% CO2) for 4 days, after which the cell culture solution was transferred to a 1.5 mL micro test tube (manufactured by Eppendorf Co., Ltd.), and the cell-attached GEM was accumulated while gently pipetting on the above-mentioned magnetic stand, and the supernatant was removed. 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 for 10 minutes at 37° C. 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added to 200 μL of the cell suspension obtained here, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a portion of the cell suspension, after which the number of live cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.).

[0184] As a result, it was confirmed that the GEMs with mesenchymal stem cells attached thereto can be cultured in a suspended state by using the medium composition, and that the cells can grow efficiently in the medium composition. Moreover, it was confirmed that the medium composition has a superior effect of promoting cell growth compared to existing media that do not contain deacylated gellan gum. It was also confirmed that the mesenchymal stem cell-attached GEMs can be accumulated from the medium composition by using magnetic force, and that the mesenchymal stem cells can be recovered from the GEMs. Table 31 shows the cell numbers when mesenchymal stem cells were cultured on GEM for 4 days in media containing or not containing deacylated gellan gum.

[0185] [Table 31]

[0186] Reference Test Example 26: 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 sodium alginate solution (manufactured by PG Research Co., Ltd.) so as to have a concentration of 400,000 cells / mL, and human recombinant laminin 511 (manufactured by Veritas Co., Ltd.) was further added to have a concentration of 5 μg / mL to prepare a cell suspension. This cell suspension was collected with a 5 mL syringe (manufactured by Terumo Co., Ltd.) equipped with a sonde, and then a 22G injection needle (manufactured by Terumo Co., Ltd.) was attached to this syringe. Subsequently, 10 drops of this cell suspension were added to the wells of a 24-well flat-bottom microplate (manufactured by PG Research Co., Ltd.) to which 2 mL of calcium chloride aqueous solution (manufactured by PG Research Co., Ltd.) had been added. After leaving it at room temperature for 10 minutes, the formation of alginate beads was confirmed, and then the calcium chloride solution was removed, 2 mL of PBS was added, and the mixture was left at room temperature for 15 minutes. After removing the PBS, 2 mL of DMEM medium (manufactured by WAKO) containing 10% (v / v) fetal bovine serum was added and 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 Sansho Co., Ltd.) and 10% (v / v) fetal bovine serum, prepared using the same method as in Reference Test Example 2, or the above medium without deacylated gellan gum was added to each well, and the plates were statically cultured for 8 days in a CO2 incubator (5% CO2). The medium was replaced on the 4th day of culture.

[0187] The cultured alginate beads were transferred to a 1.5 mL micro test tube (manufactured by Eppendorf) using a 1 mL tip, and 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. The cells were then precipitated by centrifugation at 300 G for 3 minutes and the supernatant was removed. 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO Co., Ltd.) was added to the cells and incubated at 37 ° C for 5 minutes. 800 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added to 200 μL of the cell suspension obtained here, and the same amount of trypan blue staining solution (manufactured by Invitrogen Co., Ltd.) was added to a part of the cell suspension, and the number of live cells was measured using a hemocytometer (manufactured by Elma Sales Co., Ltd.).

[0188] As a result, it was confirmed that the alginate beads in which HepG2 cells are embedded can be cultured in a suspended state by using the medium composition, and that the cells grow efficiently in the medium composition. Moreover, it was confirmed that the medium composition has a superior effect of promoting cell growth compared to existing media that do not contain deacylated gellan gum. The number of cells when HepG2 cells were cultured for 8 days in alginate beads in a medium containing or not containing deacylated gellan gum is shown in Table 32. The floating state when the alginate beads in which HepG2 cells are embedded were cultured in the medium composition is shown in Figure 10.

[0189] [Table 32]

[0190] Reference Test Example 27: Cell suspension test using collagen gel capsules A: Collagen for tissue culture Cellmatrix (registered trademark) Type I-A (Cellmatrix) A: 10x concentrated DMEM / F-12 medium (A C: Reconstitution buffer (100 mL of 0.05N sodium hydroxide solution, 2.2 g of sodium bicarbonate, and 4.77 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) The collagen mixture was cooled in ice and mixed with each other at A:B:C=8:1:1. Furthermore, human recombinant laminin 511 (Veritas Corporation) was added to 5μg / mL to prepare 500μL of collagen mixed solution. HepG2 cells prepared separately were added to this mixed solution at 200000 cells / mL, and the entire amount was collected using a 1.5mL syringe (Terumo Corporation) equipped with a 25G injection needle (Terumo Corporation). Next, the cell suspension was dropped drop by drop using the above syringe into a flat-bottom tube (BM Equipment Co., Ltd.) containing 10mL of DMEM medium (WAKO Co., Ltd.) containing 10% (v / v) fetal bovine serum that had been warmed at 37℃. After incubating in a 37°C water bath for 10 minutes and confirming the formation of amorphous collagen gel capsules with a diameter of about 2 mm, deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was added to a final concentration of 0.04% in the same manner as in Reference Test Example 2, and the capsules were suspended by gentle stirring. The tubes were then subjected to static culture in a CO2 incubator (5% CO2) for 5 days.

[0191] 25 mL of PBS was added to the culture solution containing collagen gel capsules, and the collagen gel capsules were precipitated by centrifugation at 400G for 5 minutes, and the supernatant was removed. 25 mL of PBS was added again, and centrifugation was performed, and the supernatant was removed so that the remaining volume was 5 mL. 20 μL of 1% (W / V) collagenase L (Nitta Gelatin Co., Ltd.) was added to this solution, and the solution was shaken at 37°C for 2 hours. After confirming that the collagen gel had dissolved, 10 mL of PBS was added, and the cells were precipitated by centrifugation at 400G for 5 minutes, and the supernatant was removed. 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (WAKO Co., Ltd.) was added to the cells, and the solution was incubated at 37°C for 5 minutes. 4 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added to the cell suspension obtained here, and the cells were precipitated by centrifugation at 400G for 5 minutes, and the supernatant was removed. The obtained cells were suspended in 2 mL of the above medium, and an equal amount of trypan blue staining solution (Invitrogen) was added to a portion of the suspension, after which the number of viable cells was measured using a hemocytometer (Elma Sales Co., Ltd.).

[0192] As a result, it was confirmed that collagen gel capsules in which HepG2 cells are embedded can be cultured in a suspended state by using the medium composition, and that the cells can grow efficiently in the medium composition. Moreover, it was confirmed that the medium composition has a superior effect of promoting cell growth compared to existing media that do not contain deacylated gellan gum. The number of cells when HepG2 cells were cultured for 5 days in collagen gel capsules in media containing or not containing deacylated gellan gum is shown in Table 33. In addition, the floating state when collagen gel capsules in which HepG2 cells are embedded were cultured in the medium composition is shown in Figure 11.

