Cell culture scaffold material and cell culture method

Abstract

The present invention relates to: a scaffold material comprising cellulose nanofibers (CNF) into which an ionic functional group has been introduced; a cell culture substrate having the scaffold material on the surface thereof; and a method for culturing cells such as stem cells using the scaffold material.

Description

Technical Field

[0001] The invention relates to a cell culture scaffold material and a cell culture method. Background Art

[0002] In the past, the culture of stem cells (adult stem cells, embryonic stem cells, induced pluripotent stem cells, etc.) was carried out in an environment consisting of a scaffold material for cell attachment and a culture medium containing serum. For example, a material in which bovine collagen, a cell attachment component, is coated on a substrate such as polystyrene or glass is used as a scaffold material for cell culture. In addition, as serum, fetal bovine serum (FBS) and the like are usually also used for cell culture as additives important for cell proliferation. However, when using cultured stem cells for medical purposes, there is a risk that the xenobiotic-derived components used during cell culture become a source of infection for blood-borne pathogens or xenobiotic antigens. Therefore, in recent years, there is an ongoing technical development of using plant-derived materials, etc., which are safer for organisms, for scaffold materials and culture medium components.

[0003] Scaffold materials for cell culture must satisfy both biochemical affinity with cells and mechanical properties necessary for cell proliferation after attachment. However, the cell adhesion and proliferation properties of scaffold materials are difficult to predict due to numerous unknown factors. Furthermore, sufficient insights into scaffold materials suitable for stem cell culture remain elusive.

[0004] Patent Document 1 discloses that human neuronal cells encapsulated in a hydrogel containing plant-derived anionic nanofibrillated cellulose (also known as cellulose nanofibers) were cultured in a cell culture medium containing serum substitute N2. The results showed that the hydrogel supported the survival and growth of neuronal cells over a two-week period. Patent Document 1 also mentions reports that anionic polymers may induce a supportive effect on neuronal cells. However, Patent Document 1 does not describe a scaffold material that can be used for stem cell culture.

[0005] For stem cells, we hope to develop safer cell culture technologies.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. WO2018 / 234634 Summary of the Invention

[0009] Problems to be solved by the invention

[0010] An object of the present invention is to provide a stem cell culture technology using a scaffold material that can reduce the amount of serum used.

[0011] Solutions to Problems

[0012] The present inventors conducted in-depth research to solve the above-mentioned problems and found that by using surface-modified cellulose nanofibers such as sulfated cellulose nanofibers as scaffold materials for cell culture, cells can be well proliferated not only under normal serum concentration conditions, but surprisingly even under low serum or serum-free conditions. In particular, stem cells can be well proliferated while maintaining their differentiation ability, thereby completing the present invention.

[0013] That is, the present invention includes the following aspects.

[0014] [1] A cell culture substrate having a scaffold material on its surface, wherein the scaffold material comprises cellulose nanofibers (CNF) into which ionic functional groups are introduced.

[0015] [2] The cell culture substrate according to [1] above, comprising the scaffold material in a dry state.

[0016] [3] The cell culture substrate according to [1] or [2] above, which is used for culturing stem cells.

[0017] [4] The cell culture substrate according to any one of [1] to [3] above, which is used for cell culture in a serum-free medium or a low-serum medium.

[0018] [5] The cell culture substrate according to any one of [1] to [4] above, wherein the ionic functional group is an anionic functional group.

[0019] [6] The cell culture substrate according to [5] above, wherein the anionic functional group is a sulfate group or a carboxyl group.

[0020] [7] The cell culture substrate according to any one of [1] to [6] above, wherein the cellulose nanofibers into which the ionic functional groups are introduced have an ionic functional group content of 0.2 mmol / g to 10 mmol / g.

[0021] [8] The cell culture substrate according to any one of [1] to [7] above, wherein the cellulose nanofibers into which the ionic functional groups are introduced have an ionic functional group content of 0.2 mmol / g to 2 mmol / g.

[0022] [9] The cell culture substrate according to any one of [1] to [8] above, wherein the scaffold material contains 10% by weight or more of cellulose nanofibers into which ionic functional groups are introduced, based on dry weight.

[0023]

[10] The cell culture substrate according to any one of [1] to [9] above, wherein the cellulose nanofibers into which ionic functional groups are introduced have a viscosity of 500 mPa·s to 50,000 mPa·s when a viscosity measurement is made at 25°C of a CNF aqueous dispersion having a solid content concentration of 0.5% at a shear rate of 6 rpm for 10 minutes.

[0024]

[11] The cell culture substrate according to any one of [1] to

[10] above, wherein the scaffold material comprises two or more cellulose nanofibers into which anionic functional groups are introduced.

[0025]

[12] The cell culture substrate according to any one of [1] to

[11] above, wherein the scaffold material is provided on the surface of any material selected from the group consisting of resin, rubber, glass, membrane, paper, cloth, beads and silk.

[0026]

[13] The cell culture substrate according to any one of [1] to

[12] above, which is a cell culture plate having the above-mentioned scaffold material coated on its inner surface.

[0027]

[14] The cell culture substrate according to

[13] above, wherein the cell culture plate is made of plastic or glass.

[0028]

[15] A cell culture method, comprising: culturing cells using the cell culture substrate described in any one of [1] to

[14] above.

[0029]

[16] The method according to

[15] above, wherein the cells are stem cells.

[0030]

[17] The method according to

[15] or

[16] above, wherein the cells are cultured in a serum-free medium or a low-serum medium.

[0031]

[18] The method according to

[16] or

[17] above, wherein the cultured stem cells are further cultured in a differentiation induction medium to induce differentiation of the stem cells.

[0032]

[19] A scaffold material comprising cellulose nanofibers into which ionic functional groups are introduced,

[0033] The scaffold material is used for culturing stem cells.

[0034]

[20] The scaffold material according to

[19] above, wherein the culture is performed while maintaining the undifferentiated state of the stem cells.

[0035]

[21] The scaffold material according to

[19] or

[20] above, wherein the culture is carried out in a serum-free medium or a low-serum medium.

[0036] This specification incorporates the disclosure of Japanese Patent Application No. 2023-030017 and Japanese Patent Application No. 2023-158511, which are the basis of priority of this application.

[0037] Effects of the Invention

[0038] According to the cell culture technique of the present invention, cells such as stem cells can be proliferated not only under normal serum concentration conditions but also under low serum or serum-free conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Photographs showing the morphology of immortalized human bone marrow-derived mesenchymal stem cells (MSCs) after three days of culture in a medium containing 10% fetal bovine serum (FBS). Scale bar: 200 μm. A: Experiment 1 (Sample 1, unmodified CNF), B: Experiment 2 (Sample 2), C: Experiment II-1 (Sample A1), D: Experiment 3 (Sample 4), E: Experiment II-2 (Sample A2), F: Experiment 4 (Samples 2 and 4, 1:1 ratio), G: Experiment 5 (Samples 2 and 5, 1:1 ratio).

[0040] Figure 2 Photographs showing the morphology of immortalized MSCs after 3 days of culture in a low-serum medium containing 2.5% FBS. Scale bar: 200 μm. A: Experiment 6 (Sample 1, unmodified CNF), B: Experiment 7 (Sample 3), C: Experiment 8 (Sample 4), D: Experiment 9 (Samples 3 and 4, 2:1 ratio), E: Experiment 10 (Samples 3 and 4, 1:1 ratio), F: Experiment 11 (Samples 3 and 5, 2:1 ratio).

[0041] Figure 3 Photographs showing the morphology of immortalized MSCs after 3 days of culture in serum-free medium without FBS. Scale bar: 200 μm. A: Experiment 12 (Sample 1, unmodified CNF), B: Experiment 13 (Sample 3), C: Experiment II-3 (Sample A1), D: Experiment 14 (Sample 4), E: Experiment II-4 (Sample A2), F: Experiment 15 (Samples 3 and 4, 1:1).

[0042] Figure 4The photographs show the results of staining of immortalized MSCs after differentiation induction after three subcultures in a serum-free medium without FBS, with each subculture lasting three days. Scale bars in the figures: 100 μm for cartilage and adipocytes, 200 μm for osteoblasts. A, D, and G: Cells obtained by inducing differentiation of the proliferating cells obtained in Experiment 13 using an adipocyte differentiation induction medium, an osteoblast differentiation induction medium, or a chondrocyte differentiation induction medium, respectively. B, E, and H: Cells obtained by inducing differentiation of the proliferating cells obtained in Experiment 14 using an adipocyte differentiation induction medium, an osteoblast differentiation induction medium, or a chondrocyte differentiation induction medium, respectively. C, F, and I: Cells obtained by inducing differentiation of the proliferating cells obtained in Experiment 15 using an adipocyte differentiation induction medium, an osteoblast differentiation induction medium, or a chondrocyte differentiation induction medium, respectively.

[0043] Figure 5 Photographs showing the morphology of primary MSCs after 3 days of culture in serum-free medium without FBS. Scale bar: 200 μm. A: Experiment II-5 (TCPS), B: Experiment II-6 (Type I collagen-coated TCPS), C: Experiment II-7 (Sample 1, unmodified CNF), D: Experiment II-8 (Sample 3), E: Experiment II-9 (Sample 4), F: Experiment II-10 (Samples 3 and 4, 1:1). DETAILED DESCRIPTION

[0044] Hereinafter, the present invention will be described in detail.

