Hydrogel for cell culture, gel kit, method for producing cell culture product, and method for producing hydrogel for cell culture

Abstract

To provide a technique for controlling gene expression while reducing growth activity caused by unknown growth factors mixed in scaffold raw materials during cell culture using hydrogel as the scaffold.SOLUTION: The present invention provides a hydrogel for cell culture that has a cell growth activity, as calculated in terms of fibroblast growth factor-1 activity, of 100 pg / mL or less, and an elastic modulus of 0.1-500 kPa and includes a radiation-induced crosslinked structure of a hydrophilic polymer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a hydrogel for cell culture, a gel kit, a method for producing a cell culture, and a method for producing a hydrogel for cell culture. [Background technology]

[0002] The development of technology to control gene expression in cells using scaffold materials for cell culture is an urgent issue in the fields of regenerative medicine, drug discovery, and cancer treatment. In the case of plastic and glass dishes that have been commonly used for cell culture, the problem is that the characteristics of cultured cells differ from those of cells in the body because they are far removed from the in vivo environment. Therefore, in order to culture cells in an environment closer to the in vivo environment, commercially available substrates such as plastic and glass are currently used that are coated with polymer gels mainly made of synthetic resins such as polyacrylamide gel (which have a hardness close to that of soft tissues in the body), or coated with proteins such as collagen (which mimic the in vivo environment with their chemical composition). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Mitsumasa Taguchi, "Challenge to regenerative medicine with new material gelatin gel - Development of cell manipulation device using quantum beam microfabrication technology", Radiation Application Forum 2017 in Takasaki (2017) [Non-Patent Document 2] AJ Engler et al., “Matrix Elasticity Directs Stem Cell Lineage Specification.” Cell, 126, 677 (2006) Summary of the Invention [Problem to be solved by the invention]

[0004] However, when cell culture is performed using commercially available scaffold materials (coating agents), the cell culture results vary from lot to lot, and there are cases where reproducibility is poor. Furthermore, when a crosslinking agent is used in the manufacture of a scaffold material, unreacted monomers and crosslinking agents may remain in the scaffold material, and it has been pointed out that the unreacted crosslinking agents may dissolve into the cell culture medium and cause cytotoxicity.

[0005] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a technology that can control gene expression while reducing the proliferation activity caused by unidentified growth factors mixed into the scaffold raw materials in cell culture using a hydrogel scaffold. [Means for solving the problem]

[0006] The present inventors have conducted extensive research into the phenomenon in which the reproducibility of cell culture results decreases due to differences in the lots of the above-mentioned cell culture scaffold materials (commercially available coating agents), and have confirmed that growth factors may be mixed into the above-mentioned coating agents, and that the type and activity of such growth factors significantly affect the cell culture results.The inventors have then established a method for forming a scaffold by irradiating the scaffold with radiation while reducing the activity of growth factors in the scaffold material, as well as a method for producing a cell culture product.

[0007] In the following description, the term "growth factor" in the present invention is synonymous with "cell growth factor", which is a factor that affects the results of cell culture, and specific examples thereof include fibroblast growth factor-1.

[0008] According to the present invention, the following inventions are provided.

[0009] (1) The cell proliferation activity is 100 pg / mL or less in terms of fibroblast growth factor-1 activity, The elastic modulus is 0.1 to 500 kPa; A hydrogel for cell culture that has a radiation-crosslinked structure of hydrophilic polymer.

[0010] (2) The hydrogel for cell culture according to (1), wherein the cell proliferation activity, as measured by incubation of NIH-3T3 cells for 28 hours using Cell Counting Kit-8, is less than 105% compared to that of a type I collagen gel derived from bovine dermis extracted by acid treatment.

[0011] (3) The hydrogel according to (1) or (2), wherein the hydrophilic polymer is a protein.

[0012] (4) The hydrogel according to any one of (1) to (3), comprising one or more selected from the group consisting of collagen, collagen peptide, and gelatin in an amount of 1% by mass or more and 30% by mass or less relative to the hydrogel.

[0013] (5) A hydrogel according to any one of (1) to (4) having recesses and / or parallel grooves on its surface.

[0014] (6) The hydrogel according to any one of (1) to (5), for subsequent addition of a physiologically active factor separate from the hydrogel.

[0015] (7) The hydrogel according to any one of (1) to (5), further comprising one or more factors selected from the group consisting of a differentiation-inducing factor, an adhesion factor, a chemotactic factor, and an extracellular matrix.

[0016] (8) A gel kit comprising the hydrogel according to any one of (1) to (5) and a physiologically active factor separate from the hydrogel.

[0017] (9) A method for producing a cell culture, comprising culturing cells by contacting the cells with the hydrogel according to any one of (1) to (5).

[0018] (10) The method for producing a hydrogel according to (9), wherein a hydrogel having an elastic modulus and / or a surface shape corresponding to a desired gene expression is selected and used as the hydrogel.

[0019] (11) The method according to (10), wherein the gene expression is gene expression involved in cell differentiation or gene expression involved in cell growth.

[0020] (12) The method for producing a cell culture according to any one of (9) to (11), wherein the cell culture is a melanin-producing cell.

[0021] (13) The method for producing a cell culture according to any one of (9) to (11), wherein the cell culture is a phagocyte derived from the abdominal cavity.

[0022] (14) The method for producing a cell culture according to any one of (9) to (11), wherein the cell culture is cardiomyocytes.

[0023] (15) The method for producing a cell culture according to any one of (9) to (11), wherein the cell culture is a breast cancer cell.

[0024] (16) The method for producing a cell culture according to any one of (9) to (11), wherein the cell culture is a uterine cancer cell.

[0025] (17) The method for producing a cell culture according to any one of (9) to (16), wherein the cell culture is a cell mass.

[0026] (18) A method for producing a hydrogel for cell culture, comprising the steps of: For an aqueous solution containing 0.1 to 70% by mass of a hydrophilic polymer, An irradiation step of irradiating with radiation at a dose of 1 to 1000 kGy, The irradiated hydrogel has a cell proliferation activity of 100 pg / mL or less in terms of fibroblast growth factor-1 activity, The elastic modulus of the hydrogel after irradiation is 0.1 to 500 kPa; The method for producing a hydrogel for cell culture, wherein the hydrogel after irradiation has a radiation-crosslinked structure of a hydrophilic polymer.

[0027] (19) The method for producing a hydrogel for cell culture according to (18), wherein the cell proliferation activity of the irradiated hydrogel is less than 105% as compared to that of a type I collagen gel derived from bovine dermis extracted by acid treatment, as determined by incubation of NIH-3T3 cells for 28 hours using Cell Counting Kit-8.

[0028] (20) The method according to (18) or (19), wherein the aqueous solution containing the hydrophilic polymer contains a cell growth factor at a concentration of 0 to 1000 ng / mL.

[0029] (21) The method according to any one of (18) to (20), further comprising a pH adjustment step of adjusting the pH to 6 to 8 following the irradiation step.

[0030] (22) The method according to (21), wherein the pH adjustment step is an adjustment step using any one of a buffer solution, a culture medium, and water.

[0031] (23) The method according to any one of (18) to (22), further comprising a surface processing step of pressing a mold having parallel grooves at predetermined intervals on its surface.

[0032] (24) The method according to any one of (18) to (23), wherein the hydrophilic polymer is a protein.

[0033] (25) The manufacturing method according to any one of (18) to (24), wherein the irradiated hydrogel contains one or more selected from the group consisting of collagen, collagen peptide, and gelatin in an amount of 1% by mass or more and 30% by mass or less relative to the hydrogel.

[0034] (26) The method according to any one of (18) to (25), further comprising the step of adding a physiologically active factor after the irradiation. Effect of the Invention

[0035] According to the present invention, a production technique is provided for stably controlling gene expression in cell culture and producing a cell culture. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a graph showing the relationship between gamma ray dose and elastic modulus. [Diagram 2] FIG. 1 shows the relationship between the amount of FGF1 added and proliferation activity. [Diagram 3] FIG. 1 shows the relationship between the amount of FGF1 added and proliferation activity. [Figure 4] FIG. 1 shows the relationship between the amount of FGF1 added and proliferation activity. [Diagram 5] FIG. 1 shows the regulation of differentiation in phagocyte cultures derived from mouse peritoneal cavity. [Figure 6] FIG. 1 shows morphological regulation in mouse melanoma-derived B16F10 cell cultures. [Figure 7] FIG. 1 shows morphological regulation in cultures of HeLa cells derived from human cervical cancer. [Figure 8] FIG. 1 shows the proliferation rate in culture of HeLa cells derived from human cervical cancer. [Figure 9] FIG. 13 shows the effect of pH adjustment treatment after radiation exposure on the FGF1 concentration in the hydrogel. [Figure 10] FIG. 1 shows the effect of the presence or absence of a pH adjustment treatment after irradiation of an FGF1-containing hydrogel on the cell proliferation activity after cell culture using the hydrogel. [Figure 11] FIG. 1 shows the effect of radiation dose on polymer concentration in hydrogels. [Figure 12] FIG. 1 shows the results of post-addition of laminin to a hydrogel according to one embodiment of the present invention. [Figure 13] FIG. 1 shows the results of culturing mouse malignant melanoma-derived B16F10 cells using a hydrogel according to one embodiment of the present invention. [Figure 14] FIG. 1 shows the results of culturing mouse malignant melanoma-derived B16F10 cells using a hydrogel according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Hereinafter, an embodiment of the present invention will be described.

[0038] (1) Hydrogel for cell culture The hydrogel for cell culture (hydrogel for cell culture scaffold) of the present invention is a hydrogel for cell culture having a cell proliferation activity of 100 pg / mL or less in terms of fibroblast growth factor-1 activity, an elastic modulus of 0.1 to 500 kPa, and a radiation-crosslinked structure of a hydrophilic polymer.

[0039] Hereinafter, the hydrogel for cell culture of the present invention will also be simply referred to as the "hydrogel of the present invention."

[0040] In the present invention, a hydrogel is a material that contains water and hardens into a gel, and is formed by crosslinking constituent molecules of hydrophilic polymers such as proteins so as to bridge each other. As will be described later, crosslinking by radiation utilizes the inherent high energy of radiation and does not require a so-called crosslinking agent (also called, for example, a thermal crosslinking agent (polymerization initiator) or an ultraviolet crosslinking agent (photopolymerization initiator)), and therefore forms a radiation-induced crosslinked structure that does not contain any crosslinking agent.

[0041] By having such a radiation-crosslinked structure, the hydrogel state can be maintained even when left under cell culture conditions.

[0042] The hydrogel preferably has a water content of typically 10% by mass or more (preferably 30% by mass or more). The upper limit of the water content is not particularly limited and may be appropriately set to, for example, 99% by mass or less.

[0043] The hydrogel may preferably contain a hydrophilic polymer in an amount of, for example, 1% by mass or more, preferably 3% by mass or more. The upper limit of the amount of the hydrophilic polymer in the hydrogel is not particularly limited, and may preferably be, for example, 50% by mass or less, preferably 40% by mass or less.

[0044] (Radiation-crosslinked structure of hydrophilic polymer) Although there is no particular limitation, radiation crosslinking is a process in which active sites are generated on polymer chains by irradiation, and the polymer chains form three-dimensional networks by bonding in X- or T-shapes from these sites. A distinctive feature of radiation crosslinking is that it proceeds at room temperature or below without the use of additives such as crosslinking agents, and it is used to gel materials, improve heat resistance, and impart shape memory properties.

