A complex comprising a hydrogel and mature skeletal muscle cells, a method for producing the same, and a method for producing a culture comprising skeletal muscle cells.

Abstract

Realization of a technology that can obtain mature skeletal muscle cells by culturing and differentiating myoblasts. [Solution] The composite of the present disclosure is a composite comprising a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and mature skeletal muscle cells adhering to the hydrogel.

Description

Technical Field

[0001] The present invention relates to a complex containing a hydrogel and mature skeletal muscle cells, a method for producing the same, and a method for producing a culture containing mature skeletal muscle cells.

Background Art

[0002] Muscle mass or muscle strength decreases due to various causes such as aging, inactivity, and disease. This causes a decline in motor function or metabolic function and increases the risk of bedriddenness or disease. Therefore, the development of a good treatment method for preventing or treating the decrease in muscle mass or muscle strength is required. If such a treatment method is established, it is expected to lead to the solution of problems such as the soaring medical costs in an aging society, the increase in the number of care recipients, and the shortening of the healthy life expectancy.

[0003] In the development of such a treatment method, it is necessary to stably produce mature skeletal muscle cells that can verify candidate drugs or means acting on muscle mass, muscle strength, or metabolic function effectively and in a short period under culture. However, the culture technique has not been established and is currently under research and development.

[0004] For example, Patent Document 1 and Non-Patent Document 1 disclose that myoblasts were cultured using a hydrogel for cell culture having a radiation crosslinked structure of a hydrophilic polymer.

[0005] In Non-Patent Document 2, in order to obtain myotube cells, the use of a gel having an elastic modulus closer to that of a living body has been studied as compared with a generally used plastic dish. In addition, in Non-Patent Document 3, skeletal muscle cells are cultured on a physical gel made of collagen derived from a living body.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007] [Non-Patent Document 1] TG Oyama et al., Collagen hydrogels with controllable combined cues of elasticity and topography to regulate cellular processes, Biomed Mater. 2021 Jun 11;16(4). [Non-Patent Document 2] Takahiro Suzuki, Verification of the mechanism of characteristic acquisition of muscle stem cells that changes according to muscle fiber type, Sapporo Foundation for the Promotion of Biological Sciences Research Grant Report, 2021. [Non-Patent Document 3] Tomoya Uchimura, et al., A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs, Cell Rep Med. 2021 Jun 4;2(6):100298. [Overview of the project] [Problems that the invention aims to solve]

[0008] Determining whether myoblasts have differentiated into mature skeletal muscle cells cannot be done solely by cell morphology; it is necessary to check the expression levels of muscle contraction-related genes and metabolic-related genes, which are markers for mature skeletal muscle cells. However, prior art literature has not confirmed the expression levels of muscle contraction-related genes and metabolic-related genes in cultured cells. Therefore, there is no data in prior art literature that demonstrates actual differentiation from myoblasts into mature skeletal muscle cells.

[0009] One aspect of the present invention is the realization of a technology that can obtain mature skeletal muscle cells by culturing and differentiating myoblasts. [Means for solving the problem]

[0010] As a result of diligent research, the inventors discovered that differentiation into mature skeletal muscle cells is possible by using a specific hydrogel, and thus completed the present invention.

[0011] To solve the aforementioned problems, a composite according to one aspect of the present invention is a composite comprising a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and mature skeletal muscle cells adhering to the hydrogel.

[0012] A method for producing a composite according to one aspect of the present invention includes the steps of contacting myoblasts with a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and differentiating the myoblasts into mature skeletal muscle cells.

[0013] A method for producing a culture containing mature skeletal muscle cells according to one aspect of the present invention includes the steps of contacting myoblasts with a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer; differentiating the myoblasts into mature skeletal muscle cells; and recovering a culture containing mature skeletal muscle cells from the hydrogel. [Effects of the Invention]

[0014] According to one aspect of the present invention, mature skeletal muscle cells can be obtained by culturing and differentiating myoblasts. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the proliferation ratio of myoblasts after 24 hours in Example 2. [Figure 2] This figure shows the survival rate of myoblasts in Example 2. [Figure 3] This figure shows the observation results of cells on day 5 of differentiation in Example 3. [Figure 4] This is a diagram showing the observation results of cells on the 6th day of differentiation in Example 3. [Figure 5] This is a diagram showing the expression levels of each muscle contraction function-related gene in Example 4. [Figure 6] This is a diagram showing the expression levels of each metabolic function-related gene in Example 4. [Figure 7] This is a diagram showing the observation results of cells on the 6th day of differentiation and the cell orientation of each gel culture substrate in Example 5. [Figure 8] This is a diagram showing the cell differentiation rate of each gel culture substrate in Example 5.

