Hydrogel, cell culture production method, and hydrogel production method

Abstract

To provide hydrogels crosslinked by irradiation that have both gel hardness and fibrous structure more similar to those of in vivo tissues and contains collagen.SOLUTION: In one aspect of the present invention, a hydrogel has a compressive modulus of elasticity of 3 kPa to 500 kPa, contains fibrous collagen, and has a radiation crosslinking structure that crosslinks the collagen. In another aspect of the present invention, a method for producing a hydrogel includes: a neutralization pH adjustment step of adjusting the pH of a collagen solution containing 0.1 to 70 mass% collagen molecules to a pH greater than 5 and less than 8 to obtain a neutralized collagen solution; an irradiation step of irradiating the neutralized collagen solution with radiation at a dose of 1 to 1000 kGy; and a heating step of heating the irradiated neutralized collagen solution to 30°C to 45°C to allow the collagen molecules in the neutralized collagen solution to self-assemble into fibrils.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] Collagen is abundantly present in animal skin, bones, tendons, and muscles, etc., and forms a gel with a fibrous structure to construct the framework of the cell surrounding environment (extracellular matrix). Therefore, collagen extracted from cows, pigs, etc. has been used as a material for cell culture substrates for a long time. When the extracted and purified collagen is adjusted from a state dispersed in an acidic solution to neutral conditions and physiological salt concentration, which are physiological conditions, and heated to near body temperature, the molecules associate with each other to form a fibrous structure and form a physical gel. Although this fibrous physical gel is similar to the structure of collagen in vivo, both its concentration and hardness are much lower than those in vivo.

[0003] Under such circumstances, techniques for obtaining a collagen gel having a concentration and hardness comparable to those in vivo, particularly a production technique without using a cross-linking agent, have been studied. For example, as in Patent Document 1 and Non-Patent Document 1, a technique using a radiation cross-linking reaction by electron beam or gamma ray is known as a method for obtaining a protein gel with a high concentration comparable to that in vivo without using a cross-linking agent. Also, for example, as in Non-Patent Document 2 and Non-Patent Document 3, examples of irradiating a physical gel of collagen with gamma rays or electron beams to attempt cross-linking of collagen molecules have also been reported.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] However, in the prior arts described in Non-Patent Documents 2 and 3, the collagen concentration and gel hardness could not be increased to the same level as those of tissues in vivo. The prior arts described in Patent Document 1 and Non-Patent Document 1 still left room for further study from the perspective of reproducing the fibrous structure of hydrophilic molecules in vivo.

[0007] One aspect of the present invention aims to provide a collagen-containing hydrogel in which both the gel hardness and the fibrous structure are more similar to those of tissues in vivo, in a hydrogel cross-linked by radiation irradiation. [Means for Solving the Problems]

[0008] In order to solve the above problems, a hydrogel according to one aspect of the present invention has a compressive elastic modulus of 3 kPa or more and 500 kPa or less, contains fibrillated collagen, and has a radiation cross-linked structure that cross-links the collagens with each other.

[0009] Also, a method for producing a cell culture according to one aspect of the present invention cultures cells by bringing the cells into contact with the above-described hydrogel.

[0010] Also, a method for producing a hydrogel according to one aspect of the present invention includes a neutralization pH adjustment step of adjusting a collagen solution containing 0.1 to 70% by mass of collagen molecules to a pH greater than 5 and less than 8 to obtain a neutralized collagen solution, an irradiation step of irradiating the neutralized collagen solution with radiation having a dose of 1 to 1000 kGy, and a heating step of heating the irradiated neutralized collagen solution to 30°C or higher and 45°C or lower to fibrillate the collagen molecules in the neutralized collagen solution.

Advantages of the Invention

[0011] According to one aspect of the present invention, a physical gel of collagen having suitable physical properties can be realized as a biocompatible material.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] 〔Hydrogel〕 Hereinafter, the hydrogel according to an embodiment of the present invention will be described in detail. The hydrogel according to one aspect of the present invention has a compressive elastic modulus of 3 kPa or more and 500 kPa or less. Controlling the hydrogel according to this embodiment to be in the range of the same compressive elastic modulus as the cell surrounding environment in the living body is preferable from the viewpoint of mimicking the in vivo environment.