[0045] The hydrophilic polymer is the same as a known hydrophilic polymer, specifically, a polymer having a hydrophilic group in the molecule.

[0046] Examples of the hydrophilic group include a hydroxyl group, an amino group, a carboxyl group, an ether group, an acyl group, and a sulfo group. That is, the hydrophilic polymer is a polymer having at least one, and preferably two or more, such hydrophilic groups in the molecule.

[0047] The molecular weight of the hydrophilic polymer is not particularly limited, and may be appropriately selected from a range of, for example, 150 to 2,000,000. Typically, a range of 1,000 to 1,000,000 may be appropriately selected. In addition, for polymers with the same composition, the higher the molecular weight, the higher the elastic modulus of the hydrogel tends to be. In this specification, unless otherwise specified, "molecular weight" means weight average molecular weight. In addition, such weight average molecular weight may be measured by a conventionally known size exclusion chromatogram.

[0048] Examples of the hydrophilic polymer include hydrophilic polymers derived from natural products, such as proteins, peptides, polysaccharides, and nucleic acids, or derivatives thereof. "Derived from natural products" means that the polymers can be obtained by extraction or purification from natural products (earth resources, typically living organisms, such as animals, plants, and fungi), and is not limited to being natural products themselves. For example, synthetic proteins artificially synthesized using extracts or purified products from natural products as raw materials are included in the hydrophilic polymers derived from natural products (hereinafter also referred to as natural polymers). Here, the synthetic proteins include both proteins synthesized in a cell-based protein synthesis system and proteins synthesized in a cell-free protein synthesis system.

[0049] Specific examples of the above-mentioned hydrophilic polymers derived from natural products include polysaccharides such as dextrin, dextran, chitin, chitosan, agar, agarose, gellan gum, xanthan gum, karaya gum, carrageenan, cellulose, and starch; proteins such as collagen, gelatin, fibrin, albumin, laminin, keratin, ovalbumin, myosin, globulin, and peptides; and nucleic acids such as DNA and RNA.

[0050] In addition, the above-mentioned hydrophilic polymer derived from natural products may be used as a raw material for hydrogels by using only a single subtype, or may be used as a raw material for hydrogels by combining a plurality of different subtypes. For example, collagen is known to have subtypes such as type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. Therefore, one or more of these subtypes of collagen may be used in combination. Type I collagen is present in the greatest amount in the living body, and is therefore preferred in that it can be obtained relatively cheaply. Type IV collagen is a collagen present in the basement membrane of the skin, and is therefore preferred in that it can be obtained relatively easily.

[0051] Here, the above-mentioned protein means a polymer in which a plurality of amino acids are bound by peptide bonds, and is not limited by the number of amino acids constituting the protein. For example, it includes peptides consisting of two or three or more amino acids. In this specification, the term "peptide" refers to a polymer consisting of 2 to 2,000 amino acids.

[0052] In addition, the derivatives of the hydrophilic polymers (natural polymers) derived from natural products are not particularly limited, and examples thereof include derivatives in which the polymers derived from natural products are substituted with lower alkyl groups, lower alkoxyalkyl groups, or hydroxy-lower alkyl groups.Specific examples include natural polymer derivatives selected from the group consisting of lower alkyl group-substituted cellulose derivatives, lower alkoxyalkyl group-substituted cellulose derivatives, hydroxy-lower alkyl group-substituted cellulose derivatives, lower alkoxyalkyl group-substituted chitosan derivatives, lower alkoxyalkyl group-substituted chitin derivatives, lower alkoxyalkyl group-substituted starch derivatives, and lower alkoxyalkyl group-substituted carrageenan derivatives.

[0053] The hydrophilic polymer may be an artificially synthesized synthetic polymer (synthetic resin). Conventionally known hydrophilic polymers can be used without particular limitation, and examples thereof include synthetic polymers selected from the group consisting of polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polymethacrylamide, and polyethylene glycol. Alternatively, polymers having an artificially designed sequence may also be included in the synthetic molecule. Examples include artificial proteins (including peptides), nucleic acids, and polysaccharides having an artificially designed sequence.

[0054] The hydrogel may be one in which only one of the hydrophilic polymers is cross-linked (bonded) to one another, or two or more hydrophilic polymers are cross-linked (bonded) to one another.

[0055] From the viewpoint of culturing in an environment close to the living environment of the target cells, it is preferable to select a hydrophilic polymer derived from an animal or its derivative when the purpose of use is to culture cells derived from an animal, and a hydrophilic polymer (hereinafter also referred to as a bio-derived polymer) or its derivative obtained by extraction or purification from a living body (animal living body) is more preferable, although there is no particular limitation thereto. For example, a protein material with a low degree of purification extracted from a living body may be used as the hydrophilic polymer. Specifically, a mixture of multiple subtypes of collagen, or a mixture of multiple proteins such as collagen and laminin, or a mixture of different polymers such as proteins and polysaccharides, or proteins and nucleic acids, may be used as the raw polymer for the hydrogel for cell culture scaffold.

[0056] For example, when cells cultured on the above-mentioned hydrogel for cell culture scaffold are transplanted into a living body, it is preferable to select a biocompatible hydrophilic polymer, taking into consideration the possibility that the hydrogel for scaffold may be mixed into the cell population for transplantation. Examples of such biocompatible hydrophilic polymers include the above-mentioned bio-derived polymers. Polymers mainly composed of proteins, such as gelatin and collagen, can be decomposed and absorbed by enzymes such as collagenase and protease in the recipient (donor), and are therefore preferable as raw materials for the hydrogel for scaffold for culturing cells for transplantation.

[0057] (Elasticity Modulus) The elastic modulus (hereinafter, sometimes referred to as hardness for convenience) is defined by a known method for measuring a stress-strain curve in accordance with JIS K 6272 or the like, and is measured, for example, by the compressive elastic modulus. That is, the hydrogel of the present invention includes a hydrogel having a compressive elastic modulus of 0.1 to 500 kPa. In the case of a hydrogel for use as a cell culture scaffold intended for mammalian cells, a hydrogel having a compressive elastic modulus in the range of preferably 1 kPa to 200 kPa, more preferably 3 kPa to 60 kPa can be suitably used.

[0058] Cultured cells are cells isolated from various tissues in the body. Here, it is known that the elastic modulus of tissues varies from one another, with bone having an elastic modulus of GPa or more, cartilage having an elastic modulus of MPa or more (for example, about 1 to 100 MPa), and soft tissue having an elastic modulus of 0.1 to 500 kPa (for example, about 50 kPa). That is, in order to culture the target cultured cells in an environment close to in vivo, it is necessary to culture the target cells in vitro using a scaffold material with an elastic modulus that reflects the elastic modulus of the tissue from which the target cells are derived.

[0059] In other words, when culturing desired cells in a cell culture, the elastic modulus of the hydrogel used as a scaffold can be selected to match that of the tissue from which the desired cells are isolated. Specifically, the hydrogel of the present invention, which has a compressive elastic modulus of 0.1 to 500 kPa, is suitable for culturing cells derived from soft tissues such as nerves and muscles.

[0060] (Cell proliferation activity) In addition, the hydrogel for cell culture scaffold of the present invention reduces the influence of growth factors contained in the natural polymer base material (hereinafter, sometimes exemplified as proteins) so as to obtain stable cell culture results. The influence of growth factors can be quantitatively defined by using fibroblast growth factor-1 (FGF1) as a growth factor for evaluation.

[0061] Specifically, the hydrogel of the present invention has a cell proliferation activity, calculated as FGF1 activity, of 100 pg / mL or less, preferably 50 pg / mL or less, and more preferably 10 pg / mL or less.

[0062] As described above, the effect of a growth factor can be quantitatively defined by measuring the proliferation of the cells for evaluation. For example, the cell proliferation activity evaluated in a system using NIH-3T3 cells can be used as an index. Specifically, when NIH-3T3 cells (hereinafter sometimes referred to as 3T3 cells), which are cultured cells isolated from mouse fetal skin, are used as the cells for evaluation, the proliferation activity can be less than 105% compared to that of a hydrogel made using a hydrophilic polymer that has been confirmed in advance to have no growth factor activity (typically does not contain growth factors).

[0063] The absence of growth factor activity in the hydrophilic polymer can be confirmed by, for example, a purity test using a high performance liquid chromatography mass spectrometer (LC-MS) or by a reporter assay to confirm the activity of growth factors that may be contained therein.

[0064] Furthermore, a hydrophilic polymer can be used in which the activity of growth factors that may be mixed in has been inactivated by subjecting the polymer to an acidic (or alkaline) environment or heating at high temperatures. For example, it is known that the activity of growth factors in type I collagen derived from bovine dermis extracted by acid treatment (Tri-D manufactured by Nippi Corporation) is inactivated by the acid treatment, so Tri-D collagen can be used as a reference scaffold.

[0065] The proliferation activity may be measured using a cell proliferation measurement reagent (e.g., Cell Counting Kit-8, Dojindo Laboratories), or the number of cells before and after culture may be measured and compared using a commercially available cell counter, or the number of cells before and after culture may be measured and compared under a microscope using a hemocytometer.

[0066] The cell culture time for evaluating the proliferation activity is not particularly limited, but may be evaluated, for example, after 28 hours of culture (24 hours of incubation followed by a 4-hour color reaction).

[0067] Specifically, a predetermined number (amount) of predetermined cells for evaluation can be seeded on a hydrogel for cell culture scaffold and cultured for a predetermined time for evaluation. A simple evaluation can be performed by measuring the absorbance at a predetermined wavelength by absorptiometry, and estimating the cell number from a correlation graph (calibration curve) between absorbance and cell number obtained in advance. In this case, when seeding the cells, a water-soluble tetrazolium salt (e.g., C 20 H 13 NO 11 By adding S2Na, the reduction reaction accompanying cell proliferation can be measured by the increase in absorbance at a specific wavelength, and the increase in cell number can be evaluated with good reproducibility. This measurement may be performed using a commercially available measurement kit, Cell Counting Kit-8 (manufactured by Dojindo Laboratories, Inc.). In addition, the absorbance can be measured using any of a microplate reader, an absorbance meter, and a spectrophotometer.

[0068] In the hydrogel for cell culture scaffolds of the present invention, 3T3 cells are used as the cells to be evaluated, and 90 μL of each are seeded into multiple wells at a concentration of 5,000 cells / well, and pre-cultured for 24 hours (culture conditions: 5% carbon dioxide atmosphere, 37°C). Next, 10 μL / well of Cell Counting Kit-8, which is a water-soluble tetrazolium salt solution, is added. After that, the main culture is carried out for 4 hours in an incubator, and a color reaction is performed (culture conditions: 5% carbon dioxide atmosphere, 37°C). Next, the absorbance at 450 nm is measured using a microplate reader (total incubation for 28 hours).

[0069] Here, a gel made by a similar method using a highly pure natural polymer that does not contain growth factors (for example, a type I collagen gel derived from bovine dermis extracted by acid treatment) is used as a reference sample, and its absorbance at 450 nm is compared. Taking advantage of the fact that absorbance increases as cells proliferate, the proliferation activity can be defined by dividing the absorbance of the sample incubated for a total of 28 hours by the absorbance of the reference sample and expressing the result as a percentage (%). The hydrogel for cell culture scaffolds of the present invention is defined by such an evaluation method and has a proliferation activity of less than 105% after 28 hours of incubation.