Mode for Carrying Out the Invention

[0016] Hereinafter, one aspect of the present invention will be described in detail. Unless otherwise specified in this specification, "A~B" representing a numerical range is intended to mean "A or more and B or less".

[0017] 〔Composite Containing Hydrogel and Mature Skeletal Muscle Cells〕 The composite according to one aspect of the present invention contains mature skeletal muscle cells and a hydrogel.

[0018] [Mature Skeletal Muscle Cells] In this specification, mature skeletal muscle cells are, on the 6th day after culturing myoblasts in a differentiation medium, compared with myoblasts (control cells) cultured for 6 days in a differentiation medium on a general polystyrene dish, the expression levels of both the MYH2 gene and the MYH7 gene are increased by 1.3 times or more, and the expression levels of all of the GLUT4 gene, the Myoglobin gene, and the PGC1α gene are increased by 1.5 times or more. Also, in this specification, "high maturity" refers to a state in which, compared with myoblasts (control cells) cultured for 6 days in a differentiation medium on a polystyrene dish, the expression levels of both the MYH2 gene and the MYH7 gene are increased by 1.3 times or more, and the expression levels of all of the GLUT4 gene, the Myoglobin gene, and the PGC1α gene are increased by 1.5 times or more.

[0019] The differentiation medium may be any known medium used for differentiation into mature skeletal muscle cells. Examples of differentiation media include media supplemented with non-essential amino acids (such as L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, and L-proline).

[0020] The MYH2 (myosine heavy chain 2; MyHC IIa) and MYH7 (myosine heavy chain 7; MyHC I) genes are genes related to muscle contraction function. Skeletal muscle is broadly classified into fast-twitch and slow-twitch fibers, with the MYH2 gene being related to fast-twitch fibers and the MYH7 gene being related to slow-twitch fibers.

[0021] The GLUT4 (glucose transporter type 4) gene, the Myoglobin gene, and the PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) gene are metabolic function-related genes. The expression levels of these genes increase with myoblast maturation and affect myoblast metabolism.

[0022] In this specification, "compressive modulus" is defined by a known stress-strain curve measurement method in accordance with JIS K 6272. For example, the compressive modulus may be calculated based on the stress-strain curve from the hydrogel surface to 10% of its thickness. It can be measured by a known stress-strain curvature measurement method. The measurement conditions for the compressive modulus may be, for example, a compression rate of 50 μm / s and a maximum test load of 2 N. The compressive modulus may also be measured immediately after removing the hydrogel from a 37°C incubator to a room temperature environment (15°C to 25°C).

[0023] In one aspect of the present invention, mature skeletal muscle cells included in the complex are adhered to a hydrogel described later. The complex may also contain other cells besides mature skeletal muscle cells. Examples of other cells include myoblasts, skeletal muscle cells in the process of maturing, fibroblasts, nerve cells, tendon cells, and the like.

[0024] The proportion of mature skeletal muscle cells among the total number of cells in the complex according to one aspect of the present invention may be, for example, 20% or more, 30% or more, 40% or more, or 50% or more. Alternatively, the proportion of mature skeletal muscle cells among the total number of cells in the complex may be, for example, 100% or less, 90% or less, or 80% or less.

[0025] [Hydrogel] A hydrogel contained in a composite according to one aspect of the present invention has a radiation-crosslinked structure of a hydrophilic polymer.

[0026] In this specification, a hydrogel is a gel that has hardened into a gel-like state by encapsulating water, and is composed of hydrophilic polymer molecules, such as proteins, that are cross-linked to each other.

[0027] Radiation crosslinking, while not specifically defined, is a process in which radiation irradiation generates active sites on polymer chains. These active sites then act as starting points for X-shaped or T-shaped linkages within the polymer chain, forming a three-dimensional network structure. A key characteristic of radiation crosslinking is that it proceeds at room temperature or below without the use of additives such as crosslinking agents. It is applied to material gelation, improved heat resistance, and the imparting of shape memory properties.

[0028] Hydrogels with a radiation-crosslinked structure of hydrophilic polymers constitute a radiation-crosslinked structure that does not contain any crosslinking agent. Therefore, they can maintain their hydrogel state even when left unattended under cell culture conditions.

[0029] (Hydrophilic polymer) The hydrophilic polymer refers to a polymer having hydrophilic groups in its molecule. Examples of hydrophilic groups include hydroxyl groups, amino groups, carboxyl groups, ether groups, acyl groups, and sulfo groups. In other words, the hydrophilic polymer is a polymer having at least one, preferably two or more, such hydrophilic groups in its molecule.

