Method for uniform seeding of cells

Abstract

The present invention provides a method for uniformly seeding cells or tissues on the cell adhesion surface inside a container, comprising the steps of: (step 1) uniformly dispersing cells or tissues in a plastic fluid; and (step 2) adhering cells or tissues to the cell adhesion surface inside the container.

Description

Technical Field

[0001] The present invention relates to a method for uniformly seeding cells onto the cell adhesion surface inside a container (with the same modifications contemplated below). Background Technology

[0002] In recent years, the development of disease treatment technologies using cell therapy has been progressing globally. For cell therapy to be effective, technologies capable of producing high-quality cells in large quantities are needed.

[0003] Cells can be proliferated by seeding them into containers and culturing them. However, uneven seeding of cells on the cell adhesion surface inside the container causes problems such as inconsistent passage timing in each container, significant variations in the culture environment within each container, and uneven cell quality. Therefore, uniform seeding techniques are crucial for mass cell production. However, in conventional cell suspensions, ripples occur during seeding, causing a shift in the distribution of cells, which is problematic.

[0004] To date, methods for uniformly distributing cells into aliquots using plastic fluids have been reported (Patent Document 1). However, these methods refer to distributing cells in a manner that makes the cell number uniform, and then cryopreserving the uniformly distributed aliquots; they do not envision seeding the distributed cells at a uniform density onto the inner surface of a container. Furthermore, the inventors of this application have reported the efficient and effective culture of cells in suspension using a culture medium containing polysaccharides (Patent Document 2).

[0005] Existing technical documents Patent documents Patent Document 1: WO2020 / 036180 Patent Document 2: WO2014 / 017513 Summary of the Invention

[0006] The problem that the invention aims to solve The purpose of this invention is to provide a novel method for seeding cells or tissues at a uniform density on the cell adhesion surface inside a cell culture container (to achieve uniform seeding density).

[0007] Methods for solving problems The inventors of this application conducted in-depth research on the above-mentioned issues and discovered that by uniformly dispersing cells in a plastic fluid, and then distributing the plastic fluid containing the uniformly dispersed cells to a cell culture container, the cells or tissues adhere to the inner surface of the container, thereby enabling extremely uniform seeding of cells on the cell adhesion surface inside the cell culture container. Based on this insight, further research was conducted, and the present invention was completed.

[0008] That is, the present invention is as follows. [1] A method for uniformly seeding cells or tissues onto the cell adhesion surface inside a container, comprising the following steps: (Step 1) The step of uniformly dispersing cells or tissues in a plastic fluid; and (Step 2) The step of adhering cells or tissues to the cell adhesion surface inside the container. [2] The method described in [1] includes, in step 2, a step of reducing the yield stress of the plastic fluid. [3] The method described in [2] is characterized in that the yield stress of the plastic fluid is reduced by diluting the plastic fluid. [4] The method described in [2] is characterized in that the yield stress of the plastic fluid is reduced by the temperature change of the plastic fluid. [5] The method as described in any one of [1] to [4] further includes, between step 1 and step 2, a step of distributing the uniformly dispersed plastic fluid containing cells obtained in step 1 to other containers.

[0014] Invention Effects According to the present invention, cells can be uniformly seeded on the cell adhesion surface inside the cell culture vessel. Therefore, according to the present invention, the problem of different passage timing in each vessel can be avoided, and in addition, large quantities of cells of uniform quality can be cultured. Attached Figure Description

[0015] [ Figure 1 ] Figure 1 A schematic diagram for outlining the subject matter of the present invention.

[0016] [ Figure 2 ] Figure 2 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue, uniformly dispersed in a plastic fluid containing κ-CG, in Experimental Example 1, seeded in a cell culture dish, and then the yield stress of the plastic fluid was reduced by changing the temperature of the plastic fluid from room temperature to 37°C. In the table, the vertical axis represents the number of pixels, and the horizontal axis represents brightness (…). Figure 3 The same applies afterwards.

[0017] [ Figure 3 ] Figure 3To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue in Experimental Example 2, which were seeded in a cell culture dish and then the plastic fluid was diluted with cell culture medium to reduce the yield stress of the plastic fluid.

[0018] [ Figure 4 ] Figure 4 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue, uniformly dispersed in a gelatin-containing plastic fluid, in Experimental Example 3, when these cells were seeded in a cell culture dish and the yield stress of the plastic fluid was reduced by changing the temperature of the plastic fluid from room temperature to 37°C.

[0019] [ Figure 5-1 ] Figure 5-1 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue, uniformly dispersed in a plastic fluid containing κ-CG, in Experimental Example 4, seeded in a cell culture dish, and then the yield stress of the plastic fluid was reduced by changing the temperature of the plastic fluid from room temperature to 37°C.

[0020] [ Figure 5-2 ] Figure 5-2 for Figure 5-1 The following image is a continuation of the previous one.

[0021] [ Figure 6 ] Figure 6 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue uniformly dispersed in a plastic fluid containing κ-CG in Experimental Example 5, when the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid.

[0022] [ Figure 7 ] Figure 7 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue, uniformly dispersed in a plastic fluid containing κ-CG, in Experimental Example 6, seeded in a cell culture dish, and then the yield stress of the plastic fluid was reduced by changing the temperature of the plastic fluid from room temperature to 37°C.

[0023] [ Figure 8 ] Figure 8 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue uniformly dispersed in a plastic fluid containing κ-CG in Experimental Example 7, when the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid.

[0024] [ Figure 9 ] Figure 9 To illustrate the adhesion state of normal human skin fibroblasts (NHDF) uniformly dispersed in a plastic fluid containing κ-CG in Experimental Example 8, seeded on a cell culture plate, and then the yield stress of the plastic fluid was reduced by changing the temperature of the plastic fluid from room temperature to 37°C.

[0025] [ Figure 10 ] Figure 10 To illustrate the adhesion state of mesenchymal stem cells derived from human adipose tissue, uniformly dispersed in a plastic fluid containing κ-CG, in Experimental Example 9, seeded onto a cell culture plate, and then the yield stress of the plastic fluid was reduced by changing the temperature of the plastic fluid from room temperature to 37°C.

[0026] [ Figure 11 ] Figure 11 This figure illustrates the results of using 253G1 as the human induced pluripotent stem cell line in Experiment 11. It should be noted that in the histogram of the Voronoi region area, the vertical axis represents frequency (represented as Freq in the figure), and the horizontal axis represents relative area (…). Figures 12-14 (The same applies).

[0027] [ Figure 12 ] Figure 12 The figure shows the results when 1383D2 was used as a human induced pluripotent stem cell line in Experiment 11.

[0028] [ Figure 13 ] Figure 13 The figure shows the results when RPChiPS771 was used as a human induced pluripotent stem cell line in Experiment 11.

[0029] [ Figure 14 ] Figure 14 A graph showing the results of test example 13. Detailed Implementation

[0030] The present invention will now be described in detail.

[0031] A method for uniformly seeding cells or tissues onto the cell adhesion surface inside the container. This invention provides a method for uniformly seeding cells or tissues onto the cell adhesion surface inside a container (hereinafter sometimes referred to as "the method of this invention"), comprising the following steps: (Step 1) The step of uniformly dispersing the cells in the plastic fluid; and (Step 2) The step of adhering cells or tissues to the cell adhesion surface inside the container.

[0032] In the method of the present invention, the cell is not particularly limited and can be derived from animal cells or plant cells.

[0033] In one aspect of the present invention, the cells can be adhesive cells.