[0045] The present invention relates to a cell culture substrate using surface-modified cellulose nanofibers as a scaffold, and a cell culture method using the cell culture substrate. More specifically, the present invention relates to a scaffold material comprising surface-modified cellulose nanofibers, a cell culture substrate having the scaffold material on its surface, and a cell culture method comprising culturing cells using the scaffold.

[0046] In the present invention, cellulose nanofibers (CNFs) are fibers obtained by dissociating (defibration) cellulose fibers into nanometer-sized fiber widths. CNFs have many unique characteristics, such as high strength and high specific surface area, and have attracted much attention in various technical fields in recent years.

[0047] In the present invention, surface-modified CNF is used as a scaffold material. The surface-modified CNF may preferably be surface-chemically modified CNF. More specifically, the surface-modified CNF may be CNF into which ionic functional groups have been introduced. It is known that the introduction of ionic functional groups onto the surface of CNF can promote nanofiberization through electrostatic repulsion on the fiber surface.

[0048] Examples of CNFs introduced with ionic functional groups include CNFs introduced with either anionic functional groups or cationic functional groups, or both. In the present invention, such CNFs introduced with ionic functional groups are preferably used in scaffold materials for cell culture.

[0049] As CNFs into which anionic functional groups have been introduced (also referred to as "anion-modified CNFs"), it is not limited to the following, and CNFs into which anionic functional groups such as sulfate groups, carboxyl groups, carboxymethyl groups, phosphate groups, and phosphite groups have been introduced can be cited. It should be noted that CNFs into which sulfate groups, carboxyl groups, carboxymethyl groups, phosphate groups, and phosphite groups have been introduced are sometimes also referred to as sulfated CNFs, carboxylated CNFs, carboxymethylated CNFs, phosphated CNFs, and phosphite-modified CNFs, respectively. In one embodiment, the CNFs into which anionic functional groups have been introduced can be CNFs into which sulfate groups or carboxyl groups have been introduced. As an example of CNFs into which sulfate groups have been introduced, sulfated cellulose nanofibers S-CNFs (trademark) (Yokogawa Electric Corporation) can be cited. As an example of CNFs into which carboxyl groups have been introduced, TEMPO oxidized cellulose nanofibers (TOCNFs) can be cited.

[0050] Examples of CNFs into which a cationic functional group is introduced (also referred to as "cation-modified CNFs") include, but are not limited to, CNFs into which a cationic functional group such as a quaternary ammonium group is introduced.

[0051] In the present invention, CNFs introduced with ionic functional groups may have an average fiber width of 1 nm to 200 nm, preferably 1 nm to 50 nm, more preferably 1 nm to 16 nm or 1 nm to 10 nm, for example, 1 nm to 5 nm, 3 nm to 9 nm, 3 nm to 16 nm, 5 nm to 10 nm, 6 nm to 9 nm, 6 nm to 16 nm, 5 nm to 7 nm, 7 nm to 9 nm, or 7 nm to 10 nm. In the present invention, the average fiber width of each type of CNF is calculated as the arithmetic mean of the fiber widths of at least 50 randomly selected fibers measured under a microscope.

[0052] In the present invention, CNFs introduced with ionic functional groups may have an average fiber length of 0.05 μm to 1000 μm, preferably 0.1 μm to 50 μm, more preferably 0.1 μm to 10 μm or 0.1 μm to 5 μm, for example, 0.2 μm to 10 μm, 0.2 μm to 5 μm, 0.2 μm to 3 μm, 0.1 μm to 1 μm, or 0.2 μm to 1 μm. In the present invention, the average fiber length of each type of CNF is calculated as the arithmetic mean of the fiber lengths of at least 50 randomly selected fibers measured under a microscope.

[0053] The amount of ionic functional groups (e.g., anionic functional groups such as sulfate, carboxyl, carboxylmethyl, phosphate, and phosphite groups; or cationic functional groups such as quaternary ammonium groups) in the CNF into which ionic functional groups are introduced can be 0.1 mmol or more per 1 g of the CNF, more preferably 0.2 mmol / g or more, for example, 0.1 mmol / g to 10 mmol / g, 0.2 mmol / g to 10 mmol / g, or 0.3 mmol / g to 10 mmol / g. In one embodiment, the amount of ionic functional groups (e.g., anionic functional groups such as sulfate, carboxyl, carboxylmethyl, phosphate, and phosphite groups; or cationic functional groups such as quaternary ammonium groups) in the CNF into which ionic functional groups are introduced can be 0.5 mmol or more per 1 g of the CNF, for example, 0.5 mmol / g to 10 mmol / g, 0.5 mmol / g to 5 mmol / g, or 2 mmol / g to 5 mmol / g, but is not limited thereto. In one embodiment, the amount of ionic functional groups (e.g., anionic functional groups such as sulfate, carboxyl, carboxymethyl, phosphate, and phosphite groups; or cationic functional groups such as quaternary ammonium groups) in the CNF into which ionic functional groups are introduced can be 0.8 mmol or more per 1 g of the CNF, for example, 0.8 mmol / g to 5 mmol / g.In one embodiment, the amount of ionic functional groups (e.g., anionic functional groups such as sulfate groups, carboxyl groups, carboxymethyl groups, phosphate groups, and phosphite groups; or cationic functional groups such as quaternary ammonium groups) in the CNF into which the ionic functional groups are introduced may be 2 mmol or less, for example, 1 mmol or less, for example, 0.1 mmol / g to 2 mmol / g, 0.2 mmol / g to 2 mmol / g, 0.3 mmol / g to 2 mmol / g, 0.5 mmol / g to 2 mmol / g, 0.7 mmol / g to 2 mmol / g, 0.8 mmol / g to 2 mmol / g, 0.9 mmol / g to 2 mmol / g, 10 mmol / g to 2 mmol / g, 15 mmol / g to 2 mmol / g, 16 mmol / g to 2 mmol / g, 17 mmol / g to 2 mmol / g, 18 mmol / g to 2 mmol / g, 19 mmol / g to 2 mmol / g, 20 mmol / g to 2 mmol / g, 21 mmol / g to 2 mmol / g, 22 mmol / g to 2 mmol / g, 23 mmol / g to 2 mmol / g, 24 mmol / g to 2 mmol / g, 25 mmol / g to 2 mmol / g, 26 mmol / g to 2 mmol / g, 27 mmol / g to 2 mmol / g, 28 mmol / g to 2 mmol / g, 29 mmol / g to 30 mmol / g, 31 mmol / g to 30 mmol / g, 32 mmol / g to 30 mmol / g, 33 mmol / g to 30 mmol / g, 34 mmol / g to 35 1mmol / g~2mmol / g, 1.2mmol / g~2mmol / g, 0.1mmol / g~1.5mmol / g, 0.2mmol / g~1.5mmol / g, 0.3mmol / g~1.5mmol / g, 0.5mmol / g~1.5mmol / g , 0.7mmol / g~1.5mmol / g, 0.8mmol / g~1.5mmol / g, 1mmol / g~1.5mmol / g, 1.2mmol / g~1.5mmol / g, 0.1mmol / g~1.3mmol / g, 0.2mmol / g~1.3m mol / g, 0.3mmol / g~1.3mmol / g, 0.5mmol / g~1.3mmol / g, 0.7mmol / g~1.3mmol / g, 0.8mmol / g~1.3mmol / g, 1mmol / g~1.3mmol / g, 1.2mmol / g ~1.3mmol / g, 0.1mmol / g~1mmol / g, 0.2mmol / g~1mmol / g, 0.3mmol / g~1mmol / g, 0.5mmol / g~1mmol / g, 0.7mmol / g~1mmol / g, 0.8mmol / g~1m The amount of ionic functional groups introduced into CNF can be increased by, for example, increasing the amount of the reagent for introducing the ionic functional groups or further extending the reaction time.

[0054] The viscosity of the aqueous dispersion of CNF into which the ionic functional groups are introduced is measured at a viscosity value of 1 mPa·s or more, 5 mPa·s or more, 10 mPa·s or more, 30 mPa·s or more, 80 mPa·s or more, 200 mPa·s or more, 500 mPa·s or more, or 700 mPa·s or more, preferably 5 mPa·s to 5000 mPa·s, for example, 10 mPa·s to 5000 mPa·s, 80 mPa·s to 5000 mPa·s, 100 mPa·s to 5000 mPa·s, or 100 mPa·s to 5000 mPa·s. ·s, 80mPa·s~3000mPa·s, 100mPa·s~3000mPa·s, 200mPa·s~3000mPa·s, 200mPa·s~2500mPa·s, 200mPa·s~1200mPa·s, 500mPa·s~4000mPa·s, 500mPa·s~2500mPa·s, 500mPa·s~1200mPa·s, 700mPa·s~4000mPa·s, 700mPa·s~3000mPa·s, 700mPa·s~2500mPa·s or 700mPa·s~1200mPa·s, but is not limited thereto. In addition, the viscosity of the aqueous dispersion of CNF into which the ionic functional groups are introduced may be 10 mPa·s or more, 30 mPa·s or more, 100 mPa·s or more, 200 mPa·s or more, 300 mPa·s or more, 400 mPa·s or more, 500 mPa·s or more, 1000 mPa·s or more, or 5000 mPa·s or more, preferably 30 mPa·s to 50,000 mPa·s, more preferably 200 mPa·s to 50,000 mPa·s or 500 mPa·s to 50,000 mPa·s. Pa·s, for example, can be 100mPa·s~50000mPa·s, 200mPa·s~50000mPa·s, 400mPa·s~50000mPa·s, 500mPa·s~50000mPa·s, 1000mPa·s~50000mPa·s, 5000mPa·s~20000mPa·s, 5000mPa·s~10000mPa·s, 5000mPa·s~8000mPa·s, 1000mPa·s~30000mPa·s, 1000mPa·s~20000mPa·s or 1000mPa·s~10000mPa·s, but is not limited thereto.

