Method for uniformly seeding cells

Abstract

The present invention provides a method for uniformly seeding cells or tissue onto a cell attachment surface on the inner side of a container. The method comprises: (step 1) a step for uniformly dispersing cells or tissue in a plastic fluid; and (step 2) a step for causing cells or tissue to attach to the cell attachment surface on the inner side of the container.

Description

How to seed cells uniformly

[0001] The present invention relates to a method for uniformly seeding cells onto a cell adhesive surface (assuming similar modifications below) inside a vessel.

[0002] In recent years, the development of cell therapy-based disease cure technologies has been progressing worldwide. To carry out cell therapy, the technology to mass-produce high-quality cells is essential.

[0003] Cells can be grown by seeding them in containers and culturing them. However, uneven seeding of cells on the cell adhesion surface inside the container can lead to problems such as different passage times for each container and significant changes in the culture environment in each container, resulting in inconsistent quality of the produced cells. Therefore, technology for uniformly seeding cells is extremely important for mass cell production. However, with conventional cell suspensions, waves are generated in the suspension when cells are seeded, which causes a problem of uneven distribution of the cells.

[0004] A method for uniformly distributing cells into aliquots using a plastic fluid has been reported (Patent Document 1). However, this method is directed to distributing cells so that the cell number is uniform and then cryopreserving the uniformly distributed cells into the aliquots, and does not anticipate seeding the distributed cells at a uniform density on the inner surface of a container. Furthermore, the present inventors have reported that cells can be efficiently cultured in a suspension state by using a medium containing polysaccharides (Patent Document 2).

[0005] WO2020 / 036180WO2014 / 017513

[0006] An object of the present invention is to provide a novel method for seeding cells or tissues at a uniform density (for achieving a uniform seeding density) onto the inner cell adhesive surface of a cell culture vessel.

[0007] As a result of intensive research into the above-mentioned problems, the present inventors have discovered that cells can be seeded extremely uniformly onto the cell adhesion surface inside a cell culture vessel by uniformly dispersing cells in a plastic fluid, distributing the plastic fluid with the cells uniformly dispersed therein into a cell culture vessel, and then adhering the cells or tissues to the inner surface of the vessel. Based on this finding, further research has led to the completion of the present invention. That is, the present invention is as follows.

[0008] [1] A method for uniformly seeding cells or tissues onto a cell adhesive surface inside a container, comprising the following steps: (Step 1) uniformly dispersing cells or tissues in a plastic fluid, and (Step 2) adhering cells or tissues to the cell adhesive surface inside the container. [2] The method of [1], which comprises a step of reducing the yield stress of the plastic fluid in Step 2. [3] The method of [2], characterized in that the reduction in the yield stress of the plastic fluid is achieved by diluting the plastic fluid. [4] The method of [2], characterized in that the reduction in the yield stress of the plastic fluid is achieved by changing the temperature of the plastic fluid. [5] The method of any of [1] to [4], further comprising a step of distributing the plastic fluid in which the cells have been uniformly dispersed obtained in Step 1 into another container between Steps 1 and 2.

[0009] According to the present invention, cells can be uniformly seeded onto the cell adhesion surface inside the cell culture vessel, thereby avoiding the problem of different passage timings for different vessels and enabling the mass cultivation of cells of uniform quality.

[0010] Figure 1 is a schematic diagram outlining the problem to be solved by the present invention. Figure 2 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing κ-CG, seeded on a cell culture dish in Test Example 1, and then the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid. In the graph, the vertical axis represents the number of pixels, and the horizontal axis represents brightness (the same applies to Figure 3 and subsequent figures). Figure 3 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing DAG / ALG, seeded on a cell culture dish in Test Example 2, and then the yield stress of the plastic fluid was reduced by diluting the plastic fluid with cell culture medium. Figure 4 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing gelatin, seeded on a cell culture dish in Test Example 3, and then the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid. Figure 5-1 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing κ-CG, seeded on a cell culture dish in Test Example 4, and then the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid. Figure 5-2 is a continuation of Figure 5-1. Figure 6 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing κ-CG, seeded on a cell culture dish in Test Example 5, and then the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid. Figure 7 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing κ-CG in Test Example 6, seeded on a cell culture dish, and then the temperature of the plastic fluid was changed from room temperature to 37°C to reduce the yield stress of the plastic fluid.Figure 8 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing κ-CG when seeded onto a cell culture dish and the temperature of the plastic fluid was then changed from room temperature to 37°C to reduce the yield stress of the plastic fluid (Test Example 7). Figure 9 is a diagram and graph showing the adhesion state of normal human dermal fibroblasts (NHDF) uniformly dispersed in a plastic fluid containing κ-CG when seeded onto a cell culture plate and the temperature of the plastic fluid was then changed from room temperature to 37°C to reduce the yield stress of the plastic fluid (Test Example 8). Figure 10 is a diagram and graph showing the adhesion state of human adipose tissue-derived mesenchymal stem cells uniformly dispersed in a plastic fluid containing κ-CG when seeded onto a cell culture plate and the temperature of the plastic fluid was then changed from room temperature to 37°C to reduce the yield stress of the plastic fluid (Test Example 9). Figure 11 shows the results of Test Example 11, where 253G1 was used as the human induced pluripotent stem cell line. In the histogram of Voronoi region area, the vertical axis represents Frequency (represented as Freq in the figure) and the horizontal axis represents Relative area (the same applies to Figures 12 to 14). Figure 12 shows the results of Test Example 11, where 1383D2 was used as the human induced pluripotent stem cell line. Figure 13 shows the results of Test Example 11, where RPChiPS771 was used as the human induced pluripotent stem cell line. Figure 14 shows the results of Test Example 13.

[0011] The present invention will be described in detail below.

[0012] Method for uniformly seeding cells or tissues onto a cell adhesive surface inside a container The present invention provides a method for uniformly seeding cells or tissues onto a cell adhesive surface inside a container (hereinafter, sometimes referred to as the "method of the present invention"), which comprises the following steps: (Step 1) uniformly dispersing cells in a plastic fluid; (Step 2) adhering the cells or tissues to the cell adhesive surface inside the container.

[0013] In the method of the present invention, the cells are not particularly limited, and may be cells of animal origin or cells of plant origin.

[0014] In one embodiment of the present invention, the cells may be adherent cells.

[0015] In the method of the present invention, examples of adhesive cells include, but are not limited to, stem cells, progenitor cells, somatic non-stem cells, primary culture cells, cell lines, cancer cells, etc. Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples of adhesive stem cells include, but are not limited to, somatic stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, neural stem cells, hematopoietic stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Mesenchymal stem cells are stem cells that have the ability to differentiate into all or some of the following: bone cells, chondrocytes, and adipocytes. Mesenchymal stem cells are present 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. Progenitor cells are cells that are in the process of differentiating from the stem cells into specific somatic cells or germ cells. Examples of adhesive progenitor cells include, but are not limited to, preadipocytes, precursor cardiac muscle cells, precursor endothelial cells, neural progenitor cells, hepatic progenitor cells, pancreatic progenitor cells, and kidney progenitor cells. Examples of adhesive somatic non-stem cells include, but are not limited to, fibroblasts, osteocytes, bone pericytes, 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, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, and melanocytes. Primary culture cells refer to cells that have been isolated from a living body and seeded and are in a culture state until the first passage is performed. Primary culture 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, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain, or nerve tissue. A cell line refers to cells that have acquired the ability to proliferate indefinitely through artificial manipulation outside of a vitro system.The adherent cells in the method of the present invention are preferably stem cells or progenitor cells, and more preferably mesenchymal stem cells or induced pluripotent stem cells (iPS cells), adipose-derived stem cells contained in stromal vascular fibril (SVF), or preadipocytes.

[0016] The origin of the adherent cells in the method of the present invention is not particularly limited, and they may be cells derived from either animals or plants. Examples of animals include, but are not limited to, fish, amphibians, reptiles, birds, pancrustaceans, hexapods, and mammals, with mammals being preferred. 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. Plants are not particularly limited, as long as the collected cells can be cultured in liquid. Examples of such plants include, but are not limited to, plants that produce herbal medicines (e.g., saponin, alkaloids, berberine, scopolin, plant sterols, etc.) (e.g., ginseng, periwinkle, henbane, coptis, belladonna, etc.), plants that produce pigments and polysaccharides (e.g., anthocyanins, safflower pigments, madder pigments, saffron pigments, flavones, etc.) that are used as raw materials for cosmetics and foods (e.g., blueberries, safflower, madder, saffron, etc.), and plants that produce pharmaceutical ingredients.