[0070] That is, in a preferred embodiment of the hydrogel for cell culture scaffolds of the present invention, the proliferation activity of NIH-3T3 cells when incubated for 28 hours using Cell Counting Kit-8 is less than 105% compared to type I collagen gel derived from bovine dermis extracted by acid treatment.

[0071] The effects of the growth factors can be quantitatively defined by using fibroblast growth factor-1 (FGF1) as a growth factor for evaluation. For example, a reference hydrogel is prepared by adding a predetermined amount of FGF1 to a desired hydrogel for cell culture scaffolds, and the activity of the growth factor mixed into the target hydrogel for cell culture scaffolds can be evaluated by converting it into the amount of FGF1 and comparing it with a calibration curve of the cell proliferation rate when cells are cultured in the reference hydrogel for a predetermined time.

[0072] Alternatively, the activity of the growth factor mixed into the target hydrogel for cell culture scaffold may be evaluated in terms of the amount of FGF1 by comparing the cell proliferation rate when FGF1 is added to the culture medium with a standard curve.

[0073] The hydrogel for cell culture scaffolds of the present invention has a reduced effect of growth factors contained in the natural polymer used as the base material. Specifically, the cell proliferation activity is suppressed to a low activity of 100 pg / mL or less in terms of FGF1, and therefore gene expression and differentiation state can be stably controlled. Preferably, if the proliferation activity value as defined above is suppressed to a low activity of less than 105%, gene expression and differentiation state can be stably controlled.

[0074] Thus, the hydrogel of the present invention has an elastic modulus suitable for the desired cell culture and reduces the effects of physiologically active factors (growth factors) contained in the hydrophilic polymer base material, and when used as a cell culture scaffold, it can stably control gene expression and differentiation state.

[0075] In particular, when a protein, which is a hydrophilic polymer derived from a natural product (typically derived from a living organism), is used as the hydrophilic polymer, the bioactive factors (growth factors) contained in the protein tend to affect the culture results. However, the hydrogel of the present invention reduces the effects of bioactive factors (growth factors) derived from the living organism, even when the above-mentioned protein is used as the hydrophilic polymer.

[0076] [Preferred embodiment of the hydrogel of the present invention] Preferred embodiments of the present invention will now be described.

[0077] In a preferred embodiment, the cell culture surface of the hydrogel may have a desired unevenness, for example, parallel grooves, recesses (such as approximately circular recesses), or a combination thereof.

[0078] Here, the grooves constituting the parallel grooves may be straight lines or dotted lines. The interval between the parallel grooves is arbitrary, but preferably 0.5 to 1000 μm. The interval between the parallel grooves may be constant or variable. The depth of the parallel groove recess is arbitrary, but preferably 0.5 to 500 μm.

[0079] The recess may have any shape, for example, a dot shape. For example, it may have a substantially circular shape (typically a circle) with a diameter of 10 μm to 1000 μm. With such a recess, a plurality of cells (for example, a cell population, a colony, a fertilized egg) can be captured and cultured in the recess.

[0080] Also, for example, a well may be formed with a diameter of 10 to 100 μm. By setting the diameter of the well in this range, the number of cells that can be captured in the well can be limited. For example, a well may be formed with a diameter of 20 to 30 μm.

[0081] A culture scaffold having such recesses can be suitably used for the purpose of isolating, retaining and culturing an average of about 1 to 10 cells (typically an average of 1 to 3 cells, preferably an average of 1 cell) per recess.

[0082] The hydrogel of the present invention is suitable for culturing cell clusters (spheroids, spheres). When used for culturing cell clusters, the hydrogel of the present invention may be provided with recesses of a size according to the diameter of the cell clusters, for example, recesses with a diameter of 50 to 1000 μm, 100 to 800 μm, 10 to 200 μm, or 20 to 100 μm may be provided. The depth of the recess may be 10 to 1000 μm in terms of the deepest distance from the hydrogel surface. The hydrogel may have one recess or multiple recesses (for example, 2 to 10,000 recesses). When multiple recesses are provided, the longest distance between adjacent recesses may be 10 to 10,000 μm.

[0083] In another preferred embodiment, the elastic modulus of the hydrogel may be non-uniform. For example, it can be set to be non-uniform in the thickness direction. For example, it may be set to be composed of a plurality of hydrogel layers with different elastic moduli, or it may be a gradient gel in which the elasticity of the cell culture surface is set low and the elasticity is set high in the depth direction (thickness direction). In this way, by setting the elastic modulus to be non-uniform in the thickness direction (typically the elastic modulus of the cell culture surface is set low), for example, cells can easily penetrate into the hydrogel, and three-dimensional culture can be realized.

[0084] Alternatively, the elastic modulus may be set to be non-uniform in the horizontal direction. For example, a portion may be provided with an elastic modulus different from that of the surroundings, or a gradient gel may be used in which the elastic modulus is set to gradually decrease in a specific direction. Setting the elastic modulus to be non-uniform in the horizontal direction in this way can be used, for example, in a method for separating cells that prefer a scaffold with a predetermined elastic modulus.

[0085] In another preferred embodiment, the hydrogel of the present invention may be a gel containing a polymer at a higher concentration than conventional ones. For example, the hydrogel may contain 1% by mass to 30% by mass of a polymer. Specifically, the hydrogel of the present invention may contain 1% by mass to 30% by mass of one or more selected from the group consisting of collagen, gelatin, and collagen peptide.

[0086] The above concentration is higher than that contained in general neutralized collagen or commercially available hydrogels derived from biological extracts (such as Matrigel). According to the present invention, a hydrogel containing such a high concentration of polymer can be obtained by irradiation.

[0087] The hydrogel containing the above-mentioned concentrations of polymers can be obtained by irradiating a solution of each polymer, the concentration of which has been appropriately adjusted, with air saturation as necessary. The conditions for the method for producing the hydrogel can be those listed in the following [(4) Method for producing hydrogel for cell culture].

[0088] [Post-addition of various physiologically active factors to the hydrogel of the present invention] By adding (post-adding) a physiologically active factor separate from the hydrogel to the hydrogel of the present invention, the hydrogel can be selectively endowed with a desired effect according to the added component and to a desired degree. Therefore, the present invention also includes a hydrogel for post-adding a physiologically active factor separate from the hydrogel. Examples of physiologically active factors to be post-added to the hydrogel include cell growth factors, differentiation-inducing factors, adhesion factors, chemotactic factors, extracellular matrices, etc., and one or more selected from these may be added.

[0089] The cell growth factor to be added later to the hydrogel of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include hepatocyte growth factor, platelet-derived growth factor, granulocyte colony-stimulating factor, epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor, transforming growth factor, nerve growth factor, bone morphogenetic factor, etc.

[0090] The differentiation-inducing factor to be added to the hydrogel of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include Activin, TGF β2, HGF (Hepatocyte growth factor), Dex (Dexamethasone), Rac1 (RAS-related C3 botulinus toxin substrate 1), Zfp521 (Zinc finger protein 521), etc.

[0091] The adhesion factor to be added to the hydrogel of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include integrin, nephronectin, laminin, fibronectin, tenascin, fibulin, EMILIN, QBRICK, osteopontin, polydom, MAEG, fibronogen, etc.

[0092] The chemotactic factor to be added to the hydrogel of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include CCL21, fMLP, leukotriene B4, IL-8, C5a, leukotriene B4 (LTB4), platelet activating factor (PAF), etc.

[0093] The extracellular matrix to be added later is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include collagen, laminin, fibronectin, elastin, polysaccharides, and the like.

[0094] The amount of physiologically active factors (cell growth factors, differentiation-inducing factors, adhesion factors, chemotactic factors, extracellular matrices, etc.) added to the hydrogel of the present invention is not particularly limited and may be appropriately selected depending on the type of factor added.

[0095] The present invention also includes a hydrogel in which one or more factors selected from the group consisting of a differentiation-inducing factor, an adhesion factor, a chemotactic factor, and an extracellular matrix are added to the above-mentioned hydrogel of the present invention. The differentiation-inducing factor, the adhesion factor, the chemotactic factor, and the extracellular matrix may be appropriately selected from the examples of the physiologically active factors to be added later and added.

[0096] [Gel kit containing the hydrogel of the present invention] By adding (post-adding) a physiologically active factor (e.g., cell growth factor and / or extracellular matrix) separate from the hydrogel to the hydrogel of the present invention, a desired action according to the added component can be selectively imparted to the hydrogel to a desired extent. Therefore, the present invention also includes a gel kit containing the hydrogel of the present invention and a physiologically active factor (e.g., cell growth factor and / or extracellular matrix) separate from the hydrogel. The physiologically active factor contained in these gel kits can be one or more selected from cell growth factors, differentiation induction factors, adhesion factors, chemotactic factors, extracellular matrices, etc.

[0097] The cell growth factor that can be contained in the gel kit of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include hepatocyte growth factor, platelet-derived growth factor, granulocyte colony-stimulating factor, epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor, transforming growth factor, nerve growth factor, bone morphogenetic factor, etc.

[0098] The differentiation-inducing factor that can be contained in the gel kit of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include Activin, TGF β2, HGF (Hepatocyte growth factor), Dex (Dexamethasone), Rac1 (RAS-related C3 botulinus toxin substrate 1), Zfp521 (Zinc finger protein 521), etc.

[0099] The adhesion factor that can be contained in the gel kit of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include integrin, nephronectin, laminin, fibronectin, tenascin, fibulin, EMILIN, QBRICK, osteopontin, polydom, MAEG, fibronogen, etc.

[0100] The chemotactic factor that can be contained in the gel kit of the present invention is not particularly limited, and may be any component used in cell culture, etc. Examples of such components include CCL21, fMLP, leukotriene B4, IL-8, C5a, leukotriene B4 (LTB4), platelet activating factor (PAF), etc.

[0101] The extracellular matrix that can be contained in the gel kit of the present invention is not particularly limited, and may include any component used in cell culture, etc. Examples of such components include collagen, laminin, fibronectin, elastin, polysaccharides, etc.

[0102] In the gel kit of the present invention, the mass ratio between the hydrogel of the present invention and a physiologically active factor (e.g., a cell growth factor and / or an extracellular matrix) separate from the hydrogel is not particularly limited and may be appropriately selected depending on the use of the gel kit.

[0103] The method of using the gel kit of the present invention is not particularly limited, but a physiologically active factor (e.g., cell growth factor and / or extracellular matrix) separate from the hydrogel can be added to the hydrogel of the present invention at a desired timing, and then the hydrogel can be used for the production of a cell culture product described below. The method of addition, etc. is appropriately selected depending on the use of the gel kit and the type of physiologically active factor (e.g., cell growth factor and extracellular matrix).

[0104] (2) Method for producing cell culture The method for producing a cell culture of the present invention is a method for culturing cells by contacting the cells to be cultured with the hydrogel of the present invention as a scaffold. In particular, it is preferable to select and use a hydrogel having an elastic modulus and / or surface shape corresponding to the desired gene expression or differentiation state. The gene expression may be the expression of a gene involved in cell differentiation or the expression of a gene involved in cell growth.

[0105] The "elastic modulus and / or surface shape corresponding to the desired gene expression or differentiation state" is appropriately selected depending on the type of gene to be expressed.