[0055] In addition, in this specification, % indicating concentration refers to weight percentage (weight %) unless otherwise specified, and may also be expressed as weight / weight % (w / w %).

[0056] In one embodiment, the CNF having imported an ionic functional group can enumerate the CNF with a crystallinity of more than 10%.The crystallinity of the CNF having imported an ionic functional group can be calculated by Segal's method.The crystallinity of the CNF having imported an ionic functional group is preferably more than 20%, more preferably more than 30%, for example, it can be 10%~100%, 20%~100%, 40%~100%, 50%~100%, 10%~80%, 20%~80%, 30%~80%, 40%~80%, 50%~80%, 60%~80%, 70%~80%, 50%~78%, 60%~78%, 60%~70%, 40%~70% or 50%~60%, but is not limited thereto.

[0057] In one embodiment, the CNF into which the ionic functional group is introduced may be a CNF having a light transmittance of 10% or more at a wavelength of 600 nm. The light transmittance of the CNF into which the ionic functional group is introduced at a wavelength of 600 nm may preferably be 15% or more, more preferably 20% or more, for example, 10% to 90%, 15% to 90%, 10% to 88%, 15% to 88%, 15% to 80%, 15% to 78%, 20% to 85%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 64% to 90%, 65% to 90%, 70% to 90%, 60% to 88%, 64% to 88%, 66% to 88%, 67% to 88%, 68% to 88%, 69% to 90%, 71% to 90%, 72% to 90%, 73% to 88%, 74% to 88%, 76% to 88%, 77% to 88%, 78% to 88%, 79% to 90%, 80% to 88%, 81% to 88%, 82% to 88%, 83% to 88%, 84% to 88%, 86% to 88%, 87% to 88%, 88% to 88%, 89% to 90%, 90% to 90%, 91% to 90%, 91% to 90%, 92% to 90%, 93% to 90%, 94% to 90%, 95% to 90%, 96% to 90%, 97% to 90%, 98% 5%~88%, 70%~88%, 60%~85%, 64%~85%, 65%~85%, 70%~85%, 75%~85%, 60%~82%, 64%~82%, 65%~82%, 70%~82%, 75%~82%, 60%~80%, 64%~80%, 65%~80%, 70%~80%, 75%~80%, 60%~78%, 64%~78%, 65%~78%, 70%~78%, or 60%~70%, but is not limited thereto.

[0058] In one embodiment, the CNF into which the ionic functional group is introduced contains 0.5 mmol / g to 1 mmol / g, preferably 0.7 mmol / g to 1 mmol / g, for example, 0.88 mmol / g of the ionic functional group (eg, carboxyl group). The CNF into which the ionic functional groups are introduced may contain 0.5 mmol / g to 1 mmol / g, preferably 0.7 mmol / g to 1 mmol / g, for example 0.88 mmol / g of ionic functional groups (e.g., carboxyl groups), and have an average fiber width of 7 nm to 10 nm (typically 8 nm), an average fiber length of 0.2 μm to 5 μm, a crystallinity of 70% to 80% (typically 77%), and / or a light transmittance of 75% to 80% (typically 78%), and / or a viscosity of its aqueous dispersion of 2500 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 16000 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, TOCNF such as the TOCNF shown as Sample 2 in Examples 1 and 2 described later, but is not limited thereto.

[0059] In one embodiment, the CNF into which the ionic functional group is introduced contains 0.5 mmol / g to 1.5 mmol / g, preferably 1.2 mmol / g to 1.5 mmol / g, for example, 1.45 mmol / g of the ionic functional group (eg, carboxyl group). The CNF into which the ionic functional groups are introduced may contain 0.5 mmol / g to 1.5 mmol / g, preferably 1.2 mmol / g to 1.5 mmol / g, for example 1.45 mmol / g of ionic functional groups (e.g., carboxyl groups), and have an average fiber width of 1 nm to 5 nm (typically 3 nm), an average fiber length of 0.2 μm to 1 μm, a crystallinity of 60% to 78% (typically 70%), and / or a light transmittance of 70% to 88% (typically 85%), and / or a viscosity of its aqueous dispersion of 1200 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 9000 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, TOCNF such as the TOCNF shown as Sample 3 in Examples 1 and 2 described later, but is not limited thereto.

[0060] In one embodiment, the CNF into which the ionic functional group is introduced contains 0.1 mmol / g to 1 mmol / g, preferably 0.2 mmol / g to 0.4 mmol / g, for example 0.27 mmol / g of the ionic functional group (eg, sulfate group). The CNF into which the ionic functional groups are introduced may contain 0.1 mmol / g to 1 mmol / g, preferably 0.2 mmol / g to 0.4 mmol / g, for example 0.27 mmol / g of ionic functional groups (e.g., sulfate groups), and have an average fiber width of 1 nm to 50 nm (typically 16 nm), an average fiber length of 0.2 μm to 10 μm, a crystallinity of 60% to 80% (typically 71%), and / or a light transmittance of 60% to 70% (typically 64%), and / or a viscosity of its aqueous dispersion of 200 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 1200 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, sulfated CNF such as the sulfated CNF shown as Sample A0 in Examples 1 and 2 described later, but is not limited thereto.

[0061] In one embodiment, the CNF into which the ionic functional groups are introduced contains 0.1 mmol / g to 1 mmol / g, preferably 0.5 mmol / g to 0.7 mmol / g, for example 0.56 mmol / g of the ionic functional groups (eg, sulfate groups). The CNF into which the ionic functional groups are introduced may contain 0.1 mmol / g to 1 mmol / g, preferably 0.5 mmol / g to 0.7 mmol / g, for example 0.56 mmol / g of ionic functional groups (e.g., sulfate groups), and have an average fiber width of 1 nm to 10 nm (typically 8 nm), an average fiber length of 0.2 μm to 5 μm, a crystallinity of 60% to 70% (typically 67%), and / or a light transmittance of 70% to 82% (typically 73%), and / or a viscosity of its aqueous dispersion of 700 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 5500 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, sulfated CNF such as the sulfated CNF shown as Sample A1 in Examples 1 and 2 described later, but is not limited thereto.

[0062] In one embodiment, the CNF into which the ionic functional groups are introduced contains 0.5 mmol / g to 1 mmol / g, preferably 0.7 mmol / g to 1 mmol / g, for example 0.85 mmol / g of the ionic functional groups (eg, sulfate groups). The CNF into which the ionic functional groups are introduced may contain 0.5 mmol / g to 1 mmol / g, preferably 0.7 mmol / g to 1 mmol / g, for example 0.85 mmol / g of ionic functional groups (e.g., sulfate groups), and have an average fiber width of 1 nm to 10 nm (typically 6 nm), an average fiber length of 0.2 μm to 1 μm, a crystallinity of 50% to 60% (typically 55%), and / or a light transmittance of 70% to 90% (typically 80%), and / or a viscosity of its aqueous dispersion of 800 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 6100 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, sulfated CNF such as the sulfated CNF shown as Sample 4 in Examples 1 and 2 described later, but is not limited thereto.

[0063] In one embodiment, the CNF into which the ionic functional groups are introduced contains 0.5 mmol / g to 2 mmol / g, preferably 1 mmol / g to 2 mmol / g, for example, 1.38 mmol / g of the ionic functional groups (eg, sulfate groups). The CNF into which the ionic functional groups are introduced may contain 0.5 mmol / g to 2 mmol / g, preferably 1 mmol / g to 2 mmol / g, for example 1.38 mmol / g of ionic functional groups (e.g., sulfate groups), and have an average fiber width of 1 nm to 5 nm (typically 3 nm), an average fiber length of 0.2 μm to 0.5 μm, a crystallinity of 40% to 80% (typically 46%), and / or a light transmittance of 70% to 90% (typically 88%), and / or a viscosity of its aqueous dispersion of 2600 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 12000 mPa·s (viscosity value measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, sulfated CNF such as the sulfated CNF shown as sample A2 in Examples 1 and 2 described later, but is not limited thereto.