[0017] In the method of the present invention, a tissue is a structural unit in which cells with several types of different properties and functions are assembled in a certain pattern. Examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, nervous tissue, etc. Examples of plant tissues include meristem, epidermal tissue, assimilation tissue, mesophyll tissue, conductive tissue, mechanical tissue, parenchyma tissue, dedifferentiated cell mass (callus), etc. In the method of the present invention, tissues may also include spheroids and organoids.

[0018] As used herein, the term "plastic fluid" refers to a fluid that requires a yield stress in order to flow, i.e., a fluid that has a yield value. Plastic fluids may be Bingham or non-Bingham fluids.

[0019] In the method of the present invention, the plastic fluid is not particularly limited as long as it can retain cells or tissues without sedimentation in a stationary state. However, the yield value of the plastic fluid is preferably 5 mPa or more from the viewpoint of retaining cells or tissues in a dispersed state, and preferably 500 mPa or less from the viewpoint of operability. The yield value of the plastic fluid can be measured by a method known per se. For example, it can be derived by measurement using a rheometer (manufactured by Anton Paar, model: MCR301, cone rotor: CP75-1). In one embodiment, 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 from the viewpoint of operability. The viscosity of the plastic fluid can also be measured by a method known per se. For example, it can be measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1°34' x R24, rotation speed 10 to 100 rpm).

[0020] The plastic fluid can be prepared by a method known per se, for example, by mixing a polysaccharide with a liquid that is not a plastic fluid.

[0021] Polysaccharides capable of transforming a non-plastic liquid into a plastic fluid are known. Examples include polysaccharides formed by polymerizing 10 or more monosaccharides or oligosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.), and more preferably, acidic polysaccharides having anionic functional groups. The acidic polysaccharides referred to herein are not particularly limited as long as they have anionic functional groups in their structure, but may include, for example, polysaccharides having uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides having sulfate or phosphate groups in part of their structure, or polysaccharides having both structures. These polysaccharides may be naturally occurring polysaccharides, polysaccharides produced by microorganisms, polysaccharides produced by genetic engineering, or polysaccharides artificially synthesized using enzymes. More specifically, acidic polysaccharides include those composed of one or more of the group consisting of carrageenan, deacylated gellan gum (hereinafter sometimes referred to as "DAG"), alginic acid (hereinafter sometimes referred to as "ALG"), gellan gum, rhamsan gum, diutan gum, hyaluronic acid, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof. Examples of salts include, but are not limited to, salts of alkali metals such as lithium, sodium, and potassium, salts of alkaline earth metals such as calcium, barium, and magnesium, and salts of aluminum, zinc, copper, iron, ammonium, organic bases, and amino acids.

[0022] The polysaccharide used in the present invention may preferably be, but is not limited to, carrageenan, deacylated gellan gum, alginic acid, diutan gum, hyaluronic acid, or salts thereof.

[0023] Multiple types of polysaccharides (e.g., two types) can be used in combination. The type of polysaccharide combination is not particularly limited, but preferably, the combination includes DAG or a salt thereof. That is, suitable polysaccharide combinations include DAG or a salt thereof, and polysaccharides other than DAG or a salt thereof (e.g., ALG, xanthan gum, locust bean gum, methylcellulose, diutan gum, or salts thereof). Specific polysaccharide combinations include, but are not limited to, DAG and sodium alginate, DAG and xanthan gum, DAG and locust bean gum, DAG and methylcellulose, DAG and diutan gum, xanthan gum and locust bean gum, etc.

[0024] The concentration of polysaccharides required to turn a liquid that is not a plastic fluid into a plastic fluid depends on the type of polysaccharide, but may be set appropriately within a range that allows the plastic fluid to uniformly disperse cells or tissues, and it is preferable that the concentration be such that sterilization by filtration is possible.

[0025] For example, when carrageenan is used as the polysaccharide, the content may 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), even more preferably 0.01 to 0.05% (weight / volume), and most preferably 0.02 to 0.04% (weight / volume).

[0026] When a combination of DAG or a salt thereof with a polysaccharide other than DAG or a salt thereof is used, the concentration of DAG or a salt thereof is, for example, 0.005 to 0.02% (weight / volume), preferably 0.01 to 0.02% (weight / volume), and the concentration of the polysaccharide other than DAG or a salt thereof is, for example, 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). Specific examples of combinations in concentration ranges are as follows:

[0027] DAG or its salt: 0.005 to 0.08% (preferably 0.01 to 0.04%) (weight / volume) Polysaccharides other than DAG Sodium alginate: 0.01 to 0.2% (weight / volume) Xanthan gum: 0.01 to 0.2% (weight / volume) Locust bean gum: 0.01 to 0.2% (weight / volume) Methylcellulose: 0.01 to 0.2% (weight / volume) (preferably 0.2 to 0.4%) (weight / volume) Diutan gum: 0.01 to 0.2% (weight / volume) Carboxymethylcellulose: 0.01 to 0.2% (weight / volume)

[0028] In another embodiment of the present invention, a plastic fluid can be prepared using deacylated gellan gum and alginic acid. When using such a combination, the concentration of deacylated gellan gum or a salt thereof can be 0.002 (wt / vol)% or more, preferably 0.003 (wt / vol)% or more, and 0.01 (wt / vol)% or less, preferably 0.009 (wt / vol)% or less. The concentration of alginic acid or a salt thereof can be 0.004 (wt / vol)% or more, preferably 0.005 (wt / vol)% or more, and 0.1 (wt / vol)% or less, preferably 0.02 (wt / vol)% or less, more preferably 0.015 (wt / vol)% or less.

[0029] In another embodiment, the amount of alginic acid or a salt thereof is 1 part by mass or more, preferably 2 parts by mass or more, per part by mass of deacylated gellan gum or a salt thereof. In one embodiment, the amount of alginic acid or a salt thereof can be, for example, 1 to 4 parts by mass, preferably 1 to 3 parts by mass, and more preferably 1 to 2 parts by mass, per part by mass of deacylated gellan gum or a salt thereof.

[0030] The concentration of the polysaccharide can be calculated by the following formula: Concentration (%) = Weight of polysaccharide (g) / Volume of plastic fluid (ml) × 100

[0031] [Metal Cations] In one embodiment, metal cations, for example, divalent metal cations (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), preferably calcium ions, may be added to the plastic fluid used in the method of the present invention. In particular, when a water-soluble polysaccharide such as deacylated gellan gum is used to convert a liquid that is not a plastic fluid into a plastic fluid, it may be preferable to add monovalent and / or divalent metal cations as needed. This is because the inclusion of metal cations causes the water-soluble polysaccharide such as deacylated gellan gum to aggregate via the metal cations, forming nanofibers in the composition, which then construct a three-dimensional network, thereby enabling cells or tissues to be uniformly dispersed.

[0032] In another embodiment, nanofibers made of polysaccharides may be used to prepare the plastic fluid.

[0033] By adding nanofibers made of polysaccharides to a liquid that is not a plastic fluid, the liquid can be made into a plastic fluid.

[0034] As used herein, 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 achieve a flotation effect, and may not lead to improvement in the properties of the composition containing them.

[0035] The aspect ratio (L / D) of the nanofibers used in the present 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. An aspect ratio of less than 2 may result in a lack of dispersibility in the composition, and a lack of sufficient floating ability. An aspect ratio of more than 500 means that the fiber length becomes extremely large, which may increase the viscosity of the composition and impair operability, such as filling or transferring the liquid composition into a container or introducing it into a culture tank. Furthermore, the composition may become less transparent to visible light, leading to a decrease in transparency and potentially making it difficult to observe cells or tissues over time.