[0034] In the method of this invention, the term "adhesive cell" is not particularly limited, and examples include stem cells, precursor cells, adult non-stem cells, primary cultured cells, cell lines, and cancer cells. Stem cells are cells that possess both the ability to self-replicate and the ability to differentiate into multiple cell lineages. Examples of adhesive stem cells are not limited to the following, but include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, neural stem cells, hematopoietic stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and other adult stem cells. Mesenchymal stem cells refer to stem cells capable of differentiating into all or several types of osteocytes, chondrocytes, and adipocytes. Mesenchymal stem cells exist at low frequencies in tissues such as bone marrow, peripheral blood, umbilical cord blood, and adipose tissue, and can be isolated from these tissues using known methods. Precursor cells are cells in the intermediate stage of differentiation from the aforementioned stem cells into specific somatic cells or germ cells. Examples of adhesive progenitor cells include, but are not limited to, those such as preadipocytes, precardiomyocytes, preendothelial cells, neural progenitor cells, hepatic progenitor cells, pancreatic progenitor cells, and renal progenitor cells. Examples of adhesive adult non-stem cells include, but are not limited to, those such as fibroblasts, osteocytes, periosteal cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, heart cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic β cells, and melanocytes. Primary cultured cells refer to cells in the state of culture between cell / tissue seeding from an organism and the first passage. Primary cultured cells can be cells collected from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testes, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain, or nerve tissue. A cell line refers to cells that have acquired unlimited proliferative capacity through artificial manipulation outside a living organism. The adhesive cells in the method of this invention are preferably stem cells or precursor cells, more preferably mesenchymal stem cells or induced pluripotent stem cells (iPS cells), adipose-derived stem cells contained in vascular matrix components (SVF), or adipose-derived precursor cells.

[0035] The source of the adhesive cells in the method of this invention is not particularly limited and can be any cells from animals and plants. As for animals, there are no limitations; examples include fish, amphibians, reptiles, birds, pancrustaceans, hexapods, mammals, etc., with mammals being preferred. Examples of mammals are not limited and include rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypuses, dolphins, whales, dogs, cats, goats, cattle, horses, sheep, pigs, elephants, marmosets, squirrel monkeys, macaques, chimpanzees, humans, etc. As for plants, there are no particular limitations as long as the collected cells can be cultured in liquid. Examples of plants that produce medicinal materials (such as saponins, alkaloids, berberine, scopolamine, phytosterols, etc.) include medicinal ginseng, periwinkle, hyoscyamine, coptis, belladonna, etc.; plants that produce pigments and polysaccharides used as raw materials for cosmetics and food (such as anthocyanins, safflower pigments, madder pigments, crocin pigments, flavonoids, etc.) include blueberries, safflower, madder, saffron, etc.; or plants that produce raw materials for pharmaceuticals, etc., but are not limited to these.

[0036] The tissue in the method of this invention is a structural unit composed of several cells with different characteristics and functions assembled in a certain way. Examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, and nervous tissue. Examples of plant tissues include dividing tissue, epidermal tissue, assimilating tissue, mesophyll tissue, vascular tissue, mechanical tissue, parenchyma tissue, and dedifferentiated cell masses (callus). The tissue in the method of this invention may also include spheroids, organoids, etc.

[0037] In this specification, "plastic fluid" refers to a fluid that requires yield stress to flow, i.e., a fluid with a yield value. Plastic fluids can be Bingham fluids or non-Bingham fluids.

[0038] In the method of the present invention, the plastic fluid is not particularly limited as long as it is a plastic fluid that can retain cells or tissues without sedimentation in a static state. Regarding the yield value of the plastic fluid, it is preferably 5 mPa or more from the viewpoint of dispersing and retaining cells or tissues, and preferably 500 mPa or less from the viewpoint of operability. The yield value of the plastic fluid can be determined using methods known per se. As an example, it can be derived by measuring using a rheometer (Anton Paar, model: MCR301, conical rotor: CP75-1).

[0039] In another aspect, from an operational point of view, the viscosity of the plastic fluid used in the method of the present invention is preferably 1.5 to 200 mPa·s at 4 to 25°C. Furthermore, the viscosity of the plastic fluid can also be measured using methods known to exist. For example, it can be measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, conical rotor: standard rotor 1°34'×R24, rotational speed 10 to 100 rpm).

[0040] Plastic fluids can be prepared using methods known to them. For example, plastic fluids can be prepared by mixing polysaccharides into a liquid that is not a plastic fluid.

[0041] Polysaccharides capable of transforming non-plastic fluids into plastic fluids are known. Examples include polysaccharides composed of more than 10 monosaccharides and oligosaccharides (e.g., trioses, tetras, pentoses, hexoses, heptaoses, etc.), and more preferably, acidic polysaccharides possessing anionic functional groups. The acidic polysaccharides described herein are not particularly limited as long as they possess anionic functional groups in their structure. Examples include polysaccharides containing uronic acids (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides with sulfate or phosphate groups locally in their structure, or polysaccharides possessing both of these structures. These polysaccharides can also be naturally occurring, microbially produced, genetically engineered, or artificially synthesized using enzymes. More specifically, acidic polysaccharides include substances composed of one or more of the following groups: carrageenan, deacylated gellan gum (hereinafter sometimes referred to as "DAG"), alginate (hereinafter sometimes referred to as "ALG"), gellan gum, rhamsan gum, diutangum, hyaluronic acid, hexuronic acid, fucoidan, pectin, pectic acid, pectin ester acid, heparan sulfate, heparin, heparitin sulfate, keratin sulfate, chondroitin sulfate, dermatin sulfate, rhamnose sulfate, and their salts. It should be noted that salts include, for example, salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; or salts of aluminum, zinc, steel, iron, ammonium, organic bases, and amino acids, but are not limited to these.

[0042] The polysaccharides used in this invention may preferably be carrageenan, deacylated gellan gum, alginate, dette gum, hyaluronic acid, or their salts, but are not limited thereto.

[0043] Polysaccharides can be used in combination in multiple ways (e.g., two types). The types of polysaccharides in a combination are not particularly limited, but preferably include DAG or its salts. That is, preferred combinations of polysaccharides include DAG or its salts, and polysaccharides other than DAG or its salts (e.g., ALG, xanthan gum, locust bean gum, methylcellulose, diter gum, or their salts). Specific examples of polysaccharide combinations include DAG with sodium alginate, DAG with xanthan gum, DAG with locust bean gum, DAG with methylcellulose, DAG with diter gum, xanthan gum and locust bean gum, etc., but are not limited to these.

[0044] The concentration of polysaccharides used to convert non-plastic fluids into plastic fluids depends on the type of polysaccharide, but it can be appropriately set within a range that allows the plastic fluid to uniformly disperse cells or tissues, preferably at a concentration that enables filtration-based sterilization.

[0045] For example, when carrageenan is used as a polysaccharide, the content can be 0.001 to 0.1% (weight / volume), preferably 0.005 to 0.08% (weight / volume), more preferably 0.005 to 0.06% (weight / volume), further preferably 0.01 to 0.05% (weight / volume), and most preferably 0.02 to 0.04% (weight / volume).

[0046] Furthermore, when using a combination of DAG or its salts with polysaccharides other than DAG or its salts, the concentration of DAG or its salts can be exemplified as 0.005 to 0.02% (weight / volume), preferably 0.01 to 0.02% (weight / volume), and the concentration of polysaccharides other than DAG or its salts can be exemplified as 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). The following combinations can be cited as specific concentration ranges.

[0047] DAG or its salt: 0.005–0.08% (preferably 0.01–0.04%) (by weight / volume) Polysaccharides other than DAG Sodium alginate: 0.01–0.2% (by weight / volume) Xanthan gum: 0.01–0.2% (by weight / volume) Locust bean gum: 0.01–0.2% (by weight / volume) Methylcellulose: 0.01–0.2% (by weight / volume) (preferably 0.2–0.4% (by weight / volume)) Dieter adhesive: 0.01~0.2% (by weight / volume) Carboxymethyl cellulose: 0.01–0.2% (by weight / volume) In another aspect of the present invention, a plastic fluid can be prepared using deacylated gellan gum and alginate. When using this combination, the concentration of deacylated gellan gum or its salt can be 0.002% (wt / volume) or more, preferably 0.003% (wt / volume) or more, and can be 0.01% (wt / volume) or less, preferably 0.009% (wt / volume) or less. Furthermore, the concentration of alginate or its salt can be 0.004% (wt / volume) or more, preferably 0.005% (wt / volume) or more, and can be 0.1% (wt / volume) or less, preferably 0.02% (wt / volume) or less, more preferably 0.015% (wt / volume) or less.

[0048] In another embodiment, the alginate or its salt is set to 1 or more parts by mass, preferably 2 or more parts by mass, relative to 1 part by mass of deacylated gellan gum or its salt. In one embodiment, the alginate or its salt can be set to, for example, 1 to 4 parts by mass, preferably 1 to 3 parts by mass, and more preferably 1 to 2 parts by mass, relative to 1 part by mass of deacylated gellan gum or its salt.