[0064] In one embodiment, the CNF into which the ionic functional groups are introduced contains 0.5 mmol / g to 5 mmol / g, preferably 2 mmol / g to 5 mmol / g, for example, 2.56 mmol / g of the ionic functional groups (eg, sulfate groups). The CNF into which the ionic functional groups are introduced may contain 0.5 mmol / g to 5 mmol / g, preferably 2 mmol / g to 5 mmol / g, for example 2.56 mmol / g of ionic functional groups (e.g., sulfate groups), and have an average fiber width of 1 nm to 5 nm (typically 3 nm), an average fiber length of 0.2 μm to 0.5 μm, a crystallinity of 30% to 80% (typically 40%), and / or a light transmittance of 70% to 90% (typically 90%), and / or a viscosity of its aqueous dispersion of 30 mPa·s (viscosity measured at 25°C after 10 minutes at a shear rate of 60 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%) and / or 100 mPa·s (viscosity measured at 25°C after 10 minutes at a shear rate of 6 rpm for a CNF aqueous dispersion having a solid content concentration of 0.5%), but is not limited thereto. The CNF into which the ionic functional group is introduced may be, for example, sulfated CNF such as the sulfated CNF shown as Sample 5 in Examples 1 and 2 described later, but is not limited thereto.

[0065] The CNF having introduced an ionic functional group can be manufactured by a known method, or can be obtained as a commercially available product of surface-modified cellulose nanofibers. The CNF having introduced an ionic functional group can be manufactured by various biomass raw materials containing cellulose such as pulp, cotton, wood, bamboo, rice straw, rice husk, agricultural residues (skins, leaves, stems, etc. of vegetables and fruits), herbs, seaweed, bacterial cellulose. In one embodiment, the CNF having introduced an ionic functional group can be formed by introducing an ionic functional group into a CNF of plant origin. In one embodiment, the CNF having introduced an ionic functional group can be manufactured in the following manner: by subjecting a biomass raw material containing cellulose represented by pulp or cotton to chemical treatments such as oxidation, acid treatment, and ion exchange, and then nanofiberizing the cellulose by physical fiberization such as stirring. The pulp used in the manufacture of the CNF having imported the ionic functional group is not particularly limited, and can be NBKP (Nadelholz bleached kraft pulp) as the pulp extracted from coniferous trees, LBKP (Laubholz bleached kraft pulp) as the pulp extracted from broad-leaved trees, or cotton pulp manufactured by cotton. As the cotton used in the manufacture of the CNF having imported the ionic functional group, cotton linter can be used. Alternatively, the CNF having imported the ionic functional group can also be made by using a biological method such as a microorganism or a plant cell such as a bacterium.

[0066] The present invention provides a cell culture substrate having a scaffold material comprising CNFs introduced with ionic functional groups on its surface. In the present invention, a "cell culture substrate" refers to a solid product or component that supports cell attachment and / or proliferation during cell culture. In the present invention, a "scaffold material" refers to a material that provides a support for promoting cell attachment and proliferation during cell culture.

[0067] The scaffold material of the present invention may be composed solely of CNFs into which ionic functional groups have been introduced, or may be a composition comprising CNFs into which ionic functional groups have been introduced and other materials or components. The scaffold material may comprise solely CNFs into which ionic functional groups have been introduced as a scaffold, or may comprise other materials as a scaffold in addition to CNFs into which ionic functional groups have been introduced. The scaffold material may comprise one type of CNFs into which ionic functional groups have been introduced, or may comprise two or more types of CNFs into which ionic functional groups have been introduced. The scaffold material may comprise at least one of CNFs into which anionic functional groups have been introduced and CNFs into which cationic functional groups have been introduced, or may comprise both CNFs into which anionic functional groups have been introduced and CNFs into which cationic functional groups have been introduced, or may comprise CNFs into which both anionic functional groups and cationic functional groups have been introduced. The scaffold material may comprise two or more types of CNFs into which either or both anionic functional groups or cationic functional groups have been introduced. In one embodiment, the scaffold material may include two or more CNFs into which anionic functional groups have been introduced. In one embodiment, the scaffold material may include CNFs into which sulfate groups have been introduced and CNFs into which carboxyl groups have been introduced. For example, the scaffold material may include CNFs into which sulfate groups have been introduced and CNFs into which carboxyl groups have been introduced in any weight ratio, for example, in a weight ratio of 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1 or 1:1 to 2:1. In one embodiment, the scaffold material may include two or more CNFs into which the same ionic functional groups have been introduced in different amounts. In the case where the scaffold material includes two or more CNFs into which ionic functional groups have been introduced, the weighted average value of the entire CNFs may be calculated based on the characteristic values ​​determined for the various types of CNFs into which ionic functional groups have been introduced, specifically, for example, the content of ionic functional groups, average fiber width, average fiber length, viscosity (viscosity of the aqueous dispersion of CNFs), crystallinity and transmittance, and the weighted average value may be used as a value representing the characteristics of the entire CNFs. The weighted average value here is the average value of the characteristic value calculated by adding the weight corresponding to the weight ratio of the various types of CNFs introduced with ionic functional groups contained in the scaffold material. In the case where the scaffold material contains more than two types of CNFs introduced with ionic functional groups, the weighted average value of these CNFs as a whole related to the above characteristics preferably falls within the numerical ranges described above for each characteristic, but is not limited thereto. For CNFs introduced with ionic functional groups contained in the scaffold material (more than two types), at least one of the CNFs introduced with ionic functional groups may be included, wherein at least one of the above characteristics falls within the above numerical ranges; at least one of the above characteristics may fall within the above numerical ranges in all types of CNFs; or, for at least one characteristic, the weighted average value of all types of CNFs may fall within the above numerical ranges.The present invention also provides a scaffold material comprising CNFs into which ionic functional groups have been introduced.

[0068] In addition to CNFs into which ionic functional groups have been introduced, the scaffold material may further comprise unmodified CNFs (CNFs that have not been surface-modified) and / or other modified CNFs (modified CNFs other than CNFs into which ionic functional groups have been introduced). In one embodiment, the scaffold material may comprise: at least one of CNFs into which anionic functional groups have been introduced and CNFs into which cationic functional groups have been introduced, and unmodified CNFs and / or other modified CNFs. In one embodiment, the scaffold material may comprise: at least one selected from CNFs into which sulfate groups have been introduced and CNFs into which carboxyl groups have been introduced, and unmodified CNFs and / or other modified CNFs. For example, the scaffold material may comprise: CNFs into which ionic functional groups have been introduced, and unmodified CNFs and / or other modified CNFs in any weight ratio, for example, in a weight ratio of 1:9 to 9:1, 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1 to 2:1. In one embodiment, the scaffold material can include: at least one selected from CNFs introduced with sulfate groups and CNFs introduced with carboxyl groups, and unmodified CNFs and / or other modified CNFs in any weight ratio, for example, it can include them in a weight ratio of 1:9~9:1, 1:5~5:1, 1:3~3:1, 1:2~2:1 or 1:1~2:1. In the case of a scaffold comprising CNFs introduced with ionic functional groups and unmodified CNFs and / or other modified CNFs, a weighted average value of the overall mixture of the CNFs introduced with ionic functional groups and the unmodified CNFs and / or other modified CNFs is calculated based on the property values ​​determined for each type of CNFs introduced with ionic functional groups, unmodified CNFs, or other modified CNFs, specifically, based on the values ​​of properties selected from the group consisting of ionic functional group content (for unmodified CNFs and other modified CNFs, this is assumed to be 0 mmol / g), average fiber width, average fiber length, viscosity (viscosity of an aqueous CNF dispersion), crystallinity, and transmittance. This weighted average value is used as a value representing the properties of the CNF mixture as a whole. The weighted average value here is the average value of the property values ​​calculated by adding weights corresponding to the weight ratios of the respective types of CNFs introduced with ionic functional groups and unmodified CNFs and / or other modified CNFs contained in the scaffold. In the case where the scaffold material comprises CNFs into which ionic functional groups have been introduced, and unmodified CNFs and / or other modified CNFs, for at least one or all of the above-mentioned properties (selected from the content of ionic functional groups, average fiber width, average fiber length, viscosity, crystallinity and transmittance), the weighted average value related to the property of the mixture of CNFs into which ionic functional groups have been introduced and unmodified CNFs and / or other modified CNFs as a whole preferably falls within the numerical range described above for CNFs into which ionic functional groups have been introduced.In one embodiment, when the scaffold comprises CNFs introduced with ionic functional groups, and unmodified CNFs and / or other modified CNFs, the weighted average value of the ionic functional group content of the entire mixture of the CNFs introduced with ionic functional groups and the unmodified CNFs and / or other modified CNFs preferably falls within the numerical range described above for the ionic functional group content of the CNFs introduced with ionic functional groups. In this case, as long as the weighted average value for a given characteristic falls within the numerical range described above for CNFs introduced with ionic functional groups, the CNFs introduced with ionic functional groups, unmodified CNFs, or other modified CNFs contained in the scaffold do not necessarily fall within the numerical range described above for CNFs introduced with ionic functional groups.

[0069] The scaffold material may contain 10% or more, preferably 15% or more, more preferably 25% or more, for example, 10-100%, 10-90%, 30-100%, 30-70%, 50-100%, or 50-70% by weight of CNFs introduced with ionic functional groups, on a dry weight basis. In a preferred embodiment, when such a scaffold material comprises two or more types of CNFs, the weighted average value of the characteristic values ​​determined for each type of CNF (CNFs introduced with ionic functional groups, unmodified CNFs, or other modified CNFs) contained in the scaffold material, such as the value of the characteristic selected from the group consisting of the content of ionic functional groups, average fiber width, average fiber length, viscosity (viscosity of the aqueous dispersion of CNFs), crystallinity, and transmittance, for the entire CNF mixture, falls within the numerical ranges described above for CNFs introduced with ionic functional groups.