[0030] Furthermore, the molecular weight of the hydrophilic polymer is not particularly limited; for example, a polymer in the range of 150 to 2,000,000 may be appropriately selected and used. Typically, a polymer in the range of 1,000 to 1,000,000 may be appropriately selected and used. For polymers with the same composition, the higher the molecular weight, the greater the compressive modulus of the hydrogel tends to be. In this specification, unless otherwise specified, "molecular weight" refers to the weight-average molecular weight, and such weight-average molecular weight may be measured by a conventionally known size exclusion chromatogram.

[0031] Examples of the hydrophilic polymers include hydrophilic polymers derived from natural products or their derivatives, such as proteins, peptides, polysaccharides, and nucleic acids. "Derived from natural products" means that they 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 are included in the category of hydrophilic polymers derived from natural products (hereinafter also referred to as natural polymers). Synthetic proteins include both proteins synthesized in cell-based protein synthesis systems and proteins synthesized in cell-free protein synthesis systems.

[0032] The term "protein" refers to a macromolecule formed by the linkage of multiple amino acids via 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, "peptide" specifically refers to a macromolecule consisting of two to 2,000 amino acids.

[0033] Examples of hydrophilic polymers derived from natural products include, for example, 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, fibronectin, keratin, ovalbumin, myosin, globulin, and peptides; and nucleic acids such as DNA or RNA.

[0034] The hydrophilic polymer derived from natural products may be used as a raw material for hydrogels using only a single subtype, or a combination of multiple 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 can be used in combination. Type I collagen is preferred because it is the most abundant type in the body and can be obtained relatively inexpensively. Type IV collagen is also preferred because it is the collagen found in the basement membrane of the skin and can be obtained relatively easily.

[0035] Furthermore, the derivatives of the hydrophilic polymer (natural polymer) derived from the natural product are not particularly limited, but examples include derivatives obtained by substituting the natural product-derived polymer with a lower alkyl group, a lower alkoxyalkyl group, or a hydroxy lower alkyl group. Specifically, 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.

[0036] The hydrophilic polymer may be an artificially synthesized synthetic polymer (synthetic resin). Conventionally known hydrophilic polymers can be used without particular limitation, but examples include synthetic polymers selected from the group consisting of polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polymethacrylamide, and polyethylene glycol. Furthermore, polymers having artificially designed sequences are also included in the category of synthetic polymers, and examples include artificial proteins (including peptides), nucleic acids, and polysaccharides having artificially designed sequences.

[0037] The hydrogel may be formed by cross-linking (bonding) only one of the hydrophilic polymers, or by cross-linking (bonding) two or more hydrophilic polymers.

[0038] From the viewpoint of achieving culture in an environment similar to the survival environment of mature skeletal muscle cells, it is preferable to select an animal-derived hydrophilic polymer or a derivative thereof, and more preferably a hydrophilic polymer obtained by extraction or purification from a living organism (animal living organism) (hereinafter also referred to as a bio-derived polymer) or a derivative thereof. Examples of bio-derived polymers include gelatin, collagen, or collagen peptides.

[0039] Furthermore, when a composite according to one aspect of the present invention is used as a transplantable member, the hydrophilic polymer is preferably a hydrophilic polymer having biocompatibility and biodegradability, and more preferably a bio-derived polymer, in that it eliminates the need to remove the hydrogel from the composite. The biodegradability refers to the ability to be degraded by enzymes such as collagenase or protease of the target to be transplanted.

[0040] (Compression modulus of hydrogels) The compressive modulus of the hydrogel is between 10 kPa and 130 kPa. By having the compressive modulus within this range, mature skeletal muscle cells can be obtained from myoblasts with increased expression levels of muscle contraction function-related genes (MYH2 gene, MYH7 gene) and metabolic function-related genes (GLUT4 gene, Myoglobin gene, PGC1α gene).

[0041] In terms of increasing the expression levels of muscle contraction function-related genes and metabolic function-related genes, it is preferable that the compressive modulus of the hydrogel be 10 kPa or more and 100 kPa or less (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kPa).

[0042] Furthermore, the compressive modulus of the hydrogel may be made 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 multiple hydrogel layers with different compressive moduli, or it may be a gradient gel in which the elastic force of the cell culture surface is set to be low and the elastic force is set to be high in the depth direction (thickness direction). In this way, by setting the elastic modulus to be non-uniform in the thickness direction (typically low in the elastic modulus of the cell culture surface), for example, cells can penetrate the hydrogel more easily, and three-dimensional culture can be achieved. Furthermore, the compressive modulus may be set to be non-uniform in the horizontal direction. For example, there may be areas with different compressive moduli from the surroundings, or it may be a gradient gel in which the compressive modulus gradually decreases in a specific direction.