[0036] The average fiber diameter (D) of the nanofibers can be determined, for example, as follows: First, a collodion support membrane manufactured by Oken Shoji Co., Ltd. is subjected to a hydrophilization treatment for 3 minutes using an ion cleaner (JIC-410) manufactured by JEOL Ltd., and several drops of the nanofiber dispersion to be evaluated (diluted with ultrapure water) are dropped onto the membrane, followed by drying at room temperature. This is observed with a transmission electron microscope (TEM, H-8000) manufactured by Hitachi, Ltd. (10,000x magnification) at an accelerating voltage of 200 kV, and the obtained image is used to measure the fiber diameter of each of 200 to 250 nanofiber specimens, and the number average value is taken as the average fiber diameter (D).

[0037] The average fiber length (L) is determined as follows: The nanofiber dispersion to be evaluated is diluted with pure water to 100 ppm, and the nanofibers are uniformly dispersed using an ultrasonic cleaner. This nanofiber dispersion is cast onto a silicon wafer whose surface has been previously hydrophilized using concentrated sulfuric acid, and dried at 110°C for 1 hour to obtain a sample. The obtained sample is observed with a scanning electron microscope (SEM, JSM-7400F) manufactured by JEOL Ltd. (2,000x magnification), and the fiber length of each of 150 to 250 nanofiber specimens is measured using an image, and the number average value is taken as the average fiber length (L).

[0038] When the nanofibers used in the present invention are mixed with a liquid that is not a plastic fluid, the nanofibers are uniformly dispersed in the liquid while maintaining their primary fiber diameter, and are effective in substantially retaining cells or tissues and preventing their sedimentation without substantially increasing the viscosity of the liquid. The viscosity of a liquid containing nanofibers can be evaluated using a tuning fork vibro viscometer (SV-1A, A&D Company Ltd.) at 25°C.

[0039] Polysaccharides that can be used as raw materials for nanofibers include not only naturally occurring polysaccharides but also substances produced by microorganisms, substances produced by genetic engineering, and substances artificially synthesized using enzymes or chemical reactions. The polysaccharides that make up the nanofibers used in the present invention are preferably naturally occurring substances (i.e., substances extracted from nature) or substances obtained by modifying such substances through chemical or enzymatic reactions.

[0040] In one embodiment of the present invention, the polysaccharides constituting the nanofibers include, but are not limited to, celluloses such as cellulose and hemicellulose.

[0041] Cellulose is a natural polymeric compound formed by β-1,4-glucosidic bonds between D-glucopyranose, a six-membered ring of glucose. Examples of raw materials include plant-derived cellulose, such as wood, bamboo, hemp, jute, kenaf, cotton, and agricultural and food waste. Alternatively, cellulose produced by microorganisms or animals, such as bacterial cellulose, Cladophora, Glaucocystis, Valonia, and ascidian cellulose, can be used. Plant-derived cellulose consists of extremely fine fibers called microfibrils, which are further bundled together to form a higher-order structure consisting of fibrils, lamellae, and fiber cells. Bacterial cellulose is formed by cellulose microfibrils secreted from fungal cells, which form a fine mesh structure with their original thickness.

[0042] 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. Celluloses suitable for use in the present invention include cotton cellulose, bacterial cellulose, kraft pulp cellulose, microcrystalline cellulose, etc. Kraft pulp cellulose is particularly suitable for use because of its high flotation ability.

[0043] Nanofibers made of polysaccharides can be prepared by a method known per se.

[0044] For example, in the case of cellulose nanofibers, nanofibers are usually obtained by pulverizing the raw material. Although there are no particular limitations on the pulverization method, in order to refine the raw material to the fiber diameter and fiber length described below that meet the objectives of the present invention, a method that can generate strong shear force, such as a high-pressure homogenizer, a grinder (millstone), or a media-agitating mill such as a bead mill, is preferred.

[0045] Among these, it is preferable to use a high-pressure homogenizer for the micronization, and it is desirable to use a wet milling method such as that disclosed in JP-A-2005-270891 and Japanese Patent No. 5232976. Specifically, the raw materials are milled by spraying a dispersion liquid in which the raw materials are dispersed from a pair of nozzles at high pressure and causing the nozzles to collide with each other, and this can be done using, for example, a Starburst System (a high-pressure milling device manufactured by Sugino Machine Co., Ltd.) or a Nanovaita (a high-pressure milling device manufactured by Yoshida Kikai Kogyo Co., Ltd.).

[0046] When the raw material is pulverized (pulverized) using the above-mentioned high-pressure homogenizer, the degree of pulverization and 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 pumping 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 will not be sufficiently pulverized, and the expected effect of the pulverization may not be obtained.

[0047] Furthermore, 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, productivity is low, and if it is higher than 30% by mass, the pulverization efficiency is low, and the desired nanofibers cannot be obtained. The number of micronization (pulverization) treatments is not particularly limited and 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, sufficient micronization is achieved with about 10 to 100 treatments, while when the raw material concentration is 1 to 10% by mass, about 10 to 1,000 treatments are required. Furthermore, when the concentration is higher than 30% by mass, several thousand treatments or more are required, and the viscosity increases to a level that makes handling difficult, making this method industrially unrealistic.

[0048] The concentration of nanofibers in the plastic fluid used in the method of the present invention is not particularly limited as long as it can convert a liquid that is not a plastic fluid into a plastic fluid, but is typically 0.0001% to 1.0% (wt / vol), for example, 0.0005% to 1.0% (wt / vol), preferably 0.001% to 0.5% (wt / vol), more preferably 0.005% to 0.1% (wt / vol), and even more preferably 0.005% to 0.05% (wt / vol). For example, in the case of cellulose nanofibers, the concentration is typically 0.0001% to 1.0% (wt / vol), for example, 0.0005% to 1.0% (wt / vol), preferably 0.001% to 0.5% (wt / vol), more preferably 0.01% to 0.1% (wt / vol), and even more preferably 0.01% to 0.05% (wt / vol). In the case of pulp cellulose nanofibers among cellulose nanofibers, the lower limit of the concentration is preferably 0.01% (wt / vol) or more, 0.015% (wt / vol) or more, 0.02% (wt / vol) or more, 0.025% (wt / vol) or more, or 0.03% (wt / vol) or more. Furthermore, in the case of pulp cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (wt / vol) or less, or 0.04% (wt / vol) or less. In the case of microcrystalline cellulose nanofibers, the lower limit of the concentration is preferably 0.01% (wt / vol) or more, 0.03% (wt / vol) or more, or 0.05% (wt / vol) or more. In another aspect, the lower limit of the microcrystalline cellulose nanofiber concentration is preferably 0.03% (wt / vol) or more, or 0.05% (wt / vol) or more. Furthermore, in the case of microcrystalline cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (wt / vol) or less. Water-insoluble nanofibers such as cellulose nanofibers typically do not substantially increase the viscosity of plastic fluids at a concentration of 0.1% (weight / volume) or less.

[0049] The concentration of nanofibers in the composition can be calculated using the following formula: Concentration (%) (weight / volume) = Weight of nanofibers (g) / Volume of plastic fluid (ml) × 100

[0050] In yet another embodiment, in addition to the polysaccharides or nanofibers made of polysaccharides described above, substances that form a three-dimensional network structure in a liquid and thereby change the flow properties of the liquid (hereinafter, sometimes referred to as "other substances") can be used to prepare the plastic fluid. Examples of other substances include, but are not limited to, gelatin, collagen, fibrin, laminin, elastin, Matrigel, and hyaluronic acid.

[0051] The concentration of other substances to turn a liquid that is not a plastic fluid into a plastic fluid depends on the type of substance, but may be set appropriately within a range that allows the plastic fluid to uniformly disperse cells or tissues.

[0052] For example, when gelatin is used as the other substance, the content may 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).

[0053] In preparing the plastic fluid used in the present invention, a polysaccharide, a polysaccharide nanofiber, or other substance capable of changing the flow properties of a liquid is added to a non-plastic fluid to prepare the plastic fluid. The non-plastic fluid to which the polysaccharide, the polysaccharide nanofiber, or other substance capable of changing the flow properties of a liquid is added is not particularly limited as long as it does not affect the survival of cells or tissues. Examples of non-plastic fluid liquids include, but are not limited to, water, buffer solutions, and liquid media for cell or tissue culture. In a preferred embodiment, the non-plastic fluid liquid may be a buffer solution.