[0049] It should be noted that the concentration of polysaccharides can be calculated using the following formula.

[0050] Concentration (%) = Weight of polysaccharide (g) / Volume of plastic fluid (ml) × 100 [Metal cations] In one approach, metal cations, such as divalent metal cations (calcium ions, magnesium ions, zinc ions, iron ions, and copper ions, etc.), are added to the plastic fluid used in the method of the present invention, with calcium ions being preferred. Especially when using water-soluble polysaccharides such as deacylated gellan gum to convert a non-plastic fluid into a plastic fluid, it is sometimes preferable to add monovalent and / or divalent metal cations as needed. This is because by including metal cations, water-soluble polysaccharides such as deacylated gellan gum aggregate through the metal cations to form nanofibers in the composition, which constructs a three-dimensional network, thereby enabling the uniform dispersion of cells or tissues.

[0051] In another approach, nanofibers containing polysaccharides can also be used in the preparation of plastic fluids.

[0052] By adding nanofibers containing polysaccharides to a non-plastic fluid, it is possible to transform a non-plastic fluid into a plastic fluid.

[0053] In this specification, nanofibers refer to fibers with an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofibers used in this 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, there is a concern that the nanofibers may be too fine to achieve a suspension effect, potentially preventing the improvement of the properties of the composition containing them.

[0054] The aspect ratio (L / D) of the nanofibers used in this invention is obtained by dividing the average fiber length by the average fiber diameter, and is typically 2 to 500, preferably 5 to 300, and more preferably 10 to 250. When the aspect ratio is less than 2, the dispersibility in the composition is insufficient, raising concerns about inadequate suspension. When it exceeds 500, the fiber length becomes extremely large, thus increasing the viscosity of the composition and hindering operability such as filling, transferring, or adding the liquid composition to a culture tank. Furthermore, the composition becomes less transparent to visible light, leading to reduced transparency and potentially making long-term observation of cells or tissues difficult.

[0055] It should be noted that the average fiber diameter (D) of the nanofibers can be determined, for example, by the following method. First, a collodion support film manufactured by Oken Shoji Co., Ltd. was subjected to hydrophilication treatment for 3 minutes using an IonCleaner (JIC-410) manufactured by Nippon Electronics Co., Ltd. A few drops of the nanofiber dispersion (diluted with ultrapure water) as the evaluation object were added, and the film was dried at room temperature. The nanofibers were observed using a transmission electron microscope (TEM, H-8000) (10,000x) manufactured by Hitachi, Ltd. at an accelerating voltage of 200kV. Using the obtained images, the diameter of each nanofiber was measured for a sample of 200 to 250 nanofibers, and the average of these measurements was taken as the average fiber diameter (D).

[0056] Furthermore, the average fiber length (L) was determined as follows: The nanofiber dispersion was diluted with pure water to a concentration of 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 pre-treated with concentrated sulfuric acid for hydrophilicity, 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) (2000x magnification) manufactured by Nippon Electron Co., Ltd., the length of each nanofiber was measured for a sample size of 150–250 fibers, and the average of these measurements was taken as the average fiber length (L).

[0057] The nanofibers used in this invention have the following effects: when mixed with a non-plastic fluid, the nanofibers are uniformly dispersed in the liquid while maintaining the primary fiber diameter, without substantially increasing the viscosity of the liquid, and can substantially retain cells or tissues, preventing them from settling.

[0058] The viscosity of liquids containing nanofibers can be evaluated using a tuning fork viscometer (SV-1A, A&D Company Ltd.) at 25°C.

[0059] Polysaccharides, which are considered raw materials for nanofibers, include not only naturally occurring polysaccharides, but also substances produced by microorganisms, substances produced through genetic engineering, or substances artificially synthesized using enzymes or chemical reactions. The polysaccharides constituting the nanofibers used in this invention are preferably naturally occurring substances (i.e., substances extracted from nature) or substances obtained through chemical or enzymatic modifications.

[0060] In one aspect of the present invention, the polysaccharides constituting the nanofibers may include cellulose, hemicellulose, and other cellulose-based substances, but are not limited thereto.

[0061] Cellulose is a natural polymer compound formed by the β-1,4 glycosidic bond of D-glucanose, which forms the 6-membered ring of glucose. As raw materials, it can be derived from plants such as wood, bamboo, hemp, jute, hibiscus, cotton, agricultural crops, and food residues, or from microorganisms such as Cladophora, glaucophytes, Valonia, and sea squirts. In the case of plant-derived cellulose, very fine fibers called microfibrils further bundle together, forming higher-order structures such as fibrils, lamellae, and fibroblasts. Bacterial cellulose, on the other hand, is a network structure formed from the microfibrils of cellulose secreted by bacterial cells, maintaining their original thickness.

[0062] In this invention, high-purity cellulose raw materials such as cotton and bacterial cellulose can be used directly as raw materials. Other plant-derived cellulose materials are preferably separated and purified. The preferred cellulose materials used in this invention are cotton cellulose, bacterial cellulose, kraft pulp cellulose, and microcrystalline cellulose. Kraft pulp cellulose is particularly preferred due to its high suspending effect.

[0063] Nanofibers containing polysaccharides can be prepared using methods already known.

[0064] As an example, in the case of cellulose nanofibers, nanofibers are typically obtained by pulverizing the raw material. The pulverization method is not limited, but in order to refine the fibers to the fiber diameter and length described later for the purposes of this invention, methods that can obtain strong shear forces, such as high-pressure homogenizers, grinding machines (mortars), or media stirring mills like bead mills, are preferred.

[0065] Among these methods, a high-pressure homogenizer is preferred for micronization, and it is desirable to use a wet pulverization method as disclosed in, for example, Japanese Patent Application Publication No. 2005-270891 and Japanese Patent No. 5232976. Specifically, this can be achieved using an apparatus that pulverizes the raw material by spraying a dispersion formed by dispersing the raw material from a pair of nozzles at high pressure and causing them to collide, such as the Starburst System (high-pressure pulverizing apparatus manufactured by Sugino Machine Limited) and NanoVater (high-pressure pulverizing apparatus manufactured by Yoshida Machinery Co., Ltd.).

[0066] When using the aforementioned high-pressure homogenizer to refine (pulverize) the raw material, the degree of refinement and homogenization depends on the pressure pumped into the ultra-high-pressure chamber of the high-pressure homogenizer, the number of times the material passes through the ultra-high-pressure chamber (processing times), and the concentration of the raw material in the aqueous dispersion. The pumping pressure (processing pressure) is typically 50–250 MPa, preferably 150–245 MPa. If the pumping pressure is below 50 MPa, the refinement of the nanofibers becomes insufficient, raising concerns that the desired effect cannot be achieved through refinement.

[0067] Furthermore, the concentration of the raw material in the aqueous dispersion during micronization is 0.1% to 30% by mass, preferably 1% 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; if the concentration is higher than 30% by mass, the pulverization efficiency is low, and the desired nanofibers cannot be obtained. The number of micronization (pulverization) processes is not particularly limited, although it also depends on the concentration of the raw material in the aqueous dispersion. However, when the raw material concentration is 0.1% to 1% by mass, approximately 10 to 100 processes are sufficient for adequate micronization, while at 1% to 10% by mass, approximately 10 to 1000 processes are required. Moreover, at high concentrations exceeding 30% by mass, thousands of processes are required, and the viscosity increases to a level that hinders operation, making it impractical from an industrial perspective.

[0068] The concentration of nanofibers in the plastic fluid used in the method of the present invention is not particularly limited as long as it is a concentration that can convert a non-plastic fluid liquid into a plastic fluid. It is 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 0.005% to 0.1% (weight / volume), and even more preferably 0.005% to 0.05% (weight / volume).

[0069] For example, in the case of cellulose nanofibers, the content is typically 0.0001% to 1.0% (weight / volume), such as 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).

[0070] In the case of pulp cellulose nanofibers, the lower limit of the concentration 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. Furthermore, in the case of pulp cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (weight / volume) or less, or 0.04% (weight / volume) or less.