[0106] For example, when the gene to be expressed is a melamine gene, the preferred elastic modulus and surface shape of the gel depending on the cells used for gene expression are as follows:

[0107] When using mouse malignant melanoma-derived B16F10 cells: the elastic modulus of the hydrogel is preferably 5 to 48 kPa, and the surface shape is preferably flat.

[0108] When using HeLa cells derived from human cervical cancer-1: To form HeLa cell spheroids, the elastic modulus of the hydrogel is preferably 1 kPa to 30 kPa (more preferably about 5 kPa), and the surface shape is preferably flat.

[0109] In the case of using HeLa cells derived from human cervical cancer-2: In order for HeLa cells to adopt a two-dimensional form, the elastic modulus of the hydrogel is preferably about 30 kPa to 500 kPa (more preferably about 48 kPa) and the surface shape is preferably flat. Alternatively, the elastic modulus of the hydrogel is preferably about 5 kPa to 500 kPa or more (more preferably 5 kPa to 48 kPa) and the surface shape preferably has parallel grooves (preferably linear unevenness with a groove width of 1 μm to 10 μm, more preferably linear unevenness with a groove width of 5 μm).

[0110] The cells to be cultured on the hydrogel scaffold are not particularly limited, and desired cells can be selected. Examples include human-derived cells, non-human mammal-derived cells, non-mammalian animal-derived cells, and plant-derived cells. Such cells may be cell lines or primary culture cells, or transgenic cells into which a specific gene has been introduced. For example, the hydrogel scaffold for cell culture of the present invention may be suitably applied to the production of pluripotent stem cells such as ES cells and iPS cells, or the induction of differentiation.

[0111] Here, the term "cells" is not limited to single cells existing independently of each other, but includes cell populations (e.g., cell clusters, tissues) in which multiple cells are gathered. In other words, the hydrogel of the present invention can be used as a culture scaffold for various cultures such as colony culture, cell mass (spheroid, sphere) culture, and tissue culture.

[0112] As will be shown in the examples described later, it is possible to culture various animal and plant cells, such as melanin-producing cells, phagocytes derived from the abdominal cavity, cardiac muscle cells, nerve cells, liver cells, kidney cells, pancreatic cells, stem cells, antibody-producing cells, osteoclasts, epithelial cells, fibroblasts, breast cancer cells, breast cancer cells, brain tumor cells, uterine cancer cells, cervical cancer cells, pancreatic cancer cells, myoblasts, various stem cells (ES cells, etc.), etc. The cells to be cultured may include tissue fragments, etc.

[0113] Specifically, for example, when culturing phagocytes derived from the peritoneal cavity of a mouse, by using a hydrogel having an elastic modulus of approximately 16 kPa (typically approximately 13 to 19 kPa) as a scaffold, the cells can proliferate in an undifferentiated state without undergoing differentiation, and can be produced as undifferentiated cells.

[0114] Furthermore, for example, when culturing HeLa cells derived from human cervical cancer, by using a hydrogel having an elastic modulus of approximately 1 to 16 kPa as a scaffold, the cells can grow three-dimensionally and cell masses (spheroids, spheres) can be produced.

[0115] The environment in which the cells are cultured may be under known environmental conditions, for example, culture may be performed in an atmosphere of 5% CO2 at 37° C. Furthermore, a known CO2 incubator may be used as the culture device (environmental device).

[0116] The cultured cell culture can be isolated from the scaffold by known methods.

[0117] That is, the present invention can provide hydrogels for cell culture scaffolds for culturing melanin-producing cells, hydrogels for cell culture for culturing undifferentiated phagocytes derived from the peritoneal cavity, hydrogels for culturing cardiomyocytes, hydrogels for culturing osteoclasts, hydrogels for culturing epithelial cells, hydrogels for culturing fibroblasts, hydrogels for culturing breast cancer cells, hydrogels for culturing breast adenocarcinoma cells, hydrogels for culturing brain tumor cells, hydrogels for culturing uterine cancer cells, hydrogels for culturing pancreatic cancer cells, hydrogels for culturing iPS cells, hydrogels for culturing ES cells, hydrogels for culturing MUSE cells, hydrogels for culturing stem cells, hydrogels for culturing nerve cells, hydrogels for culturing blood cells, and hydrogels for culturing myoblasts.

[0118] (3) Hydrogel precursor for cell culture scaffold The precursor of the hydrogel for cell culture scaffolds of the present invention is an aqueous solution containing 0.1 to 70 mass % of a hydrophilic polymer, for example, a protein.

[0119] Although the concentration of the hydrophilic polymer in the precursor is not particularly limited, the rate of radiation crosslinking per unit volume increases when a high-concentration precursor is irradiated with radiation, and therefore hydrogels with high elastic modulus tend to have a low water content. Therefore, the concentration of the hydrophilic polymer in the precursor can be determined from the desired elastic modulus and water content.

[0120] The precursor of the hydrogel may contain a physiologically active factor, such as a growth factor, i.e., even if the aqueous solution of the precursor of the hydrogel contains a physiologically active factor, the activity of the factor may be reduced by the action of radiation to provide a hydrogel.

[0121] Here, as the hydrophilic polymer, a polymer derived from a natural product (a natural polymer, typically a polymer derived from a living body) may also be used. The term "natural polymer" is defined as above. Examples of the natural polymer include collagen, which is abundant in the extracellular matrix of multicellular animals, gelatin obtained by thermally denaturing collagen, and collagen peptides.

[0122] The above-mentioned polymers derived from natural products may contain biologically active factors (growth factors). In particular, hydrophilic polymers derived from living organisms, particularly proteins isolated from living organisms, may contain biologically active factors (growth factors). A certain amount of growth factors remain even after the precursors are crosslinked to form a hydrogel, and in cell culture using the hydrogel as a scaffold, they become an element that destabilizes or inhibits the results of cell culture, including gene expression and differentiation state of cultured cells.

[0123] Therefore, it is preferable that the substrate is a polymer (e.g., a protein) with a low content of growth factors, in other words, with high purity. On the other hand, there is a production problem in that the purification of a large amount of a high-purity protein requires a large amount of cost.

[0124] Growth factors include basic fibroblast growth factor (bFGF or FGF2), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF), which promote cell proliferation and differentiation.

[0125] However, by appropriately subjecting the hydrogel precursor to a radiation irradiation step and a treatment for reducing the activity of physiologically active factors (typically a pH adjustment treatment step) described later, gene expression of cultured cells can be stably performed in cell culture using the hydrogel as a scaffold, even if the natural polymer contains a certain amount of growth factors. Specifically, assuming irradiation with radiation at a dose of 5 kGy as an example, the precursor can contain a growth factor having a proliferation activity equivalent to that of 0 to 1000 ng / mL of fibroblast growth factor when converted into fibroblast growth factor. In one embodiment, the precursor can contain a growth factor having a proliferation activity equivalent to that of 0 to 500 ng / mL of fibroblast growth factor. In another embodiment, the precursor can contain a growth factor having a proliferation activity equivalent to that of 0 to 100 ng / mL of fibroblast growth factor. In another embodiment, the precursor can contain a growth factor having a proliferation activity equivalent to that of 0 to 10 ng / mL of fibroblast growth factor.

[0126] In addition, an increase in the radiation dose can increase the amount of growth factor that can be tolerated. Therefore, in the case of irradiation with a dose of more than 5 kGy, the precursor can contain a growth factor having a proliferation activity equivalent to that of more than 1000 ng / mL of fibroblast growth factor when converted into fibroblast growth factor.

[0127] (4) Manufacturing method of hydrogel for cell culture The method for producing the hydrogel for cell culture (hydrogel for cell culture scaffold) of the present invention includes a step of irradiating the precursor with radiation at a dose of 1 to 1000 kGy. The hydrogel of the present invention can be preferably produced by this production method.

[0128] Here, radiation is a concept including electromagnetic waves with wavelengths shorter than ultraviolet rays, electron beams, and ion beams, and electromagnetic waves include X-rays and gamma rays. The radiation irradiation device can be any known device that generates these types of radiation.

[0129] (Radiation irradiation process) In the radiation irradiation step, the precursor is continuously or intermittently irradiated with radiation at a dose of 1 to 1000 kGy using a radiation irradiation device. The lower limit of the dose may be 2 kGy or more, 5 kGy or more, 10 kGy or more, 100 kGy or more, or 500 kGy or more, and the upper limit of the dose may be 500 kGy or less, 300 kGy or less, 200 kGy or less, or 100 kGy or less. In one embodiment, radiation irradiation at a dose of 5 to 200 kGy can be preferably used.

[0130] Here, the dose is the amount of radiation irradiated per unit time, integrated over time, and can be measured using a dosimeter that is sold for each type of radiation. In addition, it is desirable to set the irradiation energy of the radiation irradiator to about 100 keV to 10 MeV.

[0131] When the precursor is irradiated with radiation, hydrophilic polymers (e.g. proteins) are partially cleaved to generate radicals, and a crosslinking reaction occurs, forming a gel that encapsulates water (the formation of a hydrogel). When the radiation irradiation is completed, the crosslinking reaction immediately stops. The crosslink density during the reaction depends on the dose of radiation used, and as a result, the elastic modulus of the gel also depends on the dose of radiation.

[0132] In addition, the higher the crosslink density of the hydrophilic polymer, the lower the water content (the higher the content of hydrophilic polymer in the hydrogel). Therefore, the content of hydrophilic polymer in the hydrogel also depends on the dose of radiation applied.

[0133] Figure 1 shows an example of the relationship between gamma ray dose and elastic modulus of cross-linked hydrogel for gelatin derived from cow and pig. Although the tendency differs slightly depending on the type of gelatin, it can be seen that there is an approximately linear relationship between the dose and elastic modulus between gamma ray doses of 0 to 100 kGy. In other words, the radiation dose can be determined according to the required elastic modulus of the hydrogel.

[0134] During radiation irradiation, it is desirable that the environmental temperature be 4 to 50° C. and the dissolved oxygen concentration in the precursor be 0 to 40 mg / L.

[0135] The environmental temperature during radiation irradiation may be appropriately set depending on the properties of the hydrophilic polymer. For example, when collagen is used, radiation crosslinking can be efficiently performed by irradiating at a low temperature of about 4 to 25°C, and when gelatin is used, radiation crosslinking can be efficiently performed by irradiating in a temperature environment of 10 to 30°C.

[0136] In addition, the dissolved oxygen in the precursor during radiation irradiation has the effect of capturing radicals. In other words, by increasing the dissolved oxygen concentration in the precursor, the crosslinking density is reduced, and as a result, the elastic modulus of the hydrogel can be reduced. Specific methods for increasing the dissolved oxygen concentration in the precursor include, for example, irradiating the precursor with radiation in an environment with a high oxygen concentration, leaving the precursor under a high oxygen concentration, and bubbling oxygen into the precursor.

[0137] In addition, by lowering the dissolved oxygen concentration in the precursor, the crosslinking density is increased, and as a result, the elastic modulus of the hydrogel can be increased.Specific methods for reducing the dissolved oxygen concentration in the precursor include irradiating the precursor with radiation in a nitrogen environment and bubbling nitrogen into the precursor.