[0054] In step 1 of the present 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. For example, uniform dispersion 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. As used herein, "uniformly" refers not only to a state in which the cells or tissues are completely uniformly dispersed, but also to a state in which the cells or tissues are not completely uniform (i.e., not in the entire area in which they are present), but are uniformly dispersed over most of the area (e.g., 50% or more of the entire area, 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 area). Furthermore, as used herein, "homogenization" refers not only to a state in which the cells or tissues are completely uniformly dispersed, but also to a state in which the cells or tissues are brought closer to that state. For example, in this specification, cells or tissues are referred to as homogenized when they are more uniformly dispersed than in embodiments that do not use a plastic fluid.

[0055] 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 adhesive surface inside the container. The method for adhering the cells or tissues to the cell adhesive surface inside the container is not particularly limited. One example is a method in which an external force exceeding the yield stress is generated between the cell adhesive surface and the cells or tissues. Examples of the external force include gravity, which is constantly generated, centrifugal force generated by rotating the container, and electrostatic force generated by electric charge.

[0056] In one embodiment of the present invention, in step 2 of the present invention, the cells or tissues may be adhered to the cell adhesive surface inside the container by lowering the yield stress of the plastic fluid in which the cells or tissues obtained in step 1 are uniformly dispersed. The yield stress is lowered to the extent that the cells or tissues uniformly dispersed in the plastic fluid are unable to maintain their dispersed state and sink to the bottom of the container. Those skilled in the art can easily determine how much the yield stress needs to be lowered to cause the cells or tissues to sink.

[0057] The method for reducing the yield stress of the plastic fluid is not particularly limited, and any method may be used as long as it does not substantially adversely affect the survival or proliferation of the dispersed cells or tissues.

[0058] For example, the yield stress of a plastic fluid can be reduced by adding a liquid that is not a plastic fluid to the plastic fluid to dilute the plastic fluid and thereby reduce the concentration of polysaccharides and nanofibers made of polysaccharides.

[0059] The non-plastic fluid liquid added to dilute the plastic fluid may be any liquid that does not substantially adversely affect the survival or proliferation of cells or tissues, including, but not limited to, cell culture media and buffer solutions.

[0060] Additionally, a reduction in the yield stress of a plastic fluid may be achieved by changing the temperature of the plastic fluid.

[0061] For example, adding polysaccharides to a liquid that is not a plastic fluid can turn it into a plastic fluid, but it is known that the gelation temperature of polysaccharides varies depending on the concentration. Therefore, by appropriately adjusting the concentration of the added polysaccharide, it is possible to prepare a plastic fluid that becomes a plastic fluid at room temperature (15-25°C) but is no longer a plastic fluid at a temperature suitable for cell culture (30-39°C). When using such a plastic fluid, step 1 of the method of the present invention (and the distribution step described in detail below) can be performed at room temperature, and in step 2, the plastic fluid in which cells or tissues are uniformly dispersed (or the plastic fluid distributed into a separate cell container) obtained in step 1 can be subjected to a temperature suitable for cell culture, thereby reducing the yield stress of the plastic fluid.

[0062] The production of a plastic fluid having such temperature characteristics is known to those skilled in the art.

[0063] In one aspect, the method of the present invention further comprises, between steps 1 and 2, a step of distributing the plastic fluid in which the cells obtained in step 1 are uniformly dispersed into another container (hereinafter, this step may be referred to as the "distribution step").

[0064] In step 1, a plastic fluid with cells uniformly dispersed therein is prepared and then distributed into containers such as cell culture dishes. In step 2, cells or tissues can be seeded evenly onto the cell adhesive surfaces inside multiple containers.

[0065] The number of containers used in the dispensing step may be one or more.

[0066] The vessels used in the present invention are not particularly limited as long as they can be used to adhere and culture cells. Examples of such vessels include, but are not limited to, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, trays, tissue culture dishes, culture flasks, culture bags, and culture tubes. Furthermore, the materials used for these culture vessels are not particularly limited. Examples include glass, polyvinyl chloride, cellulose-based polymers such as ethyl cellulose and acetyl cellulose, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, polyethylene, polybutadiene, poly(ethylene-vinyl acetate) copolymer, poly(butadiene-styrene) copolymer, poly(butadiene-acrylonitrile) copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, polychloroprene, styrene resin, chlorosulfonated polyethylene, ethylene vinyl acetate, and acrylic block copolymers.

[0067] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0068] Preparation Example 1: Preparation of a medium composition containing κ-carrageenan. One part by weight of κ-carrageenan (hereinafter referred to as κ-CG) (WR-80-J, Sansho Corporation) and 99 parts by weight of purified water were added to a glass medium bottle, suspended by stirring, and then autoclaved for 20 minutes at 121°C to prepare a sterile 1% by weight aqueous κ-CG solution. Next, in a safety cabinet, 48.5 mL of Mesenchymal Stem Cell Growth Medium 2 (#C-28009, Takara Bio Inc.) was dispensed into a 50 mL conical tube (MS-56500, Sumitomo Bakelite Co., Ltd.) and 1.5 mL of the sterile 1% by weight aqueous κ-CG solution prepared above was mixed by pipetting to prepare a 0.03% by weight κ-CG-containing medium composition.

[0069] Preparation Example 2: Preparation of a medium composition containing deacylated gellan gum and sodium alginate. 1 part by weight of sodium alginate (hereinafter referred to as ALG) (Kimica Algin IL-2, manufactured by Kimica Co., Ltd.) and 99 parts by weight of purified water were added to a glass medium bottle, suspended by stirring, and then autoclaved for 20 minutes to prepare a 1% by weight aqueous solution of ALG. Similarly, a 1% by weight aqueous solution of deacylated gellan gum (hereinafter referred to as DAG) (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) was prepared. The ALG and DAG aqueous solutions were dispensed into conical tubes at a ratio of 2:1 (v / v), and thoroughly mixed and homogenized by pipetting using a disposable syringe equipped with a syringe needle to prepare an ALG / DAG mixture. Next, preparation was carried out in a safety cabinet using a medium preparation kit (FCeM (registered trademark)-series Preparation Kit, manufactured by Nissan Chemical Industries, Ltd.). 49.5 mL of mesenchymal stem cell growth medium 2 (#C-28009, manufactured by Takara Bio Inc.) was dispensed into a 50 mL conical tube (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.), and an adapter cap, a component of the kit, was attached. The tip of a disposable syringe filled with 0.5 mL of the ALG / DAG mixture was fitted into the cylindrical portion of the adapter cap and connected. The syringe plunger was manually pressed, and the ALG / DAG mixture in the syringe was forcefully ejected into the container and instantly mixed with the medium, thereby preparing a 0.01% by mass DAG / ALG-containing medium composition.

[0070] Preparation Example 3: Preparation of medium composition containing gelatin. 49.5 mL of mesenchymal stem cell growth medium 2 (#C-28009, manufactured by Takara Bio Inc.) was dispensed into a 50 mL conical tube (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.), and 0.5 g of gelatin powder (beMatrix gelatin LS-H, manufactured by Nitta Gelatin Co., Ltd.) was added. This was heated in a 37°C water bath for 30 minutes to fully dissolve, and then passed through a 0.22 μm sterilization filter to prepare a sterile medium composition containing 1.00% by mass of gelatin.

[0071] [Preparation Example 4] Preparation of cell fixative and cell staining solution The cell fixative was prepared by adding 10.5 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) to 3.5 mL of 4% paraformaldehyde phosphate buffer (163-20145, Fujifilm Wako Pure Chemical Industries, Ltd.) and mixing by pipetting. The cell staining solution was prepared by dissolving 0.09 parts by mass of crystal violet (038-17792, Fujifilm Wako Pure Chemical Industries, Ltd.) in 10 parts by mass of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) and then mixing with 89.91 parts by mass of purified water.