[0070] In addition, the scaffold material may further contain additives that are permissible in cell culture (i.e., non-cytotoxic), such as carriers, excipients, preservatives, pH adjusters, etc. The scaffold material preferably has biocompatibility that does not adversely affect cell survival, growth, and proliferation.

[0071] In one embodiment, the cell culture substrate having a scaffold material comprising the CNF having imported an ionic functional group on the surface has a scaffold material comprising the CNF having imported an ionic functional group on the surface of any solid material (for example, a scaffold material consisting of the CNF having imported an ionic functional group or a scaffold material comprising the CNF having imported an ionic functional group and the CNF without modification of the CNF). The solid material can be, for example, resin (thermoplastic resins such as plastics, thermosetting resins, light-curing resins, etc.), rubber, glass, film, paper, cloth (non-woven fabrics, etc.), beads, silk (hollow fibers, etc.). Such solid material can be, for example, cell culture containers such as cell culture plates, flasks, culture bags, or cell culture / observation products such as cover slips, slides, etc. The cell culture / observation products such as cell culture plates, flasks, culture bags, or cover slips, slides, etc. can be plastics such as polystyrene systems, or glass systems, but are not limited to these. It should be noted that, for the purposes of the present invention, “made of polystyrene”, “made of plastic” or “made of glass” means that the target article, such as a cell culture container, a cell culture / observation product, contains polystyrene, plastic or glass as the main component (i.e., the component that is the largest in terms of weight ratio in the composition of the article), but does not exclude the situation where it further contains components or members other than these components as a secondary component. In one embodiment, an article containing polystyrene, plastic or glass “as the main component” can be, for example, an article in which more than 60%, more than 80% or more than 90% of the weight of the article is composed of polystyrene, plastic or glass, respectively. It should be noted that other similar expressions related to the composition and raw materials of the article also follow the same explanation as above.

[0072] In the present invention, "a scaffold material having CNFs with introduced ionic functional groups on its surface" refers to a scaffold material having CNFs with introduced ionic functional groups attached or fixed to the surface of a cell culture substrate. In one embodiment, "a scaffold material having CNFs with introduced ionic functional groups on its surface" refers to a state in which CNFs with introduced ionic functional groups are attached or fixed to the surface of a cell culture substrate as a scaffold material. In another embodiment, "a scaffold material having CNFs with introduced ionic functional groups on its surface" refers to a state in which, when using a scaffold material comprising CNFs with introduced ionic functional groups and unmodified CNFs, CNFs with introduced ionic functional groups and unmodified CNFs (a mixture) are attached or fixed to the surface of a cell culture substrate as a scaffold material. "The surface of a cell culture substrate" refers to at least a portion of the surface of a cell culture substrate, which is a portion that can come into contact with cells when the cell culture substrate is used for cell culture. The scaffold material (or, for example, CNF having an ionic functional group, or CNF having an ionic functional group and unmodified CNF having been introduced) can be physically or chemically attached or fixed to the surface of the solid material of the cell culture substrate. For example, the scaffold material (or, for example, CNF having an ionic functional group, or CNF having an ionic functional group and unmodified CNF having been introduced) can be coated on the surface of the solid material of the cell culture substrate. Alternatively, the scaffold material can be present on the surface of the cell culture substrate in the form of an aerogel or porous foam made from a scaffold material comprising CNF having an ionic functional group introduced. In one embodiment, the cell culture substrate having a scaffold material comprising CNF having an ionic functional group introduced on the surface can be a cell culture plate coated with a scaffold material on the inner surface. The "inner surface" of the cell culture plate refers to the surface of the receiving portion (preferably the bottom) on the inner side of the plate for culturing cells. Such a cell culture plate can be made of plastics such as polystyrene, or can be made of glass, but is not limited to these. The cell culture plate is not limited to the following, for example, it can be a culture dish, a 6-well plate, a 12-well plate, a 24-well plate, a 96-well plate, etc. In another embodiment, the scaffold material itself can be formed into a solid material (for example, a film, a nonwoven fabric, etc., but not limited thereto), thereby manufacturing a cell culture substrate with a scaffold material on the surface. In another embodiment, the composite material formed by mixing the scaffold material and other materials (for example, resins such as plastics, but not limited thereto) can be formed into a solid material (for example, a resin member or a resin product, but not limited thereto), thereby manufacturing a cell culture substrate with a scaffold material on the surface.

[0073] In one embodiment, the scaffold material can exist in a dry state on the surface of the cell culture substrate. That is, the cell culture substrate of the present invention can be a cell culture substrate having a scaffold material comprising CNF having imported ionic functional groups in a dry state on the surface. In the cell culture substrate with the scaffold material of the present invention in a dry state on the surface, the scaffold material does not exist as an aqueous dispersion (for example, an aqueous dispersion), and is not a hydrogel. In the cell culture substrate with the scaffold material in a dry state on the surface, the scaffold material can be an aerogel, a porous foam. For example, the cell culture substrate can be dried by making the scaffold material as an aqueous dispersion (for example, coated with the scaffold material on the surface) having CNF having imported ionic functional groups on the surface, thereby making the cell culture substrate having a scaffold material comprising CNF having imported ionic functional groups in a dry state on the surface. The water content of the scaffold material in the dry state is typically 50% by weight or less, preferably 30% by weight or less, more preferably 20% by weight or less, for example, 10% by weight or less or 5% by weight or less.

[0074] The cell culture substrate of the present invention having a scaffold material containing CNFs with introduced ionic functional groups on its surface is preferably sterilized before use in cell culture. Any sterilization method may be used, for example, sterilization by ultraviolet (UV) irradiation.

[0075] The cell culture substrate of the present invention has a scaffold material containing CNFs into which ionic functional groups are introduced on the surface, which can support high-level proliferation of cells by being used for cell culture. The present invention also provides a cell culture method, which includes using the cell culture substrate of the present invention to culture cells. More specifically, the cell culture substrate of the present invention can be used to culture cells in a culture medium in a manner that enables cells to adhere to the scaffold material on the surface of the cell culture substrate of the present invention, thereby suitably culturing cells. The cell culture method of the present invention can achieve effective cell proliferation.

[0076] In the cell culture method of the present invention, cells can be inoculated into the cell culture substrate of the present invention and cultured in a culture medium (preferably a liquid culture medium). The inoculated cells can adhere to a scaffold containing CNFs containing ionic functional groups in the cell culture substrate and proliferate. The scaffold material containing CNFs containing ionic functional groups in the cell culture substrate can be in a non-dried state during cell culture, for example, it can form an aqueous dispersion.

[0077] In the present invention, by using the cell culture substrate of the present invention, cells can be proliferated and cultured not only under normal serum concentration conditions, but also under low serum or serum-free conditions. That is, the cell culture method of the present invention can be a method comprising culturing cells in a medium with a normal serum concentration, or a method comprising culturing cells in a low serum medium or serum-free medium.

[0078] In general cell culture, animal serum is typically added to a basal medium containing nutrients such as amino acids, vitamins, nucleic acids or their precursors, a carbon source such as glucose, and inorganic salts. Animal serum is typically added at a concentration of 5% to 20% relative to the culture medium.

[0079] In the present invention, "normal serum concentration medium" refers to a medium containing serum at a concentration of 5% or more suitable for cell culture. As the normal serum concentration medium, any medium that can be used for cell culture, preferably animal cell culture, can be used.

[0080] In the present invention, "reduced serum medium" refers to a medium that can be used for cell culture and contains animal serum at a concentration greater than 0% and less than 5%, for example, greater than 2% and less than 4%. In one embodiment, the reduced serum medium is a liquid medium.

[0081] In the present invention, "serum-free medium" refers to a medium that does not contain animal serum and can be used for cell culture. In one embodiment, the serum-free medium is a liquid medium.

[0082] Regarding the culture medium of the present invention, "serum" refers to serum that can be used to promote cell culture, and generally refers to animal serum, and examples thereof include, but are not limited to, fetal bovine serum (FBS), calf serum, horse serum, and human serum.

[0083] The culture medium such as the normal serum concentration medium, low serum medium or serum-free medium used in the cell culture of the present invention can include any basal medium. As basal medium, it is not limited to the following, and for example, Dulbecco's modified Eagle medium (DMEM), DMEM / F12 medium, Eagle's minimum essential medium (MEM), Ham's F-12 medium (Ham's F-12), RPMI1640 medium, etc. can be listed. In one embodiment, as basal medium, a commercially available culture medium represented by MSH medium such as MSH-BM (Shimadzu Diagnostics Co., Ltd.) can be used. The culture medium such as the normal serum concentration medium, low serum medium or serum-free medium used in the cell culture of the present invention can include more or less the components contained in the basal medium.

[0084] In the culture medium such as the common serum concentration culture medium, low serum culture medium or serum-free culture medium used in the cell culture of the present invention, in addition to the basal medium, other components can also be further included. The culture medium used in the cell culture of the present invention, for example, can further include growth factors such as fibroblast growth factor (FGF). As fibroblast growth factor (FGF), it is not limited to the following, and for example, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF) or keratinocyte growth factor (KGF) etc. can be enumerated. FGF, for example, can be recombinant human FGF.

