Method for the production of a culture medium composition for the suspension culture of adherent cells

Abstract

Provided is a method for producing a culture medium composition for suspension culture of adherent cells, the method comprising the following steps: step (i), loading extracellular matrix onto nanofibers composed of non-water-soluble polysaccharides, and step (ii), adding the extracellular matrix-loaded nanofibers obtained in step (i) to a culture medium.

Description

Technical Field

[0001] This invention relates to a method for manufacturing a culture medium composition for suspension culture of adhesive cells, etc. Background Technology

[0002] In recent years, methods for transplanting and injecting cells into living organisms have been developed, primarily in the medical and cosmetic fields. Among these methods, adult stem cells and progenitor cells have attracted attention due to their lower risk of cancer and shorter differentiation time compared to pluripotent stem cells.

[0003] When utilizing these cells, a large supply of cells in good condition is required. As a method, it is known to culture stem cells, etc., in a state of adhesion to microcarriers, etc., to promote their proliferation.

[0004] However, currently available microcarriers have been found to have the following problems: they tend to settle in the culture medium under static conditions, thus requiring stirring during culture. This stirring can cause collisions between microcarriers, leading to cell death. Furthermore, cell proliferation efficiency is insufficient, and further improvements are needed.

[0005] The inventors of this application have developed a culture medium composition for culturing plant and animal cells and / or tissues in a suspension state using nanofibers such as polysaccharides that have improved dispersibility in water (Patent Document 1).

[0006] The inventors of this application have also discovered that nanofibers containing non-water-soluble polysaccharides can serve as a common carrier for various operations of adhesive cells, including i) suspension culture, ii) differentiation induction, iii) transport or preservation under non-freezing conditions, iv) transplantation, and v) recovery of physiologically active substances from culture supernatant (Patent Document 2, Patent Document 3).

[0007] Existing technical documents

[0008] [Patent Literature]

[0009] [Patent Document 1] WO2015 / 111686

[0010] [Patent Document 2] WO2017 / 175751

[0011] [Patent Document 3] WO2018 / 182016 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] The subject of this invention is to provide technology related to the mass production of adhesive cells such as adult stem cells and progenitor cells.

[0014] Methods for solving problems

[0015] To address the aforementioned issues, the inventors of this application conducted repeated and in-depth research, discovering that using a substance obtained by loading an extracellular matrix onto nanofibers composed of insoluble polysaccharides as a carrier substrate for suspending and culturing adhesive cells can highly efficiently promote the proliferation of these cells. Furthermore, it was found that by mixing nanofibers loaded with the extracellular matrix into a liquid culture medium and culturing adhesive cells while they are adhered to the nanofibers, the cells can be cultured in suspension under static conditions, or scaled up by simply adding a fresh culture medium composition without cell stripping treatment based on trypsin or similar methods. Based on these insights, the inventors of this application continued their research, thereby completing this invention.

[0016] That is, the present invention is as follows.

[0017] [1] A method for manufacturing a culture medium composition for suspension culture of adhesive cells, the method comprising the following steps:

[0018] Step (i) involves loading the extracellular matrix onto nanofibers composed of water-insoluble polysaccharides.

[0019] Step (ii) involves adding the extracellular matrix-loaded nanofibers obtained in step (i) to the culture medium.

[0020] [2] The method as described in [1], wherein the non-water-soluble polysaccharide is at least one selected from the group consisting of chitin, chitosan, cellulose, and hemicellulose.

[0021] [3] The method as described in [1] or [2], wherein the extracellular matrix is ​​at least one selected from the group consisting of collagen, fibronectin, hydrin, laminin, RGD sequence, and cadherin.

[0022] [4] The method described in any of [1] to [3], wherein the amount of extracellular matrix carried by the nanofibers is 0.01 to 50 mg of extracellular matrix per 1 g of nanofibers.

[0023] [5] The method described in any of [1] to [4], wherein the non-water-soluble polysaccharide is chitin.

[0024] [6] The method as described in [5], wherein chitosan nanofibers are added in step (ii).

[0025] [7] As described in [6], the content ratio (by weight) of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is chitin nanofibers carrying the extracellular matrix: chitosan nanofibers = 1:0.5~20.

[0026] [8] The method described in any of [1] to [7], wherein the extracellular matrix is ​​bernectin.

[0027] [9] A composition for adding to a culture medium, comprising chitin nanofibers and chitosan nanofibers carrying an extracellular matrix.

[0028]

[10] The composition as described in [9], wherein the extracellular matrix is ​​at least one selected from the group consisting of collagen, fibronectin, hyalin, laminin, RGD sequence, and cadherin.

[0029]

[11] The composition as described in [9] or

[10] , wherein the amount of extracellular matrix carried by the chitin nanofibers is 0.01 to 50 mg of extracellular matrix per 1 g of chitin nanofibers.

[0030]

[12] The composition as described in any of [9] to

[11] , wherein the ratio (by weight) of the chitin nanofibers carrying the extracellular matrix to the chitosan nanofibers contained in the culture medium composition is chitin nanofibers carrying the extracellular matrix: chitosan nanofibers = 1:0.5 to 20.

[0031]

[13] The composition as described in any of [9] to

[12] , wherein the extracellular matrix is ​​hydrin.

[0032]

[14] A culture medium composition for suspension culture of adhesive cells, comprising any one of the compositions described in [9] to

[13] .

[0033] The effects of the invention

[0034] According to the present invention, by culturing adhesive cells in a state of adhesion to nanofibers composed of insoluble polysaccharides carrying an extracellular matrix, suspension culture can be performed in a static state without the need for shaking, rotating, or other operations. Furthermore, based on the effect of the extracellular matrix, cell proliferation can be promoted.

[0035] Furthermore, according to the present invention, it is possible to maintain the culture of adhesive cells and to proliferate them while maintaining their characteristics. Attached Figure Description

[0036] [ Figure 1 ] Figure 1 This image shows a phase-contrast microscopy photograph of an aqueous dispersion of chitin nanofibers after the addition of CBB staining solution.

[0037] [ Figure 2 ] Figure 2 A phase-contrast microscopy image showing an aqueous dispersion of chitin nanofibers / chitosan nanofibers after the addition of CBB staining solution.

[0038] [ Figure 3 ] Figure 3 This image shows a phase-contrast microscopy photograph of an aqueous dispersion of chitin nanofibers loaded with vitrin after the addition of CBB staining solution.

[0039] [ Figure 4 ] Figure 4 A phase-contrast microscopy image showing an aqueous dispersion of chitin / chitosan nanofibers loaded with vitrin after the addition of CBB staining solution. Detailed Implementation

[0040] The present invention will now be described in detail.

[0041] 1. Method for manufacturing culture medium composition

[0042] The present invention provides a method for manufacturing a culture medium composition for suspending adhesive cells, comprising the following steps (hereinafter, sometimes referred to as "the manufacturing method of the present invention"):

[0043] Step (i) involves loading the extracellular matrix onto nanofibers composed of water-insoluble polysaccharides.

[0044] Step (ii) involves adding the extracellular matrix-loaded nanofibers obtained in step (i) to the culture medium.

[0045] In the manufacturing method of the present invention, the so-called adhesive cells refer to cells that require support such as container walls to survive and proliferate.

[0046] In the manufacturing method of this invention, the term "adhesive cell" is not particularly limited and can include, for example, stem cells, progenitor 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 cells of multiple other systems. Examples of adhesive stem cells include, but are not limited to, 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 are 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. Progenitor 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, precursor adipocytes, precursor cardiomyocytes, precursor endothelial 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, 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 used in the manufacturing method of this invention are preferably stem cells or progenitor cells, more preferably mesenchymal stem cells.