[0072] Test Example 1: Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (container used: 15 cm adherent cell culture dish (culture area 152 cm ２) Human adipose tissue-derived mesenchymal stem cells (Cellsource) (hereafter referred to as ADSCs) were suspended in mesenchymal stem cell medium 2 and dispensed into 50 mL conical tubes (MS-56500, Sumitomo Bakelite Co., Ltd.) at 1 million cells per tube. After 3 minutes of centrifugation at 300 x G, the supernatant was removed. Next, 30 mL of a 0.03% by mass κ-CG-containing medium composition was added, and the ADSCs were suspended by pipetting. For comparison, ADSCs were suspended in 30 mL of mesenchymal stem cell medium 2 using the same procedure as above. After allowing to stand for 10 minutes, each ADSC suspension medium was transferred to a 15 cm adherent cell culture dish (MS-10150, Sumitomo Bakelite Co., Ltd.) and the cells were seeded. This process was carried out at room temperature (15-30°C). Next, after culturing for two days at 37°C under 5% carbon dioxide conditions, the supernatant was removed under each condition, and 15 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added. After gentle shaking, the supernatant was removed. Subsequently, 14 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. After the supernatant was removed, 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, the mixture was gently shaken, and the supernatant was removed. Finally, 3 mL of the prepared cell staining solution was added and allowed to stand for 3 minutes. After the supernatant was removed, 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, the mixture was gently shaken, and the supernatant was removed. This procedure was repeated twice, and then photographs were taken with a light source placed on the bottom. Furthermore, the same treatment was performed on cells after four days of culture to evaluate the cell adhesion state on each culture day. Furthermore, the cell distribution in each sample was analyzed using image analysis software. The details of the analysis conditions are shown below.

[0073] <Analysis conditions> Analysis software: Image J (NIH) Filters: median Type: 16 bit Analyze: Calculate standard deviation from histogram (number of pixels present for each brightness)

[0074] The results are shown in Figure 2. In both the cell staining images after 2 days and 4 days of culture, it was confirmed that, while in the control, uneven distribution of cells adhered to the bottom surface was observed, with the addition of κ-CG, uneven distribution was suppressed and the distribution of cells adhered to the bottom surface was clearly uniform.

[0075] [Test Example 2] Seeding test of human adipose tissue-derived mesenchymal stem cells using a DAG / ALG-containing medium composition (container used: 15 cm adherent cell culture dish (culture area 152 cm) ２ )) ADSCs (Cellsource) were suspended in mesenchymal stem cell medium 2 and dispensed into 50 mL conical tubes (MS-56500, Sumitomo Bakelite Co., Ltd.) at 1 million cells per tube. After 3 minutes of centrifugation at 300 x G, the supernatant was removed. Next, 15 mL of a 0.01% by mass DAG / ALG-containing medium composition was added, and the ADSCs were resuspended by pipetting. For comparison, ADSCs were suspended in 15 mL of mesenchymal stem cell medium 2 using the same procedure as above. After allowing to stand for 10 minutes, 15 mL of each ADSC suspension medium was transferred to a 15 cm adherent cell culture dish (MS-10150, Sumitomo Bakelite Co., Ltd.), and the cells were seeded by diluting with 15 mL of mesenchymal stem cell medium 2. This process was carried out at room temperature (15-30°C). Next, after culturing for 2 or 4 days at 37°C under 5% carbon dioxide conditions, the supernatant was removed, and 15 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added. After gentle shaking, the supernatant was removed. Next, 14 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, the mixture was gently shaken, and the supernatant was removed. Finally, 3 mL of the prepared cell staining solution was added and allowed to stand for 3 minutes. After removing the supernatant, 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, the mixture was gently shaken, and the supernatant was removed. This procedure was repeated twice, and then photographs were taken with a light source placed on the bottom. Regarding the state of cell adhesion on each culture day, the cell distribution in each sample was analyzed using image analysis software from photographs taken on the second or fourth day of culture. The analysis conditions were the same as in Test Example 1.

[0076] The results are shown in Figure 3. In both the cell staining images after 2 days and after 4 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the DAG / ALG-containing medium composition showed suppressed uneven distribution and clearly uniform distribution of cells adhered to the bottom surface.

[0077] [Test Example 3] Cell seeding test using gelatin-containing medium composition (container used: 15 cm adherent cell culture dish (culture area 152 cm) ２ ) Human adipose tissue-derived mesenchymal stem cells (Cellsource) (hereafter referred to as ADSCs) were suspended in mesenchymal stem cell medium 2 and dispensed into 50 mL conical tubes (MS-56500, Sumitomo Bakelite Co., Ltd.) at 1 million cells per tube. After 3 minutes of centrifugation at 300 x G, the supernatant was removed. Next, 30 mL of a 1.00% by mass gelatin-containing medium composition was added, and the ADSCs were suspended by pipetting. For comparison, ADSCs were suspended in 30 mL of mesenchymal stem cell medium 2 using the same procedure as above. After allowing to stand for 10 minutes, each ADSC suspension medium was transferred to a 15 cm adherent cell culture dish (MS-10150, Sumitomo Bakelite Co., Ltd.) and the cells were seeded. This process was carried out at room temperature (15-30°C). Next, after culturing for 3 days at 37°C under 5% carbon dioxide conditions, the supernatant was removed, and 15 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added. After gentle shaking, the supernatant was removed. Subsequently, 14 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. After the supernatant was removed, 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, the mixture was gently shaken, and the supernatant was removed. Finally, 3 mL of the prepared cell staining solution was added and allowed to stand for 3 minutes. After the supernatant was removed, 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, the mixture was gently shaken, and the supernatant was removed. This procedure was repeated twice, and then photographs were taken with a light source placed on the bottom. The cell distribution of each sample was analyzed using image analysis software from the photographs taken on the third day of culture. The analysis conditions were the same as those in Test Example 1.

[0078] The results are shown in Figure 4. In the stained cell images after 3 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the gelatin-containing medium composition showed less uneven distribution and the distribution of cells adhering to the bottom surface was clearly uniform.

[0079] [Test Example 4] Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (vessel used: 5-layer adherent cell culture flask (culture area 875 cm) ２)) Human adipose tissue-derived mesenchymal stem cells (hereinafter referred to as ADSCs) (Cellsource) were suspended in Mesenchymal Stem Cell Medium 2 and dispensed into 50 mL conical tubes (MS-56500, Sumitomo Bakelite Co., Ltd.) at 5 million cells per tube. After 3 minutes of centrifugation at 300 x G, the supernatant was removed. Next, 50 mL of 0.03% (w / v) κ-CG-blended medium composition was added, and the ADSCs were suspended by pipetting. This was transferred in 25 mL portions to 100 mL conical tubes (2355-100, AGC Techno Glass Co., Ltd.), diluted by adding 50 mL of 0.03% (w / v) κ-CG-blended medium composition, and the ADSCs were resuspended by pipetting. Additionally, for comparison, ADSCs were suspended in Mesenchymal Stem Cell Medium 2 using the same procedure as above. After allowing to stand for 10 minutes, each ADSC suspension medium was transferred to an upright five-layer adherent cell culture flask (353144, Corning). The flask was tilted 45° so that the mixing port was facing downwards, and the cell suspension was mixed. The flask was then re-uprighted to distribute the cell suspension evenly among each layer. The flask was then tilted 45° so that the mixing port was facing upwards, and the flask was then inverted and allowed to stand with the culture surface facing downwards. The cells were then seeded into the culture vessel. This process was carried out at room temperature (15-30°C). Next, the cells were cultured for one day at 37°C under 5% carbon dioxide conditions. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 150 mL of mesenchymal stem cell medium 2 was added to each layer to equalize the volume of each layer for a medium change. After an additional three days of culture, the supernatant was removed, and 50 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries) was added. The flask was then gently shaken, and the supernatant was removed. Next, 70 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was removed, and 50 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added. After gentle shaking, the supernatant was removed. Next, 30 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes, after which the supernatant was removed. Subsequently, 50 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was gently shaken. This washing procedure of removing the supernatant was repeated twice. Finally, each layer of the culture vessel was cut with an ultrasonic cutter (Z0-41, Nissin EM Co., Ltd.), and photographs of each stage were taken with a light source placed on the bottom.The cell distribution in each sample was analyzed using image analysis software from the photographs taken on day 4 of the culture. The analysis conditions were the same as in Test Example 1.

[0080] The results are shown in Figure 5. In the stained cell images after 4 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the κ-CG-containing medium composition showed suppressed uneven distribution and a clearly uniform distribution of cells adhered to the bottom surface.