[0085] In one embodiment, the culture medium such as a medium with a normal serum concentration, a low serum medium or a serum-free medium used in the cell culture of the present invention does not contain a serum replacement such as N2.

[0086] In one embodiment, the serum-free medium used in the cell culture of the present invention can be a xeno-free medium (a medium that does not contain xenogeneic components) and / or an animal-free medium (a medium that does not contain animal-derived components).

[0087] In one embodiment, the culture medium such as a normal serum concentration medium, a low serum medium or a serum-free medium used in the cell culture of the present invention can be a culture medium obtained by replacing at least a portion of the xenogeneic or animal-derived components with corresponding components of the same species or non-animal origin (e.g., components produced by plants, fungi (yeast, etc.) or bacteria through recombinant methods or naturally produced components). For example, as a component of the culture medium, albumin produced by human cells is generally used, but it can also be replaced with albumin produced by plants, fungi or bacteria. For example, for a serum-free culture medium having a composition containing xenogeneic or animal-derived components, an xeno-free and / or animal-free culture medium obtained by replacing the xenogeneic or animal-derived components with corresponding components of the same species or non-animal origin can also be used in the cell culture of the present invention.

[0088] By using the cell culture method of the cell culture substrate of the present invention, cells, preferably animal cells, can be proliferated and cultured. The animal cells to be cultured are preferably mammalian cells, for example, human cells.

[0089] The culture conditions of cells using the cell culture substrate of the present invention can be set according to the cells to be cultured. Generally, cells can be cultured at 36°C to 38°C (typically 37°C) and 4% to 6% (typically 5%) CO2.

[0090] In one embodiment, the animal cell to be cultured can be a stem cell. According to the present invention, the cell culture substrate of the present invention can be used to suitably culture stem cells. More specifically, by using the cell culture substrate of the present invention to culture stem cells in a culture medium in a manner that enables cells to adhere to the scaffold material on the surface of the cell culture substrate of the present invention, stem cells can be suitably cultured. The stem cells cultured in the present invention can be adult stem cells or embryonic stem cells. Stem cells can be, for example, pluripotent stem cells such as mesenchymal stem cells (MSC), induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or tissue stem cells that can only differentiate into a specific cell type within a narrower range. Stem cells can be primary cells or immortalized cells. "Immortalized cells" refer to cells that can survive, culture, and have the ability to continue dividing beyond the cell division limit over a longer period of time compared to primary cells. Immortalized cells can be obtained by establishing a tumor cell line, or by mutation of the cell, chromosomal abnormality, or by introducing genes or mutations that contribute to immortalization. As mesenchymal stem cells, it is not limited to the following, and for example, mesenchymal stem cells (for example, immortalized mesenchymal stem cells) derived from bone marrow, adipose tissue, deciduous tooth pulp, or umbilical cord or cord blood can be enumerated. Stem cells can be obtained from cell banks or cell preservation institutions such as JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank; National Research and Development Corporation Medical Foundation Health Nutrition Research Institute, Japan), but the source of stem cells is not limited thereto. According to the cell culture method using the cell culture substrate of the present invention, stem cells can be kept in an undifferentiated state and propagated while maintaining differentiation ability. Therefore, the cell culture using the cell culture substrate of the present invention is particularly suitable for the cultivation of stem cells. That is, the present invention can be a stem cell culture method using a cell culture substrate.

[0091] The present invention also provides a scaffold material of the present invention comprising CNFs introduced with ionic functional groups, and a cell culture substrate of the present invention having the scaffold material on its surface, for use in culturing stem cells, particularly preferably in the aforementioned serum-free or low-serum medium. In one embodiment, the scaffold material of the present invention and the cell culture substrate of the present invention having the scaffold material on its surface can be used to culture stem cells while maintaining the stem cells in an undifferentiated state.

[0092] The culture conditions of stem cells using the cell culture substrate of the present invention can be set according to the stem cells to be cultured. Generally, stem cells can be cultured at 36°C to 38°C (typically 37°C) and 4% to 6% (typically 5%) CO2.

[0093] In the culture of stem cells using the cell culture substrate of the present invention, after the stem cells are maintained in an undifferentiated state and cultured, the differentiation of the stem cells can be induced. For example, the differentiation of the stem cells can be induced by culturing the cultured stem cells in a differentiation induction medium. The present invention also provides a method for culturing stem cells, which includes culturing stem cells using the cell culture substrate of the present invention, and further includes inducing the differentiation of the cultured stem cells. For example, by culturing the stem cells in a differentiation induction medium, the differentiation of the stem cells can be induced. By using a differentiation induction medium that can induce differentiation into target cells, the stem cells can be differentiated into target cells. For example, by culturing mesenchymal stem cells using differentiation induction mediums specifically for adipocytes, osteoblasts, and chondrocytes, differentiation from the stem cells into adipocytes, osteoblasts, and chondrocytes can be induced, respectively, to generate adipocytes, osteoblasts, and chondrocytes. As a differentiation induction medium for stem cells, a known differentiation induction medium can be prepared and used, or a commercially available differentiation induction medium can be used. The present invention also relates to a method for inducing differentiation of stem cells, which includes: after culturing the stem cells using the cell culture substrate of the present invention, culturing the stem cells in a differentiation induction medium, thereby inducing the differentiation of the stem cells. The cell culture substrate of the present invention can also be used for culturing stem cells in a differentiation induction medium.

[0094] Example

[0095] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples.

[0096] [Example 1] Preparation of cellulose nanofibers (CNF)

[0097] (1) Unmodified CNF

[0098] As unmodified CNF, cellulose nanofibers (BiNFi-s cellulose WFo-10002; SUGINOMACHINE Co., Ltd., Japan) were purchased and used as sample 1.

[0099] (2) Preparation of TOCNF

[0100] 0.13 mmol of 2,2,6,6-tetramethylpiperidinium-N-oxide (TEMPO) and 10 mmol of sodium bromide were dissolved in water to obtain 250 mL of an aqueous solution. In order to introduce carboxyl groups into the pulp and perform nanofiberization, 3 g of pulp (bleached kraft softwood pulp (NBKP), Nippon Paper Industries, Ltd., Japan) was added to the aqueous solution and stirred until the pulp was uniformly dispersed. After adjusting the temperature of the obtained mixture to 20°C, 32 mmol of an aqueous sodium hypochlorite solution (FUJIFILM Wako Pure Chemical Co., Ltd., Japan) was added to start the oxidation reaction. During the oxidation reaction, the temperature of the reaction system was maintained at 20°C, and the pH was maintained at 10 by gradually adding a 5% aqueous sodium hydroxide solution. After reacting for 2 hours, centrifugation was performed and the supernatant was removed.

[0101] 300 g of distilled water was added to the precipitate and stirred until uniformly dispersed, followed by centrifugation and removal of the supernatant for washing. The same steps were repeated for a total of three washes. After three washes, distilled water was added to the carboxylated pulp containing water to a solids concentration of 0.5%, and then treated with a mixer (G5200, Biolomix) to obtain a 0.5% concentration of TEMPO oxidized cellulose nanofiber (TOCNF) aqueous dispersion (sample 2). TOCNF is a chemically modified CNF with carboxyl groups introduced on the surface.

[0102] In addition, a TOCNF aqueous dispersion having a concentration of 0.5% was obtained by the same method as above except that the oxidation reaction time was changed from 2 hours to 4 hours. This dispersion was used as Sample 3.

[0103] It is considered that the amount of carboxyl groups introduced into TOCNF increases depending on the length of the oxidation reaction time.

[0104] (3) Preparation of sulfated CNF

[0105] 90 g of dimethyl sulfoxide (DMSO), 10 g of acetic anhydride, and 0.3 g of sulfuric acid were added to a 300 mL sample bottle, and stirred at room temperature of 23° C. for about 30 seconds using a magnetic stirrer to prepare a reaction solution.

[0106] Next, 3 g of pulp (bleached softwood kraft pulp (NBKP), Nippon Paper Industries, Ltd., Japan) was added to the reaction solution and stirred for another 4 hours at room temperature of 23°C to allow for a sulfation reaction. After stirring, 200 g of distilled water was added to the defibration solution containing the pulp to terminate the reaction. Subsequently, a 5% aqueous sodium hydroxide solution was added until the pH reached 7 to neutralize the reaction solution. The solution was then centrifuged and the supernatant removed.

[0107] 300 g of distilled water was added to the precipitate and stirred until uniformly dispersed, followed by centrifugation and removal of the supernatant for washing. The same steps were repeated for a total of three washes. After three washes, distilled water was added to the sulfated pulp containing water to a solids concentration of 0.5%, and then treated with a mixer (G5200, Biolomix) to obtain a 0.5% concentration of sulfated cellulose nanofiber (sulfated CNF) aqueous dispersion (sample 4). Sulfated CNF is a chemically modified CNF with sulfate groups introduced on the surface.

[0108] A 0.5% concentration sulfated CNF aqueous dispersion was obtained as Sample 5 by the same method as above except that the amount of sulfuric acid used was changed from 0.3 g to 1 g.