[0043] (Water content of hydrogel) The water content of the hydrogel is, for example, 10% by mass or more, and preferably 30% by mass or more. The upper limit of the water content of the hydrogel is not particularly limited and may be set appropriately, for example, to 99% by mass or less.

[0044] The hydrogel contains, for example, 1% by mass or more of hydrophilic polymer, preferably 3% by mass or more. The upper limit of the hydrophilic polymer content in the hydrogel is not particularly limited, but is, for example, 50% by mass or less, preferably 40% by mass or less, and more preferably 30% by mass or less.

[0045] (Preferred embodiment of hydrogel) The hydrogel may have at least one of the following on its surface: an uneven surface and parallel grooves.

[0046] By having the aforementioned parallel grooves on the surface of the hydrogel, the dysmorphism of mature skeletal muscle cells differentiated from myoblasts can be suppressed. In this specification, dysmorphism of mature skeletal muscle cells refers to the transformation of mature skeletal muscle cells into branched fibrous cells.

[0047] The grooves constituting the parallel grooves may be straight lines or dotted lines. In order to obtain mature skeletal muscle cells oriented in one direction, the spacing between the parallel grooves is more preferably 1 μm or more, even more preferably 2 μm or more, and even more preferably 3 μm or more. Furthermore, in order to further suppress dysmorphism of mature skeletal muscle cells, the spacing between the parallel grooves is preferably 200 μm or less, more preferably 50 μm or less, and even more preferably 30 μm or less.

[0048] In order to obtain mature skeletal muscle cells oriented in one direction, the depth of the groove recess of the parallel groove is preferably 0.5 μm or more, and more preferably 1 μm or more. Furthermore, in order to further suppress dysmorphism of mature skeletal muscle cells, the depth of the groove recess of the parallel groove is preferably 5 μm or less, more preferably 4 μm or less, and even more preferably 3 μm or less.

[0049] By having the aforementioned uneven surfaces on the surface of the hydrogel, cells can be seeded and cultured in the depressions. In this specification, having uneven surfaces on the surface of the hydrogel means having at least one depression and at least one protrusion on the surface of the hydrogel. The shape of the uneven surfaces can be any shape, for example, it may be dot-shaped or substantially circular. Furthermore, a hydrogel having uneven surfaces on its surface can be suitably used, for example, for the purpose of isolating and holding an average of 1 to 10 cells per depression for culture.

[0050] A preferred embodiment of the hydrogel included in the complex according to one aspect of the present invention is a hydrogel having a compressive modulus of 10 kPa or more and 100 kPa or less, and having at least one of a surface with irregularities and parallel grooves. This hydrogel can be used to obtain a complex containing mature skeletal muscle cells in which the expression levels of muscle contraction function-related genes and metabolic function-related genes are further increased, and orientation and morphology are controlled. A compressive modulus of 10 kPa or more and 50 kPa or less is more preferable, and 10 kPa or more and 30 kPa or less, in that it has unidirectional orientation and further suppresses dysmorphism of mature skeletal muscle cells.

[0051] The hydrogel contained in the composite according to one aspect of the present invention can be obtained, for example, by irradiating solutions of each hydrophilic polymer with appropriately adjusted concentrations. For example, the hydrogel contained in the composite according to one aspect of the present invention can be produced in accordance with the hydrogel production method described in Patent Document 1.

[0052] Furthermore, a hydrogel having at least one of the uneven surface and parallel grooves on its surface can also be manufactured in accordance with the hydrogel manufacturing method described in Patent Document 1.

[0053] [Uses of the composite] A composite included in one aspect of the present invention can be used as a medical component such as a transplant component, a component for pharmacological testing (for example, a component for drug screening), an experimental component, and a component for the production of cultured meat, etc.

[0054] The mature skeletal muscle cells contained in the complex according to one aspect of the present invention exhibit high expression levels of fast-twitch muscle-related genes and slow-twitch muscle-related genes. Since a decrease in muscle mass or strength can occur specifically in fast-twitch or slow-twitch muscles depending on the type of cause, the complex according to one aspect of the present invention can be used in the research and development of therapeutic methods that can address a decrease in muscle mass or strength.

[0055] Furthermore, complexes containing mature skeletal muscle cells with controlled orientation and morphology can be used as transplant components where the orientation of muscle tissue needs to be considered.

[0056] [Method for producing the composite] A method for producing a complex according to one aspect of the present invention includes the steps of contacting myoblasts with the hydrogel and differentiating the myoblasts into mature skeletal muscle cells. The hydrogel has already been described and will not be repeated here.