[0047] The source of the adhesive cells in the manufacturing method of this invention is not particularly limited, and can be any cells from animals and plants. As for animals, there are no limitations, and examples include fish, amphibians, reptiles, birds, pancrustaceans, hexapods, mammals, etc., with mammals being preferred. Examples of mammals are not limited, and examples include rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypuses, dolphins, whales, dogs, cats, goats, cattle, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, macaques, chimpanzees, and humans. 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 substances (such as saponins, alkaloids, berberine, scopolamine, phytosterols, etc.) include medicinal ginseng, periwinkle, hyoscyamine, coptis, belladonna, etc.; plants that produce pigments and polysaccharides (such as anthocyanins, safflower pigments, madder pigments, crocin pigments, flavonoids, etc.) used as raw materials for cosmetics and food include blueberries, safflower, madder, saffron, etc.; and plants that produce pharmaceutical raw materials, but are not limited thereto. The culture medium manufactured using the method of the present invention is preferably used for adhesive cells of mammals.

[0048] 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.

[0049] In the manufacturing method of the present invention, the aspect ratio (L / D) of the nanofibers used can be obtained by the average fiber length / average fiber diameter, and is not particularly limited. It is usually 2 to 500, preferably 5 to 300, and more preferably 10 to 250.

[0050] In this specification, the average fiber diameter (D) of the nanofibers was determined as follows. First, a collodion support membrane manufactured by Oken Shoji Co., Ltd. was subjected to hydrophilication treatment for 3 minutes using Ion Cleaner (JIC-410) manufactured by Nippon Electronics Co., Ltd. A few drops of the nanofiber dispersion of the evaluation object (diluted with ultrapure water) were then added and dried at room temperature. The nanofibers were observed using a transmission electron microscope (TEM, H-8000) (10,000x) manufactured by Hitachi, Ltd., with an accelerating voltage of 200 kV. Using the obtained images, the diameter of each nanofiber was measured for a sample of 200–250 nanofibers, and the arithmetic mean was taken as the average fiber diameter (D).

[0051] Furthermore, the average fiber length (L) was calculated as follows. The nanofiber dispersion for evaluation 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 to achieve hydrophilicity, and dried at 110°C for 1 hour to obtain a sample. Using images obtained from the obtained sample observed with a scanning electron microscope (SEM, JSM-7400F) (2000x) manufactured by NJE Ltd, the length of each fiber was measured for a sample size of 150–250 nanofibers, and the arithmetic mean of these measurements was taken as the average fiber length (L).

[0052] In a preferred embodiment, the nanofibers have the following effects: when mixed with a liquid culture medium, the nanofibers are uniformly dispersed in the liquid while maintaining their primary fiber diameter, without substantially increasing the viscosity of the liquid, and substantially retaining the cells attached to the nanofibers, preventing them from settling.

[0053] The nanofibers used in the manufacturing method of this invention are composed of non-water-soluble polysaccharides. Sugars refer to sugar polymers composed of 10 or more monosaccharides (such as trioses, tetras, pentoses, hexoses, heptaoses, etc.).

[0054] Examples of non-water-soluble polysaccharides include cellulose, hemicellulose, and other cellulose-based substances; chitin, chitosan, and other chitin-based substances, but are not limited to these. Chitin or chitosan is preferred as the non-water-soluble polysaccharide, and chitin is more preferred. It should be noted that in this specification, "nanofibers composed of chitin" is sometimes referred to as "chitin nanofibers." The same applies to other non-water-soluble polysaccharides.

[0055] Chitosan-like substances refer to one or more sugars selected from the group consisting of chitin and chitosan. The main sugar units constituting chitin and chitosan are N-acetylglucosamine and glucosamine, respectively. Generally speaking, chitosan-like substances with a high content of N-acetylglucosamine and poor solubility in acidic aqueous solutions are called chitin, while chitosan-like substances with a high content of glucosamine and soluble in acidic aqueous solutions are called chitosan. In this specification, for convenience, sometimes substances with N-acetylglucosamine accounting for more than 50% of the constituent sugars are called chitin, and those with less than 50% are called chitosan.

[0056] As raw materials for chitin, various biological resources such as shrimp, crab, insects, shellfish, and mushrooms can be used. The chitin used in this invention can be chitin with an α-type crystal structure, such as that derived from crab or shrimp shells, or chitin with a β-type crystal structure, such as that derived from cuttlebone. Crab and shrimp shells are often treated as industrial waste, and from the viewpoint of easy availability and efficient utilization, they are preferred as raw materials. However, to remove proteins, ash, and other impurities, deproteinization and deashing steps are required. Therefore, in this invention, purified chitin that has undergone matrix removal treatment is preferred. Purified chitin is commercially available. As raw materials for chitin nanofibers used in this invention, chitin with either an α-type or β-type crystal structure can be used, with α-type chitin being preferred.

[0057] By pulverizing the aforementioned polysaccharides, polysaccharide nanofibers can be obtained. The pulverization method is not limited; however, to achieve the fiber diameter and length required for this invention, methods that generate strong shear forces, such as high-pressure homogenizers, grinding machines (mortars), or media stirring mills like bead mills, are preferred.

[0058] 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) or NanoVater (high-pressure pulverizing apparatus manufactured by Yoshida Machinery Kogyo Co., Ltd.).

[0059] 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 (number of processes), and the concentration of the raw material in the aqueous dispersion. The pumping pressure (processing pressure) is not particularly limited, but is typically 50–250 MPa, preferably 100–200 MPa.

[0060] Furthermore, the concentration of the raw material in the aqueous dispersion during the micronization process is not particularly limited, typically ranging from 0.1% to 30% by mass, preferably from 1% to 10% by mass. The number of micronization (pulverization) processes is not particularly limited, but depends on the concentration of the raw material in the aforementioned aqueous dispersion. When the raw material concentration is 0.1% to 1% by mass, approximately 10 to 100 processes are sufficient for adequate micronization; however, when the concentration is 1% to 10% by mass, approximately 10 to 1000 processes may be required.

[0061] The viscosity of the aqueous dispersion during the aforementioned micronization process is not particularly limited. For example, in the case of α-chitosan, the viscosity range of the aqueous dispersion is 1–100 mPa·s, preferably 1–85 mPa·s (measured using a tuning fork vibration viscometer at 25°C (SV-1A, A&D Company Ltd.)). In the case of chitosan, the viscosity range of the aqueous dispersion is 0.7–30 mPa·s, preferably 0.7–10 mPa·s (measured using a tuning fork vibration viscometer at 25°C (SV-1A, A&D Company Ltd.)).

[0062] Methods for preparing nanofibers are described in WO2015 / 111686A1, etc.

[0063] In the manufacturing method of this invention, in the first step, an extracellular matrix is ​​loaded onto nanofibers composed of insoluble polysaccharides. In this specification, "nanofiber-loaded" extracellular matrix refers to a state in which the nanofibers and the extracellular matrix are attached or adsorbed without being mediated by chemical covalent bonds. The loading of the extracellular matrix based on nanofibers can be achieved through intermolecular forces, electrostatic interactions, hydrogen bonds, hydrophobic interactions, etc., but is not limited to these. Furthermore, the state of nanofiber-loaded extracellular matrix can be described as a state in which the nanofibers and the extracellular matrix are in contact without being mediated by chemical covalent bonds, or a state in which the nanofibers and the extracellular matrix form a complex without being mediated by chemical covalent bonds.

[0064] In the first step of the manufacturing method of the present invention, the extracellular matrix supported on the nanofibers is not particularly limited, as long as the desired effect can be obtained. Examples include collagen (collagen I to XIX), fibronectin, brecciatin, laminin (lamin-1 to 12), RGD sequences, cadherin, etc. The choice of extracellular matrix varies depending on the type of cells to be proliferated, and those skilled in the art can make appropriate selections. For example, in the case of mesenchymal stem cells, brecciatin is preferred as the extracellular matrix. In addition, when the brecciatin is derived from human brecciatin, brecciatin with an amino acid sequence (hereinafter referred to as aa sequence) of 20-398 (sequence number 1) or 62-478 (sequence number 2) is preferred. When using brecciatin derived from non-human sources, a region corresponding to a fragment of human brecciatin can be used.