[0071] In the case of microcrystalline cellulose nanofibers, the lower limit of the concentration is preferably 0.01% (weight / volume) or more, 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more. Alternatively, the lower limit of the concentration of microcrystalline cellulose nanofibers is preferably 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more. Furthermore, in the case of microcrystalline cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (weight / volume) or less.

[0072] For non-water-soluble nanofibers such as cellulose nanofibers, a concentration of less than 0.1% (by weight / volume) will generally not substantially increase the viscosity of plastic fluids.

[0073] It should be noted that the concentration of nanofibers in the composition can be calculated using the following formula.

[0074] Concentration (%) (weight / volume) = weight of nanofibers (g) / volume of plastic fluid (ml) × 100 In another approach, in the preparation of plastic fluids, in addition to the aforementioned polysaccharides or nanofibers containing polysaccharides, substances that form a three-dimensional network structure in the liquid, thereby altering the liquid's flow properties (hereinafter sometimes referred to as "other substances") can also be used. Examples of other substances include, but are not limited to, gelatin, collagen, fibroin, laminin, elastin, matrix gel, and hyaluronic acid.

[0075] The concentration of other substances used to convert non-plastic fluids into plastic fluids also depends on the type of substance, but it can be appropriately set within the range that allows the plastic fluid to uniformly disperse cells or tissues.

[0076] For example, when gelatin is used as another substance, it can be 0.5 to 5% by mass, preferably 0.5 to 4% by mass, more preferably 0.5 to 3% by mass, even more preferably 0.7 to 2% by mass, and most preferably 0.8 to 1.5% by mass (e.g., 1% by mass).

[0077] In the preparation of the plastic fluid used in this invention, a plastic fluid is prepared by adding polysaccharides, polysaccharide-containing nanofibers, or other substances that can alter the flow properties of a non-plastic fluid to a liquid. The non-plastic fluid to which polysaccharides, polysaccharide-containing nanofibers, or other substances that can alter the flow properties of a liquid are added is not particularly limited, as long as it does not affect the survival of cells or tissues. Examples of non-plastic fluids include water, buffer solutions, and liquid culture media for culturing cells or tissues, but are not limited to these. In a preferred embodiment, the non-plastic fluid may be a buffer solution.

[0078] In step 1 of this invention, cells or tissues are uniformly dispersed in a plastic fluid. The method for uniformly dispersing cells or tissues in a plastic fluid is not particularly limited. As an example, this can be achieved by mixing a liquid containing cells or tissues with a plastic fluid, and then mixing the mixture using a micropipette or the like. It should be noted that the term "uniformly" in this specification refers not only to a state where cells or tissues are completely and equally dispersed, but also to a state where cells or tissues, while not completely (i.e., in all areas where they exist) equally dispersed, are equally dispersed in most of their areas (e.g., in 50% or more, preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the total area). Furthermore, the term "homogenization" in this specification refers not only to a state where cells or tissues are completely and equally dispersed, but also to a state where cells or tissues are brought close to that state. For example, in this specification, when cells or tissues are more evenly dispersed compared to embodiments that do not use plastic fluids, it is also described as cells or tissues being homogenized.

[0079] Furthermore, in step 2 of the present invention, the cells or tissues uniformly dispersed in the plastic fluid prepared in step 1 are adhered to the cell adhesion surface on the inner side of the container. The method for adhering the cells or tissues to the cell adhesion surface on the inner side of the container is not particularly limited. As an example, a method that generates an external force exceeding the yield stress between the cell adhesion surface and the cells or tissues can be cited. Examples of such external forces include gravity, centrifugal force generated by rotating the container, and electrostatic force generated by electrical charge.

[0080] In one embodiment of the invention, step 2 can also be achieved by reducing the yield stress of the plastic fluid in which cells or tissues are uniformly dispersed, obtained in step 1, thereby causing the cells or tissues to adhere to the cell adhesion surface on the inner side of the container. The yield stress is reduced to the point where the cells or tissues uniformly dispersed in the plastic fluid can no longer maintain their dispersion and settle to the bottom of the container. Those skilled in the art can easily determine the extent to which the yield stress needs to be reduced before the cells or tissues settle.

[0081] There are no particular limitations on the methods used to reduce the yield stress of plastic fluids, as long as they do not substantially have an adverse effect on the survival or proliferation of dispersed cells or tissues. Any method can be used.

[0082] For example, as a method to reduce the yield stress of a plastic fluid, it can be achieved by diluting the plastic fluid by adding a non-plastic fluid liquid to it, thereby reducing the concentration of polysaccharides and polysaccharide-containing nanofibers.

[0083] The non-plastic fluid liquid added for the purpose of diluting the plastic fluid can be any liquid, as long as it does not substantially have an adverse effect on the survival or proliferation of cells or tissues. Examples of such liquids include, but are not limited to, cell culture media and buffer solutions.

[0084] In addition, the reduction of yield stress in plastic fluids can sometimes be achieved by changing the temperature of the plastic fluid.

[0085] For example, a plastic fluid can be made by adding polysaccharides to a liquid that is not a plastic fluid, but it is known that the gelation temperature of the polysaccharides varies depending on their concentration. Therefore, by appropriately adjusting the concentration of the added polysaccharides, a plastic fluid can be prepared that is plastic at room temperature (15–25°C), but no longer plastic at temperatures suitable for cell culture (30–39°C). Using this plastic fluid, the yield stress of the plastic fluid can be reduced by performing step 1 of the method of the present invention (and the dispensing steps described in detail below) at room temperature, followed by step 2, where the plastic fluid (or the plastic fluid dispensed into other cell containers) uniformly dispersed with cells or tissue obtained in step 1 is supplied to a temperature suitable for cell culture.

[0086] It should be noted that the manufacture of plastic fluids with the above-mentioned temperature characteristics is known to those skilled in the art.

[0087] In one embodiment, the method of the present invention further includes, between step 1 and step 2, a step of distributing the uniformly dispersed plastic fluid containing cells obtained in step 1 to other containers (hereinafter sometimes referred to as the "distribution step").

[0088] In step 1, a plastic fluid in which cells are uniformly dispersed is prepared and then distributed to containers such as cell adhesion dishes. In step 2, cells or tissues are evenly seeded on the cell adhesion surfaces inside multiple containers.

[0089] The allocation step can use one or more containers.

[0090] It should be noted that the containers used in this invention are not particularly limited as long as they are capable of allowing cells to adhere and be cultured. Examples of such containers include multi-plate dishes, microplates, microplates, multi-plates, multi-well plates, chambered slides, trays, tissue culture dishes, culture flasks, culture bags, or culture tubes, but are not limited to these. Furthermore, the materials of these culture containers are not particularly limited. Examples include glass, cellulose polymers such as polyvinyl chloride, ethyl cellulose, and acetyl cellulose, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, polyethylene, polybutadiene, poly(ethylene-vinyl acetate) copolymer, poly(butadiene-styrene) copolymer, poly(butadiene-acrylonitrile) copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methyl methacrylate) copolymer, polychloroprene, styrene resin, chlorosulfonated polyethylene, ethylene vinyl acetate, acrylic block copolymers, and other plastics.

[0091] The invention will be further illustrated in the following embodiments, but the invention is not limited by these examples.

[0092] Example [Preparation Example 1] Preparation of a culture medium composition containing κ-carrageenan One part by weight of κ-carrageenan (hereinafter referred to as κ-CG) (WR-80-J, Sansho Co., Ltd.) and 99 parts by weight of purified water were added to a glass culture medium bottle. After stirring and suspending, the mixture was autoclaved (121°C, 20 minutes) to prepare a sterile 1% by weight κ-CG aqueous solution. Next, 48.5 mL of mesenchymal stem cell proliferation medium 2 (#C-28009, TakaraBio Inc.) was dispensed into a 50 mL conical tube (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) in a biosafety cabinet. 1.5 mL of the sterile 1% by weight κ-CG aqueous solution prepared above was mixed by pipetting, thereby preparing a culture medium composition containing 0.03% by weight κ-CG.