[0138] Furthermore, by irradiating a precursor with a low dissolved oxygen concentration under high oxygen concentration, it is possible to change the dissolved oxygen concentration between the surface in contact with oxygen (typically the cell culture surface) and other parts. By controlling the oxygen concentration, it is possible to make only the surface of the hydrogel used for cell culture (only the surface in contact with the cells to be cultured) have a desired elastic modulus, and to make the other parts have a higher elastic modulus than the surface. For example, in the case of a flat hydrogel, it is possible to make the basal part have a high elastic modulus and only the surface used for cell culture have a low elastic modulus.

[0139] As described above, the method for producing a hydrogel for cell culture scaffold includes a step of irradiating with radiation (amount of radiation: 1 to 1000 kGy). In the radiation irradiation step, crosslinking occurs in the hydrophilic polymer by a predetermined radiation irradiation, and a predetermined elastic modulus is imparted. At this time, the high energy of the radiation also acts on the growth factor in the hydrogel precursor, and the activity of the growth factor can be reduced. That is, radiation irradiation has the effect of both crosslinking the hydrophilic polymer and reducing the activity of the bioactive factor, so that a hydrogel for scaffold can be provided by a simple method. The method for producing a hydrogel for cell culture scaffold by irradiation with radiation is particularly suitable when the activity of the bioactive factor contained in the precursor is relatively low. Furthermore, the radiation irradiation is also excellent from the viewpoint of being able to sterilize (sterilize) the hydrogel for cell culture scaffold.

[0140] (Various treatments before and after radiation exposure) The method for producing a hydrogel for use as a cell culture scaffold may further include a treatment to reduce the activity of a bioactive factor (eg, a growth factor).

[0141] For example, the activity of physiologically active factors (e.g., growth factors) in a hydrogel crosslinked by irradiation can be reduced by adjusting the pH (pH adjustment step). After irradiation, the hydrogel can be used as a scaffold for cell culture with reduced activity of physiologically active factors by adjusting the pH to 6 to 8 using a basic solution or a buffer solution.

[0142] As the basic solution or buffer solution that can be used in the pH adjustment step after irradiation, various commercially available buffer solutions (e.g., PBS), culture media, or water can be selected. The culture media may contain phenolsulfonephthalein (phenol red).

[0143] For example, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium (MEM), Eagle's minimum essential medium alpha modified (α-MEM), Glasgow's minimum essential medium (GMEM), Iscove's modified Dulbecco's medium, nutrient mixture F-12 Ham's F-12, RPMI-1640, etc. can be used.

[0144] The temperature conditions for the pH adjustment step after radiation exposure are not particularly limited, but may be preferably 4 to 60°C, more preferably 32 to 38°C.

[0145] Heat treatment may be performed as the activity reduction treatment of the physiologically active factor. The activity of the physiologically active factor can be irreversibly inactivated by exposure to a high temperature environment. This heat treatment may be performed either before or after the radiation irradiation step. For example, the activity of the physiologically active factor in the precursor can be reduced by subjecting the precursor to a heat treatment before radiation irradiation. Although not particularly limited, heating at 50°C or higher for 1 minute or more is sufficient. The upper limit of the heating temperature is not particularly limited, but may be, for example, 60°C or lower, and the upper limit of the heating time may be 5 minutes or less.

[0146] Furthermore, as a treatment for reducing the activity of the physiologically active factor, the precursor may be subjected to an acid treatment to adjust the pH of the precursor to pH 5 or less (preferably pH 4 or less) or an alkali treatment to adjust the pH to 8 or more (preferably pH 9 or more) prior to irradiation. The activity of a physiologically active factor can be irreversibly inactivated by exposure to an acidic or alkaline environment. In other words, the activity of the physiologically active factor in the precursor can be reduced by performing such an acid or alkali treatment.

[0147] The method for reducing the activity of these physiologically active factors may be appropriately selected depending on the properties of the hydrophilic polymer.

[0148] For example, when gelatin is selected as the hydrophilic polymer, a method of heat-treating a precursor can be suitably adopted. By adopting this method, the treatment of dissolving gelatin in an aqueous solution and the treatment of reducing the activity of a physiologically active factor can be simultaneously realized, and a hydrogel for a cell culture scaffold can be easily produced.

[0149] For example, when collagen is selected as the hydrophilic polymer, it is preferable to adjust the pH of the hydrogel obtained by radiation irradiation. Typically, collagen is liable to be denatured when exposed to a high temperature environment, which makes crosslinking by radiation irradiation difficult. For this reason, it is preferable to select a treatment for reducing the activity of growth factors after radiation irradiation (e.g., a pH adjustment treatment or a heat treatment) rather than a heat treatment of the precursor before radiation irradiation.

[0150] As described above, a preferred embodiment of the method for producing a hydrogel for cell culture scaffold includes a step of irradiating with radiation and a step of reducing the activity of a physiologically active factor. By including both the radiation irradiation step and the step of reducing the activity of a physiologically active factor, the growth factors contained in the precursor can be significantly reduced, and the proliferation activity of the hydrogel as defined above can be suppressed to less than 105%.

[0151] Here, the hydrogel production method of the present invention, which has both a radiation irradiation step and a physiologically active factor activity reducing step, exerts an effect equal to or greater than the sum of the growth factor reducing effects of the two steps. That is, when attempting to reduce the growth factor activity to a predetermined level or lower only by radiation irradiation, the radiation irradiation may cause the elastic modulus of the hydrogel to exceed the desired elastic modulus. In other words, the reduction in growth factor activity acts in the direction of increasing the radiation dose, which is an obstacle when attempting to obtain a gel with a small elastic modulus of about several kPa, for example, for culturing nerve cells.

[0152] Furthermore, it may be difficult to reduce the activity of growth factors to a desired level only by treatment to reduce the activity of physiologically active factors.

[0153] Therefore, by having both the radiation irradiation step and the activity reducing step of the physiologically active factor, it becomes easy to simultaneously ensure the elastic modulus required for culturing the desired cells while effectively reducing the activity of the growth factor. Furthermore, as the activity reducing step of the physiologically active factor, the pH adjustment is preferable in that the pH of the scaffold for cell culture can be made optimal for cell culture.

[0154] The above-mentioned treatments for reducing the activity of physiologically active factors may be carried out either alone or in combination of two or more types.

[0155] In this way, by carrying out both the radiation irradiation step and the activity reduction step of the physiologically active factor, the gene expression and the differentiation state of the cultured cells can be stably controlled even if the precursor contains a certain amount of growth factors. Specifically, as described above, the precursor can contain 0 to 1000 ng / mL of growth factors. This eliminates the need to use highly purified raw materials as hydrophilic polymers, and allows the use of hydrophilic polymers (e.g., proteins) that are widely available, thereby expanding the range of material choices and reducing material costs.

[0156] In addition to the above-mentioned radiation irradiation step and the activity inactivation step of the bioactive factor, a surface processing step for processing the shape of the hydrogel surface may be added. An example of such a surface processing step is a method of pressing a mold of a predetermined shape onto the hydrogel precursor. Since the hydrogel precursor of the present invention has a relatively low elastic modulus, the shape of the mold can be transferred to the hydrogel by irradiating the hydrogel with the mold pressed against it. The surface shape of such a mold may be a shape corresponding to the shape of the desired cell culture surface of the hydrogel. For example, a mold having parallel grooves at a predetermined interval, or a mold having convex portions of a predetermined size and interval is exemplified. In cell culture, the parallel grooves have the effect of giving cells a certain shape and a certain activity directionality. In addition, the concave portions, which are formed by transferring the convex portions of the mold to the hydrogel, can exert the effect of playing the role of microwells for isolating and holding the desired cells in cell culture.

[0157] The material of the mold is not particularly limited as long as it is a water-insoluble material, and it is possible to use silicon, glass, synthetic resin, etc. Since the mold is peeled off after pressing, it is preferable that the mold is flexible, and synthetic resin is desirable.

[0158] The parallel grooves may be straight or dotted. The parallel grooves may have any interval, but preferably have an interval of 0.5 to 1000 μm. The parallel grooves may have any interval, but preferably have an interval of 0.5 to 500 μm. The parallel grooves may have any depth, but preferably have an interval of 0.5 to 500 μm.

[0159] The convex portion may have any shape, for example, a dot shape. For example, it may have a substantially circular shape (typically a circle) with a diameter of 10 μm to 1000 μm. By transferring such a convex portion, a concave portion with a diameter of 10 μm to 1000 μm can be formed. With such a concave portion, a plurality of cells (for example, a cell population, a colony, a fertilized egg) can be captured and cultured in the concave portion.

[0160] Alternatively, for example, a convex portion having a diameter of 10 μm to 100 μm may be transferred to form a concave portion having a diameter of 10 to 100 μm. By setting the size of the concave portion within this range, the number of cells that can be captured in the concave portion can be limited. For example, a convex portion having a diameter of 20 to 30 μm may be transferred to form a concave portion having a diameter of 20 to 30 μm.

[0161] A culture scaffold having such recesses can be suitably used for the purpose of isolating, retaining and culturing an average of about 1 to 10 cells (typically an average of 1 to 3 cells, preferably an average of 1 cell) per recess.

[0162] In consideration of the above, in one embodiment, when producing a hydrogel (collagen gel) with an elastic modulus of 5 to 60 kPa for culturing fibroblasts and cervical cancer cells, a sample under the following conditions is irradiated with gamma rays and subjected to pH adjustment treatment.

[0163] Molecular weight of hydrophilic polymer (collagen): 600,000 or less (for example, 200,000 to 600,000, preferably 400,000 to 500,000) Concentration of hydrophilic polymer (collagen) solution: 0.1 to 5.0% by mass Dissolved oxygen concentration: 0~8mg / L Gamma ray dose: 1-100 kGy (preferably 2-50 kGy), The solution temperature during gamma ray irradiation is 4 to 37°C (preferably 4 to 25°C), pH adjustment treatment: DMEM medium, 37℃, 10 to 180 minutes.

[0164] In another embodiment, when producing a hydrogel (gelatin gel) having an elastic modulus of 1 to 200 kPa for culturing melanin-producing cells, phagocytes derived from the peritoneal cavity, cardiomyocytes, osteoclasts, epithelial cells, fibroblasts, cardiomyocytes, breast cancer cells, breast adenocarcinoma cells, brain tumor cells, uterine cancer cells, cervical cancer cells, pancreatic cancer cells, or myoblasts, a sample is prepared under the following conditions, and gamma ray irradiation after pretreatment is selected.

[0165] Molecular weight of hydrophilic polymer (gelatin): 200,000 or less (for example, 100,000 to 170,000) Concentration of hydrophilic polymer (gelatin) solution: 0.1 to 70% by mass Dissolved oxygen concentration: 0~8mg / L Gamma ray dose: 1-300 kGy (preferably 2-200 kGy) The solution temperature during gamma ray irradiation is 4 to 37°C (preferably 10 to 30°C).

[0166] pH adjustment treatment: DMEM medium, 50℃ for 10 to 180 minutes.

[0167] As described above in the [Gel kit containing the hydrogel of the present invention], by adding a physiologically active factor to the hydrogel of the present invention, the hydrogel can be endowed with a desired effect according to the added component.

[0168] Therefore, a step of adding a physiologically active factor may be carried out after the radiation exposure step.

[0169] Details of the physiologically active factors (eg, cell growth factors and extracellular matrices) to be added are the same as those explained above in [Gel kit containing the hydrogel of the present invention]. EXAMPLES

[0170] The present invention will be described in detail below with reference to examples, but it should be understood that the present invention is not limited to these examples.