[0057] (The process of bringing myoblasts into contact with the hydrogel) Myoblasts can be brought into contact with the hydrogel by seeding them onto the hydrogel. Any myoblasts known to those skilled in the art can be used. Examples of myoblasts include those derived from skeletal muscle stem cells of humans and non-human animals. Examples of non-human animals include laboratory animals such as mice, rats, rabbits, and guinea pigs; pets such as dogs and cats; and livestock such as birds, pigs, cows, goats, sheep, and horses. Furthermore, the myoblasts may be derived from skeletal muscle stem cells induced from pluripotent stem cells such as C2C12 cells or L6 cells, ES cells (embryonic stem cells), or iPS cells (induced pluripotent stem cells).

[0058] The hydrogel may be coated with culture medium before or after contact between the hydrogel and myoblasts. Any known culture medium used for myoblast culture can be used. Examples of culture media include Dulbecco's modified Eagle medium (DMEM), Eagle's minimal essential medium (MEM), Eagle's minimal essential medium α-modified (α-MEM), Glasgow's minimal essential medium (GMEM), I-Iskoff modified Dulbecco's medium, nutrient mixture F-12 Ham (Ham's F-12), and RPMI-1640.

[0059] To promote the proliferation of myoblasts, the culture medium may contain physiologically active factors (e.g., cell growth factors, differentiation-inducing factors, adhesion factors, chemotactic factors, extracellular matrix, etc.).

[0060] The number of myoblasts to be in contact with the hydrogel can be appropriately selected depending on the intended use of the complex. For example, 5 × 10⁶ cells seeded. 4 pieces / cm 2 The myoblasts may be brought into contact with the hydrogel as described below.

[0061] After contacting the myoblasts with the hydrogel, it is preferable to culture and proliferate the myoblasts at 37°C before the step of differentiating the myoblasts into mature skeletal muscle cells, so that the area percentage (cell occupancy area percentage, confluence) of myoblasts occupying the hydrogel is 70% or more.

[0062] (The process of differentiating myoblasts into mature skeletal muscle cells) By contacting myoblasts with a hydrogel and then replacing the culture medium with a differentiation medium, the myoblasts can be differentiated into mature skeletal muscle cells.

[0063] After replacing the culture medium with a differentiation medium, myoblasts can be differentiated into mature skeletal muscle cells by culturing them for three days or more.

[0064] [Method for producing cultures containing mature skeletal muscle cells] A method for producing a culture containing mature skeletal muscle cells according to one aspect of the present invention includes the steps of contacting myoblasts with the hydrogel, differentiating the myoblasts into mature skeletal muscle cells, and recovering the culture containing mature skeletal muscle cells from the hydrogel. The steps of contacting myoblasts with the hydrogel and differentiating the myoblasts into mature skeletal muscle cells have already been described and will not be repeated here.

[0065] (Steps for recovering cultures containing mature skeletal muscle cells from hydrogels) The culture containing mature skeletal muscle cells can be detached from the hydrogel and recovered from the hydrogel by enzymatic treatment with a proteolytic enzyme (e.g., trypsin) or mechanical treatment such as pipetting.

[0066] The culture containing the mature skeletal muscle cells can be used, for example, in therapeutic applications (e.g., transplantation, regenerative medicine), pharmacological testing methods (e.g., drug screening methods), and as material for cultured meat.

[0067] A kit for producing the complex or a culture containing the mature skeletal muscle cells is also included in one aspect of the present invention. The kit is used in a method for producing the complex or a method for producing a culture containing the mature skeletal muscle cells, and includes the hydrogel.

[0068] The kit may further include at least one of the following: a bioactive factor different from the hydrogel, a culture medium (e.g., a growth medium, a differentiation medium), and myoblasts. The kit may also include instructions describing the manufacturing procedure of the complex. These instructions may be written or printed on paper or other media, or attached to electronic media such as magnetic tape, a computer-readable disk, or a CD-ROM.

[0069] 〔summary〕 The composite according to embodiment 1 of the present invention comprises a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and mature skeletal muscle cells adhering to the hydrogel.

[0070] In the composite according to embodiment 2 of the present invention, the compressive modulus may be 10 kPa or more and 100 kPa or less, as in embodiment 1.

[0071] In the composite according to embodiment 3 of the present invention, the hydrogel may have at least one of the uneven surface and parallel grooves on its surface, as in embodiment 1 or 2.

[0072] In the composite according to embodiment 4 of the present invention, in any one of embodiments 1 to 3, the hydrophilic polymer may be one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

[0073] A method for producing a composite according to aspect 5 of the present invention includes the steps of contacting myoblasts with a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and differentiating the myoblasts into mature skeletal muscle cells.

[0074] In the method for manufacturing the composite according to embodiment 6 of the present invention, the compressive modulus may be 10 kPa or more and 100 kPa or less, as described in embodiment 5.