[0014] In this specification, the "compressive elastic modulus" is defined by a known stress-strain curve measurement method according to JIS K 6272. For example, the compressive elastic modulus may be calculated based on the stress-strain curve from the surface of the hydrogel to 10% of the thickness.

[0015] The measurement conditions of the compressive elastic modulus may be, for example, a compression speed of 50 μm / s and a maximum test load of 2 N. The compressive elastic modulus may be measured promptly after taking the hydrogel out of a 37°C incubator into a room temperature environment (25 degrees).

[0016] Unless otherwise specified in this specification, "A to B" representing a numerical range is intended to be a range including A and B themselves, that is, "A or more and B or less".

[0017] If the compressive elastic modulus is within the above range, it can be appropriately changed according to the use of the hydrogel and the like. For example, the compressive elastic modulus of the hydrogel may be changed with reference to the compressive elastic modulus of the tissue to be mimicked. From the viewpoint of realizing a hydrogel closer to the in-vivo cell surrounding environment, the compressive elastic modulus is preferably 5 kPa or more, and preferably 8 kPa or more. Also, from the same viewpoint, it is preferably 400 kPa or less, and preferably 300 kPa or less.

[0018] The hydrogel according to this embodiment contains fibrotic collagen. This configuration is preferable from the viewpoint of reproducing the structure of collagen present in the living body.

[0019] The collagen contained in the hydrogel of this embodiment preferably contains at least one of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. In particular, the collagen preferably contains type I collagen. This configuration is preferable from the viewpoints of reproducing the in-vivo state and being available at a relatively low cost. The collagen contained in the hydrogel preferably contains type I collagen as the main component, and particularly preferably contains only type I collagen as the collagen. In this specification, when simply expressed as "collagen", it is intended to be collagen having a triple helix structure and does not include denatured collagen with disorder in the triple helix structure.

[0020] The hydrogel according to this embodiment has an optical density at a wavelength of 405 nm of 0.3 or more. The hydrogel containing fibrillar collagen is turbid, and if the optical density of the hydrogel at a wavelength of 405 nm is 0.3 or more, it can be distinguished from a hydrogel substantially free of fibrillar collagen. The optical density at a wavelength of 405 nm can be measured by a conventionally known method using an arbitrary densitometer. For example, the optical density at a wavelength of 405 nm can be measured with a microplate reader that has prepared a 125 μL sample in a 96-well microplate and kept it warm at 37°C.

[0021] The optical density of the hydrogel at a wavelength of 405 nm increases as the amount of fibrillar collagen contained therein increases. The optical density can be appropriately changed according to the use of the hydrogel or the like. From the viewpoint of realizing a fibrillar hydrogel closer to the in-vivo cell surrounding environment, the optical density at a wavelength of 405 is preferably 0.5 or more, and more preferably 0.7 or more. On the other hand, there is no particular limitation on the upper limit of the optical density of the hydrogel at a wavelength of 405 nm. For example, the optical density at a wavelength of 405 nm may be 2.0 or less.

[0022] The hydrogel according to this embodiment has a radiation crosslinked structure that crosslinks collagens. The hydrogel having such a configuration is preferable because it can increase the compression elastic modulus regardless of the effect of the crosslinking agent. As the crosslinked structure of the hydrogel according to this embodiment, it is preferable that other crosslinked structures have substantially no function. Further, in the hydrogel according to this embodiment, it is preferable that the number of other crosslinked structures with respect to the number of radiation crosslinked structures is small. For example, the number of other crosslinked structures with respect to the number of radiation crosslinked structures in the hydrogel is preferably 10% or less, more preferably 5% or less, and the smaller the number, the more preferable. For example, the hydrogel preferably contains only a radiation crosslinked structure as the crosslinked structure. Such a configuration can be realized by producing without using a crosslinking agent. The hydrogel containing only a radiation crosslinked structure as the crosslinked structure is preferable because it does not contain a crosslinking agent component.