[0171] In the following, the pH adjustment treatment performed on a gel that has not been irradiated is also referred to as a "neutralization pH adjustment treatment," and the pH adjustment treatment performed on a gel after radiation is also referred to as a "post-irradiation pH adjustment treatment."

[0172] Reference Example 1: Effects of radiation exposure on proteins including growth factors, and effects of radiation exposure and post-exposure pH adjustment The following two types of hydrogels were prepared, and the amount of growth factor present in the hydrogels was evaluated from the fluorescence intensity of antibody staining. Gel 2 corresponds to the hydrogel of the present invention.

[0173] (Gel 1-1) Protein containing growth factors irradiated with gamma rays (Gel 1-2) Protein containing growth factors was irradiated with gamma rays and then pH adjusted (post-irradiation pH adjustment). Materials and Methods A high-purity bovine dermis-derived type I collagen solution (collagen gel cell culture kit Tri-D manufactured by Nippi Corporation, the same applies below) was added to DMEM medium supplemented with the growth factor fibroblast growth factor-1 (FGF1, R&D Systems, 232-FA-025 / CF) at concentrations of 0, 30, 300, and 3000 ng / mL. The mixture ratio was collagen solution:DMEM = 2:1. Next, 100 μL of the resulting mixture was placed in a glass base dish, and the temperature was immediately raised to 37°C in a CO2 incubator to fiberize the collagen.

[0174] By the above procedure, neutralized collagen gels containing 0, 10, 100, and 1000 ng / mL of FGF1 as a protein containing a growth factor were obtained.

[0175] Each neutralized collagen gel was stained with FGF1 antibody (Santa Cruz Biotechnology, sc-55520) and secondary antibody (Invitrogen, A32723) and observed for fluorescence under the same conditions to obtain a calibration curve showing the relationship between neutralized collagen gels with different FGF1 contents and fluorescence intensity.

[0176] Then, a neutralized collagen gel containing 1000 ng / mL of FGF1 was irradiated with 1 kGy of gamma rays (corresponding to gel 1-1), and a neutralized collagen gel containing 1000 ng / mL of FGF1 was irradiated with 1 kGy of gamma rays and then adjusted to pH 7.4 with PBS at 37°C or higher (corresponding to gel 1-2) were obtained. These gels were subjected to immunostaining in the same manner as above, and the fluorescence intensity was measured.

[0177] <Result> The measurement results of the fluorescence intensity after immunostaining are shown in Figure 9. In Figure 9, a higher fluorescence intensity value indicates a higher FGF1 concentration in the gel.

[0178] As shown in Figure 9, the FGF1 concentration was reduced in all gels. Furthermore, the fluorescence intensity of Gel 1-2 was lower than that of Gel 1-1 and was lower than the detection limit (200 ng / mL). From these results, it was found that the FGF1 concentration can be further reduced by adjusting the pH after radiation exposure, and even if the raw material contains FGF1 at a high concentration of 1000 ng / mL, the amount can be reduced to below the detection limit.

[0179] (Reference Example 2) Evaluation of the proliferation activity of NIH-3T3 cells when a protein containing growth factors is used as a scaffold material (effect of neutral pH adjustment treatment) After adjusting the pH of the growth factor-containing protein, the temperature was raised to create a hydrogel scaffold. 3T3 cells, a fibroblast cell line derived from mouse fetal skin, were cultured on the scaffold and their proliferation activity was evaluated.

[0180] Materials and Methods A high-purity bovine dermis-derived type I collagen solution was prepared as a high-purity collagen solution, to which FGF1 was added at concentrations of 0, 10, and 100 ng / mL. 10 μL of this was placed in a 96-well microplate, 5 μL of colorless DMEM medium was added, and the temperature was raised to 37°C in a CO2 incubator, and the pH was adjusted to pH = 7.1 to 7.5. As a result, the collagen in the collagen solution was fibrous, and a neutralized collagen gel was obtained. The neutralized collagen gel corresponds to a product obtained by neutralizing and pH-adjusting a protein containing growth factors.

[0181] Using this neutralized collagen gel as a scaffold, 3T3 cells were seeded at a concentration of 5,000 cells / well (90 μL), cultured for 24 hours, and Cell Counting Kit-8 (Dojindo Laboratories, Inc.) was added at 10 μL / well. After that, a color reaction was carried out in an incubator for 4 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0182] <Result> Figure 2 shows the change in absorbance as a function of FGF1 concentration, i.e., the change in proliferation activity, with the result without FGF1 being taken as 100%. It is clear that proliferation activity increases with increasing FGF1 concentration, indicating that 3T3 cells are affected by growth factors.

[0183] The results indicate that when a protein material of biological origin containing one or more known or unknown growth factors is used as a culture substrate, cell culture is influenced by the growth factors.

[0184] It was also shown that the inhibition of growth factors was insufficient with pH adjustment treatment using 5 μL of DMEM medium.

[0185] (Reference Example 3) Evaluation of the proliferation activity of 3T3 cells when a protein containing growth factors is used as a scaffold material (effect of radiation treatment) We used a growth factor-containing protein as a precursor, irradiated it with gamma rays, cultured 3T3 cells, a type of fibroblast derived from mouse fetal skin, and evaluated their proliferation activity.

[0186] Materials and Methods Fibroblast growth factor (FGF1) was added to a colorless DMEM solution containing 10% fetal bovine serum (FBS) at concentrations of 0, 1, and 10 ng / mL, and irradiated with 5 kGy of gamma rays. 10 μL of each solution was then added to a 96-well microplate, and 90 μL of 3T3 cells were seeded at a concentration of 5,000 cells / well. After culturing for 24 hours, 10 μL / well of Cell Counting Kit-8 was added, and the color reaction was carried out in an incubator for 4 hours. The absorbance at 450 nm was measured using a microplate reader.

[0187] <Result> Figure 3 shows the change in absorbance as a function of FGF1 concentration, i.e., the change in proliferation activity, with the result without FGF1 set at 100%. It is clear that proliferation activity increases with increasing FGF1 concentration, indicating that 3T3 cells are affected by growth factors.

[0188] These results indicate that even if a biomaterial containing one or more known or unknown growth factors is irradiated with gamma rays and used as a culture substrate, the cells will be affected by the growth factors derived from the substrate, and the culture results will not be stable.

[0189] It was also shown that the suppression of growth factors was insufficient with 5 kGy gamma irradiation.

[0190] Example 1: Evaluation of the proliferation activity of 3T3 cells when a protein containing growth factors is used as a scaffold material (effects of radiation irradiation and post-irradiation pH adjustment treatment) A hydrogel was produced by irradiating a precursor protein containing growth factors with gamma rays, and the pH was adjusted at 37°C or higher. The hydrogel was then used as a scaffold to culture 3T3 cells, a fibroblast cell line derived from mouse fetal skin, and the proliferation activity was evaluated.

[0191] Materials and Methods FGF1 was added to a high-purity bovine dermis-derived type I collagen solution at concentrations of 0, 10, and 100 ng / mL, and 10 μL was placed in a 96-well microplate. After irradiating with 5 kGy of gamma rays, colorless DMEM medium was added and pH adjustment treatment (post-irradiation pH adjustment treatment) was performed at 37 °C to pH 7.1 to 7.5. After removing the colorless DMEM medium used for pH adjustment treatment, 3T3 cells were seeded at a concentration of 5,000 cells / well in 90 μL, cultured for 24 hours, and Cell Counting Kit-8 was added at 10 μL / well. After that, the color reaction was performed in an incubator for 4 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0192] <Result> Figure 4 shows the change in absorbance as a function of FGF1 concentration, i.e., the change in proliferation activity, with the result without the addition of FGF1 set at 100%. The proliferation activity was less than 105% regardless of the concentration of FGF1, indicating that 3T3 cells were not affected by the growth factor.

[0193] These results indicate that when a biomaterial containing one or more known or unknown growth factors is irradiated with gamma rays and subjected to pH adjustment treatment at 37°C or higher before being used for culture, i.e., when the hydrogel obtained by the present invention is used as a culture substrate, the cultured cells are not affected by the growth factors contained in the substrate and the culture results are stable.

[0194] Furthermore, these results and the above (Reference Example 1) show that even when a protein containing growth factors is used as a raw material, the growth factors can be inactivated to a level that does not affect the cell culture results by radiation exposure treatment and pH adjustment treatment (post-irradiation pH adjustment treatment).

[0195] Example 2: Cultivation of mouse peritoneal-derived phagocytes in hydrogel scaffolds The proliferation of mouse peritoneal phagocytes seeded and cultured on the hydrogel scaffold was directly observed using an optical microscope. The state of phagocyte proliferation was observed from the obtained images.

[0196] Materials and Methods 10 wt% of porcine gelatin was added to 1 L of distilled water and thoroughly stirred at 50℃ to obtain an aqueous gelatin solution. This solution was then transferred to a 35 mm dish and physically gelled and air-saturated at 20℃. It was then irradiated with gamma rays at 25℃, at a dose rate of 10 kGy / h, and at a dose of 20 kGy. The resulting gelatin crosslinked hydrogel was confirmed to have an elastic modulus of 16 kPa from the stress-strain curve in a compression test.

[0197] RPMI-10% FCS medium and a population of 1,000,000 mouse peritoneal phagocytes were added to the obtained hydrogel, and the cells were cultured at 37°C under 5% CO2, and the growth state was periodically observed using a phase contrast microscope.

[0198] The mouse peritoneal phagocyte population was prepared by administering thioglycollate medium intraperitoneally to mice, collecting peritoneal exudate cells from the mice four days later, culturing the cells in RPMI medium containing 10% FCS in a polystyrene dish for one day, and then isolating the cells attached to the dish. The cells were then cultured for a further seven days and then detached from the dish with trypsin.

[0199] <Result> The microscopic images obtained are shown in Figure 5. When using polystyrene dishes, which are used in ordinary culture experiments, most of the cells in the phagocyte population differentiate and spread over the surface of the dish, attaching firmly and losing their ability to proliferate; even if transplanted into a living organism, they quickly lose their ability to function. The spherical cells scattered on the surface of the spread cells are poorly differentiated cells with high proliferative capacity, and although they are suitable for transplantation, the number obtained is small (Figure 5(a)).

[0200] On the other hand, when a population of phagocytes is cultured on a cross-linked gelatin with an elastic modulus of 16 kPa, a very small proportion of the cells attach to the surface and elongate (Fig. 5(b), elongated, linear cells), while the majority of the cells attach to the surface like clusters, maintaining their spherical shape. With further culture, the number of spherical cells continues to grow and increase (Fig. 5(c)), making it possible to prepare large quantities of transplantable, spherical, less differentiated cells with the ability to proliferate.

[0201] The hydrogel of the present invention has been shown to be capable of growing a population of less differentiated or undifferentiated cells, and is closely related to the preparation of cell sources in regenerative medicine.

[0202] Example 3: Cultivation of melanoma cells in hydrogel scaffolds The proliferation of melanoma cells seeded and cultured on the hydrogel scaffolds and the pigmentation due to melanin production were directly observed using an optical microscope, and the amount of melanin production was measured by absorbance.