[0093] [Preparation Example 2] Preparation of a culture medium composition comprising deacylated gellan gum and sodium alginate One part by weight of sodium alginate (hereinafter referred to as ALG) (KIMICA ALGIN IL-2, manufactured by KIMIA Co., Ltd.) and 99 parts by weight of purified water were added to a glass culture medium bottle. After stirring and suspending, the mixture was autoclaved (121°C, 20 minutes) to prepare a 1% by weight ALG aqueous solution. A 1% by weight deacylated gellan gum (hereinafter referred to as DAG) aqueous solution (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) was prepared in the same manner. The ALG and DAG aqueous solutions were dispensed into conical tubes at a ratio of 2:1 (v / v) and carefully mixed by pipetting and aspiration using a disposable syringe equipped with a needle to homogenize the mixture, thus preparing an ALG / DAG mixture. Next, the mixture was prepared using a culture medium preparation kit (FCeM (registered trademark) - series Preparation Kit, manufactured by Nissan Chemical Co., Ltd.) in a biosafety cabinet. Dispense 49.5 mL of mesenchymal stem cell proliferation medium 2 (#C-28009, Takara Bio Inc.) into a 50 mL conical tube (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) and cap it with the adapter cap, which is a component of the kit. Connect the syringe by inserting the tip of a disposable syringe filled with 0.5 mL of ALG / DAG mixture into the cylindrical part of the adapter cap. By manually pressing the plunger of the syringe, the ALG / DAG mixture in the syringe is injected into the container with strong force, instantly mixing it with the culture medium, thereby preparing a culture medium composition containing 0.01% by mass DAG / ALG.

[0094] [Preparation Example 3] Preparation of a culture medium composition containing gelatin Dispense 49.5 mL of mesenchymal stem cell proliferation medium 2 (#C-28009, Takara Bio Inc.) into a 50 mL conical tube (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.), and add 0.5 g of gelatin powder (beMatrixgelatin LS-H, manufactured by Nitta Gelatin Co., Ltd.). Heat in a 37°C water bath for 30 minutes to fully dissolve the gelatin, then pass the liquid through a 0.22 μm sterile filter to prepare a sterile culture medium composition containing 1.00% by weight gelatin.

[0095] [Preparation Example 4] Preparation of Cell Fixation Solution and Cell Staining Solution Cell fixative was prepared by adding 10.5 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) to 3.5 mL of 4% paraformaldehyde-phosphate buffer (163-20145, manufactured by FUJIFILM Wako Pure Chemical Corporation) and mixing by pipetting. Cell staining solution was prepared by dissolving 0.09 parts by weight of crystal violet (038-17792, manufactured by FUJIFILM Wako Pure Chemical Corporation) in 10 parts by weight of ethanol (manufactured by FUJIFILM Wako Pure Chemical Corporation) and mixing the resulting solution with 89.91 parts by weight of purified water.

[0096] [Experimental Example 1] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: 15cm adhesion cell culture dish (culture area 152cm²)). 2 )) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and aliquoted into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) at a rate of 1 million cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 30 mL of a culture medium composition containing 0.03% κ-CG was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were resuspended in 30 mL of mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, each ADSC suspension was transferred to a 15 cm cell adhesion dish (MS-10150, manufactured by Sumitomo Bakelite Co., Ltd.) for cell seeding. All the above steps were performed at room temperature (15–30°C). After culturing at 37°C and 5% carbon dioxide for 2 days, the supernatant was removed under various conditions. 15 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, gently shaken, and the supernatant was removed again. Next, 14 mL of the prepared cell fixative was added, and the mixture was allowed to stand for 15 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, gently shaken, and the supernatant was removed again. Finally, 3 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, gently shaken, and the supernatant was removed again. This process was repeated twice, and photographs were taken with a light source placed on the bottom surface. In addition, cells cultured for 4 days underwent the same treatment to evaluate cell adhesion status at each culture day. Furthermore, image analysis software was used to analyze cell distribution in each sample. Details of the analysis conditions are shown below.

[0097] <Analysis Conditions> Analysis software: ImageJ (NIH) • Filter: Median Filter Type: 16-bit Analysis: Calculate the standard deviation based on the histogram (the number of pixels present at each brightness level). The results are shown in Figure 2In random cell staining images after 2 days and 4 days of culture, a noticeable difference in the density of cells adhering to the bottom surface was observed compared to the control group. The bias was suppressed by adding κ-CG, and the distribution of cell adhesion on the bottom surface became significantly more uniform.

[0098] [Experimental Example 2] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing DAG / ALG (container: 15cm adhesion cell culture dish (culture area 152cm²)). 2 )) ADSCs (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and dispensed into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) at a concentration of 1 million cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 15 mL of a culture medium composition containing 0.01% DAG / ALG was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were suspended in 15 mL of mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, each ADSC suspension was transferred in 15 mL to a 15 cm cell adhesion dish (MS-10150, manufactured by Sumitomo Bakelite Co., Ltd.), and further diluted with 15 mL of mesenchymal stem cell culture medium 2 before seeding. All the above steps were performed at room temperature (15–30°C). Next, after culturing at 37°C and 5% carbon dioxide for 2 or 4 days, the supernatant was removed, and 15 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed again. Then, 14 mL of the prepared cell fixation solution was added, and the mixture was allowed to stand for 15 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was gently shaken. Finally, 3 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was gently shaken. This process was repeated twice, and photographs were taken with a light source placed on the bottom surface. Regarding cell adhesion status at each culture day, cell distribution in each sample was analyzed using image analysis software based on photographs taken on day 2 or day 4 of each culture. The analysis conditions were the same as in Experiment 1.

[0099] The results are shown in Figure 3 In random cell staining images after 2 days and 4 days of culture, a noticeable difference in the density of cells adhering to the bottom surface was observed compared to the control group. The bias of the samples inoculated with the DAG / ALG-containing culture medium composition was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0100] [Experimental Example 3] Cell inoculation experiment using a gelatin-containing culture medium composition (container: 15cm adhesion cell culture dish (culture area 152cm²)). 2 )) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and aliquoted into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) at a rate of 1 million cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 30 mL of a culture medium composition containing 1.00% gelatin was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were resuspended in 30 mL of mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, each ADSC suspension was transferred to a 15 cm cell adhesion dish (MS-10150, manufactured by Sumitomo Bakelite Co., Ltd.) for cell seeding. All the above steps were performed at room temperature (15–30°C). After culturing at 37°C and 5% carbon dioxide for 3 days, the supernatant was removed, and 15 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. The mixture was gently shaken, and the supernatant was removed again. Next, 14 mL of the prepared cell fixation solution was added, and the mixture was allowed to stand for 15 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was gently shaken, and the supernatant was removed again. Finally, 3 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was gently shaken, and the supernatant was removed again. This process was repeated twice, and photographs were taken with a light source placed on the bottom surface. Cell distribution in each sample was analyzed using image analysis software based on the photographs taken on day 3 of culture. The analytical conditions are the same as those in Experiment 1.

[0101] The results are shown in Figure 4 In the cell staining images after 3 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. The bias of the sample inoculated with the gelatin-containing culture medium composition was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0102] [Experimental Example 4] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: 5-layer culture flask for adhesion cells (culture area 875 cm²)). 2 )) Mesenchymal stem cells (hereinafter referred to as ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and aliquoted into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) at a concentration of 5 million cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 50 mL of a culture medium composition containing 0.03% (w / v) κ-CG was added, and the ADSCs were resuspended by pipetting. 25 mL of each ADSC were transferred to 100 mL conical tubes (2355-100, manufactured by AGC Techno Glass Co., Ltd.), and each tube was diluted by adding 50 mL of a culture medium composition containing 0.03% (w / v) κ-CG. The ADSCs were then resuspended again by pipetting. As a comparison, ADSCs were resuspended in mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, with the adhered cells in a 5-layer culture flask (353144, Corning), transfer each ADSC suspension medium separately, tilting the flask at a 45° angle with the mixing port facing down to mix the cell suspension. Then, again, stand the flask upright to evenly distribute the cell suspension among the layers, tilting the flask at a 45° angle with the mixing port facing up and the culture surface facing down to seed the cells into the culture vessel. The above steps were performed at room temperature (15–30°C). Next, culture at 37°C with 5% CO2 for 1 day. After confirming cell adhesion to the culture vessel, remove the supernatant and add 150 mL of mesenchymal stem cell culture medium 2 to each layer to replace the medium, ensuring equal volume. After further culturing for 3 days, remove the supernatant, add 50 mL of D-PBS (045-29795, FUJIFILM Wako Pure Chemical Corporation), gently shake, and remove the supernatant. Next, add 70 mL of the prepared cell fixation solution and let stand for 15 minutes. Remove the supernatant, add 50 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation), gently shake, and remove the supernatant. Then, repeat the following washing procedure twice: add 30 mL of the prepared cell staining solution, let stand for 3 minutes, and remove the supernatant. Next, add 50 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation), gently shake, and remove the supernatant. Finally, use an ultrasonic cutter (Z0-41, manufactured by Nisshin EM Co., Ltd.) to cut each layer of the culture vessel, and take photographs of each segment with a light source placed on the bottom.Cell distribution in each sample was analyzed using image analysis software based on photographs taken on day 4 of culture. The analysis conditions were the same as in Experiment 1.