[0081] Test Example 5: Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (container used: adherent cell culture flask (culture area 225 cm) ２) Human adipose tissue-derived mesenchymal stem cells (hereinafter referred to as ADSCs) (Cellsource) were suspended in Mesenchymal Stem Cell Medium 2 and dispensed into 50 mL conical tubes (MS-56500, Sumitomo Bakelite Co., Ltd.) at 1.5 million cells per tube. After 3 minutes of centrifugation at 300 x G, the supernatant was removed. Next, 45 mL of 0.03% (w / v) κ-CG blended medium composition was added, and the ADSCs were suspended by pipetting. For comparison, ADSCs were also suspended in Mesenchymal Stem Cell Medium 2 using the same procedure as above. After allowing to stand for 10 minutes, each ADSC suspension medium was transferred to an adherent cell culture flask (MS-21800, Sumitomo Bakelite Co., Ltd.) and the cells were seeded. This process was carried out at room temperature (15-30°C). Next, the cells were cultured for one day under conditions of 37°C and 5% carbon dioxide. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 45 mL of mesenchymal stem cell medium 2 was added to each well to perform a medium change. After culturing for another one or three days, the supernatant was removed, 25 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after gentle shaking, the supernatant was removed. Next, 21 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was removed, and 45 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after gentle shaking, the supernatant was removed. Next, 5 mL of the prepared cell staining solution was added, and after allowing to stand for 3 minutes, the supernatant was removed. Next, 45 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was gently shaken. The supernatant was then removed and the washing procedure was repeated twice. Finally, photographs were taken with a light source placed on the bottom surface. The cell distribution in each sample was analyzed using image analysis software from the photographs taken on the second or fourth day of culture. The analysis conditions were the same as in Test Example 1.

[0082] The results are shown in Figure 6. In both the cell staining images after 2 days and after 4 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the κ-CG-containing medium composition showed suppressed uneven distribution and clearly uniform distribution of cells adhered to the bottom surface.

[0083] Test Example 6: Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (vessel used: 6-well flat-bottom plate for adherent cell culture (culture area 9.5 cm) ２ Human adipose tissue-derived mesenchymal stem cells (hereinafter referred to as ADSCs) (Cellsource) were suspended in Mesenchymal Stem Cell Medium 2 and dispensed into 15 mL conical tubes (50015, SPL Life Sciences) at a concentration of 360,000 cells per tube. After centrifugation at 300 x G for 3 minutes, the supernatant was removed. Next, 12 mL of 0.03% (w / v) κ-CG formulated medium composition was added, and the ADSCs were suspended by pipetting. For comparison, ADSCs were also suspended in Mesenchymal Stem Cell Medium 2 using the same procedure as above. After allowing to stand for 10 minutes, each ADSC suspension medium was dispensed into a 6-well flat-bottom adherent cell culture multiwell plate (353046, Corning) at 2 mL per well. This process was carried out at room temperature (15-30°C). Next, the cells were cultured for one day under conditions of 37°C and 5% carbon dioxide. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 3 mL of mesenchymal stem cell medium 2 was added to each well for medium replacement. After an additional three days of culture, the supernatant was removed, 1 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the cells were gently shaken and the supernatant was removed. Next, 1.5 mL of the prepared cell fixation solution was added and the cells were left to stand for 15 minutes. The supernatant was removed, 1 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the cells were gently shaken and the supernatant was removed. Next, 3 mL of the prepared cell staining solution was added, and the cells were left to stand for three minutes, after which the supernatant was removed. Next, 1 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the cells were gently shaken and the supernatant was removed. This washing procedure was repeated twice. Finally, images were taken using a cell morphology analysis imager CELL3IMAGER DUOS2 (manufactured by SCREEN), and the cell distribution in each sample on day 4 of culture was analyzed using image analysis software. The analysis conditions were the same as in Test Example 1.

[0084] The results are shown in Figure 7. In the stained cell images after 4 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the κ-CG-containing medium composition showed suppressed uneven distribution and a clearly uniform distribution of cells adhered to the bottom surface.

[0085] [Test Example 7] Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (container used: 96-well flat-bottom plate for adherent cell culture (culture area 0.32 cm) ２Human adipose tissue-derived mesenchymal stem cells (hereinafter referred to as ADSCs) (Cellsource) were suspended in Mesenchymal Stem Cell Medium 2 and dispensed into 15 mL conical tubes (50015, SPL Life Sciences) at a concentration of 360,000 cells per tube. After centrifugation at 300 x G for 3 minutes, the supernatant was removed. Next, 12 mL of 0.03% (w / v) κ-CG formulated medium composition was added, and the ADSCs were suspended by pipetting. For comparison, ADSCs were suspended in Mesenchymal Stem Cell Medium 2 using the same procedure as above. After allowing to stand for 10 minutes, each ADSC suspension medium was dispensed into a 96-well flat-bottomed multiwell plate for adherent cell culture (MS-8096F, Sumitomo Bakelite Co., Ltd.) at 125 μL per well. This process was carried out at room temperature (15-30°C). Next, the cells were cultured for one day under conditions of 37°C and 5% carbon dioxide. After confirming cell adhesion to the culture vessel, the supernatant was removed, and 200 μL of mesenchymal stem cell medium 2 was added to each well to perform a medium change. After a further two days of culture, the supernatant was removed, 100 μL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after gentle shaking, the supernatant was removed. Next, 150 μL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was removed, and 100 μL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after gentle shaking, the supernatant was removed. Next, 300 μL of the prepared cell staining solution was added, and after allowing to stand for 3 minutes, the supernatant was removed. Next, 100 μL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was gently shaken. This washing procedure of removing the supernatant was repeated twice. Finally, images were taken using a cell morphology analysis imager, CELL3IMAGER DUOS2 (SCREEN), and the cell distribution of each sample on days 1 and 3 of culture was analyzed using image analysis software. The analysis conditions were the same as in Test Example 1.

[0086] The results are shown in Figure 8. In the cell staining images after 1 and 3 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the κ-CG-containing medium composition showed suppressed uneven distribution and clearly uniform distribution of cells adhered to the bottom surface.

[0087] Test Example 8 Seeding test of normal human skin fibroblasts using a κ-CG-containing medium composition (vessel used: 15 cm adherent cell culture dish (culture area 152 cm) ２ )) Normal human dermal fibroblasts (manufactured by Takara Bio Inc.) (hereinafter referred to as NHDF) were suspended in Fibroblast Growth Medium 2 (C-23020, manufactured by Takara Bio Inc.) and dispensed into 50 mL conical tubes (MS-56500, manufactured by Sumitomo Bakelite Co., Ltd.) at 1 million cells per tube. After centrifugation at 300 x G for 3 minutes, the supernatant was removed. Next, 30 mL of a medium composition containing 0.03% by mass of κ-CG prepared using Fibroblast Growth Medium 2 using the same procedure as in Preparation Example 1 was added, and the NHDF was suspended by pipetting. For comparison, NHDF was suspended in 30 mL of Fibroblast Growth Medium 2 using the same procedure as above. After standing for 10 minutes, each NHDF suspension medium was transferred to a 15 cm adherent cell culture dish (MS-10150, manufactured by Sumitomo Bakelite Co., Ltd.) and cells were seeded. All steps up to this point were carried out at room temperature (15-30°C). Next, after culturing for 3 days at 37°C under 5% carbon dioxide conditions, the supernatant was removed, 15 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was gently shaken. The supernatant was then removed. Next, 14 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was then removed, and 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was gently shaken. The supernatant was then 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. This washing procedure, in which 30 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, gently shaken, and the supernatant was removed, was repeated twice. Finally, a photograph was taken with a light source placed on the bottom. The cell distribution in each sample was analyzed using image analysis software from the photographs taken. The analysis conditions were the same as in Test Example 1.

[0088] The results are shown in Figure 9. In the stained cell images after 3 days of culture, it was confirmed that, while in the control, uneven distribution of cells adhered to the bottom surface was observed, with the addition of κ-CG, uneven distribution was suppressed and the distribution of cells adhered to the bottom surface was clearly uniform.