[0075] In the method for producing the composite according to embodiment 7 of the present invention, in embodiment 5 or 6, the hydrogel may have at least one of the uneven portion and parallel grooves on its surface.

[0076] In the method for producing a composite according to embodiment 8 of the present invention, in any one of embodiments 5 to 7, the hydrophilic polymer may be one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

[0077] A method for producing a culture according to aspect 9 of the present invention is a method for producing a culture containing mature skeletal muscle cells, comprising the steps of: contacting myoblasts with a hydrogel having a compressive modulus of 10 kPa or more and a radiation crosslinked structure of a hydrophilic polymer; differentiating the myoblasts into mature skeletal muscle cells; and recovering a culture containing mature skeletal muscle cells from the hydrogel.

[0078] In the method for producing a culture according to embodiment 10 of the present invention, the compressive modulus may be 10 kPa or more and 100 kPa or less, as described in embodiment 9.

[0079] In the method for producing a culture according to embodiment 11 of the present invention, in embodiment 9 or 10, the hydrogel may have at least one of the uneven surface and parallel grooves on its surface.

[0080] In the method for producing a culture according to embodiment 12 of the present invention, in any one of embodiments 9 to 11, the hydrophilic polymer may be one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

[0081] The embodiments of the present invention will be further described in detail below, with reference to the following examples. Of course, the present invention is not limited to the following embodiments, and it goes without saying that various forms are possible in terms of details. Furthermore, the present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included in the technical scope of the present invention. In addition, all the documents mentioned herein are incorporated by reference. [Examples]

[0082] [Example 1] Preparation of hydrogel culture substrate having radiation crosslinking structure To prepare hydrogels with radiation-crosslinked structures, we selected gelatin, a highly purified form of type I collagen, which is the main component of the extracellular matrix surrounding skeletal muscle cells in vivo. First, Japanese Pharmacopoeia gelatin GLS250 (Nitta Gelatin) was dissolved in ultrapure water in a 50°C water bath to obtain 10 wt% or 15 wt% aqueous solutions. The gelatin aqueous solutions were poured into polystyrene dishes, and PDMS (polydimethylsiloxane) molds were placed on top to impart surface irregularities to the gel culture substrate. The prepared samples were sealed in gas barrier bags with an oxygen absorber and left to stand overnight at 20°C to physically gel. Then, they were irradiated with cobalt-60 gamma rays at a dose rate of 5 kGy / h in a 15°C environment. After irradiation, the PDMS molds were removed, PBS was poured in, and the mixture was incubated overnight at 37°C to obtain radiation-crosslinked gelatin gels with a flat or desired surface shape. The compressive modulus of radiation-crosslinked gelatin gel was adjusted by controlling the concentration of the gelatin aqueous solution and the amount of gamma rays irradiated.

[0083] [Example 2] Culture of myoblasts using a flat hydrogel culture substrate having a radiation crosslinking structure Mouse skeletal muscle cell line C2C12 cells were seeded on radiation-crosslinked gelatin gels with compressive moduli of 10, 30, 50, 100, 160, and 230 kPa, and incubated in growth medium at 37°C and 5% CO2. For comparison with radiation-crosslinked gelatin gels, polystyrene dishes, known to have a hardness of approximately 1 GPa and commonly used in cell culture, were coated with a cell matrix containing collagen, air-dried at room temperature for 30 minutes, and washed with PBS.

[0084] At 24 or 48 hours after the start of incubation, a Hoechst 33342 solution capable of staining cell nuclei was added to the growth medium, and the number of cell nuclei was analyzed. The analysis results are shown in Figure 1. The growth ratio on the vertical axis of Figure 1 represents the number of cell nuclei at 48 hours after the start of incubation / the number of cell nuclei at 24 hours after the start of incubation. As shown in Figure 1, there was no significant difference in the myoblast growth ratio per 24 hours between polystyrene dishes and radiation-crosslinked gelatin gels of various elastic moduli.

[0085] Furthermore, at 24 or 48 hours, calcein-AM solution, which stains only living cells, was added to the growth medium to a final concentration of 1 μM, and propidium iodide, which stains only dead cells, was added to a final concentration of 1 μg / mL. The ratio of living cells to (living cells + dead cells) was then analyzed as the survival rate. The analysis results are shown in Figure 2. No significant difference was observed in the survival rate of myoblasts that engrafted after 24 and 48 hours of culture between polystyrene dishes and radiation-crosslinked gelatin gels of various elastic moduli, confirming that over 95% of cells could survive in both cases.