[0065] In the manufacturing method of the present invention, the amount of extracellular matrix carried by the nanofibers is typically 0.001 to 50 mg per 1 g of nanofibers, preferably 0.01 to 10 mg, more preferably 0.1 to 10 mg, even more preferably 0.3 to 10 mg, even more preferably 1 to 10 mg, particularly preferably 2 to 10 mg, but not limited to these.

[0066] In the manufacturing method of the present invention, the preparation of nanofibers carrying the extracellular matrix can be carried out as follows: a dispersion obtained by dispersing nanofibers in an aqueous solvent is mixed with an aqueous solution of the extracellular matrix, and the mixture is allowed to stand for a certain period of time as needed. Examples of aqueous solvents for dispersing nanofibers include water, dimethyl sulfoxide (DMSO), etc., but are not limited to these. Water is preferred as the aqueous solvent. The aqueous solvent may contain a suitable buffer or salt. To ensure uniform contact between the extracellular matrix and the nanofibers, thorough mixing by blowing or suction is preferred. In addition, as the standing time, the mixture of the nanofiber dispersion and the aqueous solution of the extracellular matrix can be allowed to stand for 30 minutes or more, preferably 1 hour or more, more preferably 3 hours or more, further preferably 6 hours or more, even more preferably 9 hours or more, and particularly preferably 12 hours or more. There is no particular upper limit to the standing time; for example, the upper limit can be set to 48 hours or less (e.g., 36 hours or less, 24 hours or less, or 16 hours or less, etc.). There is no particular limitation on the temperature during standing, which can usually be 1 to 30°C, preferably 1 to 15°C, more preferably 2 to 10°C, and particularly preferably 2 to 5°C (e.g., 4°C).

[0067] The mixing ratio of nanofibers composed of insoluble polysaccharides to the extracellular matrix varies depending on the type of the aforementioned substances used. In terms of solid component weight, it can be, for example, 100:0.1 to 1, preferably 100:0.4 to 0.6, but is not limited to these.

[0068] The amount of extracellular matrix loaded on nanofibers composed of insoluble polysaccharides can be measured by methods such as Micro BCA assay, enzyme immunoassay (ELISA), etc., but is not limited to these.

[0069] In a preferred embodiment, nanofibers composed of insoluble polysaccharides are uniformly dispersed in a liquid culture medium, thereby suspending adhesive cells attached to the nanofibers in the liquid culture medium.

[0070] In the second step of the manufacturing method of the present invention, the culture medium in which nanofibers carrying extracellular matrix are added can be appropriately selected according to the type of adhesive cells used. For example, if the goal is to culture mammalian adhesive cells, a culture medium commonly used for culturing mammalian cells can be used. Examples of culture media used for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, McCoy's 5Amedium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagles's Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), RPMI 1640, Iscove's Modified Dulbecco's Medium (IMDM), MCDB 131, William's Medium E, IPL 41, Fischer's Medium, StemPro 34 (Invitrogen), and X-VIVO 10 (Cambrex). 15 (made by Cambrex), HPGM (made by Cambrex), StemSpan H3000 (made by STEMCELL Technologies), StemSpanSFEM (made by STEMCELL Technologies), StemlineII (made by Sigma Aldrich), QBSF-60 (made by Qualitybiological), StemProhESCSFM (made by Invitrogen), mTeSR1 or mTeSR2 culture medium (made by STEMCELL Technologies), Sf-900II (made by Invitrogen), Opti-Pro (made by Invitrogen), etc.

[0071] Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc., to the above-mentioned culture medium according to their purpose. When culturing mammalian cells, those skilled in the art may also add one or more other chemical or biological components in combination according to their purpose. Examples of components that can be added to culture media for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, thioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrix components, and various cell adhesion molecules. Examples of cytokines that can be added to culture media include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), and interleukin-14 (IL-14). Interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncogene M (OM), erythropoietin (EPO), thrombopoietin (TPO), etc., but not limited to these.

[0072] Examples of hormones that can be added to culture media include melatonin, serotonin, thyroxine, triiodothyronine, adrenaline, noradrenaline, dopamine, anti-Müllerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental prolactin, growth hormone, inhibin, insulin, and insulin-like growth factor. Leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin, but not limited to these.

[0073] Examples of growth factors that can be added to the culture medium include, but are not limited to, transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epithelial growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve cell growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease conjugate I, protease conjugate II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokines, Notch ligands (Delta1, etc.), Wnt protein, angiopoietin-like protein 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), and pleiotrophin.

[0074] Alternatively, substances obtained by artificially altering the amino acid sequences of these cytokines and growth factors through gene recombination technology can be added. Examples include IL-6 / soluble IL-6 receptor complex or HyperIL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

[0075] Examples of antibiotics that can be added to culture media include sulfonamides, penicillins, fenescillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclopecillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mezlocillin, adenocillin, cephalosporins and their derivatives, oxaquinic acid, amloxacin, timafloxacin, nalidixic acid, pyrrolizic acid, ciprofloxacin, sinofloxacin, norfloxacin, mefloxacin, rosaxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulanic acid, β-bromopenicillanic acid, and β-chloropenicillanic acid. (acid), 6-acetylmethylene-penicillinic acid, cefoxazole, sultazine, adinoshirin and sulbactam formaldehyde hudrate ester, tazobactam, aztreonam, sulfazethin, isosulfazethin, Norcardicin, phenylacetamidophosphonicacid methyl, chlorotetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0076] The mixing ratio is not particularly limited. The volume ratio of the nanofiber dispersion to the liquid culture medium (aqueous solution of the culture medium) is usually 1:99 to 99:1, preferably 10:90 to 90:10, and more preferably 20:80 to 80:20.

[0077] In this specification, cell suspension refers to a state in which cells are not adhered to the culture container (non-adhesive), regardless of whether the cells settle. Furthermore, this specification refers to the following state as "suspended static culture": when culturing cells, without external pressure, vibration, or oscillation / rotation of the liquid culture medium composition, the cells are dispersed and suspended in the liquid culture medium composition; the culture of cells and / or tissues in this state is referred to as "suspended static culture." The time for suspension in "suspended static culture" is at least 5 minutes, preferably 1 hour, 24 hours, 48 ​​hours, 6 days, or 21 days or more, but is not limited to these times as long as the suspension is maintained.

[0078] In a preferred embodiment, the culture medium composition manufactured by the method of the present invention can achieve cell suspension at at least one point within a temperature range (e.g., 0–40°C) capable of culturing cells. Preferably, the culture medium composition achieves cell suspension at at least one point within a temperature range of 25–37°C, and most preferably at 37°C.

[0079] Adhesive cells can be cultured in suspension by culturing them attached to nanofibers loaded with extracellular matrix. The nanofibers loaded with extracellular matrix exhibit an effect of suspending cells attached to them in the culture medium (preferably, suspending them in stillness) and promoting cell proliferation. The nanofibers loaded with extracellular matrix are dispersed without dissolving in the liquid culture medium or attaching to the culture vessel; therefore, when adhesive cells are cultured in this liquid culture medium composition, the adhesive cells attach to the nanofibers and are suspended in the culture medium composition. Based on this suspension effect, the number of cells per unit volume can be increased compared to monolayer culture. Furthermore, in conventional suspension cultures involving rotation and agitation, shear forces act on the cells, resulting in low cell proliferation rates, low recovery rates, or impaired cell function. However, by using the culture medium composition manufactured according to the method of this invention, cells can be cultured in a dispersed state without agitation or similar operations. Therefore, it is expected that the target adhesive cells can be easily and abundantly cultured in suspension without loss of cell function. Furthermore, in conventional culture media containing gel substrates, cell observation and recovery are difficult, or their function may be impaired during recovery. However, by using the culture medium composition manufactured according to the method of the present invention, it is expected that suspended cells can be observed and recovered without impairing their function. Additionally, conventional culture media containing gel substrates have high viscosity and are difficult to replace. However, the culture medium composition manufactured using the method of the present invention has low viscosity, thus allowing for easy replacement of the culture medium using pipettes, pumps, etc.