[0103] The results are shown in Figure 5. In the cell staining images after 4 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. The bias of the sample inoculated with the culture medium composition containing κ-CG was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0104] [Experimental Example 5] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: adhesion cell culture flask (culture area 225 cm²)). 2 )) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and aliquoted into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) at a rate of 1.5 million cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 45 mL of a culture medium composition containing 0.03% (w / v) κ-CG was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were resuspended in mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, each ADSC suspension was transferred to an adhesion cell culture flask (MS-21800, manufactured by Sumitomo Bakelite Co., Ltd.) for cell seeding. All the above steps were performed at room temperature (15–30°C). Next, the cells were cultured at 37°C and 5% CO2 for one day. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 45 mL of mesenchymal stem cell culture medium 2 was added to each container to replace the medium. After further culturing for 1 or 3 days, the supernatant was removed, and 25 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed. Next, 21 mL of the prepared cell fixation solution was added, and the mixture was allowed to stand for 15 minutes. The supernatant was removed, and 45 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed. Next, 5 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. The supernatant was then removed. Next, the following washing procedure was repeated twice: 45 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and after gentle shaking, the supernatant was removed. Finally, photographs were taken with a light source placed on the bottom surface. Cell distribution in each sample was analyzed using image analysis software based on photographs taken on day 2 or day 4 of culture. The analysis conditions were the same as in Experiment 1.

[0105] The results are shown in Figure 6 In random cell staining images after 2 days and 4 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. The bias of the sample inoculated with the culture medium composition containing κ-CG was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0106] [Experimental Example 6] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: 6-well adhesion cell culture multi-well plate (culture area 9.5 cm²)). 2 / hole)) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and aliquoted into 15 mL conical tubes (50015, manufactured by SPL Life Sciences) at a density of 360,000 cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 12 mL of a culture medium composition containing 0.03% (w / v) κ-CG was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were resuspended in mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, each ADSC suspension was aliquoted into 6-well adhesion cell culture multi-well plates (353046, manufactured by Corning) at a density of 2 mL per well. All the above steps were performed at room temperature (15–30°C). Next, the cells were cultured at 37°C and 5% carbon dioxide for one day. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 3 mL of mesenchymal stem cell culture medium 2 was added to each container to replace the culture medium. After further culturing for 3 days, the supernatant was removed, and 1 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed. Next, 1.5 mL of the prepared cell fixation solution was added, and the mixture was allowed to stand for 15 minutes. The supernatant was removed, and 1 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed. Next, 3 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. The supernatant was then removed. Next, the following washing procedure was repeated twice: 1 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and after gentle shaking, the supernatant was removed. Finally, images were taken using a CELL3IMAGER DUOS2 cell morphology imaging system (manufactured by SCREEN), and image analysis software was used to analyze the cell distribution of each sample on day 4 of culture. The analysis conditions were the same as in Experiment 1.

[0107] The results are shown in Figure 7In the cell staining images after 4 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. The bias of the sample inoculated with the culture medium composition containing κ-CG was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0108] [Experimental Example 7] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: 96-well adhesion cell culture multi-well plate (culture area 0.32 cm²)). 2 / hole)) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell culture medium 2 and aliquoted into 15 mL conical tubes (50015, manufactured by SPL Life Sciences) at a density of 360,000 cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 12 mL of a culture medium composition containing 0.03% (w / v) κ-CG was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were resuspended in mesenchymal stem cell culture medium 2 following the same procedure. After standing for 10 minutes, each ADSC suspension was aliquoted into 96-well adhesion cell culture multi-well plates (MS-8096F, manufactured by Sumitomo Bakelite Co., Ltd.) at a density of 125 μL per well. All steps were performed at room temperature (15–30°C). Next, the cells were cultured at 37°C and 5% carbon dioxide for one day. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 200 μL of mesenchymal stem cell culture medium 2 was added to each container to replace the culture medium. After further culturing for 2 days, the supernatant was removed, and 100 μL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed. Next, 150 μL of the prepared cell fixation solution was added, and the mixture was allowed to stand for 15 minutes. The supernatant was removed, and 100 μL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed. Finally, 300 μL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes before removing the supernatant. Next, the following washing procedure was repeated twice: 100 μL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and after gentle shaking, the supernatant was removed. Finally, images were taken using a CELL3IMAGER DUOS2 (manufactured by SCREEN) cell morphology analysis imaging system, and the cell distribution of each sample on day 1 and day 3 of culture was analyzed using image analysis software. The analysis conditions were the same as in Experiment 1.

[0109] The results are shown in Figure 8 In the cell staining images after 1 day and 3 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. The bias of the sample inoculated with the culture medium composition containing κ-CG was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0110] [Experimental Example 8] Inoculation test of normal human skin fibroblasts using a culture medium composition containing κ-CG (container: 15cm adhesion cell culture dish (culture area 152cm²)). 2 )) Normal human skin fibroblasts (manufactured by Takara Bio Inc.) (hereinafter referred to as NHDF) were suspended in fibroblast proliferation medium 2 (C-23020, manufactured by Takara Bio Inc.) and dispensed at a rate of 1 million cells / vial into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.). After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 30 mL of a culture medium composition containing 0.03% κ-CG, prepared using fibroblast proliferation medium 2 according to the same procedure as in Preparation Example 1, was added, and the NHDF was resuspended by pipetting. As a comparison, NHDF was also resuspended in 30 mL of fibroblast proliferation medium 2 according to the same procedure described above. After standing for 10 minutes, each NHDF suspension was transferred to a 15 cm cell adhesion culture dish (MS-10150, manufactured by Sumitomo Bakelite Co., Ltd.) for cell seeding. The above steps were performed at room temperature (15–30°C). After culturing at 37°C and 5% carbon dioxide for 3 days, the supernatant was removed, and 15 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. The mixture was gently shaken, and the supernatant was removed again. Next, 14 mL of the prepared cell fixative was added, and the mixture was allowed to stand for 15 minutes. The supernatant was removed, and 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. The mixture was gently shaken, and the supernatant was removed again. Next, 3 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. The supernatant was then removed. The following washing procedure was repeated twice: 30 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was gently shaken, and the supernatant was removed. Finally, photographs were taken with a light source placed on the bottom. The cell distribution of each sample was analyzed using image analysis software based on the photographs. The analysis conditions were the same as in Experiment 1.

[0111] The results are shown in Figure 9 In the cell staining images after 3 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. By adding κ-CG, the bias was suppressed, and the distribution of cell adhesion on the bottom surface became significantly more uniform.