[0086] The results of Example 1 confirmed that the radiation-crosslinked gelatin gel has the function of maintaining the proliferative capacity of undifferentiated myoblasts. Furthermore, the results of Example 2 confirmed that it can be used in the same way as conventional polystyrene dishes used for cell culture while minimizing the death of engrafted cells.

[0087] [Example 3] Differentiation of skeletal muscle using a flat hydrogel culture substrate having a radiation crosslinking structure C2C12 cells were seeded to 80-90% confluence (cell area ratio) on radiation-crosslinked gelatin gels with compressive moduli of 10, 30, 50, 100, 160, and 230 kPa, and incubated in growth medium at 37°C and 5% CO2. For comparison with the radiation-crosslinked gelatin gels, polystyrene dishes as shown in Example 2 were used. 24 hours after the start of incubation, the medium was replaced with a differentiation medium consisting of 95% DMEM high glucose, 2% calf serum, 1% MEM non-essential amino acid solution, 1% L-glutamine solution, and 1% penicillin-streptomycin solution, and the cells were cultured for differentiation for 5 days. Figure 3 shows the observation results of the polystyrene dishes and each gel culture substrate after 5 days of differentiation culture.

[0088] Differentiated skeletal muscle cells undergo morphological changes through the fusion of myoblasts, each containing a single nucleus, into a long, slender state called myotubes, which have multiple nuclei. As shown in Figure 3, myoblasts cultured on polystyrene dishes and on radiation-crosslinked gelatin gels with a compressive modulus of 10-100 kPa differentiated into elongated myotubes. On the other hand, on radiation-crosslinked gelatin gels with compressive moduli of 160 and 230 kPa, the myotubes detached from the gel and formed aggregates.

[0089] Next, on day 6 of differentiation, immunostaining was performed using anti-myosin heavy chain antibody, an indicator of skeletal muscle cell differentiation, as the primary antibody. The results of the immunostaining are shown in Figure 4. As shown in Figure 4, differentiated skeletal muscle cells were observed on a polystyrene dish and on a radiation-crosslinked gelatin gel with a compressive modulus of 10-100 kPa.

[0090] The results from Example 3 showed that radiation-crosslinked gelatin gel with a compressive modulus of 10-100 kPa allows skeletal muscle cells to differentiate and culture without detachment.

[0091] [Example 4] Maturation of skeletal muscle cells using a flat hydrogel culture substrate having a radiation crosslinking structure C2C12 cells were seeded in the same manner as in Example 3 on a general polystyrene dish that allows differentiation culture without detachment of skeletal muscle cells, and on radiation-crosslinked gelatin gels with compressive moduli of 10 kPa, 30 kPa, 50 kPa, 100 kPa, 150 kPa, or 230 kPa, and differentiated for 6 days. Mature skeletal muscle cells in vivo are broadly classified into fast-twitch and slow-twitch fibers, each expressing specific myosin heavy chain (MYH) genes related to muscle contraction. In fast-twitch types, MYH2 gene expression increases with maturation, while in slow-twitch types, MYH7 gene expression increases. To evaluate the maturation of skeletal muscle cells differentiated and cultured on polystyrene dishes and radiation-crosslinked gelatin gels with compressive moduli of 10-230 kPa, gene expression was analyzed. The analysis results are shown in Figure 5. In Figure 5, ">1 GPa" indicates the results for polystyrene dishes.

[0092] As shown in Figure 5, myotubes on radiation-crosslinked gelatin gels with compressive moduli of 10 kPa, 30 kPa, 50 kPa, or 100 kPa showed increased expression of both fast-twitch type MYH2 and slow-twitch type MYH7 genes compared to those on polystyrene dishes. In particular, higher expression of these genes was observed on radiation-crosslinked gelatin gels with a compressive modulus of 10 kPa.

[0093] As skeletal muscle cells mature, the gene expression of proteins related to muscle cell metabolism also increases. Gene expression of GLUT4, Myoglobin, and PGC1α, which are related to metabolism, was also analyzed. The analysis results are shown in Figure 6. In Figure 6, ">1GPa" indicates the results for polystyrene dishes.

[0094] As shown in Figure 6, the expression levels of the GLUT4, Myoglobin, and PGC1α genes increased in myotubes cultured on radiation-crosslinked gelatin gel compared to those cultured on polystyrene dishes. In particular, higher expression of these genes was confirmed on radiation-crosslinked gelatin gel at 10 kPa.

[0095] The results from Example 4 showed that radiation-crosslinked gelatin gel with a compressive modulus of 10-100 kPa increased the expression of both fast-twitch and slow-twitch muscle type genes in differentiated myotube cells. Furthermore, radiation-crosslinked gelatin gel with a compressive modulus of 10-100 kPa also increased the expression of metabolism-related genes, and it was found that differentiated skeletal muscle cells could mature more effectively compared to those cultured in conventional polystyrene dishes.