[0080] When using nanofibers loaded with extracellular matrix for suspension culture of adhesive cells, separately prepared adhesive cells are added to the culture medium composition containing the nanofibers and mixed uniformly. The mixing method is not particularly limited; examples include manual mixing such as blowing and aspiration, mixing using a magnetic stirrer, vortex mixer, microplate mixer, or shaker. After mixing, the resulting cell suspension can be cultured in a static state, or it can be cultured while rotating, shaking, or stirring as needed. The rotation speed and frequency can be appropriately set according to the purposes of those skilled in the art. For example, adhesive cells can be recovered from passage culture, dispersed to a single-cell or near-single-cell state using a suitable cell dissociation solution, and the dispersed adhesive cells can be suspended in the culture medium composition for suspension culture (preferably static suspension culture).

[0081] For cell culture temperature, it is typically 25 to 39°C, preferably 33 to 39°C (e.g., 37°C) for animal cells. For CO2 concentration, it is typically 4 to 10% by volume, preferably 4 to 6% by volume, in the culture atmosphere. The culture time can be set appropriately according to the purpose of the culture.

[0082] The culture of adhesive cells in the culture medium composition manufactured using the method of the present invention can be carried out using culture vessels commonly used for cell culture, such as culture dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, Petit's culture dishes, tissue culture dishes, multi-plates, microplates, microwell plates, multi-plates, chambered slides, tubes, trays, culture bags, roller flasks, etc. It is desirable that these culture vessels have low cell adhesion, so that adhesive cells attached to nanofibers do not adhere to the culture vessel. As a culture vessel with low cell adhesion, a culture vessel whose surface has not been artificially treated for the purpose of improving cell adhesion (e.g., coating treatment with extracellular matrix, etc.) or a culture vessel whose surface has been artificially treated for the purpose of reducing cell adhesion can be used.

[0083] When the culture medium needs to be changed, after separating the cells by centrifugation and filtration, fresh culture medium or a culture medium composition manufactured using the method of the present invention can be added to the cells. Alternatively, after appropriately concentrating the cells by centrifugation and filtration, fresh culture medium or a culture medium composition manufactured using the method of the present invention can be added to the concentrate. For example, the gravitational acceleration (G) during centrifugation is 100G to 400G, and the pore size of the filter used for filtration is 10μm to 100μm, but is not limited thereto.

[0084] Adhesive cell culture can be carried out using bioreactors and automated culture devices that can automatically perform cell inoculation, culture medium replacement, cell image acquisition, culture cell recovery, pH, temperature, oxygen concentration, etc., under mechanical control and in a closed environment, while also enabling high-density culture.

[0085] Suspension culture of adhesive cells attached to nanofibers loaded with extracellular matrix can efficiently promote their proliferation, making this suspension culture an excellent method for adhesive cell proliferation. When adhesive cells are suspended in suspension on nanofibers loaded with extracellular matrix, they are not merely localized to the bottom of the culture vessel, but rather dispersed in a three-dimensional expansion manner, thus promoting proliferation. As a result, the proliferating cells are arranged in grape-like clusters on the nanofibers. For this proliferation-promoting effect, it is sufficient that the culture medium composition contains a sufficient concentration of nanofibers to suspend the adhesive cells (i.e., to prevent adhesion of the adhesive cells to the culture vessel), rather than requiring static suspension (i.e., cells are uniformly dispersed and suspended in the liquid culture medium composition without external pressure, vibration, oscillation, rotation, etc.).

[0086] When adhesiotropic cells are cultured in suspension and proliferated while attached to nanofibers carrying an extracellular matrix, a culture medium that can maintain the characteristics of the adhesiotropic cells while promoting cell proliferation can be used as the culture medium. Depending on the type of adhesiotropic cells, those skilled in the art can appropriately select the culture medium.

[0087] In another embodiment of the manufacturing method of the present invention, chitosan nanofibers may be incorporated in addition to nanofibers carrying extracellular matrix. By using a culture medium composition containing nanofibers carrying extracellular matrix and chitosan nanofibers for suspension culture of adhesive cells, cell proliferation can be promoted while maintaining cell quality. For example, when mammalian stem cells (e.g., mesenchymal stem cells) are cultured in suspension, their differentiation capacity, homing and migration capacity can be maintained while promoting proliferation. Therefore, according to this method, high-quality stem cells can be prepared in large quantities. It should be noted that whether stem cells maintain their differentiation capacity, migration capacity, and other characteristics can be determined by known methods. Simply put, this can be easily determined by determining the expression levels of cell markers related to undifferentiation (e.g., OCT4 gene, SOX2 gene, NANOG gene, etc.) and cell markers related to migration (e.g., CXCR4 gene, etc.) in stem cells based on mRNA and / or protein levels.

[0088] In this method, in order to prepare a culture medium containing nanofibers (e.g., chitin nanofibers) and chitosan nanofibers loaded with extracellular matrix (e.g., fibronectin) in a desired ratio (by weight), the nanofibers loaded with extracellular matrix are blended with chitosan nanofibers at a ratio of 1:0.5 to 20 (preferably 1:0.5 to 10, more preferably 1:0.7 to 9, even more preferably 1:1 to 8, and still more preferably 1:2 to 7, and particularly preferably 1:3 to 6). The resulting mixture of extracellular matrix-loaded nanofibers and chitosan nanofibers can be incorporated into a liquid culture medium at a concentration of typically 0.0001–0.2% (w / v), preferably 0.0005–0.1% (w / v), more preferably 0.001–0.05% (w / v), and particularly preferably 0.006–0.05% (w / v) of total nanofibers (including extracellular matrix-loaded nanofibers and chitosan nanofibers), in which the total nanofibers are contained in the culture medium, in which case the concentration is typically 0.0001–0.2% (w / v), preferably 0.0005–0.1% (w / v), more preferably 0.001–0.05% (w / v), and particularly preferably 0.006–0.05% (w / v). Alternatively, the desired culture medium can be prepared by separately adding nanofibers (e.g., chitin nanofibers) and chitosan nanofibers loaded with the desired amount of extracellular matrix (e.g., vitrin) to the liquid culture medium and stirring thoroughly.

[0089] In one embodiment, the concentrations of the nanofibers (e.g., chitin nanofibers) and chitosan nanofibers loaded with the extracellular matrix (e.g., fibronectin) in the culture medium composition, manufactured using the method of the present invention, satisfy the following conditions:

[0090] (1) The concentration of total nanofibers (nanofibers loaded with extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.0001 to 0.2% (w / v), and the weight ratio of nanofibers loaded with extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6);

[0091] (2) The concentration of total nanofibers (nanofibers loaded with extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.0005 to 0.1% (w / v), and the weight ratio of nanofibers loaded with extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0092] (3) The concentration of total nanofibers (nanofibers carrying extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.001 to 0.05% (w / v), and the weight ratio of nanofibers carrying extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0093] or,

[0094] (4) The total concentration of nanofibers (nanofibers carrying extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.006 to 0.05% (w / v), and the weight ratio of nanofibers carrying extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0095] In other embodiments, the resulting mixture of extracellular matrix-loaded nanofibers / chitosan nanofibers can be incorporated into a liquid culture medium in such a manner that the concentration of the total nanofibers (extracellular matrix-loaded nanofibers and chitosan nanofibers) contained in the culture medium is typically 0.0001 to 1.0% (w / v), preferably 0.001 to 0.5% (w / v), more preferably 0.005 to 0.3% (w / v), and particularly preferably 0.01 to 0.1% (w / v).