[0112] [Experimental Example 9] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: 1-well adhesion cell culture plate (culture area 97.4 cm²)). 2 / hole)) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were suspended in mesenchymal stem cell proliferation medium 2 and aliquoted into 50 mL conical tubes at a rate of 600,000 cells / tube. After centrifugation at 300×G for 3 minutes, the supernatant was removed. Then, 20 mL of a culture medium composition containing 0.03% (w / v) κ-CG was added, and the ADSCs were resuspended by pipetting. As a comparison, ADSCs were resuspended in mesenchymal stem cell proliferation medium 2 following the same procedure. After standing for 10 minutes, 20 mL of the total volume was transferred to a 1-well adhesion cell culture plate (229101, manufactured by CELLTREAT Scientific Products). The above steps were performed at room temperature (15–30°C). The cells were then cultured at 37°C under 5% CO2 atmosphere for 1 day. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 20 mL of mesenchymal stem cell proliferation medium 2 was added to replace the culture medium. After further culturing for 3 days, the supernatant was removed, and 10 mL of D-PBS (045-29795, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added. After gentle shaking, the supernatant was removed again. Next, 10 mL of the prepared cell fixation solution was added, and the mixture was allowed to stand for 15 minutes. The supernatant was removed again, and 10 mL of D-PBS was added. After gentle shaking, the supernatant was removed again. Then, 3 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes. The supernatant was then removed. Next, the following washing procedure was repeated twice: 10 mL of D-PBS was added, and the mixture was gently shaken. Finally, images were taken using a CELL3IMAGER DUOS (manufactured by SCREEN) cell morphology analysis imaging system, and the cell distribution of each sample was analyzed using image analysis software.

[0113] The results are shown in Figure 10 In the cell staining images after 4 days of culture, the density of cells adhering to the bottom surface was observed relative to that in the control group. The bias of the sample inoculated with the culture medium composition containing κ-CG was suppressed, and the distribution of cell adhesion on the bottom surface was significantly more uniform.

[0114] [Experimental Example 10] Seeding experiment of mesenchymal stem cells derived from human adipose tissue using a culture medium composition containing κ-CG (container: 1-well adhesion cell culture plate (culture area 97.4 cm²)).2 / hole)) Mesenchymal stem cells (ADSCs) derived from human adipose tissue (manufactured by CellSource Co., Ltd.) were seeded at a rate of 600,000 cells in a one-well adhesion cell culture plate (229101, manufactured by CELLTREAT Scientific Products) using a culture medium composition containing 0.03% (w / v) κ-CG, in the same manner as in Example 9. As a comparison, mesenchymal stem cell proliferation medium 2 was used for seeding following the same procedure. After 4 days of culture, cells were detached using a Detach Kit (C-41220, manufactured by PromoCell). The detached cells were collected in 50 mL centrifuge tubes, centrifuged at 220 × g for 3 minutes, the supernatant was removed, and the cells were resuspended in 10 mL of mesenchymal stem cell proliferation medium 2. The cell suspension was then resuspended in trypan blue solution, and measurements were taken twice using a TC20 automated cell counter (manufactured by BIORAD), and the average value was calculated.

[0115] The results are shown in Table 1. It was confirmed that when comparing cell counts after 4 days of culture, the samples inoculated with the κ-CG-containing culture medium composition had a higher cell count than the control group. The κ-CG-containing culture medium composition suppressed cell bias, thus enabling more efficient cell proliferation.

[0116] [Table 1] Next, the cell suspension was washed with FACS buffer (D-PBS (-) with 2% FBS). PE-labeled mouse anti-human CD105 antibody (560839, BD Biosciences), BV421-labeled mouse anti-human CD73 antibody (562430, BD Biosciences), APC-labeled mouse anti-human CD90 antibody (559869, BD Biosciences), PE-labeled mouse anti-human CD34 antibody (555822, BD Biosciences), and FITC-labeled mouse anti-human CD11b antibody (562793, BD Biosciences) were added to the cells suspended in FACS buffer again, and the mixture was incubated at room temperature in the dark for 30 minutes. After washing with FACS buffer, the cell suspension was measured using FACSFortessaX-20 (BD Biosciences).

[0117] The results are shown in Table 2. For cells cultured for 4 days, the positivity rates of CD105, CD73, and CD90, as positive markers, were high, while the expression of CD34 and CD11b, as negative markers, was almost unobserved. Therefore, it was confirmed that the κ-CG-containing composition did not affect the expression of surface markers in ADSCs.

[0118] [Table 2] [Preparation Example 5] Preparation of a culture medium composition containing κ-carrageenan A 1% (w / w) κ-CG aqueous solution was prepared in the same manner as in Preparation Example 1. 48.5 mL of StemFit AK02N (manufactured by AJINOMOTO CO., INC.) was dispensed into a 50 mL conical tube (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) inside a biosafety cabinet, and 1.5 mL of the sterile 1% (w / w) κ-CG aqueous solution was mixed by pipetting, thereby preparing a culture medium composition containing 0.03% (w / w) κ-CG.

[0119] [Experimental Example 11] Seeding experiment of human induced pluripotent stem cells using a culture medium composition containing κ-CG (container: 8-well cell culture plate (culture area 10.5 cm²)). 2 / hole)) Human induced pluripotent stem cells (iPSCs) lines 253G1, 1383D2 (obtained from the iPS Cell Research Institute, Kyoto University), and RPChiPS771 (obtained from REPROCELL) were cultured using StemFit AK02N medium (manufactured by AJINOMOTO CO., INC.). The culture medium contained 0.5 μg / cm³ of [amount missing]. 2Cells were coated in iMatrix511silk (387-10131, Nippi) 6-well flat-bottomed plates (351146, Corning). After washing with D-PBS (-), 0.5 mL of TrypLE Express Enzyme (1X) (12604013, ThermoFisher Scientific) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 10 minutes. The cells were then detached and collected into 15 mL conical tubes using StemFit medium. The cells were aliquoted into 15 mL conical tubes at a rate of 8000 cells / tube, centrifuged at 200×G for 3 minutes, and the supernatant was removed. Next, 2 mL of the culture medium composition containing 0.03% (w / v) κ-CG prepared in Preparation Example 5 was added, and the iPSCs were resuspended by pneumatic aspiration. Y-27632 (manufactured by FUJIFILM Wako Pure Chemical Corporation) was then added to a final concentration of 10 μM. As a comparison, iPSCs were resuspended in StemFit AK02N medium containing 10 μM Y-27632 following the same procedure. 2 mL of the cell suspension was seeded into a medium pre-concentrated with 0.75 μg / cm³ of κ-CG. 2The cells were coated with iMatrix511 silk in Nunc rectangular culture dishes (267062, Thermo Fisher Scientific). The above steps were performed at room temperature (15–30°C). Next, the cells were cultured at 37°C with 5% CO2 for 3 hours. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 2 mL of StemFit AK02N medium containing 10 μM Y-27632 was added to each dish for medium replacement. The same method was used to replace the medium on the second and fourth days after inoculation using StemFit AK02N medium (without Y-27632). After 5 days of culture, the supernatant was removed, 2 mL of D-PBS (-) was added, and after gentle shaking, the supernatant was removed again. Next, 2 mL of the prepared cell fixative was added, and the mixture was allowed to stand for 15 minutes. The supernatant was removed, 2 mL of D-PBS (-) was added, and after gentle shaking, the supernatant was removed again. Next, 0.5 mL of the prepared cell staining solution was added, and after standing for 3 minutes, the supernatant was removed. The following washing procedure was repeated twice: 2 mL of D-PBS (-) was added, gently shaken, and the supernatant was removed. Finally, images were captured using a CELL3IMAGER DUOS (SCREEN) cell morphology analysis imager. Using Python image analysis, Voronoi diagrams were created based on the center coordinates of each colony, and the areas of the Voronoi regions were compared to analyze the colony distribution of each sample.

[0120] It should be noted that a Voronoi diagram is a graph that segments regions based on which seed point is closer to other points in the same metric space relative to multiple seed points placed at arbitrary locations. The boundary of a Voronoi region becomes part of the bisecting lines of each seed point, and the intersection of these boundaries is called a Voronoi point. For the segmented region, the farther away from surrounding seed points, the larger the area of ​​the region; the closer to surrounding seed points, the smaller the area of ​​the region. Therefore, with uniform cell seeding, the Voronoi region has a fixed area; with non-uniform cell seeding, the deviation of the Voronoi region increases. Based on this, the standard deviation of the Voronoi region area is calculated. Details of the analysis conditions are shown below.

[0121] <Analysis Conditions> Python runtime environment: JupyterLab • Libraries and functions used: OpenCV, NumPy, scipy (Voronoi functions), Shapely (Polygon functions), pandas, matplotlib • Mean filter: cv2.blur (5×5 cores) Binarization: cv2.threshold • Obtain the centroid coordinates of each settlement: cv2.connectedComponentsWithStats • Creating a Voronoi plot based on barycentric coordinates: voronoi_plot_2d • Get the coordinates of the Voronoi point: vor.vertices • Calculate the area of ​​the Voronoi region based on the coordinates of the Voronoi point: Polygon • Calculate the standard deviation for the area of ​​the Voronoi region: NumPy.std The results are shown in Figures 11-13 Image analysis of cell staining images after 5 days of culture confirmed that the standard deviation decreased in all cell lines, indicating that the use of a culture medium composition containing κ-CG reduced cell bias.