[0193] [Table 33]

[0194] Reference Test Example 28: Sphere recovery test using a filter A composition of DMEM medium (WAKO) containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.) and 10% (v / v) fetal bovine serum was prepared using the same method as in Reference Test Example 2. In addition, the same medium without deacylated gellan gum was prepared as a control. HepG2 cell spheres were prepared using the same method as in Reference Test Example 2, and the spheres were added to 1 mL of the medium prepared above so that the number of cells was 86,000, and then the medium was left to stand 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 (Becton Dickinson Co., Ltd.) with a mesh size of 40 μm, and the spheres were captured on the filter. Subsequently, 10 mL of PBS was poured into the back of the filter to collect the spheres in a 15 mL tube, and the spheres were precipitated by centrifugation at 300 G for 5 minutes. After removing the supernatant, 500 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the spheres and incubated at 37° C. for 5 minutes. 1 mL of DMEM medium containing 10% (v / v) fetal bovine serum was added to the cell suspension obtained here, and the same amount of trypan blue staining solution (manufactured by Invitrogen) was added to a part of it, and the number of live cells was measured using a hemocytometer (manufactured by Elma 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, it was confirmed that the HepG2 cell spheres could be recovered at a recovery rate equivalent to that of a medium not containing deacylated gellan gum by filtering this sphere suspension containing 0.015% deacylated gellan gum. The relative cell numbers recovered from a medium containing deacylated gellan gum when the number of HepG2 cells recovered by a filter using a medium not containing deacylated gellan gum is set to 1 are shown in Table 34.

[0195] [Table 34]

[0196] Reference Test Example 29: Cell suspension test of spheres using a mixture of various polysaccharides Using the same method as in Reference Test Example 9, xanthan gum (KELTROL CG, manufactured by Sansho Co., Ltd.), sodium alginate (Duck alginate NSPM, manufactured by Food Chemifa Co., Ltd.), locust bean gum (GENUGUM RL-200-J, manufactured by Sansho Co., Ltd.), methylcellulose (cP400, manufactured by WAKO Co., Ltd.), κ-carrageenan (GENUGEL DMEM / F-12 medium compositions were prepared by mixing and combining cellulose acetate (WR-80-J, Sansho Co., Ltd.), pectin (GENU pectin LM-102AS, Sansho Co., Ltd.) or diutan gum (KELCO CRETE DG-F, Sansho Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.). HepG2 cell spheres were prepared using the same method as in Reference Test Example 2, and several dozen spheres were added to 1 mL of the medium prepared above, which was then left to stand at 37°C, and the floating state of the sphere cells was visually observed after 1 hour and overnight. As a result, the HepG2 cell spheres It was confirmed that the cells were maintained in a floating state in all of the above medium compositions. Furthermore, it was confirmed that, in all medium compositions, the HepG2 cell spheres were precipitated and the cells could be recovered by adding twice the volume of medium and then centrifuging the cell suspension (500G, 5 minutes). The dispersion state of the spheres after one night was visually confirmed, and the results are shown in Tables 35 and 36, with a floating dispersion state being evaluated as ○, a partial precipitation / dispersion state being △, and a precipitation state being ×. Note that - in the tables indicates that the test was not performed.

[0197] [Table 35]

[0198] [Table 36]

[0199] Comparison of dispersibility of beads and cells 1 The deacylated gellan gum-containing medium and methylcellulose-containing medium prepared above (Comparative Example) were compared for the dispersion state of dextran beads Cytodex (registered trademark) 1 (GE Healthcare Life Sciences) and HeLa cell spheres. The results are shown in Tables 37 and 38. The dispersion state of Cytodex 1 and HeLa cell spheres correlate well, so Cytodex 1 can be used as a cell sphere model.

[0200] [Table 37]

[0201] [Table 38]

[0202] Comparison of dispersion of beads and cells 2 The polysaccharide and deacylated gellan gum-containing medium prepared in Reference Test Example 9 was compared with the dispersion state of polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.). The floating dispersion state was evaluated as ○, partial sedimentation / dispersion state as △, and sedimentation state as ×. The results are shown in Table 39. Since the dispersion state of polystyrene beads and HepG2 cell spheres correlate well, polystyrene beads can be used as a cell sphere model.

[0203] [Table 39]

[0204] Reference Test Example 30: Suspension culture test of rice-derived plant callus Fifty fully ripe seeds of Nipponbare rice (purchased from Koto Agricultural Cooperative Association) that had been refined by salt water selection were transferred to a 50 mL polystyrene tube (manufactured by BD Falcon) and washed with 50 mL of sterilized water. After that, the seeds were stirred in 30 mL of 70% ethanol water for 1 minute. After removing the ethanol water, 30 mL of Kitchen Haiter (Kao Corporation) was added and stirred for 1 hour. After removing the Kitchen Haiter, the seeds were washed four times with 50 mL of sterilized water. The sterilized seeds were placed on 1.5 mL / well of Murashige and Skoog basal medium (M9274, Sigma-Aldrich) containing 2 μg / mL 2,4-dichlorophenoxyacetic acid (Sigma-Aldrich) and agar (24-well flat-bottom microplate (Corning)). The seeds were cultured for 3 weeks at 30°C, 16 hours in the dark / 8 hours in the dark, and cream-colored calli (1-2 mm) that grew on the scutellum of the seeds were harvested.

[0205] Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolved by stirring while heating at 90°C, and the resulting solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.03% (w / v) in Murashige-Skoog basal medium (M9274, Sigma-Aldrich Corporation) containing 2 μg / mL 2,4-dichlorophenoxyacetic acid (Sigma-Aldrich Corporation). 15 calli prepared as above were added to 10 mL of this medium composition per flat-bottom tube (BM Equipment Co., Ltd.) and cultured with shaking at 25°C for 7 days. As a result, it was confirmed that rice-derived calli could be cultured in a floating state by using this medium composition, and that calli could be maintained in this medium composition. FIG. 12 shows the floating state of rice-derived callus when cultured in the medium composition.

[0206] [Production Example 1: Production of cellulose nanofibers derived from crystalline cellulose] After adding 396 parts by mass of pure water to 4 parts by mass of commercially available crystalline cellulose (PH-101 manufactured by Asahi Kasei Chemicals Corporation) and dispersing, the mixture was pulverized 100 times at 220 MPa using a high-pressure pulverizer (Starburst System) manufactured by Sugino Machine Co., Ltd. to obtain an aqueous dispersion of cellulose nanofibers derived from crystalline cellulose (MNC). The obtained dispersion was weighed into a petri dish and dried at 110°C for 1 hour, and the amount of residue was measured to remove moisture and measure the concentration. As a result, the cellulose concentration (solid content concentration) in the water was 1.0% by mass. This aqueous dispersion was autoclaved at 121°C for 20 minutes to sterilize.