[0096] In another embodiment, the concentrations of the nanofibers (e.g., chitin nanofibers) and chitosan nanofibers loaded with the extracellular matrix (e.g., fibronectin) in the culture medium composition, manufactured using the method of the present invention, satisfy the following conditions:

[0097] (5) The concentration of total nanofibers (nanofibers carrying extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.0001 to 1.0% (w / v), and the weight ratio of nanofibers carrying extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0098] (6) The concentration of total nanofibers (nanofibers carrying extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.001 to 0.5% (w / v), and the weight ratio of nanofibers carrying extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0099] (7) The concentration of total nanofibers (nanofibers carrying extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.005-0.3% (w / v), and the weight ratio of nanofibers carrying extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5-20 (preferably 1:0.5-10, 1:0.7-9, 1:1-8, 1:2-7, or 1:3-6);

[0100] or,

[0101] (8) The concentration of total nanofibers (nanofibers carrying extracellular matrix and chitosan nanofibers) in the culture medium composition is 0.01 to 0.1% (w / v), and the weight ratio of nanofibers carrying extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0102] When adhesiotropic cells are cultured in suspension attached to nanofibers and / or chitosan nanofibers carrying extracellular matrix, the operation of removing the cells from the culture vessel is unnecessary. Adhesiotropic cells can be passaged simply by adding fresh culture medium or the culture medium composition of the present invention to the suspension culture, or by adding all or part of the suspension culture to fresh culture medium or the culture medium composition of the present invention. Using this passage culture method, adhesiotropic cells can be passaged without removing them from the culture vessel. Furthermore, this passage culture method allows for scaling up the culture scale of adhesiotropic cells without removing them from the culture vessel. Examples of removing cells from the culture vessel include treatment with chelating agents (e.g., EDTA) and / or proteolytic enzymes (e.g., trypsin, collagenase). The above-described passage culture method is advantageous for the passage culture of adhesiotropic cells that are highly sensitive to the operation of removing cells from the culture vessel (e.g., adhesiotropic cells whose viability decreases due to removal, or adhesiotropic cells whose characteristics are easily altered due to removal). Examples of adhesive cells that are highly sensitive to manipulation of detaching cells from culture containers include, but are not limited to, stem cells (e.g., mesenchymal stem cells), progenitor cells (e.g., preadipocytes), and primary cultured cells.

[0103] For example, a chitin-degrading enzyme is added to a suspension of adhesive cells attached to chitin nanofibers, and the mixture is incubated for a sufficient time for the peeling of the adhesive cells. The incubation temperature for chitin-based enzymes is typically 20°C to 37°C. The incubation time also depends on the type of enzyme, but is usually 5 to 60 minutes.

[0104] After the chitin nanofibers decompose and the adhesive cells are detached from the nanofibers, the detached adhesive cells can be recovered by centrifuging the suspension.

[0105] Damage to the recovered adhesive cells is minimized, making them a preferred choice for functional analysis, transplantation, and other applications.

[0106] 2. Composition for adding culture medium

[0107] The present invention also provides a composition for adding a culture medium containing chitin nanofibers and chitosan nanofibers loaded with an extracellular matrix (hereinafter, sometimes referred to as "the composition of the present invention").

[0108] The compositions of the present invention are characterized by comprising chitin nanofibers and chitosan nanofibers carrying an extracellular matrix. The chitin nanofibers, chitosan nanofibers, and extracellular matrix in the compositions of the present invention are the same as those described in the manufacturing method of the present invention.

[0109] As an example of a method for preparing the composition of the present invention, the following method can be used. First, an aqueous solution of an extracellular matrix (e.g., vitrin) is mixed into a dispersion containing chitin nanofibers and an aqueous solvent, stirred, and then allowed to stand (the standing time, temperature, and other conditions are the same as described in the "Manufacturing Method of the Present Invention"). Stirring is not particularly limited and can be performed by blowing or suction. This prepares a dispersion of chitin nanofibers carrying an extracellular matrix. Next, a dispersion containing chitosan nanofibers and an aqueous solvent is added to the dispersion of chitin nanofibers carrying an extracellular matrix, and the mixture is stirred to prepare the composition of the present invention.

[0110] The amount of extracellular matrix carried by the chitin nanofibers in the composition of the present invention is not particularly limited as long as the desired effect can be obtained. The extracellular matrix is ​​usually 0.001 to 50 mg per 1 g of chitin nanofibers, preferably 0.01 to 10 mg, more preferably 0.1 to 10 mg, even more preferably 0.3 to 10 mg, even more preferably 1 to 10 mg, particularly preferably 2 to 10 mg, but is not limited to these.

[0111] In the composition of the present invention, the ratio (by weight) of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is not particularly limited as long as the desired effect can be obtained. Generally, the ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is 1:0.5 to 20 (preferably, the ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is 1:0.5 to 10, more preferably, the ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is 1:0.7 to 9, even more preferably, the ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is 1:1 to 8, even more preferably, the ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is 1:2 to 7, and particularly preferably, the ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers is 1:3 to 6).

[0112] The composition of the present invention is incorporated into a culture medium for adhesive cells. There is no particular limitation on the amount of the composition of the present invention added to the culture medium, as long as the desired effect is obtained. For example, the concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium can be added in a manner typically 0.0001–0.2% (w / v), preferably 0.0005–0.1% (w / v), more preferably 0.001–0.05% (w / v), and particularly preferably 0.006–0.05% (w / v).

[0113] In another embodiment, the composition of the present invention can be added to the culture medium in such a way that the concentration of the total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) contained in the culture medium is typically 0.0001 to 1.0% (w / v), preferably 0.001 to 0.5% (w / v), more preferably 0.005 to 0.3% (w / v), and particularly preferably 0.01 to 0.1% (w / v).

[0114] 3. Culture medium composition

[0115] The present invention also provides a culture medium composition for suspension culture of adhesive cells, which comprises the composition of the present invention (hereinafter, sometimes also referred to as "the culture medium composition of the present invention").

[0116] The culture medium composition of the present invention can be prepared by mixing the composition of the present invention with a cell culture medium. The cell culture medium can be appropriately determined according to the type of adhesive cells being cultured. Examples of culture media exemplified in the "Manufacturing Method of the Invention" can be cited as culture media for preparing the culture medium composition of the present invention.

[0117] In one embodiment, the chitin nanofibers and chitosan nanofibers carrying extracellular matrix (e.g., hydrin) in the culture medium composition of the present invention satisfy the following conditions:

[0118] (1) The total concentration of nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.0001 to 0.2% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6);

[0119] (2) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.0005 to 0.1% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0120] (3) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.001 to 0.05% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0121] or,

[0122] (4) The total concentration of nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.006 to 0.05% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0123] In another embodiment, the chitin nanofibers and chitosan nanofibers carrying extracellular matrix (e.g., vitrin) in the culture medium composition of the present invention meet the following conditions:

[0124] (5) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.0001 to 1.0% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0125] (6) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.001 to 0.5% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0126] (7) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.005 to 0.3% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0127] or,

[0128] (8) The total concentration of nanofibers (chitin nanofibers and chitosan nanofibers carrying the extracellular matrix) in the culture medium composition is 0.01 to 0.1% (w / v), and the weight ratio of chitin nanofibers carrying the extracellular matrix to chitosan nanofibers in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6).

[0129] The invention will be described in more detail in the following embodiments, but the invention is not limited by these examples.

[0130] Example

[0131] [Preparation Example 1]

[0132] (Preparation of an aqueous dispersion containing chitin nanofibers)

[0133] A 2% by mass chitin nanofiber aqueous dispersion prepared according to the description in Patent Document 1 (International Publication No. 2015 / 111686) was autoclaved at 121°C for 20 minutes. Then, the aqueous dispersion was mixed and suspended in sterile distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) at a concentration of 1% (w / v) to prepare an aqueous dispersion containing sterile chitin nanofibers.

[0134] [Preparation Example 2]

[0135] (Preparation of an aqueous dispersion containing chitosan nanofibers)

[0136] A 2% by mass chitosan nanofiber aqueous dispersion prepared according to the description in Patent Document 1 (International Publication No. 2015 / 111686) was autoclaved at 121°C for 20 minutes. Then, the aqueous dispersion was mixed and suspended in sterile distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) at a concentration of 1% (w / v) to prepare an aqueous dispersion containing sterile chitosan nanofibers.