[0023] In this specification, the "radiation crosslinked structure" refers to a crosslinked structure that specifically occurs when irradiated with radiation. Examples of the radiation crosslinked structure that crosslinks collagens include, for example, the crosslinked structure between tyrosines and the crosslinked structure between phenylalanines between collagen molecules. On the other hand, the "other crosslinked structure" refers to a crosslinked structure other than the radiation crosslinked structure, and examples thereof include a crosslinked structure by a crosslinking agent. The "crosslinked structure by a crosslinking agent" refers to a crosslinked structure in which amino acids in collagen molecules are crosslinked via a crosslinking agent component.

[0024] The hydrogel according to this embodiment preferably does not contain a crosslinking agent and components derived from the crosslinking agent. Here, "not containing a crosslinking agent" means that a crosslinking agent is not used in the production of the hydrogel. For example, it means that the hydrogel does not contain any crosslinking agent or components derived from the crosslinking agent at all. The crosslinking agent that crosslinks collagens is cytotoxic, and when collagen is used as a biocompatible material, it is particularly required to be reduced. Therefore, a hydrogel that does not contain a crosslinking agent and components derived from the crosslinking agent is preferable because it can be suitably used as a material during cell culture.

[0025] The hydrogel according to this embodiment preferably contains collagen in an amount of 1.5% by mass or more and 30% by mass or less with respect to the hydrogel. The content of collagen in the hydrogel affects the physical properties of the hydrogel such as the compressive elastic modulus and the amount of fibrotic collagen. Setting the content of collagen in the hydrogel within the above range is preferable from the viewpoint of obtaining a hydrogel having desired physical properties.

[0026] From the perspective of realizing a hydrogel having a collagen concentration closer to the in-vivo cell surrounding environment, the collagen content in the hydrogel is preferably 1.5% by mass or more, preferably 2% by mass or more, and more preferably 3% by mass or more with respect to the hydrogel. Also, from the perspective of realizing the state of a hydrogel encapsulating moisture, the collagen content in the hydrogel is preferably 30% by mass or less, preferably 25% by mass or less, and more preferably 20% by mass or less with respect to the hydrogel.

[0027] The water content rate of the hydrogel according to this embodiment may be 10% by mass or more, and is preferably 30% by mass or more. By the water content rate of the hydrogel not falling below the lower limit, the physical properties within the hydrogel can be suitably used when the hydrogel is used as a cell culture material. The upper limit of the water content rate is not particularly limited, and can be appropriately set to, for example, 99% by mass or less.

[0028] The hydrogel according to this embodiment is preferably a cell culture material. As described above, the hydrogel according to this embodiment has a fibrous structure, does not contain a crosslinking agent component having cytotoxicity, and has a high compression elastic modulus. Therefore, since cell culture can be suitably performed, it is preferable as a cell culture material.

[0029] Also, since the hydrogel according to this embodiment is excellent in biocompatibility, it may be used as a biocompatible material. For example, it may be transplanted into the body of an arbitrary subject in a state where a cell culture product produced using the hydrogel is attached. The hydrogel according to this embodiment not only does not contain a crosslinking agent component, but can also be decomposed by enzymes such as collagenase and protease of the subject to be transplanted, and thus can be suitably used for transplantation.

[0030] The hydrogel according to this embodiment can be used in a kit for cell culture. For example, it can be used in a kit containing the hydrogel and at least one of a bioactive factor different from the hydrogel, a culture medium, and cells.

[0031] Examples of the bioactive factors include, for example, cell growth factors, differentiation-inducing factors, adhesion factors, chemotactic factors, extracellular matrices, etc., and one or more selected from these can be added.

[0032] Examples of the cell growth factors include, for example, 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.