[0096] In conventional polystyrene dishes, the expression levels of muscle contraction function-related genes and metabolic function-related genes, which are markers for mature skeletal muscle cells, were low, indicating that differentiation into highly mature skeletal muscle cells did not occur.

[0097] Furthermore, when using radiation-crosslinked gelatin gels with a compressive modulus of 150 kPa or 230 kPa, the expression levels of genes related to myotube cell metabolism increased, while the expression level of the MYH7 gene, a slow-twitch muscle type gene, did not increase compared to conventional polystyrene dishes. This indicates that cells do not differentiate into highly mature skeletal muscle cells when using radiation-crosslinked gelatin gels with a compressive modulus of 150 kPa or 230 kPa.

[0098] Therefore, it was found that myoblasts can be differentiated into highly mature skeletal muscle cells by culturing them using radiation-crosslinked gelatin gel with a compressive modulus of 10-100 kPa.

[0099] [Example 5] Preparation of skeletal muscle cells using a hydrogel culture substrate having a surface-treated radiation-crosslinked structure A mold having linear grooves (parallel grooves) with widths of 3, 5, 10, or 50 μm was pressed into a 10 wt% or 15 wt% gelatin aqueous solution prepared in Example 1. By irradiating with radiation as in Example 1, linear groove (parallel groove) structures with widths of 3, 5, 10, or 50 μm and depths of 2 μm were fabricated on the surface of radiation-crosslinked gelatin gels with compressive moduli of 10 and 30 kPa. A flat radiation-crosslinked gelatin gel was used for comparison with the radiation-crosslinked gelatin gels on which the linear grooves were formed. C2C12 cells were seeded on each radiation-crosslinked gelatin gel with linear grooves formed, as in Example 3, and differentiated and cultured for 6 days. Immunostaining was performed using an anti-myosin heavy chain antibody as the primary antibody to visualize the differentiated skeletal muscle cells. The observation results are shown in Figure 7. As shown in Figure 7, myotubes differentiated on the flat radiation-crosslinked gelatin gel (Flat) were not uniformly oriented, and branched atypical forms were formed. On the other hand, myotubes on the radiation-crosslinked gelatin gel in which line grooves were formed showed uniform orientation and lacked branching, exhibiting the fibrous morphology seen in mature skeletal muscle cells in vivo.

[0100] Figure 8 shows the cell differentiation rates for each radiation-crosslinked gelatin gel culture substrate. As shown in Figure 8, culturing on radiation-crosslinked gelatin gels with line grooves (3, 5, 10, 50 μm) increased the differentiation efficiency of skeletal muscle cells compared to culturing on flat radiation-crosslinked gels (Flat).

[0101] The results from Example 5 showed that radiation-crosslinked gelatin gel can efficiently differentiate and culture skeletal muscle cells while preventing dysmorphism by forming line grooves on its surface, thus enabling aligned cells. [Industrial applicability]

[0102] A composite according to one aspect of the present invention can be used, for example, as a medical component such as a transplant component, a pharmacological testing component such as a drug screening component, an experimental component, and a component for the production of cultured meat.

Claims

1. A hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, A complex comprising mature skeletal muscle cells adhering to the hydrogel.

2. The composite according to claim 1, wherein the compressive modulus is 10 kPa or more and 100 kPa or less.

3. The composite according to claim 1 or 2, wherein the hydrogel has at least one of a surface with an uneven surface and parallel grooves.

4. The composite according to claim 1 or 2, wherein the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

5. A step of contacting myoblasts with a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation-crosslinked structure of a hydrophilic polymer, and A method for producing a complex, comprising the step of differentiating the myoblasts into mature skeletal muscle cells.

6. The method for producing the composite according to claim 5, wherein the compressive modulus is 10 kPa or more and 100 kPa or less.

7. The method for producing the composite according to claim 5 or 6, wherein the hydrogel has at least one of the uneven portion and the parallel groove on its surface.

8. The method for producing a composite according to claim 5 or 6, wherein the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

9. A step of contacting myoblasts with a hydrogel having a compressive modulus of 10 kPa or more and 130 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer. The process of differentiating the myoblasts into mature skeletal muscle cells, and A method for producing a culture containing mature skeletal muscle cells, comprising the step of recovering the culture containing mature skeletal muscle cells from the hydrogel.

10. The method for producing a culture according to claim 9, wherein the compressive modulus is 10 kPa or more and 100 kPa or less.

11. The method for producing a culture according to claim 9 or 10, wherein the hydrogel has at least one of an uneven surface and parallel grooves on its surface.

12. The method for producing a culture according to claim 9 or 10, wherein the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

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