[0207] [Production Example 2: Production of pulp-derived cellulose nanofibers] After adding 145 parts by mass of pure water to 5 parts by mass of commercially available kraft pulp (LBKP manufactured by Oji F-Tex Co., Ltd., solid content 89% by mass) and dispersing, the mixture was ground 9 times at 1500 rpm using a stone mill type grinding device (mass colloider) manufactured by Masuko Sangyo Co., Ltd. to prepare a pulp slurry. The pulp slurry was treated 300 times at 220 MPa using a high pressure grinding device (Starburst System) manufactured by Sugino Machine Co., Ltd. to obtain an aqueous dispersion of nanocellulose (PNC). The obtained dispersion was weighed into a petri dish and dried at 110 ° C for 1 hour, the moisture was removed, the amount of residue was measured, and the concentration was measured. As a result, the cellulose concentration (solid content concentration) in the water was 1.6% by mass. This aqueous dispersion was autoclaved at 121 ° C for 20 minutes and sterilized.

[0208] [Production Example 3: Production of chitin nanofibers] 20 parts by mass of commercially available chitin powder (manufactured by Koyo Chemical Co., Ltd.) was dispersed in 980 parts by mass of pure water, and then crushed 200 times at 245 MPa using a high-pressure crushing device (Starburst System) manufactured by Sugino Machine Co., Ltd. to obtain an aqueous dispersion of chitin nanofibers (CT). The resulting dispersion was weighed into a petri dish and dried at 110°C for 1 hour, and the amount of residue was measured to remove moisture and measure the concentration. As a result, the chitin concentration (solid concentration) in the water was 2.0% by mass. This aqueous dispersion was autoclaved at 121°C for 20 minutes to sterilize.

[0209] [Test Example 1: Measurement of average fiber diameter D and average fiber length L] The average fiber diameter (D) of the nanofibers was determined as follows. First, a collodion support membrane manufactured by Oken Shoji Co., Ltd. was subjected to hydrophilization treatment for 3 minutes using an ion cleaner (JIC-410) manufactured by JEOL Ltd., and several drops of the nanofiber dispersion liquid (diluted with ultrapure water) prepared in Production Examples 1 to 3 were dropped onto the membrane and dried at room temperature. This was observed with a transmission electron microscope (TEM, H-8000) (10,000x) manufactured by Hitachi Ltd. at an accelerating voltage of 200 kV, and the obtained image was used to measure the fiber diameter of each of the 200 to 250 nanofiber specimens, and the number average value was taken as the average fiber diameter (D). The average fiber length (L) was determined by diluting the nanofiber dispersion prepared in the manufacturing example with pure water to 100 ppm, and uniformly dispersing the nanofibers using an ultrasonic cleaner. This nanofiber dispersion was cast onto a silicon wafer whose surface had been hydrophilized using concentrated sulfuric acid, and dried at 110°C for 1 hour to obtain a sample. The obtained sample was observed with a scanning electron microscope (SEM, JSM-7400F) (2,000x) manufactured by JEOL Ltd., and the fiber length of each of the 150 to 250 nanofiber specimens was measured using an image of the sample. The number average value was determined as the average fiber length (L). The average fiber diameter D and average fiber length L of the nanofibers obtained in Production Examples 1 to 3 were determined, and the aspect ratio L / D was calculated from these values. The results are shown in Table 40.

[0210] [Table 40]

[0211] [Examples 1 to 4] The medium compositions shown in Table 41 below were prepared using the nanofiber dispersions and deacylated gellan gum aqueous solutions prepared in Production Examples 1 to 3 described above. First, sterile water was added to the cellulose nanofibers MNC, PNC, and chitin nanofibers prepared in Production Examples 1 to 3 to dilute them to 1% (w / v) aqueous dispersions. Meanwhile, 99 parts by volume of sterile water was added to 1 part by mass of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Corporation: DAG), and the mixture was dissolved and sterilized by autoclaving at 121°C for 20 minutes to prepare a 1% (w / v) aqueous solution of deacylated gellan gum. One volume part of the above 1% (w / v) dispersion or aqueous solution was placed in a 50 mL conical tube, 49 volumes of sterile water was added, and pipetted until homogenized. 50 volumes of 2x concentration DMEM (high glucose, Aldrich, containing a specified amount of sodium bicarbonate) sterilized through a 0.22 μm filter was added and mixed by pipetting to prepare a medium composition with a nanofiber concentration of 0.01% (w / v). Similarly, a medium composition was prepared by adding a nanofiber dispersion or an aqueous solution of deacylated gellan gum to a desired final concentration of 0.01 to 0.1% (w / v).

[0212] [Example 5 and Comparative Examples 2 to 5] 1 part by mass of κ-carrageenan (GENUGEL WR-80-J, manufactured by Sansho Co., Ltd.: Car) (Example 5), locust bean gum (GENUGUM RL-200-J, manufactured by Sansho Co., Ltd.: LBG) (Comparative Example 2), xanthan gum (KELTROL CG, manufactured by Sansho Co., Ltd.: Xan) (Comparative Example 3), diutan gum (KELCO CRETE DG-F, manufactured by Sansho Co., Ltd.: DU) (Comparative Example 4), and sodium alginate (Duck alginate NSPM, manufactured by Food Chemifa Co., Ltd.: Alg) (Comparative Example 5) were added to 99 parts by mass of sterilized water and dissolved and sterilized by autoclaving at 121°C for 20 minutes. The polysaccharide solutions prepared as described above were added in the same manner as in Examples 1 to 4 to prepare medium compositions having final concentrations of 0.03, 0.05, 0.07, and 0.1% (w / v).

[0213] [Test Example 2: Evaluation of Floating Action 1] Polystyrene beads (Polysciences Inc., 200-300 μm) were added to the medium compositions of Examples 1 to 5 and Comparative Examples 2 to 5, and after confirming that the beads were uniformly dispersed in the medium compositions by vortex stirring, the medium compositions were left to stand at room temperature (25° C.) for one day, and the dispersion state of the beads was visually confirmed. The state in which the beads were uniformly suspended in the medium composition was evaluated as ◎, the state in which some supernatant was generated was evaluated as ○, and the state in which the beads had settled was evaluated as ×. The results are shown in Table 41.

[0214] As a result, the medium compositions of Examples 1 to 4 exhibited the effect of floating the beads. In Example 5, the beads were able to float at room temperature, but when heated to 37° C. In Comparative Examples 2 to 5, the beads completely settled to the bottom.