[0033] Examples of the differentiation-inducing factors include, for example, Activin, TGF β2, HGF (Hepatocyte growth factor), Dex (Dexamethasone), Rac1 (RAS-related C3 botulinus toxin substrate 1), Zfp521 (Zinc finger protein 521), etc.

[0034] Examples of the adhesion factors include, for example, integrin, nephronectin, laminin, fibronectin, tenascin, fibulin, EMILIN, QBRICK, osteopontin, polydom, MAEG, fibronogen, etc.

[0035] Examples of the chemotactic factors include, for example, CCL21, fMLP, leukotriene B4, IL-8, C5a, leukotriene B4 (LTB4), platelet-activating factor (PAF), etc.

[0036] Examples of the extracellular matrices include, for example, collagen, laminin, fibronectin, elastin, polysaccharides, etc.

[0037] Examples of the culture medium include Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (MEM), α-Modified Eagle's Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium, Nutrient Mixture F-12 Ham (Ham’s F-12), and RPMI-1640.

[0038] The type of cells contained in the above kit is not particularly limited. For example, the cells contained in the kit may be the cells described later in (Method for producing cell culture).

[0039] The hydrogel according to the present embodiment is a collagen gel having a fibrous structure with a collagen concentration and hardness similar to those in vivo. Collagen in vivo is known to abnormally increase in concentration due to aging and various diseases such as cancer, fibrosis, and vascular diseases, thereby stiffening the cellular microenvironment. By using the hydrogel according to the present embodiment as a cell culture substrate, it becomes possible to investigate how the fibrous structure, concentration, and hardness of collagen affect the onset and malignancy of diseases. For example, the hydrogel according to the present embodiment can be used as a research material (e.g., a substrate for cell culture) for elucidating the pathological conditions of diseases affected by collagen abnormalities and denaturation, or for developing drugs for treating these diseases. Therefore, the hydrogel can be applied to research and development in the medical field. Thus, for example, it also contributes to the achievement of Goal 3, "Ensure healthy lives and promote well-being for all," of the Sustainable Development Goals (SDGs) proposed by the United Nations.

[0040] (Method for producing cell culture) The method for producing a cell culture according to an embodiment of the present invention is a method for culturing cells by bringing the cells into contact with the above-described hydrogel. In the present specification, the "cells" are not limited to a state in which single cells exist independently of each other, but include a cell population (e.g., cell cluster, tissue) in which a plurality of cells are aggregated. That is, the hydrogel of the present embodiment can be used as a culture scaffold for various cultures such as colony culture, cell mass (spheroid, sphere) culture, and tissue culture.

[0041] The cells cultured on the hydrogel are not particularly limited, and desired cells can be selected. Examples of the cells cultured on the hydrogel include cells derived from humans, cells derived from mammals other than humans, cells derived from animals other than mammals, cells derived from plants, and the like. Such cells may be cell lines or primary cultured cells, or genetically engineered cells into which a predetermined gene has been introduced.

[0042] More specifically, the cells cultured on the hydrogel include, for example, melanin-producing cells, peritoneal-derived phagocytes, cardiomyocytes, neurons, hepatocytes, renal cells, pancreatic cells, stem cells, antibody-producing cells, osteoclasts, epithelial cells, fibroblasts, breast cancer cells, mammary gland cancer cells, brain tumor cells, uterine cancer cells, cervical cancer cells, pancreatic cancer cells, myoblasts, and various stem cells (such as ES cells). The cells to be cultured may include tissue pieces and the like.

[0043] Cell culture may be performed under known conditions. For example, it may be culture in an atmosphere of 5% CO2 and 37°C. Also, as the culture apparatus (environmental apparatus), a known CO2 incubator can be used.

[0044] The cultured cell culture can be separated from the above hydrogel by a known method.

[0045] 〔Method for producing hydrogel〕 Hereinafter, the method for producing a hydrogel according to an embodiment of the present invention will be described in detail. The method for producing a hydrogel according to this embodiment includes a neutralization pH adjustment step, an irradiation step, and a heating step.