[0089] Test Example 9 Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (container used: 1-well adherent cell culture plate (culture area 97.4 cm) ２ Human adipose tissue-derived mesenchymal stem cells (hereinafter referred to as ADSCs) (Cellsource) were suspended in Mesenchymal Stem Cell Growth Medium 2 and dispensed into 50 mL conical tubes at 600,000 cells per tube. After 3 minutes of centrifugation at 300 x G, the supernatant was removed. Next, 20 mL of a 0.03% (w / v) κ-CG-containing medium composition was added, and the ADSCs were suspended by pipetting. For comparison, ADSCs were also suspended in Mesenchymal Stem Cell Growth Medium 2 using the same procedure as above. After allowing to stand for 10 minutes, the entire 20 mL volume was transferred to a 1-well flat-bottom adherent cell culture plate (229101, CELLTREAT Scientific Products). The steps up to this point were carried out at room temperature (15-30°C). Next, the cells were cultured for one day under conditions of 37°C and 5% carbon dioxide. After confirming adhesion of the cells to the culture vessel, the supernatant was removed, and 20 mL of mesenchymal stem cell growth medium 2 was added to each well to perform a medium change. After an additional three days of culture, the supernatant was removed, 10 mL of D-PBS (045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after gentle shaking, the supernatant was removed. Next, 10 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was removed, 10 mL of D-PBS was added, and after gentle shaking, the supernatant was removed. Next, 3 mL of the prepared cell staining solution was added, and after allowing to stand for 3 minutes, the supernatant was removed. Next, 10 mL of D-PBS was added, and after gentle shaking, the supernatant was removed. This washing procedure was repeated twice. Finally, images were taken using a cell morphology analysis imager CELL3IMAGER DUOS (manufactured by SCREEN), and the cell distribution in each sample was analyzed using image analysis software.

[0090] The results are shown in Figure 10. In the cell staining image after 4 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the κ-CG-containing medium composition showed suppressed uneven distribution and clearly uniform distribution of cells adhered to the bottom surface.

[0091] Test Example 10: Seeding test of human adipose tissue-derived mesenchymal stem cells using a κ-CG-containing medium composition (container used: 1-well adherent cell culture plate (culture area 97.4 cm) ２ In the same manner as in Test Example 9, 600,000 human adipose tissue-derived mesenchymal stem cells (hereinafter referred to as ADSCs) (Cellsource) were seeded in a 1-well flat-bottom adherent cell culture plate (229101, CELLTREAT Scientific Products) using a 0.03% (w / v) κ-CG-containing medium composition. For comparison, cells were seeded using mesenchymal stem cell proliferation medium 2 using the same procedure. After 4 days of culture, the cells were detached using a DetachKit (C-41220, PromoCell). The detached cells were collected in a 50 mL centrifuge tube and centrifuged at 220 × g for 3 minutes. The supernatant was then removed and the cells were suspended in 10 mL of mesenchymal stem cell proliferation medium 2. The cell suspension was suspended in trypan blue solution, and counted twice using a TC20 fully automated cell counter (BIORAD), and the average value was calculated.

[0092] The results are shown in Table 1. Comparing the cell counts after 4 days of culture, the cells seeded using the κ-CG-containing medium composition had a higher cell count than the control, demonstrating that the κ-CG-containing medium composition suppresses uneven cell distribution, enabling more efficient cell proliferation.

[0093]

[0094] Subsequently, the cell suspension was washed with FACSBuffer (2% FBS-supplemented D-PBS(-)). PE-labeled mouse anti-human CD105 antibody (560839, BDBiosciences), BV421-labeled mouse anti-human CD73 antibody (562430, BDBiosciences), APC-labeled mouse anti-human CD90 antibody (559869, BDBiosciences), PE-labeled mouse anti-human CD34 antibody (555822, BDBiosciences), and FITC-labeled mouse anti-human CD11b antibody (562793, BDBiosciences) were added to the cells again suspended in FACSBuffer, and the mixture was incubated at room temperature for 30 minutes in the dark. After washing with FACSBuffer, the cell suspension in FACSBuffer was measured using FACSFortessaX-20 (manufactured by BDBiosciences).

[0095] The results are shown in Table 2. After 4 days of culture, the cells showed high positivity for the positive markers CD105, CD73, and CD90, but almost no expression of the negative markers CD34 and CD11b. This confirmed that the κ-CG-containing composition does not affect the expression of surface markers in ADSCs.

[0096]

[0097] Preparation Example 5 Preparation of a medium composition containing κ-carrageenan A 1% by mass aqueous solution of κ-CG was prepared in the same manner as in Preparation Example 1. In a safety cabinet, 48.5 mL of StemFit AK02N (Ajinomoto Co., Inc.) was dispensed into a 50 mL conical tube (MS-56500, Sumitomo Bakelite Co., Ltd.), and 1.5 mL of a sterile 1% by mass aqueous solution of κ-CG was mixed by pipetting to prepare a medium composition containing 0.03% by mass of κ-CG.

[0098] Test Example 11: Seeding test of human induced pluripotent stem cells using a κ-CG-containing medium composition (vessel used: 8-well cell culture plate (culture area 10.5 cm) ２Human induced pluripotent stem cells (hereinafter referred to as iPSCs) 253G1 and 1383D2 strains (obtained from the Center for iPS Cell Research and Application, Kyoto University), and RPChiPS771 strain (obtained from ReproCell) were cultured in StemFit AK02N medium (manufactured by Ajinomoto Co., Inc.). For the culture, iMatrix511 silk (387-10131, manufactured by Nippi Co., Ltd.) was added at 0.5 μg / cm ２ A 6-well flat-bottom plate (351146, Corning) coated to a thickness of 100 μm was used. The obtained cells were washed with D-PBS(-), and then 0.5 mL / well of TrypLE Express Enzyme (1X) (12604013, Thermo Fisher Scientific) was added. The plate was incubated at 37°C and 5% carbon dioxide for 10 minutes to detach the cells. The obtained cells were collected in StemFit medium and placed in a 15 mL conical tube. The cells were dispensed into 15 mL conical tubes at 8,000 cells per tube, and centrifuged at 200×G for 3 minutes, after which the supernatant was removed. Next, 2 mL of the 0.03% (w / v) κ-CG-containing medium composition prepared in Preparation Example 5 was added, and the iPSCs were suspended by pipetting. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was then added to a final concentration of 10 μM. For comparison, iPSCs were suspended in StemFit AK02N medium containing 10 μM Y-27632 using the same procedure as above. 2 mL of the cell suspension was pre-suspended in 0.75 μg / cm ２The cells were seeded onto Nunc Rectangular Dishes (267062, Thermo Fisher Scientific) coated with iMatrix511 silk so that the cell density was 100%. The steps up to this point were carried out at room temperature (15-30°C). Next, the cells were cultured for 3 hours under 5% carbon dioxide conditions at 37°C. After confirming adhesion of the cells 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 to perform a medium change. Similarly, on the day after seeding and on the fourth day, a medium change was performed with StemFit AK02N medium (without Y-27632). After culturing for 5 days, the supernatant was removed, 2 mL of D-PBS(-) was added, and the cells were gently shaken, and the supernatant was then removed. Next, 2 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was removed, and 2 mL of D-PBS(-) was added. After gentle shaking, the supernatant was removed. Next, 0.5 mL of the prepared cell staining solution was added, and the mixture was allowed to stand for 3 minutes, after which the supernatant was removed. Subsequently, 2 mL of D-PBS(-) was added, and the mixture was gently shaken. This washing procedure of removing the supernatant was repeated twice. Finally, images were taken using a cell morphology analysis imager, CELL3IMAGER DUOS (manufactured by SCREEN), and image analysis was performed using Python to create a Voronoi diagram based on the center coordinates of each colony. The colony distribution of each sample was analyzed by comparing the area of ​​the Voronoi region.

[0099] A Voronoi diagram is a diagram in which multiple kernel points placed at arbitrary positions are divided into regions based on the proximity of other points in the same metric space to each kernel point. The boundaries of the Voronoi regions are part of the bisectors of each kernel point, and the intersections of the Voronoi boundaries are called Voronoi points. The greater the distance between a divided region and its surrounding kernel points, the larger the region's area, and the closer the region is to its surrounding kernel points, the smaller the region's area. Therefore, when cells are seeded uniformly, the Voronoi region will have a constant area, but when cells are seeded unevenly, the variation in the Voronoi region will increase. Therefore, the standard deviation of the area of ​​the Voronoi region was calculated. Details of the analysis conditions are shown below.