[0215] [Table 41]

[0216] *Kappa-carrageenan (Car) in Example 5 exhibited a buoyancy effect at 25°C, but immediately lost its buoyancy and settled at 37°C, which is equivalent to the cell culture conditions. The same results were obtained at 37°C and 25°C for the other media.

[0217] [Test Example 3: Evaluation of Floating Action 2] As in Test Example 2, the medium compositions of Examples 2, 4, and 5 and Comparative Example 2 were evaluated in detail for their floating action in the low concentration range (0.01 to 0.04% (w / v)). Polystyrene beads were added, and after standing for 2 days, the dispersion state of the beads was visually confirmed. The floating / dispersed state was evaluated as ○, and the settling state was evaluated as ×. The partial settling / dispersion state was evaluated using a 10 mL Konica The bead floating rate was calculated based on the height of the floating area in the tube. The results are shown in Table 42.

[0218] [Table 42]

[0219] * indicates the bead floating rate. PNC showed floating action at a concentration of 0.015% (w / v) or more, and the medium composition of Example 4 showed floating action at a concentration of 0.015% (w / v) or more. The medium composition of Example 2 showed a stepwise improvement in floating action as the concentration increased. The medium composition of Example 5 showed a floating action at 25°C at 0.02% or more, but immediately lost its floating action at 37°C and sank (*). For the other media, the same results were obtained at 37°C and 25°C.

[0220] [Test Example 4: Viscosity of medium composition] The viscosities of the medium compositions of Examples 1 to 5 and Comparative Examples 2 to 5 were measured using a tuning fork vibro viscometer at 25° C. The results are shown in Figure 13. As a result, since the medium composition of the present invention has an extremely low content of nanofibers or thickening polysaccharides, no significant increase in viscosity was observed compared to the viscosity of general medium. Comparison with the results of Test Example 2 showed no correlation between viscosity and flotation effect.

[0221] [Test Example 5: Scanning electron microscope observation of medium composition] The medium compositions prepared in Examples 1 to 5 and Comparative Examples 3 to 4 were cast onto silicon wafers whose surfaces had been hydrophilized with concentrated sulfuric acid, dried at 110°C (room temperature only in Comparative Example 1) for 1 hour, washed with pure water to remove excess salts, and then dried again at 110°C for 1 hour to prepare samples. The aforementioned samples were observed using a JEOL Ltd. scanning electron microscope (SEM, JSM-7400F, 10,000x magnification). The observation results of the medium compositions of Examples 1 to 4 and Comparative Examples 3 to 4 are shown in Figures 14 to 21.

[0222] As a result of the observation, many fibers were observed in Examples 1 to 4 and Example 5 dried at room temperature, whereas no fibers were observed at all in Example 5 and Comparative Examples 3 and 4 dried at 110°C. The many spherical objects observed in the observation images are precipitated salt components in the medium. This result suggests that the fiber structure contained in the medium composition may contribute to the flotation effect.

[0223] [Test Example 7: Floating Action of Spheres] Human hepatoma cell line HepG2 (DS Pharma Biomedical) was suspended in DMEM medium (WAKO) containing 10% (v / v) fetal bovine serum at 50,000 cells / mL, 10 mL of the suspension was seeded in EZ SPHERE (Asahi Glass Co., Ltd.), and then cultured for 2 days in a CO2 incubator (5% CO2). 80 mL of the resulting sphere suspension was centrifuged (800 rpm, 5 minutes) to settle the spheres, and 4.5 mL of the sphere suspension was prepared by removing the supernatant. Subsequently, 10 mL of each of the medium compositions of Examples 1 to 4, Comparative Example 1, and Comparative Example 3 to 5 was placed in a 15 mL conical tube, and 100 μL of the sphere suspension of HepG2 cells was added. The spheres were dispersed by pipetting, incubated at 37° C. for 5 days, and the dispersion state of the spheres in the medium composition was visually observed. The state in which the spheres were uniformly suspended in the medium composition was evaluated as ⊚, the state in which a supernatant was generated was evaluated as ◯, and the state in which the spheres settled was evaluated as ×. The observation results of the medium compositions of Examples 1 to 5 and Comparative Examples 3 to 5 are shown in Table 43 and Figures 22 to 29.

[0224] As a result, the spheres remained floating even after 6 days of culture in the medium compositions of Examples 1 to 4. On the other hand, in the medium compositions of Example 5 and Comparative Examples 3 to 5, all the spheres settled and furthermore the spheres aggregated together.

[0225] [Table 43]

[0226] [Examples 1' to 4'] Sterile water was added to the cellulose nanofibers MNC, PNC and chitin nanofibers prepared in Production Examples 1 to 3 to prepare 1% (w / v) aqueous dispersions. On the other hand, 99 parts by volume of sterile water was added to 1 part by mass of deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.: DAG), and the mixture was dissolved and sterilized by autoclaving at 121°C for 20 minutes to prepare a 1% (w / v) aqueous solution. The 1% (w / v) fiber dispersion or deacylated gellan gum aqueous solution prepared above was added to DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd., high-glucose) containing 10% (v / v) fetal bovine serum so that the final concentrations were 0.01%, 0.03%, 0.06% and 0.1% (w / v), to prepare a medium composition.

[0227] [Example 5' and Comparative Examples 3' to 5'] 99 parts by volume of sterilized water was added to 1 part by mass of κ-carrageenan (GENUGEL WR-80-J, manufactured by Sansho Co., Ltd.: Car), xanthan gum (KELTROL CG, manufactured by Sansho Co., Ltd.: Xan), diutan gum (KELCO CRETE DG-F, manufactured by Sansho Co., Ltd.: DU), and sodium alginate (Duck alginate NSPM, manufactured by Food Chemifa Co., Ltd.: Alg), and the mixture was dissolved and sterilized by autoclaving at 121°C for 20 minutes to prepare 1% (w / v) polysaccharide aqueous solutions. The polysaccharide aqueous solutions prepared above were added to DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% (v / v) fetal bovine serum to final concentrations of 0.01%, 0.03%, 0.06%, and 0.1% (w / v) by the same procedure as in Examples 5 to 8 to prepare medium compositions.

[0228] [Test Example 8: Cell proliferation test] Human breast cancer cell line MCF-7 (DS Pharma Biomedical) and human melanoma cell line A375 (ATCC) were seeded in the medium compositions prepared in Examples 1' to 5' and Comparative Examples 3' to 5' so as to give a concentration of 33,333 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (Corning, #3474) at 150 μL per well. As a negative control, MCF7 cells or A375 cells were suspended in the same medium without nanofibers or polysaccharides and dispensed. The plate was then cultured in a stationary state for up to 6 days in a CO2 incubator (37°C, 5% CO2). 150 μL of ATP reagent (CellTiter-Glo) was added to the culture fluid after 2 and 6 days of culture. TM The cells were suspended in 10 mL of 100% cellulose ester (Luminescent Cell Viability Assay, Promega) and allowed to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The number of viable cells was determined by subtracting the luminescence value of the medium alone.