[0137] [Preparation Example 3]

[0138] (Preparation of an aqueous dispersion containing chitin nanofibers and chitosan nanofibers)

[0139] Add the chitosan nanofiber aqueous dispersion (8 mL) prepared in Preparation Example 2 to the chitin nanofiber aqueous dispersion (2 mL) prepared in Preparation Example 1, and mix by blowing and sucking to prepare an aqueous dispersion (10 mL) containing chitin nanofibers and chitosan nanofibers.

[0140] [Preparation Example 4]

[0141] (Preparation of an aqueous dispersion containing vitrin-supported chitin nanofibers)

[0142] An aqueous dispersion containing 500 μg / mL of vitrin (Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated, Thermo Fisher Scientific) was added to the 1% (w / v) chitin nanofiber aqueous dispersion prepared in Preparation Example 1. The mixture was stirred by blowing and aspiration and then incubated overnight at 4°C to prepare aqueous dispersions containing vitrin-supported chitin nanofibers with varying amounts of vitrin. The resulting aqueous dispersions containing vitrin-supported chitin nanofibers are shown in Table 1. Analysis Example 1, described below, confirmed that the chitin nanofibers were supported on vitrin.

[0143] [Table 1]

[0144]

[0145] [Preparation Example 5]

[0146] (Preparation of an aqueous dispersion containing hydrin-supported chitin nanofibers and chitosan nanofibers)

[0147] The chitosan nanofiber aqueous dispersion prepared in Preparation Example 2 (8 mL) was added to the glassne-supported chitin nanofiber aqueous dispersion prepared in Preparation Example 4 (2 mL), and mixed by blowing and suction to prepare an aqueous dispersion (10 mL) containing glassne-supported chitin nanofibers and chitosan nanofibers. The resulting aqueous dispersions containing glassne-supported chitin nanofibers and chitosan nanofibers are shown in Table 2.

[0148] [Table 2]

[0149]

[0150] [Analysis example 1]

[0151] (Staining of vitrin in the composition)

[0152] 40 μL of high-sensitivity protein staining solution (TaKaRa CBB Protein SafeStain, manufactured by Takara Bio Co., Ltd.) was added to the chitin nanofiber aqueous dispersion (100 μL) prepared in Preparation Example 1, the chitin nanofiber / chitosan nanofiber aqueous dispersion (100 μL) prepared in Preparation Example 3, the DHd511 (100 μL) prepared in Preparation Example 4, and the DHd515 (100 μL) prepared in Preparation Example 5, respectively, and allowed to stand for 10 minutes. 20 μL of each was then dropped onto a glass slide, covered with a coverslip, and observed using a phase-contrast microscope. Images of the chitin nanofiber aqueous dispersions are shown below. Figure 1 The observed images of the chitin nanofiber / chitosan nanofiber aqueous dispersion are shown in the figure. Figure 2 The observed image of DHd511 is shown in the image. Figure 3 The observed image of DHd515 is shown in the image. Figure 4 middle. Figure 1 and Figure 2 No stained areas were observed in the middle, in contrast, Figure 3 and Figure 4 Staining was observed on the chitin nanofibers. This confirms that hylocinin was physically adsorbed and supported on the surface of the chitin nanofibers using the method described in Example 4. This is believed to be due to the physical adsorption of hylocinin (not by chemical bonds) onto the chitin nanofibers. Furthermore, Figure 4It was also shown that even after the steps of Preparation Example 5, chitin nanofibers still carried hydrin.

[0153] [Analysis example 2]

[0154] (Calculation of the amount of hydrin loaded on chitin fibers)

[0155] In the aqueous dispersion of chitin nanofibers supported on hydrin prepared in Example 4, the protein contained only hydrin. Therefore, the hydrin loading was calculated using the Micro BCA Protein Assay Kit (ThermoFisher Scientific), a kit for total protein quantification, by the following method.

[0156] DHd509, 510, and 511 (1 mL each) prepared in Preparation Example 4 were injected into 1.5 mL microtubes and centrifuged (12300 × g, 5 min). The supernatant (500 μL) was filtered through a 0.45 μm filter flask (Thomson), and the filtrate (300 μL) was collected into new 1.5 mL microtubes. Following the procedure outlined in the kit, 300 μL of the Working Reagent reagent (prepared by mixing MA solution (5.0 mL) / MB solution (4.8 mL) / MC solution (0.2 mL)) was added to the collected supernatant, and the mixture was heated at 60 °C for 1 hour. After heating, 300 μL was dispensed into each well of a 96-well plate (Corning 3603), and the absorbance at 562 nm was measured using a plate reader (Infinite M200PRO, Tecan). It should be noted that the standard curve for vitrin was prepared using absorbance at 562 nm obtained by processing solutions of 1, 5, 25, 50, and 100 μg / mL in the same manner as the samples described above. Based on the amount of vitrin contained in the supernatant of the aqueous dispersion quantified from the obtained measurements, the amount of vitrin loaded on the chitin surface (physical adsorption) was calculated using the following formula.

[0157] [Total amount of added fibronectin] - [Amount of fibronectin in the supernatant of the aqueous dispersion] = [Amount of fibronectin loaded on the surface of chitin]

[0158] The results are shown in Table 3. Table 3 confirms that the ligands are loaded onto the chitin nanofibers, and it can be seen that the loading of chitin nanofibers onto the chitin nanofibers increases with the increase of the amount of ligand added.

[0159] [Table 3]

[0160]

[0161] [Experimental Example 1]

[0162] (Continuous expansion culture of mesenchymal stem cells derived from human umbilical cord in 3D culture using a culture medium composition containing fibronectin-supported chitin nanofibers and chitosan nanofibers)

[0163] The following culture medium compositions were prepared by adding DHd513, DHd514, and DHd515 prepared in Preparation Example 5 to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) used as a serum medium, respectively, to a final concentration of 0.05% (w / v). Additionally, as a control sample, a culture medium composition was prepared by adding an aqueous dispersion containing 1% (w / v) chitin nanofibers and chitosan nanofibers prepared in Preparation Example 3 to a final concentration of 0.05% (w / v).

[0164] Next, cultured mesenchymal stem cells (C-12971, manufactured by Takara Bio Inc.) derived from human umbilical cord were suspended at 16,667 cells / mL in the aforementioned culture medium compositions, and then seeded at 1.2 mL / well in 24-well flat-bottomed ultra-low adhesion microplates (Corning, #3473). The cells were cultured statically for 4 days in a CO2 incubator (37°C, 5% CO2). At seeding (day 0) and on day 4 post-seeding, 300 μL of ATP reagent (CellTiter-Glo) was added to 300 μL of the culture medium. TM The Luminescent Cell Viability Assay (Promega) was used to suspend the cells and allow them to stand at room temperature for about 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The luminescence value of the cells in the culture medium was subtracted to calculate the number of viable cells (the average of the two points).

[0165] On day 4 post-inoculation, the cell-adhering nanofibers were redispersed by pipetting. Approximately 2.4 mL of this suspension was recovered from two wells of a 24-well flat-bottomed ultra-low adhesion surface microplate. 7.6 mL of each of the aforementioned culture medium compositions was added to each well and mixed by pipetting. The mixture was then seeded and cultured in 6-well flat-bottomed ultra-low adhesion surface microplates (Corning, #3471) at total volume per well. On day 7, the cell-adhering nanofibers were redispersed by pipetting. Approximately 5 mL of this suspension was recovered from the 6-well flat-bottomed ultra-low adhesion surface microplate. 5 mL of each of the aforementioned culture medium compositions was added to each well and mixed by pipetting. The mixture was then seeded and cultured in new 6-well flat-bottomed ultra-low adhesion surface microplates (Corning, #3471) at total volume per well. The same procedure as day 7 was performed on day 10, and culture continued until day 13. For cell culture media at days 7, 10, and 13 in 6-well flat-bottomed ultra-low adhesion microplates, 500 μL of ATP reagent (CellTiter-Globe) was added. TM The Luminescent Cell Viability Assay (Promega) was prepared by suspending the cells in the medium and allowing them to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The luminescence value was subtracted from the value obtained with only the culture medium, and the number of viable cells was calculated (the average of the two measurements). Additionally, the final ATP values ​​at days 4, 7, 10, and 13 were converted using the expansion ratios used in each step.