[0046] (Neutralization pH adjustment step) The neutralization pH adjustment step is a step of adjusting the pH of a collagen solution containing collagen molecules to be greater than 5 and less than 8 to obtain a neutralized collagen solution. The collagen solution contains 0.1 to 70% by mass of collagen molecules. Performing the neutralization pH adjustment step before the irradiation step is preferable from the viewpoint of obtaining a hydrogel having a fibrous structure and a suitable compression elastic modulus.

[0047] In the neutralization pH adjustment step, the pH of the collagen solution to be adjusted may be 5.3 or more, preferably 5.5 or more, and more preferably 6 or more. Also, in the neutralization pH adjustment step, the pH of the collagen solution to be adjusted may be 7.8 or less, preferably 7.7 or less, and more preferably 7.5 or less.

[0048] The concentration of collagen molecules in the collagen solution is 0.1% by mass or more, preferably 0.2% by mass or more, and more preferably 0.3% by mass or more. On the other hand, the concentration of collagen molecules in the collagen solution is 20% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less. Setting the mass of collagen molecules in the collagen solution within this range is preferable from the viewpoint of optimizing the concentration of collagen in the hydrogel and realizing a hydrogel having desired shape and physical properties.

[0049] The collagen solution is not particularly limited, and may be an acidic solution containing type I collagen. The type I collagen may be obtained by any method, and may be, for example, type I collagen derived from a mammalian animal.

[0050] From the viewpoint of obtaining a hydrogel having suitable physical properties, the concentration of collagen molecules in the neutralized collagen solution is preferably 0.05% by mass or more, and more preferably 0.1% by mass or more.

[0051] In the neutralization pH adjustment step, the pH may be adjusted using at least one of a basic solution, a buffer solution, a medium, and water. With this configuration, the pH of an acidic solution can be suitably adjusted. In particular, in the neutralization pH adjustment step, from the viewpoint of achieving a pH and physiological salt concentration suitable for fiber formation, it is preferable to adjust the pH of the collagen solution using a basic solution and a buffered saline or a medium.

[0052] Examples of the basic solution include aqueous solutions of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydrogen carbonate, sodium carbonate, potassium carbonate, calcium carbonate, and the like.

[0053] Examples of the buffered saline include phosphate buffered saline and Tris buffered saline.

[0054] The medium may be appropriately changed according to the type of cells to be cultured. For example, Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium α-modified type (α-MEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco Medium, Nutrient Mixture F-12 Ham (Ham’s F-12), and RPMI-1640 may be used.

[0055] With respect to 1 part by mass of the collagen acidic solution, at least one of the basic solution, buffer solution, medium, and water used in the neutralization pH adjustment step is preferably 0.05 part by mass or more, more preferably 0.1 part by mass or more, and even more preferably 0.2 part by mass or more. Also, with respect to 1 part by mass of the collagen acidic solution, at least one of the basic solution, buffer solution, medium, and water used in the neutralization pH adjustment step is preferably 1 part by mass or less, more preferably 0.5 part by mass or less, and even more preferably 0.3 part by mass or less. Here, when a plurality of the basic solution, buffer solution, medium, and water are used for neutralization, the total thereof only needs to satisfy the above numerical range.

[0056] When a basic solution and a buffer solution or a medium are used in the neutralization pH adjustment step, the basic solution may be 0.025 to 0.5 parts by mass, 0.05 to 0.25 parts by mass, or 0.1 to 0.15 parts by mass with respect to 1 part by mass of the acidic collagen solution. Also, in this case, the buffer solution or the medium may be 0.025 to 0.5 parts by mass, 0.05 to 0.25 parts by mass, or 0.1 to 0.15 parts by mass with respect to 1 part by mass of the acidic collagen solution.

[0057] The temperature of the neutralization pH adjustment step is preferably 4°C or lower. For example, the neutralization pH adjustment step may be carried out at 1°C or higher and 4°C or lower. The neutralization pH adjustment step may be carried out in an ice-cooled state. Carrying out the neutralization pH adjustment step under such conditions is preferable from the viewpoint of preventing gelation of the collagen solution during the neutralization pH adjustment step.