[0229] As a result, it was confirmed that the medium composition containing PNC, MNC, and nanochitin can culture the cell aggregates in a uniformly dispersed state without excessive size, and that the cells can grow efficiently. On the other hand, the medium composition containing sodium alginate did not promote growth. The RLU values ​​(ATP measurement, luminescence intensity) of MCF7 cells after 2 and 6 days of static culture are shown in Tables 44 to 47, and the RLU values ​​after 6 days are shown in Figures 30 to 33, and the results of A375 cells are shown in Tables 48 to 51, and the RLU values ​​after 6 days are shown in Figures 34 to 37. In microscopic observation of the aggregates cultured for 2 days, the results of MCF7 cells are shown in Figure 38, and the results of A375 cells are shown in Figure 39.

[0230] [Table 44]

[0231] [Table 45]

[0232] [Table 46]

[0233] [Table 47]

[0234] [Table 48]

[0235] [Table 49]

[0236] [Table 50]

[0237] [Table 51]

[0238] [Test Example 9: Preservation test using 3T3-L1 cells] Mouse preadipocyte cell line 3T3-L1 (ATCC) was seeded on a 10 cm polystyrene petri dish using 10% FBS-containing DMEM medium and cultured in an incubator set at 5% CO2 and 37°C. When the 3T3-L1 cells were confluent, the medium was aspirated and FBS was removed with D-PBS (Wako Pure Chemical Industries, Ltd.), and 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (Wako Pure Chemical Industries, Ltd.) was added to the polystyrene petri dish. After confirming cell detachment, the cells were recovered from the petri dish by adding 10% FBS-containing DMEM medium by volume and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. Approximately 100 × 10 4To prepare a cell suspension of 100 μL of cells / mL, 100 μL of the cell suspension was added to a 1.5 mL microtube, and 100 μL each of the medium compositions of Examples 1 to 2, Examples 4 to 5, Comparative Example 3, and Comparative Example 5, which had been prepared in advance to contain 10% (v / v) FBS, was added, and the cell suspension was prepared by pipetting. The cells were stored in a sealed container at room temperature for 10 days. After 3 and 10 days, a portion of the cell suspension was diluted 1 / 10 with 10% FBS-containing DMEM medium. 100 μL of the diluted cell suspension was added to 100 μL of ATP reagent (CellTiter-Glo). TM Luminescent The cells were suspended in 100% ethanol (Promega Cell Viability Assay) and allowed to stand at room temperature for 15 minutes, after which the luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices) and the number of viable cells was determined by subtracting the luminescence value of the medium alone. A sample containing only the medium without polysaccharide was used as a negative control.

[0239] As a result, the cell viability of the negative control or the medium compositions of Comparative Example 3 and Comparative Example 5 significantly decreased in ATP value after storage at room temperature for 3 to 10 days, whereas the medium compositions of Examples 1, 2, and 4 suppressed the decrease in ATP value, demonstrating a cell protective effect. The results of the viable cell count are shown in Table 52.

[0240] [Table 52]

[0241] [Test Example 10: Preservation test using CHO-K1 cells] Chinese hamster ovary line CHO-K1-hIFNβ cells (Kitakyushu National College of Technology, (Transferred from Professor Kawahara) were seeded on a 10 cm polystyrene petri dish using 10% FBS-containing F12 medium and cultured in an incubator set at 5% CO2 and 37℃. When the CHO-K1-hIFNβ cells were confluent, the medium was aspirated and the FBS was removed with D-PBS (manufactured by Wako Pure Chemical Industries, Ltd.), and 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the polystyrene petri dish. After confirming that the cells had detached, the cells were recovered from the petri dish by adding 10% FBS-containing F12 medium and transferred to a centrifuge tube. After centrifugation at 300Xg, the supernatant was removed. Approximately 5x10 6 To prepare a cell suspension of 100 cells / mL, 25 μL of the cell suspension was added to a 1.5 mL microtube, and 25 μL of the medium compositions of Examples 2 and 4, which had been prepared in advance to contain 10% (v / v) FBS, were added, followed by pipetting to prepare a cell suspension. A portion of the cell suspension that had been stored at room temperature for 1 day in a sealed state was diluted 1 / 10 with F12 medium containing 10% FBS, and 100 μL of the diluted cell suspension was added with 100 μL of ATP reagent (CellTiter-Glo). TM Luminescent Cell Viability Assay, After adding polysaccharide-free medium (Promega), the cells were suspended and allowed to stand at room temperature for about 10 minutes, after which the luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices) and the number of live cells was determined by subtracting the luminescence value of the medium alone. A sample containing only the medium without polysaccharide was used as a negative control.

[0242] As a result, the cell viability of the negative control decreased when stored at room temperature for one day, whereas the medium compositions of Examples 2 and 4 showed ATP values ​​at the same level as at the time of seeding, demonstrating a cytoprotective effect. The results of viable cell counts are shown in Table 53.

[0243] [Table 53]

[0244] [Test Example 11: Preservation test using 3T3-L1 cells, changing the concentration of polysaccharides] Mouse preadipocyte cell line 3T3-L1 (ATCC) was seeded on a 10 cm polystyrene petri dish using 10% FBS-containing DMEM medium and cultured in an incubator set at 5% CO2 and 37°C. When the 3T3-L1 cells were 40% confluent, the medium was aspirated and FBS was removed with D-PBS (Wako Pure Chemical Industries, Ltd.), and 1 ml of a solution containing 0.25% Trypsin and 1 mM EDTA (Wako Pure Chemical Industries, Ltd.) was added to the polystyrene petri dish. After confirming cell detachment, the cells were recovered from the petri dish by adding 10% FBS-containing DMEM medium by volume and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. Approximately 100 × 10 4 To prepare a cell suspension of 100 μL per 1.5 mL microtube, 100 μL of the cell suspension was added, and 100 μL of each of the medium compositions having different polysaccharide concentrations of Examples 2, 4, and Comparative Example 5, which had been prepared in advance to contain 10% (v / v) FBS, was added, and the cell suspension was prepared by pipetting. The cells were stored in a sealed container at room temperature for 8 days. After 0, 5, and 8 days, a portion of the cell suspension was diluted 1 / 5 with 10% FBS-containing DMEM medium. 100 μL of the diluted cell suspension was added to 100 μL of ATP reagent (CellTiter-Glo). TM Luminescent The cells were suspended in 100% ethanol (Promega Cell Viability Assay) and allowed to stand at room temperature for 15 minutes, after which the luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices) and the number of viable cells was determined by subtracting the luminescence value of the medium alone. A sample containing only the medium without polysaccharide was used as a negative control.