[0203] Materials and Methods 10wt% of porcine gelatin was added to 1L of distilled water and thoroughly stirred at 50℃ to obtain an aqueous gelatin solution. This solution was then transferred to a 35mm dish and allowed to gel physically and air-saturate at 20℃. It was then irradiated with gamma rays at 25℃, at a dose rate of 10kGy / h, and at doses of 10, 20, and 40kGy. The elastic modulus of the resulting gelatin crosslinked hydrogel was confirmed to be approximately 5, 16, and 48kGy, respectively, based on the stress-strain curves obtained by compression tests. B16F10 cells derived from mouse malignant melanoma were cultured on the resulting gelatin crosslinked body and on a plastic dish used in conventional culture experiments, and were then observed over time with an optical microscope and the amount of melanin produced was quantified. The amount of melanin produced was calculated from the amount of soluble protein in the presence of 1% TritonX100 after washing the recovered cells with PBS and the amount of melanin solubilized when treated with 1N NaOH at 85°C for 30 minutes.

[0204] <Result> The obtained microscopic images are shown in Figure 6. The observed images show that no clear melanin production was observed in the cells 2D cultured on the plastic dish (Figure 6(a)), but the cells cultured on the cross-linked gelatin formed cell clusters (spheroids) regardless of the elastic modulus of the cells, which ranged from 5 to 48 kPa, and some of the cell clusters were found to produce high-density melanin (Figures 6(b) to (d)).

[0205] Quantitative analysis also showed that melanin was hardly produced under 2D culture conditions, but was significantly produced over time when cultured on crosslinked gelatin, and that the hardness of the crosslinked gelatin influenced the amount of melanin produced.

[0206] These results demonstrate that cellular gene expression can be modulated by the elastic modulus of the hydrogel, just as in vivo, and are closely related to drug discovery and cancer treatment research.

[0207] Example 4: Cultivation of human cervical cancer-derived HeLa cells in hydrogel scaffolds (1) The morphology of HeLa cells, a human cervical cancer cell line derived from human, seeded and cultured on hydrogel scaffolds with a smooth surface and a microstructure with 5μm linear irregularities (parallel grooves), was directly observed by optical microscopy. The size (adhesive area), aspect ratio, and rotation of the cells were measured from the images obtained.

[0208] Materials and Methods 10 wt% of porcine gelatin was added to 1 L of distilled water and thoroughly stirred at 50℃ to obtain an aqueous gelatin solution. This solution was then transferred to a 35 mm dish and physically gelled and air-saturated at 20℃. It was then irradiated with gamma rays at 25℃, at a dose rate of 10 kGy / h, and at doses of 10 kGy and 40 kGy. The elastic moduli of the resulting crosslinked gelatin were confirmed to be 5 kPa and 48 kPa, respectively, from the stress-strain curves in a compression test.

[0209] In addition, a mold with 5 μm linear irregularities (parallel grooves) was pressed onto the aqueous solution and irradiated in the same manner as above to produce hydrogel scaffolds with the same elastic modulus (5 kPa and 48 kPa, respectively) and 5 μm linear irregularities on the surface.

[0210] HeLa cells were cultured on the surface of the four types of hydrogel scaffolds obtained and on the surface of polystyrene dishes used in conventional cell culture experiments, and observed under an optical microscope.

[0211] <Result> The obtained microscopic images are shown in Figure 7. The observed images show that epithelial cells, HeLa, form three-dimensional cell clusters on a smooth hydrogel with a hardness of 5 kPa (Figure 7(a)). On the other hand, on a smooth hydrogel with a hardness of 48 kPa, the cells spread two-dimensionally and show a mesenchymal-like morphology (Figure 7(b)).

[0212] Even with an elastic modulus of 5 kPa, when there is a 5 μm linear unevenness, the morphology changes in response to the shape (Fig. 7(d)). In the case of an elastic modulus of 48 kPa with a 5 μm linear unevenness, the morphology changes in the same way as in the case of an elastic modulus of 5 kPa (Fig. 7(e)).

[0213] On the other hand, when cultured on a polystyrene dish without using a hydrogel scaffold, the cells spread two-dimensionally and exhibited a cobblestone-like epithelial morphology (Figure 7(c)).

[0214] These results indicate that both the elastic modulus and surface shape (surface microstructure) of the hydrogel affect cellular gene expression.

[0215] Next, Figure 8 shows the results of investigating the proliferation rate of cells cultured on hydrogels (with smooth culture surfaces) with elastic moduli of 5 to 67 kPa and on polystyrene dishes (measurements were taken on days 1 to 3, with the start of culture being taken as 1). Figure 8 shows that cells can be cultured at the same speed as on a normal plastic dish on any of the hydrogels with elastic moduli of 5 to 67 kPa. In other words, this shows that it is possible to control only gene expression while leaving the proliferation rate of the cells unchanged.

[0216] These results indicate that the gene expression and differentiation state of cells can be controlled by the elastic modulus and surface shape of the hydrogel, which is closely related to the preparation of cell sources in regenerative medicine.

[0217] (Example 5) Cultivation of HeLa cells derived from human cervical cancer in hydrogel scaffolds (2) A gelatin aqueous solution prepared under the same conditions as in Example 4 was irradiated with a 2 MeV electron beam at a dose rate of 10 kGy / time 1 to 20 times for a total dose of 10 to 200 kGy in an air atmosphere at a temperature of 25° C. When cervical cancer-derived cancer cells were seeded on the obtained protein crosslinked body, morphological changes due to elastic modulus were observed, similar to those in Example 4.

[0218] This result shows that the elastic modulus of the hydrogel can be adjusted by the dose with electron beam irradiation, as with gamma ray irradiation, and gene expression can be controlled. In other words, this shows that the present invention can be extended to all types of radiation, not just electromagnetic waves such as gamma rays.

[0219] (Examples 6 to 21) We performed culture tests of various cells on the hydrogel scaffolds under the following conditions to examine their gene expression ability. The results are shown in Table 1.

[0220] [Table 1] As shown in Table 1, although different types of cultured cells responded differently, it was demonstrated that gene expression could be controlled by appropriately selecting the elastic modulus and / or surface shape of the hydrogel scaffold.

[0221] (Reference Example 3) Comparison of remaining amino acids after radiation crosslinking and chemical crosslinking 90% by mass of distilled water was added to 10% by mass of pork gelatin having a molecular weight of 150,000, and stirred for 10 minutes at 25° C. in an air atmosphere (oxygen content approximately 21%, atmospheric pressure) to obtain an aqueous gelatin solution with a dissolved oxygen concentration of 8 mg / L.

[0222] This aqueous solution was irradiated with gamma rays at a dose rate of 10 kGy / h and a dose of 60 kGy in a Co60 irradiation facility in an air atmosphere at a temperature of 25°C to obtain a radiation-crosslinked gelatin gel.

[0223] In addition, an equal amount of an aqueous solution of glutaraldehyde (0.48% by weight), a chemical crosslinking agent, was added to the above gelatin aqueous solution and reacted at 40°C for 12 hours, after which it was washed with a 100 mM aqueous glycine solution at 50°C for 1 hour to obtain a chemically crosslinked gelatin gel.

[0224] The cross-linked gelatin gels obtained by each method were vacuum dried at 30°C for 24 hours, and approximately 1 mg was placed in a 1.5 mL small vial. They were hydrolyzed with hydrochloric acid at 110°C for 24 hours under nitrogen-saturated conditions.

[0225] As a reference, uncrosslinked gelatin was also hydrolyzed in the same manner. After neutralization with sodium hydroxide, each amino acid was fluorescently labeled with 4-Fluoro-7-nitrobenzofurazan and qualitatively and quantitatively measured by HPLC.

[0226] [Table 2] As a result of the analysis, it was revealed that, compared to untreated gelatin, glutaraldehyde treatment reduced lysine by about 20%, while gamma ray irradiation reduced the amount to about 40%, as shown in Table 2. Furthermore, radiation exposure is thought to involve phenylalanine, which forms an intermediate with a microsecond life span, in crosslinking.

[0227] The hydrogel for cell culture, the method for producing a cell culture product, and the method for producing the hydrogel for cell culture of the present invention have been described above, including examples. The above description merely shows the basic configuration of the present invention, and various applications are possible.

[0228] For example, the hydrogel for cell culture of the present invention may be used as a scaffold to be built into a cell culture vessel (cell culture container).

[0229] In addition, the cells cultured in the cell culture production method of the present invention may be cells used for culturing cells for treatment such as transplantation, or may be cells used for pharmacological testing (e.g., drug screening). These cell cultures can also be used for treatment (e.g., regenerative medicine) and pharmacological testing methods (e.g., screening methods) using cell cultures.

[0230] Furthermore, in the method for producing a hydrogel for cell culture, the formation of parallel grooves can be achieved not only by the groove forming step described above. In the radiation irradiation step, if the radiation is in the form of a beam and a radiation irradiation device capable of scanning is used, parallel grooves can also be formed by the presence or absence of radiation irradiation. For example, a hydrogel scaffold having parallel grooves can be formed by repeating a process of raster scanning a two-dimensional plane, irradiating odd-numbered lines with radiation and not irradiating even-numbered lines with radiation. When grooves are formed by this production method, the formation of grooves using a mold can be eliminated.

[0231] (Example 22) Evaluation of cell growth factor content in the hydrogel obtained by the production method according to the present invention The following two types of hydrogels were prepared, and 3T3 cells were cultured using these as scaffolds to compare the cell proliferation activity and evaluate the content of cell growth factors. Note that Gel 22-2 corresponds to the hydrogel obtained by the production method according to the present invention.

[0232] (Gel 22-1) Protein containing a specified amount of growth factor (Gel 22-2) A gel made from a protein containing a specified amount of growth factors that has been irradiated and pH adjusted (pH adjusted after irradiation). Materials and Methods A highly purified bovine dermis-derived type I collagen solution was mixed with DMEM medium supplemented with FGF1 at concentrations of 0, 0.015, 0.3, 1.5, 3, and 30 ng / mL. The mixture ratio was collagen solution:DMEM = 2:1. 40 μL of the resulting mixture was placed in a 96-well microplate and the temperature was immediately raised to 37°C in a CO2 incubator to fiberize the collagen.

[0233] By the above procedure, neutralized collagen gels containing 0, 0.005, 0.1, 0.5, 1, and 10 ng / mL of FGF1 as a growth factor-containing protein were obtained. The neutralized collagen gels correspond to Gel 22-1.

[0234] In addition, neutralized collagen gels containing various concentrations of FGF1 were irradiated with 1 kGy of gamma rays, and then DMEM medium was added to obtain hydrogels adjusted to pH 7.1 to 7.5 at 37° C. The hydrogels correspond to Gel 22-2.

[0235] 3T3 cells were seeded in gel 22-1 and gel 22-2 at a concentration of 5,000 cells / well in 60 μL each, cultured for 48 hours, and Cell Counting Kit-8 was added at 10 μL / well. After that, a color reaction was carried out in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0236] <Result> Figure 10 shows the change in absorbance depending on the FGF1 concentration in the neutralized collagen gel, i.e., the change in cell proliferation activity. Note that in Figure 10, the higher the absorbance value, the higher the cell proliferation activity of the gel.

[0237] As shown in FIG. 10, Gel 22-1 had a significantly higher cell proliferation activity than Gel 22-2.

[0238] Furthermore, in Gel 22-2, regardless of the concentration of FGF1 blended as a raw material, no tendency for the proliferation activity to increase was observed, and compared to the calibration curve obtained from Gel 22-1, the FGF1 in the gel was equivalent to 100 pg / mL or less. In other words, Gel 22-2 has a cell proliferation activity of 100 pg / mL or less in terms of FGF1 activity, which is significantly less than the amount of cell growth factor in commercially available hydrogels derived from biological extracts (such as Matrigel).