[0122] [Experimental Example 12] Seeding experiment of human induced pluripotent stem cells using a culture medium composition containing κ-CG (container: 8-well cell culture plate (culture area 10.5 cm²)). 2 / hole)) Human induced pluripotent stem cell (iPSC) lines 253G1, 1383D2, and RPChiPS771 were cultured using StemFit AK02N medium (manufactured by AJINOMOTO CO., INC.). The culture medium used was 0.5 μg / cm³. 2The iPSCs were coated with iMatrix511 silk in a 6-well flat-bottom plate (351146, Corning). After washing the obtained cells with D-PBS (-), 0.5 mL of TrypLE Express Enzyme (1X) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 10 minutes, after which the cells were detached. The obtained cells were recovered into 15 mL conical tubes using StemFit medium. The cells were aliquoted into 15 mL conical tubes at a rate of 8000 cells / tube, centrifuged at 200×G for 3 minutes, and the supernatant was removed. Next, 2 mL of the culture medium composition containing 0.03% (w / v) κ-CG prepared in Preparation Example 5 was added, and the iPSCs were resuspended by pipette. Y-27632 (FUJIFILM Wako PureChemical Corporation) was then added to a final concentration of 10 μM. Additionally, as a comparison, iPSCs were suspended in StemFit AK02N medium containing 10 μM Y-27632 following the same procedure described above. 2 mL of the cell suspension was seeded into a medium pre-mixed with 0.75 μg / cm³ of water. 2Cells were coated with iMatrix511 silk in Nunc rectangular culture dishes (267062, Thermo Fisher Scientific). The above steps were performed at room temperature (15–30°C). Cells were then cultured at 37°C with 5% CO2 for 3 hours. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 2 mL of StemFit AK02N medium containing 10 μM Y-27632 was added to each well for medium replacement. This process was repeated daily from day 2 to day 4 after inoculation, using StemFit AK02N medium (without Y-27632). On day 7, the supernatant was removed, and 1 mL / well of TrypLE Express Enzyme (1X) was added. Cells were incubated at 37°C with 5% CO2 for 10 minutes, and then detached. The resulting cells were collected into 15 mL conical tubes using StemFit medium. After washing with D-PBS (-), the cells were suspended in 4% paraformaldehyde-phosphate buffer (163-20145, manufactured by FUJIFILM Wako Pure Chemical Corporation) and fixed by incubation at room temperature for 15 minutes. Next, the cells were washed with FACS buffer (D-PBS (-) containing 2% FBS) and resuspended in FACS buffer. Cells at seed were similarly fixed and washed / resuspended with FACS buffer, and stored refrigerated until assay. Cells suspended in FACS buffer were supplemented with Alexa Fluor647-labeled mouse anti-human SSEA-4 antibody (560796, manufactured by BD Biosciences) and PE-labeled mouse anti-human TRA-1-60 antibody (560193, manufactured by BD Biosciences), and incubated at room temperature in the dark for 30 minutes. After washing three times with FACS buffer, the cell suspension was analyzed using FACS FortessaX-20 (manufactured by BD Biosciences).

[0123] The results are shown in Table 3. After 7 days of culture, the expression of SSEA-4 and TRA-1-60, which are markers of undifferentiated human iPSCs, remained at a high level, confirming that the κ-CG-containing composition did not affect the maintenance of undifferentiated iPSCs.

[0124] [Table 3] [Experimental Example 13] Seeding experiment of human induced pluripotent stem cells using a culture medium composition containing κ-CG (container: 1-well adhesion cell culture plate (culture area 97.4 cm²)).2 / hole)) Human induced pluripotent stem cell (iPSC) line RPChiPS771 was cultured using StemFit AK02N medium. The culture medium contained 0.5 μg / cm³ of [a specific substance / component]. 2 The iPSCs were coated with iMatrix511 silk in a 6-well flat-bottom plate (351146, Corning). After washing the obtained cells with D-PBS (-), 0.5 mL of TrypLE Express Enzyme (1X) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 10 minutes, after which the cells were detached. The obtained cells were recovered into 15 mL conical tubes using StemFit medium. The cells were aliquoted into 50 mL conical tubes at a rate of 80,000 cells / tube, centrifuged at 200×G for 3 minutes, and the supernatant was removed. Next, 20 mL of the culture medium composition containing 0.03% (w / v) κ-CG prepared in Preparation Example 5 was added, and the iPSCs were resuspended by pipette. Y-27632 (FUJIFILM Wako Pure Chemical Corporation) was then added to a final concentration of 10 μM. Additionally, as a comparison, iPSCs were suspended in StemFit AK02N medium containing 10 μM Y-27632 following the same procedure described above. 20 mL was inoculated into a medium pre-mixed with 0.75 μg / cm³ of Y-27632. 2Cells were cultured in a 1-well adhesive cell culture plate (229101, manufactured by CELLTREAT Scientific Products) coated with iMatrix511 silk. The above steps were performed at room temperature (15–30°C). Cells were then cultured at 37°C with 5% CO2 for 3 hours. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 20 mL of StemFit AK02N medium containing 10 μM Y-27632 was added to each well for medium replacement. Cells were then cultured again at 37°C with 5% CO2, and the same medium replacement was performed on the second and fourth days after inoculation using StemFit AK02N medium (without Y-27632). After 5 days of culture, the supernatant was removed, 10 mL of D-PBS (-) was added, and the mixture was gently shaken and then removed. Next, 10 mL of the prepared cell fixative was added, and the mixture was allowed to stand for 15 minutes. The supernatant was then removed, 10 mL of D-PBS (-) was added, and the mixture was gently shaken and then removed. Next, 5 mL of the prepared cell staining solution was added, and after standing for 3 minutes, the supernatant was removed. The following washing procedure was repeated twice: 10 mL of D-PBS (-) was added, gently shaken, and the supernatant was removed. Finally, images were captured using a CELL3IMAGER DUOS (SCREEN) cell morphology analysis imager. Using Python image analysis, Voronoi diagrams were created based on the center coordinates of each colony, and the areas of the Voronoi regions were compared to analyze the colony distribution of each sample. The analytical conditions were the same as in Example 11.

[0125] The results are shown in Figure 14 In the cell staining images after 5 days of culture, a noticeable difference in cell density was observed relative to the control group. The bias in the samples inoculated using the κ-CG-containing culture medium composition was suppressed, and the distribution of adherent cells on the bottom surface was significantly more uniform. Furthermore, image analysis also confirmed a decrease in the standard deviation of the Voronoi region area, indicating that the use of the κ-CG-containing culture medium composition reduced cell bias.

[0126] Industrial availability According to the present invention, cells can be uniformly seeded onto the cell adhesion surface inside the cell culture vessel. Therefore, the problem of inconsistent passage timing in each vessel can be avoided, and large quantities of cells of uniform quality can be cultured. Thus, the present invention is extremely useful in medical fields such as cell therapy.

[0127] This application is based on Japanese Special Application 2024-6204 (filed on January 18, 2024), the contents of which are fully contained in this specification.

Claims

1. A method for uniformly seeding cells or tissues onto the cell adhesion surface inside a container, comprising the following steps: (Step 1) The step of uniformly dispersing cells or tissues in a plastic fluid; and (Step 2) The step of adhering cells or tissues to the cell adhesion surface inside the container.

2. The method as described in claim 1, wherein, Step 2 includes a step of reducing the yield stress of the plastic fluid.

3. The method as described in claim 2, characterized in that, The yield stress of the plastic fluid is reduced by diluting the plastic fluid.

4. The method as described in claim 2, characterized in that, The yield stress of the plastic fluid is reduced by changing its temperature.

5. The method according to any one of claims 1 to 4, further comprising, between step 1 and step 2, distributing the uniformly dispersed plastic fluid containing cells obtained in step 1 to other containers.

Images