[0245] As a result, the cell viability of the negative control or the medium composition of Comparative Example 5 was significantly reduced in ATP value after storage at room temperature for 5 to 8 days, whereas the medium compositions of Examples 2 and 4 suppressed the reduction in ATP value, demonstrating a cell protective effect. The results of viable cell count are shown in Table 54.

[0246] [Table 54]

[0247] [Test Example 12: Effect on cell viability of MDCK cells] Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolved by stirring while heating at 90° C., and this aqueous solution was sterilized in an autoclave for 20 minutes at 121° C. Using this solution, medium compositions were prepared in which deacylated gellan gum was added to EMEM medium (Wako Pure Chemical Industries) containing 10% (v / v) fetal bovine serum at final concentrations of 0.005% (w / v) and 0.015%, or a medium composition not containing deacylated gellan gum was prepared. Subsequently, the canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical) was cultured overnight in a serum-free medium (starvation treatment), and then seeded in the medium composition containing the above-mentioned deacylated gellan gum to a concentration of 100,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (Corning, #3474) at 100 μL per well. Each plate was cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 15 days. 100 μL of ATP reagent (CellTiter-Glo) was added to the culture solution on the 2nd, 6th, 9th, 12th, and 15th days. TM The cells were suspended in 10 mL of 100% cellulose ester (Luminescent Cell Viability Assay, Promega) and allowed to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The number of viable cells was determined by subtracting the luminescence value of the medium alone.

[0248] As a result, it was found that culturing MDCK cells on a low-attachment plate using the medium composition of the present invention suppresses the decrease in the number of viable cells. The RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Table 55.

[0249] [Table 55]

[0250] [Test Example 13: Effect on cell viability of Vero cells] Deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) was suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w / v), then dissolved by stirring while heating at 90° C., and the resulting aqueous solution was sterilized in an autoclave for 20 minutes at 121° C. Using this solution, medium compositions were prepared in which deacylated gellan gum was added to Emedium 199 medium (Sigma) containing 5% (v / v) fetal bovine serum at final concentrations of 0.005% (w / v) and 0.015%, or a medium composition not containing deacylated gellan gum was prepared. Subsequently, monkey kidney epithelial cell line Vero (manufactured by DS Pharma Biomedical) that had been cultured overnight in serum-free medium (starvation treatment) was seeded in the medium composition containing the above-mentioned deacylated gellan gum to a concentration of 100,000 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. Each plate was cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 15 days. 100 μL of ATP reagent (CellTiter-Glo) was added to the culture medium on the 2nd, 6th, 9th, 12th, and 15th days. TM The cells were suspended in 10 mL of 100% cellulose ester (Luminescent Cell Viability Assay, Promega) and allowed to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The number of viable cells was determined by subtracting the luminescence value of the medium alone.

[0251] As a result, it was found that culturing Vero cells on a low-attachment plate using the medium composition of the present invention suppresses the decrease in the number of viable cells. The RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Table 56.

[0252] [Table 56]

[0253] [Test Example 14: Effect of each substrate on MDCK cell proliferation] The cellulose nanofiber (PNC) prepared in Production Example 2, chitin nanofiber (biomass nanofiber BiNFi-S (Binfis) 2% by mass, manufactured by Sugino Machine Co., Ltd.), and deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) were used. ) 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, and the resulting aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Medium compositions were prepared by adding cellulose nanofibers to serum-free medium KBM220 medium (Kohjin Bio Co., Ltd.) at final concentrations of 0.01% (w / v), 0.03%, and 0.1%, medium compositions by adding chitin nanofibers to serum-free medium KBM220 medium (Kohjin Bio Co., Ltd.) at final concentrations of 0.01% (w / v), 0.03%, and 0.1%, medium compositions by adding deacylated gellan gum to serum-free medium KBM220 medium (Kohjin Bio Co., Ltd.) at final concentrations of 0.005% (w / v), 0.015%, 0.03%, 0.06%, and 0.1%, and medium compositions not containing the above-mentioned base material. Subsequently, the canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical) that had been cultured overnight in a serum-free medium (starvation treatment) was seeded in a medium composition containing each of the above-mentioned substrates at 100,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (Corning, #3474) at 100 μL per well. Each plate was cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 14 days. 100 μL of ATP reagent (CellTiter-Glo) was added to the culture medium on the 3rd, 7th, 10th, and 14th days. TM The cells were suspended in 10 mL of 100% cellulose ester (Luminescent Cell Viability Assay, Promega) and allowed to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The number of viable cells was determined by subtracting the luminescence value of the medium alone.

[0254] As a result, when MDCK cells were cultured on a low-attachment plate using the medium composition of the present invention, deacylated gellan gum, nanocellulose fiber PNC, and chitin nanofiber, the addition of all substrates promoted the proliferation of MDCK cells. Among these, chitin nanofiber showed the strongest effect. The RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Table 57.

[0255] [Table 57]

[0256] [Test Example 15: Effect of chitin nanofiber on MDCK proliferation] The cellulose nanofibers (PNC) prepared in Production Example 1, chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Corporation), and deacylated gellan gum (KELCOGEL CG-LA, Sansho Corporation) were suspended in ultrapure water (Milli-Q water) to a concentration of 1% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Medium compositions were prepared by adding chitin nanofibers to serum-free KBM220 medium at final concentrations of 0.0001% (w / v), 0.0003%, 0.001%, 0.003%, 0.01%, 0.02%, and 0.03%; medium compositions were added to serum-free KBM220 medium (Kohjin Bio Co., Ltd.) with deacylated gellan gum at final concentrations of 0.005% (w / v), 0.015%, and 0.03%, and an unsupplemented medium composition not containing the above-mentioned substrate was also prepared. Subsequently, the canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical) was cultured overnight in a serum-free medium (starvation treatment), and then seeded in the medium composition containing the above-mentioned deacylated gellan gum or chitin nanofibers to a concentration of 100,000 cells / mL, and then dispensed into the wells of a 96-well flat-bottom ultra-low attachment surface microplate (Corning, #3474) at 100 μL per well. Each plate was cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 14 days. 100 μL of ATP reagent (CellTiter-Glo) was added to the culture medium on the 5th, 9th, 12th, and 15th days. TM Luminescent Cell Viability Assay, Prom. The cells were suspended in 10 ml of 100% ethanol (manufactured by ega), allowed to stand at room temperature for approximately 10 minutes, and then the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices). The luminescence value of the medium alone was subtracted, and the number of live cells was calculated as the average of three points.