[0100] <Analysis conditions> Python execution environment: JupyterLab Libraries and functions used: OpenCV, NumPy, scipy (Voronoi function), Shapely (Polygon function), pandas, matplotlib Averaging filter: cv2.blur (5x5 kernel) Binarization: cv2.threshold Obtaining the centroid coordinates of each colony: cv2.connectedComponentsWithStats Creating a Voronoi diagram based on the centroid coordinates: voronoi_plot_2d Obtaining the coordinates of Voronoi points: vor.vertices Calculating the area of ​​the Voronoi region from the coordinates of the Voronoi points: Polygon Calculating the standard deviation for the area of ​​the Voronoi region: NumPy. standard

[0101] The results are shown in Figures 11 to 13. Image analysis of the stained cell images after 5 days of culture confirmed that the standard deviation was reduced for all cell lines, and that the κ-CG-containing medium composition reduced uneven cell distribution.

[0102] Test Example 12: Seeding test of human induced pluripotent stem cells using a κ-CG-containing medium composition (container used: 8-well cell culture plate (culture area 10.5 cm) ２ Human induced pluripotent stem cells (hereinafter referred to as iPSCs) 253G1, 1383D2, and RPChiPS771 strains were cultured in StemFit AK02N medium (Ajinomoto Co., Inc.). For culture, iMatrix511 silk was added at 0.5 μg / cm ２A 6-well flat-bottom plate (351146, Corning) coated to a thickness of 100 μm was used. After washing the obtained cells with D-PBS(-), 0.5 mL of TrypLE Express Enzyme (1X) was added per well. The cells were then incubated at 37°C and 5% carbon dioxide for 10 minutes to detach the cells. The obtained cells were collected in StemFit medium and placed in a 15 mL conical tube. The cells were dispensed into 15 mL conical tubes at 8,000 cells per tube, and centrifuged at 200×G for 3 minutes, after which the supernatant was removed. Next, 2 mL of the 0.03% (w / v) κ-CG-containing medium composition prepared in Preparation Example 5 was added, and the iPSCs were suspended by pipetting. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 10 μM. For comparison, iPSCs were suspended in StemFit AK02N medium containing 10 μM Y-27632 using the same procedure as above. 2 mL of the cell suspension was pre-suspended in 0.75 μg / cm ２The cells were seeded onto Nunc Rectangular Dishes (267062, Thermo Fisher Scientific) coated with iMatrix511 silk so that the cells were 100% pure. The steps up to this point were carried out at room temperature (15-30°C). Next, the cells were cultured for 3 hours under 5% carbon dioxide conditions at 37°C. After confirming adhesion of the cells 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 to perform a medium change. Similarly, the day after seeding and every day from day 4 onwards, medium changes were carried out with StemFit AK02N medium (without Y-27632). On day 7 of culture, the supernatant was removed, and 1 mL of TrypLE Express Enzyme (1X) was added per well. The cells were incubated at 37°C and 5% carbon dioxide for 10 minutes to detach the cells. The resulting cells were collected in StemFit medium and placed in a 15 mL conical tube. After washing with D-PBS(-), they were suspended in 4% paraformaldehyde phosphate buffer (163-20145, Fujifilm Wako Pure Chemical Industries, Ltd.) and incubated at room temperature for 15 minutes for fixation. Subsequently, the cells were washed with FACSBuffer (2% FBS-supplemented D-PBS(-)) and resuspended in FACSBuffer. The cells at the time of seeding were similarly fixed and washed / resuspended in FACSBuffer, and then stored refrigerated until measurement. AlexaFluor647-labeled mouse anti-human SSEA-4 antibody (560796, BDBiosciences) and PE-labeled mouse anti-human TRA-1-60 antibody (560193, BDBiosciences) were added to the cells suspended in FACSBuffer, and the cells were incubated at room temperature for 30 minutes in the dark. After washing three times with FACSBuffer, the cell suspension suspended in FACSBuffer was measured using a FACSFortessaX-20 (BDBiosciences).

[0103] The results are shown in Table 3. After 7 days of culture, the cells maintained high levels of expression of SSEA-4 and TRA-1-60, which are markers of undifferentiation of human iPSCs, confirming that the κ-CG-containing composition does not affect the maintenance of the undifferentiated state of iPSCs.

[0104]

[0105] Test Example 13: Seeding test of human induced pluripotent stem cells using a κ-CG-containing medium composition (container used: 1-well adherent cell culture plate (culture area 97.4 cm) ２ Human induced pluripotent stem cells (hereinafter referred to as iPSC) RPChiPS771 strain were cultured in StemFit AK02N medium. For culture, iMatrix511 silk was added at 0.5 μg / cm ２ A 6-well flat-bottom plate (351146, Corning) coated to a thickness of 100 μm was used. After washing the obtained cells with D-PBS(-), 0.5 mL of TrypLE Express Enzyme (1X) was added per well. The cells were then incubated at 37°C and 5% carbon dioxide for 10 minutes to detach the cells. The obtained cells were collected in StemFit medium and placed in a 15 mL conical tube. The cells were dispensed into 50 mL conical tubes at 80,000 cells per tube, and centrifuged at 200×G for 3 minutes, after which the supernatant was removed. Next, 20 mL of the 0.03% (w / v) κ-CG-containing medium composition prepared in Preparation Example 5 was added, and the iPSCs were suspended by pipetting. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 10 μM. For comparison, iPSCs were suspended in StemFit AK02N medium containing 10 μM Y-27632 using the same procedure as above. ２The cells were seeded onto a 1-well flat-bottom adherent cell culture plate (229101, CELLTREAT Scientific Products) coated with iMatrix511 silk so that the cell density was 100%. The steps up to this point were carried out at room temperature (15-30°C). Next, the cells were cultured for 3 hours at 37°C under 5% carbon dioxide conditions. After confirming adhesion of the cells 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. The cells were further cultured at 37°C under 5% carbon dioxide conditions, and the medium was similarly replaced with StemFit AK02N medium (without Y-27632) on the day after seeding and on the fourth day. After culturing for 5 days, the supernatant was removed, 10 mL of D-PBS(-) was added, and the cells were gently shaken, followed by removal of the supernatant. Next, 10 mL of the prepared cell fixation solution was added and allowed to stand for 15 minutes. The supernatant was removed, and 10 mL of D-PBS(-) was added. After gently 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, after which the supernatant was removed. Subsequently, 10 mL of D-PBS(-) was added, gently shaken, and the washing procedure of removing the supernatant was repeated twice. Finally, images were taken using a cell morphology analysis imager CELL3IMAGER DUOS (manufactured by SCREEN). Image analysis was performed using Python to create a Voronoi diagram based on the center coordinates of each colony, and the colony distribution of each sample was analyzed by comparing the area of ​​the Voronoi region. The analysis conditions were the same as in Test Example 11.

[0106] The results are shown in Figure 14. In the stained cell images after 5 days of culture, the cells adhered to the bottom surface of the control were found to vary in density, whereas the cells seeded using the κ-CG-containing medium composition showed reduced uneven distribution, demonstrating a clearly uniform distribution of cells adhered to the bottom surface. Furthermore, the image analysis results also showed a reduction in the standard deviation of the Voronoi region area, confirming that the κ-CG-containing medium composition reduced uneven cell distribution.

[0107] According to the present invention, cells can be uniformly seeded onto the cell adhesion surface inside the cell culture vessel. Therefore, the present invention can avoid the problem of different passage timings for different vessels and allows for the mass cultivation of cells of uniform quality. Therefore, the present invention is extremely useful in the medical field, such as cell therapy.

[0108] This application is based on patent application No. 2024-6204 filed in Japan (filing date: January 18, 2024), the contents of which are incorporated in their entirety herein.

Claims

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

2. The method according to claim 1, further comprising a step of reducing the yield stress of the plastic fluid in Step 2.

3. The method according to claim 2, wherein the reduction of the yield stress of the plastic fluid is performed by diluting the plastic fluid.

4. The method according to claim 2, wherein the reduction of the yield stress of the plastic fluid is performed by changing the temperature of the plastic fluid.

5. The method according to any one of claims 1 to 4, further comprising a step of distributing the plastic fluid in which the cells obtained in Step 1 are uniformly dispersed into another container between Step 1 and Step 2.

Images