[0058] (Irradiation step) The irradiation step is a step of irradiating the neutralized collagen solution with radiation having a dose of 1 to 1000 kGy. In this specification, the "dose" is the time-integrated irradiation dose of radiation per unit time, and can be measured by a dosimeter sold for each type of radiation. In a radiation irradiation device, it is desirable to set the irradiation energy to about 100 keV to 10 MeV.

[0059] By the irradiation step, collagen is partially cleaved to generate radicals and a crosslinking reaction occurs. When the irradiation of radiation is completed, the crosslinking reaction immediately stops.

[0060] The dose of radiation irradiated in the irradiation step affects the density of the crosslinked structure of the hydrogel. Here, the density of the crosslinked structure of the hydrogel affects the physical properties of the hydrogel such as the compression elastic modulus, the concentration of collagen, and the water content. For example, when the density of the crosslinked structure increases, the compression elastic modulus of the hydrogel increases and the water content decreases, and when the density of the crosslinked structure decreases, the compression elastic modulus of the hydrogel decreases and the water content increases. Therefore, the physical properties of the hydrogel depend on the dose of radiation in the irradiation step.

[0061] The radiation dose can be appropriately adjusted to obtain a hydrogel with desired physical properties. For example, from the viewpoint of sufficiently changing the physical properties of the hydrogel by radiation irradiation, the dose is 1 kGy or more, and may be 3 kGy or more, or may be 5 kGy or more. Also, for example, from the viewpoint of avoiding decomposition and denaturation of collagen molecules due to irradiation with an excessive amount of radiation, the dose is 1000 kGy or less, and may be 500 kGy or less, may be 300 kGy or less, may be 200 kGy or less, or may be 100 kGy or less.

[0062] The dose in the irradiation step may be 1 to 200 kGy. This configuration is preferable from the viewpoint of efficiently radiation-crosslinking the neutralized collagen solution.

[0063] The irradiation step is preferably carried out at 10°C or lower from the viewpoint of preventing gelation of the neutralized collagen solution during the irradiation step. Also, for example, the irradiation step is preferably carried out at 1°C or higher from the viewpoint of preventing freezing of the neutralized collagen solution. Such temperature conditions are preferable from the viewpoint of efficiently radiation-crosslinking the neutralized collagen solution.

[0064] Dissolved oxygen in the neutralized collagen solution during the irradiation step captures radicals, thus affecting the crosslinking density of the hydrogel. The dissolved oxygen in the neutralized collagen solution during the irradiation step may be adjusted by a known method to achieve the desired physical properties of the hydrogel, and is not particularly limited. For example, the amount of dissolved oxygen in the neutralized collagen solution in the irradiation step can be 0 to 40 mg / L.

[0065] The radiation irradiation method in the irradiation step is not particularly limited. For example, radiation can be irradiated continuously or intermittently using a radiation irradiation device. For example, in the irradiation step, gamma rays may be irradiated continuously for 1 hour to 30 hours.

[0066] (Heating step) The heating step is a step of heating the neutralized collagen solution after irradiation to 30°C or higher and 45°C or lower to fibrillate the collagen molecules in the neutralized collagen solution after irradiation. By this step, since the formation of collagen fibers in the neutralized collagen solution can be promoted, a hydrogel having a fibrous structure can be obtained from the neutralized collagen solution.

[0067] The temperature of the heating step is 30°C or higher, may be 32°C or higher, or may be 35°C or higher. Also, the temperature of the heating step is 45°C or lower, may be 43°C or lower, or may be 40°C or lower. In particular, the closer to the body temperature of an animal, the easier it is for the formation of collagen fibers in the neutralized collagen solution, which is preferable.