[0109] A 0.5% concentration sulfated CNF aqueous dispersion was obtained by the same method as above except that the amount of sulfuric acid used was changed from 0.3 g to 0.2 g. This dispersion was used as sample A1.

[0110] A 0.5% concentration sulfated CNF aqueous dispersion was obtained by the same method as above except that the amount of sulfuric acid used was changed from 0.3 g to 0.6 g. This dispersion was used as sample A2.

[0111] A 0.5% concentration sulfated CNF aqueous dispersion was obtained by the same method as above except that the amount of sulfuric acid used was changed from 0.3 g to 0.1 g. This dispersion was used as sample A0.

[0112] The amount of sulfate groups introduced into the obtained sulfated CNF increases depending on the amount (concentration) of sulfuric acid in the sulfation reaction solution.

[0113] [Example 2] Analysis of CNF characteristics

[0114] The properties of the CNF obtained in Example 1 were analyzed.

[0115] (1) Quantification of the amount of functional groups (substituents) introduced

[0116] The amount of carboxyl groups introduced into TOCNF was determined by alkaline titration. Specifically, the TOCNFs of samples 2 and 3 were first dried, and after accurately weighing the dry weight, they were dispersed in water to prepare 60 mL of a 0.5-1% slurry. After adding a 0.1 M aqueous hydrochloric acid solution to the slurry to adjust the pH to approximately 2.5, a 0.05 M aqueous sodium hydroxide solution was added dropwise, and the conductivity was measured. The measurement was performed until the pH reached approximately 11. The amount of carboxyl groups (functional groups) introduced into CNF was determined based on the amount of sodium hydroxide (V mL) consumed in the neutralization stage of the weak acid where the change in conductivity is gentle, using the following formula.

[0117] Carboxyl group introduction amount (mmol / g) = V (mL) × 0.05 / TOCNF amount (g)

[0118] On the other hand, the sulfur content was quantified using combustion absorption ion chromatography (IC) to determine the amount of sulfate groups introduced into the sulfated CNF. Specifically, the sulfated CNFs of samples 4 and 5, as well as samples A0, A1, and A2, were dried. 0.01 g of the dried sulfated CNFs were then added to a magnetic boat and burned in an annular furnace (1350°C) in an oxygen atmosphere (flow rate: 1.5 L / min). The resulting gas components were absorbed by 3% hydrogen peroxide (20 mL). The resulting gas absorption liquid was diluted to 100 mL with pure water. The sulfate ion concentration (%) of the resulting diluted liquid was measured by ion chromatography, and the amount of sulfate groups introduced (mmol / g) per 1 g of sample (sulfated CNF) was calculated.

[0119] (2) Evaluation of crystallinity based on X-ray diffraction

[0120] 0.1 g of each dried product of sulfated CNF, TOCNF, and unmodified CNF was subjected to X-ray diffraction to determine the crystallinity. An X-ray diffractometer (RINT-TTR III, Rigaku Corporation, Japan) was used to measure the diffraction intensity of the lattice plane (200-degree plane) (diffraction angle 2θ = 22.6°) and the diffraction intensity of the amorphous portion (the lowest portion between the 200-degree plane and the 110-degree plane, diffraction angle 2θ = 18.5°). The crystallinity was calculated using the Segal method using the following formula.

[0121] Crystallinity of cellulose type I crystal (%) = [1 - (diffraction intensity at 18.5° / diffraction intensity at 22.6°)] × 100

[0122] (3) Determination of fiber width and fiber length

[0123] The fiber width and fiber length of sulfated CNF, TOCNF, and unmodified CNF were measured using an atomic force microscope (SPM-9700HT, Shimadzu Corporation, Japan).

[0124] 0.5% aqueous dispersions of sulfated CNF, TOCNF, and unmodified CNF were diluted with distilled water to obtain 0.001% aqueous CNF dispersions. Next, 30 μL of the 0.001% aqueous dispersion was dripped onto a natural mica (natural muscovite) substrate (15 mm × 15 mm × 0.15 mm thick) using a micropipette and allowed to air dry for 0.5 hours to prepare evaluation samples.

[0125] The fiber width and fiber length of 50 randomly selected fibers of the obtained evaluation samples were measured under an atomic force microscope, and the arithmetic mean was taken to calculate the average fiber width and average fiber length of the sulfated CNF, TOCNF, and unmodified CNF.

[0126] (4) Determination of light transmittance

[0127] 2 mL of 0.5% aqueous dispersion of sulfated CNF, TOCNF, and unmodified CNF was placed in a measuring cell, and the transmittance at a wavelength of 600 nm was measured using a transmittance meter (UV-2600, Shimadzu Corporation, Japan).

[0128] (5) Viscosity measurement

[0129] 100 g of a 0.5% aqueous dispersion of sulfated CNF, TOCNF, and unmodified CNF was degassed for 10 seconds using a degassing apparatus (Awatori Rentaro ARE-310, THINKY Co., Ltd., Japan) and then allowed to stand for 24 hours. Next, the viscosity was measured using a B-type viscometer (DV-II+, Brookfield) at speeds of 6 rpm and 60 rpm (shear rate). The viscosity was recorded three times (N=3) 10 minutes after the start of the measurement, and the average value was used as the viscosity. Viscosity measurements were performed at 25°C.

[0130] The results of the characteristic analysis of each CNF sample are summarized in Table 1.

[0131]

[0132] [Example 3] Coating of cell culture plates

[0133] Distilled water was added to a 0.5% aqueous dispersion of sulfated CNF, TOCNF, and unmodified CNF to adjust the solid content concentration to 0.25%, and the resultant dispersion was used as a coating material.

[0134] In addition, a 0.25% aqueous dispersion of TOCNF and a 0.25% aqueous dispersion of sulfated CNF were blended at the weight ratio shown in Table 2 below, and mixed by pipetting or ultrasonic homogenizer to prepare a TOCNF-sulfated CNF mixture, which was used as a coating material.

[0135] Separately, a 0.25% aqueous dispersion of unmodified CNF and a 0.25% aqueous dispersion of sulfated CNF were mixed at the weight ratio shown in Table 2 below, and the mixtures were pipetted or mixed with an ultrasonic homogenizer to prepare an unmodified CNF-sulfated CNF mixture, which was used as a coating material.

[0136] 200 μL of the prepared coating material was applied to a circular cover glass (15 pellets No. 1, C015001, Matsunami Glass Industries, Ltd., Japan) and dried overnight at room temperature to prepare a glass substrate coated with CNFs. This was used as a cell culture substrate.

[0137] [Example 4] Immortalized cell culture test

[0138] (1) Sterilization of cell culture media

[0139] The CNF-coated glass substrate prepared in Example 3 was immersed in 99.5% ethanol. It was then sterilized by irradiation with ultraviolet (UV) light for 20 minutes. The sterilized glass substrate was placed on the bottom of a 24-well plate (SPL-30024, made of polystyrene, SPL Life Sciences) using sterilized tweezers.

[0140] (2) Cell culture in normal medium or low-serum medium

[0141] Human bone marrow-derived immortalized mesenchymal stem cells (UE6E7T-11, purchased from JCRB Cell Bank under the cell number JCRB1149) were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% or 2.5% fetal bovine serum (FBS), and then the cells were detached and recovered using trypsin-ethylenediaminetetraacetic acid (EDTA).

[0142] The recovered cells were seeded into a 24-well plate at a density of 12,000 cells / well. The 24-well plate was provided with a glass substrate coated with CNF sterilized in (1) above and cultured in the same culture medium as above (DMEM (high glucose) containing 10% or 2.5% FBS) at 37°C and 5% CO2.

[0143] (3) Cell culture in serum-free medium

[0144] Human bone marrow-derived immortalized mesenchymal stem cells (UE6E7T-11, purchased from JCRB Cell Bank under cell number JCRB1149) were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% fetal bovine serum (FBS), then cultured and acclimated in serum-free medium (MSH-BM supplemented with MSH-Supplement A, Nissui Pharmaceutical Co., Ltd., Japan), and then the cells were detached and recovered using TrypLE (trademark) Express Enzyme (Thermo Fisher Scientific).

[0145] The recovered cells were seeded at a density of 12,000 cells / well in a 24-well plate equipped with the CNF-coated glass substrate sterilized in (1) above, and cultured in the same serum-free medium as above at 37°C and 5% CO2. It should be noted that the serum-free medium used here is an exogenous medium.

[0146] Under inverted microscope (DMI4000 B, Leica company), the cell after cultivating 3 days (72 hours) was observed. Use Cell Counting Kit-8 (Dongren Chemical Research Institute, Japan) to carry out the counting of cell number. Use Cellstain (registered trademark)-Double Staining Kit (Dongren Chemical Research Institute, Japan) to carry out the simultaneous staining of living cells and dead cells. Carry out the fluorescence observation of the cell after staining. According to the order of cell proliferation level from high to low, the living cell number counted is evaluated as A when being more than 40000 cells / well, is evaluated as B when being more than 20000 cells / well and less than 40000 cells / well, is evaluated as C when being less than 20000 cells / well.