[0166] The results showed that, compared with the culture medium composition containing chitin nanofibers and chitosan nanofibers without glassne loading (control sample), the culture medium composition containing glassne-loaded chitin nanofibers and chitosan nanofibers significantly promoted the proliferation of mesenchymal stem cells derived from the human umbilical cord. Furthermore, the proliferation-promoting effect depended on the amount of glassne loading. Additionally, large-scale culture could be achieved simply by adding fresh culture medium composition containing chitin nanofibers and chitosan nanofibers, without any treatment involving trypsin-based cell removal from the substrate. The converted RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Table 4.

[0167] [Table 4]

[0168]

[0169] [Experimental Example 2]

[0170] (Continuous expansion culture of mesenchymal stem cells derived from human bone marrow in 3D culture using a culture medium composition containing fibronectin-supported chitin nanofibers and chitosan nanofibers)

[0171] The following culture medium compositions were prepared by adding DHd513, DHd514, and DHd515 prepared in Preparation Example 5 to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) used as a serum medium, respectively, to a final concentration of 0.05% (w / v). Additionally, as a control sample, a culture medium composition was prepared by adding an aqueous dispersion containing 1% (w / v) chitin nanofibers and chitosan nanofibers prepared in Preparation Example 3 to a final concentration of 0.05% (w / v).

[0172] Next, cultured mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) derived from human bone marrow were suspended at a concentration of 8333 cells / mL in the aforementioned culture medium compositions, and then seeded at 1.2 mL / well in 24-well flat-bottomed ultra-low adhesion microplates (manufactured by Corning Incorporated, #3473). The cells were cultured statically in a CO2 incubator (37°C, 5% CO2). On day 4, approximately 0.6 mL of supernatant was removed from each well, and 0.6 mL of fresh mesenchymal stem cell proliferation medium was added to each well. After resuspending the cells using a pipette, they were cultured further until day 8 post-seeding. 300 μL of ATP reagent (CellTiter-Glo) was added to 300 μL of the culture medium at day 0 and day 8 post-seeding. TM The Luminescent Cell Viability Assay (Promega) was suspended and allowed to stand at room temperature for about 10 minutes. The luminescence intensity (RLU value) was measured using a FlexStation3 (Molecular Devices). The luminescence value of the culture medium alone was subtracted to calculate the number of viable cells (average of the two points).

[0173] On day 8 post-inoculation, the nanofibers adhering to the cells of the DHd515 group were redispersed by pipetting. Approximately 0.6 mL of the suspension was recovered from a 24-well flat-bottomed ultra-low adhesion surface microplate. 0.6 mL of the aforementioned DHd515-containing culture medium composition was added, and after mixing by pipetting, the mixture was seeded into 24-well flat-bottomed ultra-low adhesion surface microplates (Corning, #3471) at the total volume per well, and cultured until day 11 post-inoculation. For other sample groups, approximately 0.6 mL of the culture medium supernatant was removed from the wells, and 0.6 mL of fresh mesenchymal stem cell proliferation medium was added to each well. After resuspending the cells using a pipette, cultured until day 11 post-inoculation. On day 11, for all sample groups containing DHd515, approximately 0.6 mL of the culture medium supernatant was removed from the wells, and 0.6 mL of fresh mesenchymal stem cell proliferation medium was added to each well. After resuspending the cells using a pipette, cultured until day 15 post-inoculation. On day 15 post-inoculation, in the DHd515 and DHd514 groups, the nanofibers adhering to the cells were redispersed by pipetting. Approximately 0.6 mL of the suspension was recovered from the 24-well flat-bottomed ultra-low adhesion surface microplates. 0.6 mL of the aforementioned culture medium composition containing DHd515 or DHd514 was added to each well and mixed by pipetting. The mixture was then seeded at the total volume per well into 24-well flat-bottomed ultra-low adhesion surface microplates (Corning, #3471), and cultured until day 18 post-inoculation. For the other sample groups, approximately 0.6 mL of the culture supernatant was removed from the wells, and 0.6 mL of fresh mesenchymal stem cell proliferation medium was added to each well and resuspended by pipetting. Cultured until day 18 post-inoculation was continued. On day 18, for all sample groups containing DHd515, approximately 0.6 mL of the culture supernatant in the wells was removed, and 0.6 mL of fresh mesenchymal stem cell proliferation medium was added to each well and resuspended using a pipette. Culture continued until day 22 post-inoculation. On day 22 post-inoculation, in the DHd515 and DHd514 groups, the cell-adhesive nanofibers were redispersed by pipetting. Approximately 0.6 mL of the suspension was recovered from the 24-well flat-bottomed ultra-low adhesion surface microplate. 0.6 mL of the culture medium composition containing either DHd515 or DHd514 was added again, mixed by pipetting, and then seeded per well in a 24-well flat-bottomed ultra-low adhesion surface microplate (Corning, #3471). Culture continued until day 27 post-inoculation. For other sample groups, about 0.6 mL of culture medium was removed from the wells, and 0.6 mL of fresh mesenchymal stem cell proliferation culture medium was added to each well. After resuscitation using a pipette, the wells were cultured until day 27 post-inoculation.

[0174] For cells cultured in 24-well flat-bottomed ultra-low adhesion microplates on days 15, 22, and 27, 300 μL of ATP reagent (CellTiter-Glo) was added. TM The Luminescent Cell Viability Assay (Promega) was prepared by suspending the cells in a suspension and allowing them to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The luminescence value from the culture medium alone was subtracted to calculate the number of viable cells (the average of the two measurements). Additionally, the final ATP values ​​at days 15, 22, and 27 were converted using the expansion ratios used in each step.

[0175] The results showed that, compared with the culture medium composition containing chitin nanofibers and chitosan nanofibers without glassne loading (control sample), the culture medium composition containing glassne-loaded chitin nanofibers and chitosan nanofibers significantly promoted the proliferation of mesenchymal stem cells derived from human bone marrow. Furthermore, the proliferation-promoting effect depended on the amount of glassne loading. Additionally, large-scale culture could be achieved simply by adding fresh culture medium composition containing chitin nanofibers and chitosan nanofibers, without any treatment involving trypsin-based cell removal from the substrate. The converted RLU values ​​(ATP assay, luminescence intensity) for each culture are shown in Table 5.

[0176] [Table 5]

[0177]

[0178] [Experimental Example 3]

[0179] (Continuous expansion culture of human adipose-derived mesenchymal stem cells in 3D culture using a culture medium composition containing vitrin-supported chitin nanofibers and chitosan nanofibers, and a comparison with the effect of vitrin addition)

[0180] The following culture medium compositions were prepared by adding DHd513, DHd514, and DHd515 prepared in Preparation Example 5 to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) used as a serum medium, respectively, to a final concentration of 0.05% (w / v). Additionally, as a control sample, a culture medium composition was prepared by adding an aqueous dispersion containing 1% (w / v) chitin nanofibers and chitosan nanofibers prepared in Preparation Example 3 to a final concentration of 0.05% (w / v).

[0181] Next, cultured human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were suspended at 8333 cells / mL in each of the above-mentioned culture medium compositions, and then seeded at 1.2 mL / well in 24-well flat-bottomed ultra-low adhesion microplates (Corning, #3473). The cells were cultured statically in a CO2 incubator (37°C, 5% CO2). Alternatively, several hours after seeding with control samples, an aqueous solution containing 500 μg / mL of vitronectin (Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated, manufactured by Thermo Fisher Scientific) was added to achieve a final concentration of 0.5 μg / mL or 1.0 μg / mL, and the cells were cultured further. 0.5 μg / mL is the same amount of glassin used in DHd515, and 1.0 μg / mL is twice the amount of glassin used in DHd515. Add 300 μL of ATP reagent (CellTiter-Globe) to 300 μL of culture medium at inoculation (day 0) and on day 4 post-inoculation. TM The Luminescent Cell Viability Assay (Promega) was used to suspend the cells and allow them to stand at room temperature for about 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The luminescence value of the cells in the culture medium was subtracted, and the number of viable cells was calculated (average of the two points).