[0239] (Example 23) Preparation of high-concentration protein-containing hydrogel Hydrogels were prepared from solutions containing high concentrations of proteins (collagen, gelatin, or collagen peptides) by irradiation, and the polymer concentrations in the gels were evaluated.

[0240] Materials and Methods The following three solutions were prepared:

[0241] (1) Collagen solution A type I collagen solution derived from porcine dermis (Collagen BM, 639-30861, manufactured by Nitta Gelatin) was prepared to a concentration of 5 wt % to obtain an aqueous collagen solution.

[0242] (2) Gelatin solution 10 wt % of pork gelatin was added to 1 L of distilled water and thoroughly stirred at 50° C. to obtain an aqueous gelatin solution.

[0243] (3) Collagen peptide solution 10 wt % of porcine collagen peptide was added to 1 L of distilled water and thoroughly stirred at 50° C. to obtain an aqueous collagen peptide solution.

[0244] The above solutions were transferred to 35 mm dishes, and the collagen solution and gelatin solution were air-saturated at a temperature of 20° C., and the collagen peptide solution was air-saturated at 4° C. Then, they were irradiated with gamma rays at a temperature of 15° C. and a dose rate of 10 kGy / h to a dose of 5 kGy to 60 kGy.

[0245] The polymer concentration in the hydrogel was evaluated based on the weight of the resulting hydrogel before and after drying.

[0246] <Result> Figure 11 shows the polymer concentration in the hydrogel as a function of radiation exposure. As shown in Figure 11, the polymer concentration in the hydrogel increased with increasing radiation exposure. This result means that collagen, gelatin, or collagen peptide, the raw material of the hydrogel, gelled upon radiation exposure to form polymers. The polymer concentration in the hydrogel obtained by the above method was higher than the polymer concentration (generally 1% or less) in general neutralized collagen gels and hydrogels derived from biological extracts (such as Matrigel). This result demonstrated that the polymer concentration in the hydrogel can be adjusted by the radiation exposure dose.

[0247] (Example 24) Addition of specific components to hydrogel As described above, according to the present invention, a hydrogel with a reduced amount of growth factors contained in the raw material can be obtained. In this example, when a specific component is added to the hydrogel obtained in this way, it was examined whether the component functions on the hydrogel. Specifically, laminin (a protein involved in cell adhesion, proliferation, and differentiation), which is an extracellular matrix, was added to the hydrogel, and it was evaluated whether the gel contained laminin.

[0248] Materials and Methods A gelatin aqueous solution was obtained by adding 10 wt% of porcine gelatin to 1 L of distilled water and thoroughly stirring at 50° C. Furthermore, this solution was transferred to a 35 mm dish and physically gelled and air-saturated at a temperature of 20° C., and then irradiated with gamma rays at a temperature of 25° C., at a dose rate of 10 kGy / h, and at a dose of 20 kGy, to obtain a hydrogel (hydrogel not coated with laminin).

[0249] To the obtained hydrogel, 1 mL of 0.1 mg / mL laminin (Invitrogen 23017-015) dissolved in ultrapure water was added and stored overnight at 4°C to obtain a laminin-coated hydrogel.

[0250] Next, rat hippocampal neurons were cultured in both the non-laminin-coated and laminin-coated gels at 37°C under 5% CO2 and the growth status was periodically observed. After that, the cells were stained with laminin antibody (Abcam ab11575) and secondary antibody (Invitrogen A32731), and the presence of laminin in the hydrogel was confirmed by fluorescent observation.

[0251] <Result> The results of the fluorescence observation are shown in Figure 12. In Figure 12, (A) is the observation result of the gel that is not laminin-coated, and (B) is the observation result of the gel that is laminin-coated. If the laminin coating is functional (i.e., if the gel contains laminin), hippocampal neurons will adhere to the gel.

[0252] As shown in (A), neurons did not adhere to the uncoated gel, whereas hippocampal neurons adhered and extended processes on the laminin-coated gel, as shown in (B).

[0253] FIG. 12(C) shows the results of confirming by fluorescent staining that the laminin was actually coated on the laminin-coated gel.

[0254] From the above results, it was found that even if a given component (such as a growth factor) is added to the hydrogel of the present invention, the component functions on the hydrogel.

[0255] (Example 25) Capture and culture of single cells using hydrogel A hydrogel with depressions measuring 100 to 200 μm square was used as a scaffold, and melanoma cells were seeded into the depressions and their appearance was observed over time using an optical microscope.

[0256] Materials and Methods A gelatin aqueous solution was obtained by adding 10 wt% of porcine gelatin to 1 L of distilled water and thoroughly stirring at 50° C. Furthermore, this solution was transferred to a dish and pressed against a mold having a convex structure of 100 to 200 μm square and 100 μm high, and after physical gelation and air saturation at a temperature of 20° C., it was irradiated with gamma rays at a temperature of 15° C., at a dose rate of 5 kGy / h and a dose of 20 kGy, to produce a hydrogel with a concave surface.

[0257] B16F10 melanoma cells derived from mouse malignant melanoma were seeded into the depressions of the obtained hydrogel, and then the cells were observed over time using an optical microscope.

[0258] <Result> Figure 13 shows the results of culturing using a hydrogel with 100 μm square depressions, immediately after cell seeding. As shown in Figure 13, melanoma cells were trapped in the depressions one by one.

[0259] From these results, it was found that the hydrogel of the present invention can capture and culture a single cell. Although data is not shown, the shape of the depressions provided in the hydrogel is not limited to a square, and it was possible to culture cells of any shape.

[0260] In addition, Figure 14 shows the results of culturing using a hydrogel with 100 μm square depressions, 8 days after cell seeding. As shown in Figure 14, spheroids with different melanin production levels from melanoma cells were obtained. The fact that spheroids with different melanin production levels were obtained despite the same culture conditions means that the gene expression of each melanoma cell is different from each other.

[0261] These results demonstrate that the hydrogel of the present invention enables cells to be cultured individually on a hydrogel scaffold and enables the production of cell masses with different gene expression.

Claims

1. The cell proliferation activity is 100 pg / mL or less in terms of fibroblast growth factor-1 activity, The elastic modulus is 3 to 500 kPa, It has a radiation crosslinked structure of a hydrophilic polymer, The hydrophilic polymer comprises mammalian collagen and / or collagen peptide. Hydrogels for cell culture (excluding those containing polyvinyl alcohol).

2. The hydrogel for cell culture according to claim 1, wherein the cell proliferation activity is less than 105% as compared to a devitalized bovine dermis-derived type I collagen gel, as determined by incubation of NIH-3T3 cells for 28 hours using Cell Counting Kit-8.

3. The hydrogel according to claim 1 or 2, comprising the hydrophilic polymer in an amount of 1% by mass or more and 30% by mass or less relative to the hydrogel.

4. The hydrogel according to claim 1 , comprising 5% by mass or more and 30% by mass or less of the mammalian collagen, or 10% by mass or more and 30% by mass or less of the collagen peptide, relative to the hydrogel.

5. 5. The hydrogel according to claim 1, having recesses and / or parallel grooves on its surface.

6. The hydrogel according to claim 1 , which is used for culturing adherent cells.

7. 7. The hydrogel according to claim 1, for the subsequent addition of a physiologically active factor separate from said hydrogel.

8. The hydrogel according to any one of claims 1 to 7, further comprising one or more factors selected from the group consisting of differentiation-inducing factors, adhesion factors, chemotactic factors, and extracellular matrices.

9. A gel kit comprising the hydrogel according to claim 1 and a physiologically active factor separate from the hydrogel.

10. A method for producing a cell culture, comprising culturing cells by contacting the cells with the hydrogel according to any one of claims 1 to 8.

11. The method according to claim 10, wherein the hydrogel used is a hydrogel having an elastic modulus and / or a surface shape corresponding to a desired gene expression.

12. The method according to claim 11 , wherein the gene expression is expression of a gene involved in cell differentiation or expression of a gene involved in cell growth.

13. The method for producing a cell culture according to any one of claims 10 to 12, wherein the cell culture is a melanin-producing cell.

14. The method for producing a cell culture according to any one of claims 10 to 12, wherein the cell culture is phagocytes derived from the abdominal cavity.

15. The method for producing a cell culture according to any one of claims 10 to 12, wherein the cell culture is cardiomyocytes.

16. The method for producing a cell culture according to any one of claims 10 to 12, wherein the cell culture is a breast cancer cell.

17. The method for producing a cell culture according to any one of claims 10 to 12, wherein the cell culture is a uterine cancer cell.

18. The method for producing a cell culture according to any one of claims 10 to 17, wherein the cell culture is a cell mass.

19. A method for producing a hydrogel for cell culture, comprising the steps of: For an aqueous solution containing 0.1 to 70% by mass of a hydrophilic polymer (excluding polyvinyl alcohol), An irradiation step of irradiating with radiation at a dose of 1 to 1000 kGy, The irradiated hydrogel has a cell proliferation activity of 100 pg / mL or less in terms of fibroblast growth factor-1 activity, The elastic modulus of the hydrogel after irradiation is 3 to 500 kPa; The hydrogel after irradiation has a radiation-crosslinked structure of a hydrophilic polymer, The hydrophilic polymer comprises mammalian collagen and / or collagen peptide. A method for producing hydrogels for cell culture.

20. The content of the hydrophilic polymer in the aqueous solution in the irradiation step is 3 to 70 mass %. A method for producing the hydrogel for cell culture according to claim 19.

21. The method for producing a hydrogel for cell culture according to claim 19 or 20, wherein the cell proliferation activity of the irradiated hydrogel is less than 105% as compared to a devitalized type I collagen gel derived from bovine dermis, as determined by incubation of NIH-3T3 cells for 28 hours using Cell Counting Kit-8.

22. The method according to any one of claims 19 to 21, wherein the aqueous solution containing the hydrophilic polymer contains a cell growth factor at a concentration of 0 to 1000 ng / mL.

23. The method according to any one of claims 19 to 22, further comprising a post-irradiation pH adjustment step of adjusting the pH to 6 to 8 following the irradiation step.

24. The method according to claim 19 , further comprising a neutralization pH adjustment step of adjusting the aqueous solution containing the hydrophilic polymer to a pH of 5 or less or a pH of 8 or more before the irradiation step.

25. The method according to claim 23 or 24, wherein the pH adjustment step after the radiation exposure or the neutralization pH adjustment step is an adjustment step using any one of a buffer solution, a culture medium, and water.

26. 26. The method according to claim 19, further comprising a surface processing step of pressing a mold having parallel grooves at predetermined intervals on a surface of the mold.

27. The method according to any one of claims 19 to 26, wherein the irradiated hydrogel contains the hydrophilic polymer in an amount of 1% by mass or more and 30% by mass or less relative to the hydrogel.

28. 28. The method of claim 19, further comprising the step of adding a biologically active factor after said irradiation.

29. The method for producing a hydrogel for adherent cell culture according to any one of claims 19 to 28.

30. The dose in the irradiation step is 5 to 200 kGy; The method according to any one of claims 19 to 29, wherein the elastic modulus of the hydrogel after irradiation is 5 to 500 kPa.

31. The method according to any one of claims 19 to 29, wherein the dose in the irradiation step is 1 to 100 kGy.

Images