[0068] The heating step is performed by raising the temperature of the neutralized collagen solution to the above temperature and maintaining the neutralized collagen solution at the above temperature for a certain period of time. The time for maintaining the temperature of the neutralized collagen solution for fibrillization after raising the temperature of the neutralized collagen solution (hereinafter, also referred to as "heating time") is not particularly limited. For example, from the viewpoint of fibrillating the neutralized collagen solution, the heating time may be 10 minutes or longer, may be 15 minutes or longer, or may be 20 minutes or longer. Also, from the viewpoint of shortening the process, the heating time may be 120 minutes or shorter, may be 100 minutes or shorter, or may be 90 minutes or shorter.

[0069] <Summary> The hydrogel according to Aspect 1 of the present invention has a compressive elastic modulus of 3 kPa or more and 500 kPa or less, contains fibrillated collagen, and has a radiation cross-linked structure that cross-links the collagens.

[0070] The hydrogel according to Aspect 2 of the present invention, in Aspect 1, has an optical density at a wavelength of 405 nm of 0.3 or more.

[0071] The hydrogel according to Aspect 3 of the present invention, in Aspect 1 or 2, contains the collagen in an amount of 1.5% by mass or more and 30% by mass or less based on the hydrogel.

[0072] The hydrogel according to Aspect 4 of the present invention has a compression elastic modulus of 5 kPa or more in any of Aspects 1 to 3.

[0073] The hydrogel according to Aspect 5 of the present invention contains type I collagen as the collagen in any of Aspects 1 to 4.

[0074] The hydrogel according to Aspect 6 of the present invention is a material for cell culture in any of Aspects 1 to 5.

[0075] The method for producing a cell culture according to Aspect 7 of the present invention is a method of culturing cells by bringing the cells into contact with the hydrogel according to any of Aspects 1 to 6.

[0076] The method for producing a hydrogel according to Aspect 8 of the present invention includes a neutralization pH adjustment step of adjusting a collagen solution containing 0.1 to 70% by mass of collagen molecules to a pH greater than 5 and less than 8 to obtain a neutralized collagen solution, an irradiation step of irradiating the neutralized collagen solution with radiation having a dose of 1 to 1000 kGy, and a heating step of heating the irradiated neutralized collagen solution to 30°C or higher and 45°C or lower to fibrillate the collagen molecules in the neutralized collagen solution.

[0077] The method for producing a hydrogel according to Aspect 9 of the present invention is a method in which, in Aspect 8, in the neutralization pH adjustment step, the pH is adjusted using at least one of a basic solution, buffered physiological saline, a medium, and water.

[0078] The method for producing a hydrogel according to Aspect 10 of the present invention is a method in which, in Aspect 8 or 9, the dose in the irradiation step is 1 to 200 kGy.

[0079] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. <Example> An embodiment of the present invention will be described below.

[0080] [Example 1: Production of Hydrogel] Hydrogels N-γ, G, G-γ, and A-γ were prepared as follows. The outline of the production method of each hydrogel is shown in Fig. 1.

[0081] (Production Method of Hydrogel N-γ) To an acidic solution of type I collagen extracted from porcine tendon (3 mg / mL, Cellmatrix Type I-A: Nitta Gelatin, pH = 3), 10-fold concentrated PBS and sodium hydroxide (0.02 M) were added while cooling on ice for neutralization (neutralization pH adjustment step). At this time, the ratio of the acidic collagen solution, 10-fold concentrated PBS, and sodium hydroxide solution was 8:1:1. The pH of the obtained neutralized collagen solution was 7.4. The neutralized collagen solution was dispensed in 2 mL portions into a 12-well microplate and irradiated with gamma rays in an ice-cooled state. The gamma rays were irradiated at a dose rate of 5 kGy / h to a dose of 5 kGy or 15 kGy (irradiation step). Then, the irradiated neutralized collagen solution was heated to 37 °C and maintained for 1.5 hours to obtain a hydrogel (heating step). Each step was performed in the microplate.

[0082] As described above, the neutralization pH adjustment step, irradiation step, and heating step were performed in this order, and the obtained hydrogel was designated as hydrogel N-γ. The hydrogels obtained by irradiating gamma rays to a dose of 5 kGy or 15 kGy were designated as hydrogel N-γ5 and hydrogel N-γ15, respectively.