[0257] As a result, when MDCK cells were cultured on a low-adhesion plate using the medium composition of the present invention, deacylated gellan gum and chitin nanofiber, the addition of both substrates was found to promote the proliferation of MDCK cells. Among them, chitin nanofiber showed a proliferation promoting effect at a concentration of 0.0001% or more, and showed a particularly high effect at a concentration of 0.001% or more. The RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Table 58.

[0258] [Table 58]

[0259] [Test Example 16: Effect of chitin nanofiber on MDCK cell proliferation] Initial culture; The cellulose nanofibers (PNC) and chitin nanofibers (biomass nanofibers BiNFi-S (Binfis) 2% by mass, Sugino Machine Co., Ltd.) prepared in Production Example 2 were suspended in ultrapure water (Milli-Q water) to a concentration of 1% (w / v), and then dissolved by stirring while heating at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. A medium composition was prepared by adding chitin nanofibers to serum-free medium KBM220 medium at a final concentration of 0.01% (w / v), serum-free medium KBM220 medium (Kohjin Bio Co., Ltd.), and an unsupplemented medium composition that did not contain chitin nanofibers. Subsequently, the canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.), which had been cultured overnight in a serum-free medium (starvation treatment), was seeded in the above-mentioned medium composition supplemented with chitin nanofibers to a concentration of 75,000 cells / mL, and then the cells were cultured in a 125 ml Erlenmeyer flask (Corning The flasks were cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 6 days. The culture medium on days 0 and 6 was suspended with a pipette, and 100 μL was dispensed in three portions, and 100 μL of ATP reagent (CellTiter-Glo) was added to each suspension. TMThe cells were suspended in 10 mL of Fluorescence Cell Viability Assay (Promega) and allowed to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The number of viable cells was determined by subtracting the luminescence value of the medium alone.

[0260] Subculture; To confirm the effect on subculture, a cell suspension of MDCK cells cultured for 6 days in a medium containing 0.01% chitin nanofiber was used for the study. A cell suspension in which 3 ml of the cell suspension was mixed with 27 ml of unsupplemented medium composition to adjust the chitin nanofiber concentration to 0.001%, and a cell suspension in which 3 ml of the cell suspension was mixed with 27 ml of medium composition containing chitin nanofiber at a final concentration of 0.01% (w / v) to adjust the chitin nanofiber concentration to 0.01% were each dispensed into 125 ml Erlenmeyer flasks. The flasks were cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 14 days. After suspending the culture fluid on days 0, 7, and 14 with a pipette, 100 μL was dispensed in three portions, and 100 μL of ATP reagent (CellTiter-Glo) was added to each portion. TM The cells were suspended in 100 mL of 100% luminescent cell viability assay (Promega), allowed to stand at room temperature for approximately 10 minutes, and then the luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices). The luminescence value of the medium alone was subtracted, and the number of viable cells was calculated as the average of three points.

[0261] As a result, when MDCK cells were cultured in an Erlenmeyer flask using chitin nanofiber, which is the medium composition of the present invention, the proliferation of MDCK cells was observed. Furthermore, when the medium containing chitin nanofiber was added, proliferation of MDCK cells was observed, and it was revealed that subculture can be easily performed without treatment such as trypsin. Table 59 shows the RLU values ​​(ATP measurement, luminescence intensity) in the initial culture, and Table 60 shows the RLU values ​​(ATP measurement, luminescence intensity) in the subculture.

[0262] [Table 59]

[0263] [Table 60]

[0264] [Test Example 17: Comparison of the proliferation-promoting effect of chitin nanofibers on MDCK cells in each medium] The cellulose nanofiber (PNC) and chitin nanofiber (biomass nanofiber BiNFi-S (Binfis) 2% by mass, Suginoma Co., Ltd.) prepared in Production Example 1 were used. Chitin nanofibers were added to serum-free medium KBM220 medium (Kohjin Bio Co., Ltd.) or Cosmedium 012 medium (Cosmo Bio Co., Ltd.) at a final concentration of 0.001% (w / v) or 0.01%. Deacylated gellan gum (Kelcogel CG-LA, Sansho 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. The resulting solution was sterilized in an autoclave at 121°C for 20 minutes. A medium composition containing gellan gum and a medium composition not containing the above-mentioned base material were prepared. Subsequently, canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical) cultured overnight in serum-free medium (starvation treatment) was seeded in the medium composition containing the above-mentioned deacylated gellan gum or chitin nanofiber to a concentration of 100,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (Corning, #3474) at 100 μL per well. Each plate was cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 12 days. 100 μL of ATP reagent (CellTiter-Glo) was added to the culture solution on the 4th, 8th, and 12th days. TMThe cells were suspended in 100 mL of 100% luminescent cell viability assay (Promega), allowed to stand at room temperature for approximately 10 minutes, and then the luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices). The luminescence value of the medium alone was subtracted, and the number of viable cells was calculated as the average of three points.

[0265] As a result, when MDCK cells were cultured on a low-attachment plate using the medium composition of the present invention, deacylated gellan gum and chitin nanofiber, the addition of both substrates was found to promote the proliferation of MDCK cells. Among these, chitin nanofiber showed high proliferation ability even under conditions using Cosmedium 012 medium at a concentration of 0.001% or more. When the cells were observed under a microscope, only dispersed cell aggregates (spheres) were observed in the medium using deacylated gellan gum, whereas in the medium using chitin nanofiber, spheres and cells were observed to have grown in a grape-like cluster shape. Table 61 shows the RLU values ​​(ATP measurement, luminescence intensity) in the culture using KBM220 medium, and Table 62 shows the RLU values ​​(ATP measurement, luminescence intensity) in the culture using Cosmedium012 medium. The results of the microscopic observation after 4 days of culture are shown in Figure 40.