[0147] The results of the culture test are shown in Table 2 and Figures 1-3 . Figure 1 These are micrographs showing the results of culturing immortalized mesenchymal stem cells (immortalized MSCs) in a standard culture medium with an FBS concentration of 10%. Figure 2 The figure is a micrograph showing the results of culturing immortalized MSCs in a low-serum medium containing 2.5% FBS. Figure 3 are micrographs showing the results of culturing immortalized MSCs in a serum-free medium not containing FBS.

[0148] On the cell culture substrate coated with unmodified CNF, almost no cell proliferation was observed (Table 2, Figures 1-3 On the other hand, significant cell proliferation was confirmed on the cell culture substrate coated with sulfated CNF, TOCNF, or a mixture thereof (Table 2, Figures 1-3 ). Furthermore, significant cell proliferation was observed on cell culture substrates coated with a mixture of unmodified CNF, which showed little cell proliferation when used alone, and sulfated CNF (Table 2). Cells proliferating on cell culture substrates coated with these chemically modified CNFs remained undifferentiated. This result demonstrates that the chemically modified CNFs described above function as excellent cell culture scaffolds.

[0149] A particularly surprising result is that not only the culture under normal serum concentration conditions ( Figure 1, Table 2), even when cultured under low serum conditions ( Figure 2 , Table 2), culture under serum-free conditions ( Figure 3 Significant cell proliferation was also confirmed in Table 2. This suggests that using these chemically modified CNFs in cell culture scaffolds can reduce the amount of serum used in the culture medium, or even eliminate the need for serum. It is believed that cell culture in low-serum or serum-free medium can reduce costs and improve safety.

[0150]

[0151] This indicates that the cell culture substrate using the chemically modified CNF as a scaffold is useful for culturing immortalized cells.

[0152] [Example 5] Cultured Cell Differentiation Ability Evaluation Test

[0153] For the proliferating cells of tests 2 to 5, 7 to 11, and 13 to 15, and tests II-1 to II-4, where significant cell proliferation was confirmed, the retention of multidirectional differentiation ability was evaluated. After the cells were recovered after three proliferation and passages in each test system, differentiation induction medium was used to induce differentiation into adipocytes, osteoblasts, and chondrocytes. The induction medium for adipocytes used a culture medium supplemented with StemPro (trademark) Adipogenesis Differentiation Kit (Thermo Fisher Scientific) and penicillin-streptomycin-amphotericin B suspension (penicillin 100U / mL, streptomycin 100μg / mL, amphotericin B 0.25μg / mL). The differentiation induction medium for osteoblasts used a culture medium supplemented with 10% FBS, the above-mentioned penicillin-streptomycin-amphotericin B suspension, 0.2mM ascorbic acid, 10mM β-glycerophosphate, and 0.1μM dexamethasone in DMEM (high glucose). The chondrocyte differentiation medium used was DMEM (high glucose) supplemented with 10% FBS, the aforementioned penicillin-streptomycin-amphotericin B suspension, 1% ITS-X supplement, 50 μM ascorbic acid, 1 μM dexamethasone, and 10 ng / mL TGF-β1. After differentiation induction, adipocytes were stained with Oil Red O, osteoblasts with Alizarin Red S, and chondrocytes with Alcian Blue to confirm differentiation into each cell lineage.

[0154] The results of differentiation ability evaluation tests showed that MSCs exhibited induction of differentiation into adipocytes, osteoblasts, and chondrocytes in all test systems (Tests 2-5, 7-11, and 13-15, as well as Tests II-1-II-4). This confirmed that MSCs cultured using the chemically modified CNFs as a scaffold maintained their ability to differentiate into adipocytes, osteoblasts, and chondrocytes.

[0155] As an example of the results of the above-mentioned differentiation ability evaluation test, photographs showing the results of staining the proliferating cells collected in Tests 13 to 15 after differentiation induction are shown. Figure 4 Regarding the cells recovered in any of the experiments 13 to 15, since the fat droplets in the cells were stained with Oil Red O after the differentiation into adipocytes was induced, it was shown that they had differentiated into adipocytes ( Figure 4 A to C). In addition, regarding the cells recovered in any of the experiments 13 to 15, since calcium deposited in the calcified cells was observed after differentiation into osteoblasts was induced, it was stained with Alizarin Red S, indicating that they had differentiated into osteoblasts ( Figure 4 D to F). Regarding the cells recovered in any of the experiments 13 to 15, since the acidic mucopolysaccharide was observed to be stained with Alcian blue after the differentiation into chondrocytes was induced, it was shown that they had differentiated into chondrocytes ( Figure 4 G~I).

[0156] This result demonstrates that, in cell culture using the aforementioned chemically modified CNF as a scaffold, stem cells can be cultured while maintaining their multidirectional differentiation ability until differentiation induction treatment is performed.

[0157] [Example 6] Primary cell culture test

[0158] Primary mesenchymal stem cells (MSC-R37) derived from human iliac bone marrow were cultured using either unmodified CNF or chemically treated CNF as scaffold materials. 200 μL of the CNF-coated material, prepared according to Example 3 and having a solids concentration of 0.25%, was applied to a 24-well plate and dried at room temperature for 2 days. The 24-well plate with the scaffold was sterilized according to the method of Example 4(1) and used for MSC-R37 culture.

[0159] Furthermore, for comparison, a 24-well plate that was not coated and a 24-well plate coated with commercially available animal-derived type I collagen were used for culturing MSC-R37.

[0160] Cell culture was performed according to the method used for culturing immortalized mesenchymal stem cells in serum-free medium described in Example 4. After culturing in serum-free medium (no exogenous agents) on the above-mentioned plates for 3 days (72 hours), the number of viable cells in each well was counted, and the average value among multiple wells (average number of cells / well) was calculated.

[0161] The results are shown in Table 3 and Figure 5 Primary mesenchymal stem cells (primary MSCs) barely proliferated on untreated cell culture plates (TCPS plates) and on cell culture substrates coated with unmodified CNFs. However, significantly higher proliferation was observed on cell culture substrates coated with chemically modified CNFs ( Figure 5 , Table 3). Cell proliferation on cell culture substrates coated with chemically modified CNFs was comparable to that observed on TCPS plates coated with commercially available animal-derived type I collagen. Surprisingly, significant proliferation was observed not only for immortalized cells but also for primary cells commonly used in regenerative medicine. This demonstrates that, according to the present invention, by using chemically modified CNFs, which can be plant-derived raw materials, rather than animal-derived type I collagen, it is possible to achieve cell culture in an animal-free and / or exogenous-free environment.

[0162]

[0163] All publications, patents, and patent applications cited in this specification are incorporated herein by direct reference.

Claims

1. A cell culture substrate having a scaffold material on its surface, wherein the scaffold material comprises cellulose nanofibers (CNF) into which ionic functional groups are introduced. The cell culture substrate according to claim 1 , comprising the scaffold material in a dry state. The cell culture substrate according to claim 1 , which is used for culturing stem cells. The cell culture substrate according to claim 1 , which is used for cell culture in a serum-free medium or a low-serum medium.

5. The cell culture substrate according to claim 1, wherein The ionic functional group is an anionic functional group. The cell culture substrate according to claim 5, wherein The anionic functional group is a sulfate group or a carboxyl group.

7. The cell culture substrate according to claim 1, wherein The cellulose nanofibers into which the ionic functional groups are introduced have 0.2 mmol / g to 10 mmol / g of the ionic functional groups.

8. The cell culture substrate according to claim 1, wherein The cellulose nanofibers into which the ionic functional groups are introduced have 0.2 mmol / g to 2 mmol / g of the ionic functional groups.

9. The cell culture substrate according to claim 1, wherein The scaffold material contains 10% by weight or more of cellulose nanofibers introduced with ionic functional groups based on dry weight.

10. The cell culture substrate according to claim 1, wherein Cellulose nanofibers introduced with ionic functional groups have a viscosity of 500 to 50,000 mPa·s when a CNF aqueous dispersion having a solids concentration of 0.5% is subjected to a shear rate of 6 rpm for 10 minutes at 25° C. The cell culture substrate according to claim 1 , wherein The scaffold material includes two or more types of cellulose nanofibers into which anionic functional groups are introduced.

12. The cell culture substrate according to claim 1, wherein The support material is provided on the surface of any material selected from the group consisting of resin, rubber, glass, film, paper, cloth, beads and silk. The cell culture substrate according to claim 1 , which is a cell culture plate having an inner surface coated with the scaffold material.

14. The cell culture substrate according to claim 13, wherein Cell culture plates are made of plastic or glass.

15. A cell culture method, comprising: Cells are cultured using the cell culture substrate according to any one of claims 1 to 14.

16. The method according to claim 15, wherein The cells are stem cells.

17. The method according to claim 15, wherein: Culture cells in serum-free or reduced-serum medium.

18. The method according to claim 16, wherein The cultured stem cells are further cultured in a differentiation induction medium to induce differentiation of the stem cells.

19. A scaffold material comprising cellulose nanofibers into which ionic functional groups are introduced. The scaffold material is used for culturing stem cells.

20. The scaffold material according to claim 19, wherein The culture is performed while maintaining the undifferentiated state of the stem cells.

21. The scaffold material according to claim 19 or 20, wherein The culture is culture in a serum-free medium or a low-serum medium.

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