[0182] On day 4 post-inoculation, for control samples, approximately 0.6 mL of culture medium was removed from the wells, and 0.6 mL of fresh mesenchymal stem cell proliferation medium was added to each well. The samples were then resuspended using a pipette and cultured until day 8 post-inoculation. For other samples, the nanofibers adhering to the cells were redispersed by pipetting, and approximately 0.6 mL of the suspension was recovered from the 24-well flat-bottomed ultra-low adhesion surface microplate. 0.6 mL of each culture medium composition was added and mixed by pipetting. The mixture was then seeded per well in a 24-well flat-bottomed ultra-low adhesion surface microplate (Corning, #3473) and cultured until day 8 post-inoculation. Starting from day 8 post-inoculation, the nanofibers adhering to the cells were redispersed by blowing and aspiration at 3-4 day intervals. Approximately 0.6 mL of the suspension was recovered from the 24-well flat-bottomed ultra-low adhesion surface microplate. 0.6 mL of each culture medium composition was added and mixed by blowing and aspiration. The mixture was then inoculated into 24-well flat-bottomed ultra-low adhesion surface microplates (Corning, #3473) at the total volume per well and cultured until day 21 post-inoculation.

[0183] Add 300 μL of ATP reagent (CellTiter-Glo) to 300 μL of cell culture medium cultured in 24-well flat-bottomed ultra-low adhesion microplates on days 8, 15, and 21. TM The Luminescent Cell Viability Assay (Promega) was prepared by suspending the cells in a suspension and allowing them to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The luminescence value from the culture medium alone was subtracted to calculate the number of viable cells (the average of the two measurements). Additionally, the final ATP values ​​at days 8, 15, and 21 were converted using the expansion ratios used in each step.

[0184] The results showed that, compared with the culture medium composition containing chitin and chitosan nanofibers without glassne-loaded viscosin (control sample), the culture medium composition containing viscosin-loaded chitin and chitosan nanofibers significantly promoted the proliferation of mesenchymal stem cells derived from human adipose tissue. Furthermore, the proliferation-promoting effect depended on the amount of viscosin loaded. In addition, the proliferation-promoting effect of DHd515 was significantly higher than that of directly adding the same or twice the amount of viscosin to the culture medium using the control sample. Moreover, large-scale culture could be achieved simply by adding fresh culture medium composition containing chitin and chitosan nanofibers, without any treatment based on trypsin or similar methods to detach cells from the substrate. The converted RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Table 6.

[0185] [Table 6]

[0186]

[0187] [Experimental Example 4]

[0188] (A study on the cell proliferation effect caused by different amino acid sequences of the carried fibronectin)

[0189] To 5 mL of the 1% (w / v) chitin nanofiber aqueous dispersion prepared in Preparation Example 1, 0.5 mL of any one of the following 500 μg / mL viscolin aqueous solutions with different amino acid residues ((A) Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated, aa sequence 62-478, manufactured by Thermo Fisher Scientific; (B) PluriSTEM-XF Recombinant Vitronectin, aa sequence 20-398, manufactured by Sigma-Aldrich; (C) Animal-Free Recombinant Human Vitronectin, derived from HEK293 cells, aa sequence 20-478 (serial number 3), manufactured by PeproTech) was added. After mixing by blowing and aspiration, the mixture was incubated overnight at 4°C to prepare an aqueous dispersion containing chitin nanofibers supported by the three viscolins. Hereinafter, the aqueous dispersions containing viscosin-supported chitin nanofibers and chitosan nanofibers prepared using (A), (B), or (C) above will be referred to as aqueous dispersion A, aqueous dispersion B, or aqueous dispersion C, respectively.

[0190] Prepare the following culture medium composition: add aqueous dispersion A, aqueous dispersion B, or aqueous dispersion C to mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) used as serum medium to obtain a final concentration of 0.05% (w / v).

[0191] Next, cultured mesenchymal stem cells (C-12971, manufactured by Takara Bio Inc.) derived from human umbilical cord were suspended at 15,000 cells / mL in the aforementioned culture medium compositions, and then seeded at 10 mL / well in 6-well flat-bottomed ultra-low adhesion microplates (manufactured by Corning Incorporated, #3471). The cells were cultured statically in a CO2 incubator (37°C, 5% CO2). On day 3, approximately 5 mL of the culture supernatant in the wells was removed, and 5 mL of fresh mesenchymal stem cell proliferation medium was added to each well. After resuspending the cells using a pipette and replacing half of the medium, the cells were cultured until day 7 post-seeding. On day 7, the cell-adhering nanofibers were redispersed by blow-aspiration, and approximately 5 mL of the suspension was recovered from the 6-well flat-bottom ultra-low adhesion surface microplate. 5 mL of each of the aforementioned culture medium compositions was added and mixed by blow-aspiration. The mixture was then seeded and cultured in new 6-well flat-bottom ultra-low adhesion surface microplates (Corning, #3471) at total volume / well. The same procedure was performed on day 10, continuing culture until day 13. 500 μL of ATP reagent (CellTiter-Glo) was added to 500 μL of the cell culture medium at seeding (day 0) and on days 3, 7, 10, and 13 of culture using the 6-well flat-bottom ultra-low adhesion surface microplates. TM The Luminescent Cell Viability Assay (Promega) was prepared by suspending the cells in the medium and allowing them to stand at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was then measured using a FlexStation3 (Molecular Devices). The luminescence value at the culture medium stage was subtracted from the value at the culture medium stage to calculate the number of viable cells (average of the two values). Additionally, the final ATP values ​​at days 3, 7, 10, and 13 were converted using the scaling ratios used in each step. The converted RLU values ​​(ATP measurement, luminescence intensity) for each culture are shown in Tables 7 and 8.

[0192] As a result, glassin in (A) and (B) showed equivalent proliferative effects. On the other hand, the proliferative effect of glassin in (C) was milder than that in (A).

[0193] [Table 7]

[0194]

[0195] [Table 8]

[0196]

[0197] Industrial availability

[0198] By using polysaccharide nanofibers, which are obtained by loading the extracellular matrix onto nanofiber-like polysaccharides that are insoluble in the culture medium but suspended in the culture medium, as a carrier substrate, mesenchymal stem cells, proadipocytes, and other adhesive cells can be cultured in a static state. Under suspension culture conditions, the proliferation of cells adhering to the nanofibers is promoted, showing long-term viability.

[0199] All content described herein, including patent and patent application specifications, is incorporated herein by reference in its entirety to the same extent as expressly stated.

[0200] This application is based on Japanese Special Application 2019-125536 (filed on July 4, 2019), the contents of which are fully included in this specification.

Claims

1. A method for manufacturing a culture medium composition for suspension culture of adhesive cells, the method comprising the following steps: Step (i) involves loading hyalin onto nanofibers composed of chitin. In step (ii), chitosan nanofibers and chitin nanofibers loaded with hydrin obtained in step (i) are added to the culture medium. The amount of hylocinin loaded on chitin nanofibers ranges from 0.01 to 50 mg per 1 g of chitin nanofibers. The content ratio (by weight) of chitin nanofibers carrying hydrin and chitosan nanofibers is 1:0.5-20.

2. A composition for adding to a culture medium, comprising chitin nanofibers and chitosan nanofibers carrying hydrin. in, The amount of hylocinin loaded onto chitin nanofibers ranges from 0.01 to 50 mg per 1 g of chitin nanofibers. The ratio (by weight) of chitin nanofibers carrying vitrin to chitosan nanofibers in the culture medium composition is 1:0.5-20.

3. A culture medium composition for suspension culture of adhesive cells, comprising the composition of claim 2.

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