[0266] [Table 61]

[0267] [Table 62]

[0268] [Test Example 18: Comparison of MDCK cell proliferation effects of chitosan nanofibers and chitin nanofibers] Chitosan nanofiber (biomass nanofiber BiNFi-S, 1% by mass, Sugino Machine Corporation) and chitin nanofiber (biomass nanofiber BiNFi-S (Binfis) 2% by mass, Sugino Machine Corporation), and deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Corporation) were suspended in ultrapure water (Milli-Q water) to a concentration of 1% (w / v) as in Reference Example 1, and then the aqueous solutions prepared with stirring at 90°C were each sterilized in an autoclave at 121°C for 20 minutes. A medium composition was prepared by adding chitosan nanofiber or chitin nanofiber to serum-free medium KBM220 medium (Kohjin Bio Co., Ltd.) at a final concentration of 0.001% (w / v), 0.003%, 0.01%, or 0.03%, a medium composition was prepared by adding deacylated gellan gum to serum-free medium KBM220 medium at a final concentration of 0.015% (w / v) or 0.03%, and a medium composition without any of the above substrates was prepared. Subsequently, canine kidney tubular epithelial cell line MDCK (DS Pharma Biomedical Co., Ltd.) cultured for one day and night in a serum-free medium (starvation treatment) was seeded on the medium composition containing deacylated gellan gum, chitosan nanofiber, or chitin nanofiber to a concentration of 100,000 cells / mL, and then dispensed into wells of a 96-well flat-bottom ultra-low attachment surface microplate (Corning, #3474) at 100 μL per well. Each plate was cultured in a stationary CO2 incubator (37°C, 5% CO2) for 12 days. On the 7th and 11th days, 100 μL of ATP reagent (CellTiter-Glo) was added to the culture medium. TM The cells were suspended in 100 mL of 100% luminescent cell viability assay (Promega), allowed to stand at room temperature for approximately 10 minutes, and then the luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices). The luminescence value of the medium alone was subtracted, and the number of viable cells was calculated as the average of three points.

[0269] As a result, when MDCK cells were cultured on a low-adhesion plate using the medium composition of the present invention, chitosan nanofiber and chitin nanofiber, a higher proliferation promoting effect was observed than with deacylated gellan gum. Chitin nanofiber also showed high proliferation ability even at a concentration of 0.001%, while chitosan nanofiber showed high proliferation ability from a concentration of 0.01%. The RLU values ​​(ATP measurement, luminescence intensity) are shown in Table 63.

[0270] [Table 63]

[0271] [Test Example 19: Fresh cynomolgus monkey primary hepatocyte preservation test] The cellulose nanofibers (PNC) prepared in Production Example 2 and a 1% by mass (w / v) aqueous solution of κ-carrageenan (GENUGEL WR-80-J, manufactured by Sansho Corporation: Car) were prepared in the same manner as in Example 5 and used. Medium compositions were prepared by adding PNC or carrageenan to a final concentration of 0.03% (w / v) or 0.1% to 10% FBS-containing Williams'E medium (manufactured by Life Technologies), as well as medium compositions that did not contain the above-mentioned base material. Subsequently, fresh primary cynomolgus monkey hepatocytes (manufactured by Ina Research Co., Ltd.) were added at 2, 50 The cells were mixed with the above medium composition containing PNC or carrageenan so that the concentration was 0,000 cells / mL, and then dispensed into a cryogenic vial (manufactured by Thermo Scientific) for cell freezing. Note that primary cynomolgus monkey hepatocytes were suspended in the above medium without the substrate and dispensed. Two lots of the above procedure were carried out. The tubes were then transported in a stationary state for two days under refrigeration (approximately 4°C). After transport for two days under refrigerated conditions, the cell survival rate in the suspension was measured using trypan blue reagent (manufactured by Life Technologies).

[0272] As a result, when fresh monkey primary hepatocytes were transported under refrigeration using the medium composition PNC of the present invention, they showed a higher survival rate than when not added. On the other hand, no such effect was observed when carrageenan was used. The survival rates are shown in Table 10.

[0273] [Table 64]

[0274] [Test Example 20: Evaluation of proliferation after reseeding] Mouse preadipocyte cell line 3T3-L1 (ATCC) was seeded on a 10 cm polystyrene petri dish using 10% FBS-containing DMEM medium and cultured in an incubator set at 5% CO2 and 37°C. When the 3T3-L1 cells were confluent, the medium was aspirated and FBS was removed with D-PBS (Wako Pure Chemical Industries, Ltd.), and 1 ml of a solution containing 0.25% trypsin and 1 mM EDTA (Wako Pure Chemical Industries, Ltd.) was added to the polystyrene petri dish. After confirming cell detachment, the cells were recovered from the petri dish by adding 10% FBS-containing DMEM medium by volume and transferred to a centrifuge tube. After centrifugation at 300 × g, the supernatant was removed. Approximately 200 × 10 4 To prepare a cell suspension of 100 cells / mL, 150 μL of the cell suspension was added to a 1.5 mL microtube, and 150 μL of the medium compositions of Example 2 (PNC concentration: 0.06%) to Example 4 (DAG concentration: 0.03%) and Comparative Example 5 (Alg concentration: 0.03%), which had been prepared in advance to contain 10% (v / v) FBS, and 10% by volume FBS-containing DMEM medium as a negative control were added, and the cell suspension (approximately 100 × 10 4 cells / mL) were prepared.

[0275] After storing the cells in a sealed state at room temperature for 7 days, a portion of the cell suspension was diluted with 10% FBS-containing DMEM medium to obtain a cell density of approximately 10 × 10 4 A cell suspension of 100 cells / mL was prepared. 100 μL of the cell suspension was seeded into a 96-well multiplate (Corning) and 100 μL of ATP reagent (CellTiter-Glo) was added on the day of seeding, 1 day later, and 2 days later. TMAfter adding 500 mM NaCl (Luminescent Cell Viability Assay, Promega) and allowing to stand at room temperature for 15 minutes, the luminescence intensity (RLU value) was measured (n=5) using a FlexStation3 (Molecular Devices) and the number of viable cells was determined by subtracting the luminescence value of the medium alone.

[0276] As a result, the viable cell count (RLU value) of the negative control or the medium composition of Comparative Example 5 on the day of reseeding after 7 days of storage was significantly lower than that of the medium compositions of Examples 2 to 4. The viable cell count (RLU value) one day after reseeding increased compared to that on the day of reseeding in each of Examples 2 and 4, and the cells maintained their proliferation ability after storage. The results of the viable cell count are shown in Table 65.

[0277] [Table 65] [Industrial Applicability]

[0278] The medium composition according to the present invention exhibits an excellent cell and / or tissue suspension effect and is extremely useful for mass culturing cells and / or tissues derived from animals and plants while maintaining their functions. Furthermore, the cells and / or tissues cultured by the method of the present invention are extremely useful in fields such as efficacy and toxicity evaluation of chemical substances, pharmaceuticals, etc., mass production of useful substances such as enzymes, cell growth factors, and antibodies, and regenerative medicine for replacing organs, tissues, and cells lost due to disease or defects.

[0279] The contents of all publications, including patents and patent applications, mentioned herein are hereby incorporated by reference to the same extent as if fully set forth.

[0280] This application is based on Japanese Patent Application No. 2014-010842 (filing date: January 23, 2014), Japanese Patent Application No. 2014-123772 (filing date: June 16, 2014), Japanese Patent Application No. 2014-174574 (filing date: August 28, 2014), and Japanese Patent Application No. 2014-217761 (filing date: October 24, 2014), the contents of which are incorporated in their entirety into this specification.

Claims

[Claim 1] The invention described in the specification.

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