[0083] (Production Method of Hydrogel G) A hydrogel G was prepared in the same manner as the production method of hydrogel N-γ, except that the irradiation step was not performed and the unirradiated neutralized collagen solution was subjected to the heating step.

[0084] (Production Method of Hydrogel G-γ) For hydrogel G, it was irradiated with cobalt-60 gamma rays at 5 kGy / h at room temperature (~20 °C), and then heated again to 37 °C (heating step). Gamma rays were irradiated to a dose of 5 kGy or 15 kGy to prepare hydrogel G-γ5 and hydrogel G-γ15.

[0085] (Method for producing hydrogel A-γ) An acid-extracted type I collagen acidic solution derived from porcine tendon (3 mg / mL, Cellmatrix Type I-A: Nitta Gelatin, pH = 3) was dispensed in 1.6 mL portions into a 12-well microplate and irradiated with gamma rays while being ice-cooled. The gamma rays were irradiated at a dose rate of 5 kGy / h to a dose of 5 kGy or 15 kGy. Then, 10-fold concentrated PBS and sodium hydroxide (0.02 M) were added to the collagen acidic solution while ice-cooling to neutralize it, and a neutralized collagen solution was obtained. At this time, the weight ratio of the collagen acidic solution, 10-concentration PBS, and sodium hydroxide solution was 8:1:1. The pH of the obtained neutralized collagen solution was 7.4. Then, the neutralized collagen solution was heated to 37 °C and maintained for 1.5 hours to obtain a hydrogel (heating step). Each step was performed in the microplate.

[0086] (Results) Figure 2 shows the appearance photos of each hydrogel. Hydrogel N-γ5 and hydrogel N-γ15 were turbid and shrunk. Here, the turbid hydrogel contains the fibrous structure of collagen. Also, the shrunk hydrogel indicates an increase in the concentration of collagen. Hydrogel N-γ15 had a higher turbidity and was more shrunk than hydrogel N-γ5.

[0087] Hydrogels G, G-γ5, and G-γ15 were turbid but did not shrink. Hydrogels A-γ5 and A-γ15 shrank but were not turbid and were transparent gels.

[0088] [Example 2: Measurement of turbidity] In the heating step in the production methods of the above hydrogels N-γ, G-γ, and A-γ, the turbidity from immediately after the start of heating to 90 minutes later was measured by the optical density (OD) at a wavelength of 405 nm using a microplate reader. The type I collagen acidic solution used as the raw material was 100 μL, and the measurement was performed in a state where a total of 125 μL entered a 96-well microplate after passing through the neutralization pH adjustment step. The dose of gamma rays irradiated in the irradiation step was 5 kGy or 15 kGy.

[0089] The changes in turbidity during the heating step of hydrogels N-γ5, G-γ5, and A-γ5 are shown in Fig. 3. The turbidity (final turbidity) 90 minutes after the start of the heating step was the highest for hydrogel N-γ5. The optical density of hydrogel N-γ5 at a wavelength of 405 nm was about 0.9, exceeding 0.3. The turbidity of hydrogel G-γ5 changed little throughout the heating step. Hydrogel A-γ5 had the lowest turbidity throughout the heating step and was a transparent gel. The optical density of hydrogel A-γ5 at a wavelength of 405 nm was about 0.1, below 0.3.

[0090] The changes in turbidity during the heating step of hydrogels N-γ15, G-γ15, and A-γ15 are shown in Fig. 4. The turbidity 90 minutes after the start of the heating step was the highest for hydrogel N-γ15. The optical density of hydrogel N-γ15 at a wavelength of 405 nm was about 1.5, exceeding 0.3. The turbidity of hydrogel G-γ15 changed little throughout the heating step. Hydrogel A-γ15 had the lowest turbidity throughout the heating step and was a transparent gel. The optical density of hydrogel A-γ15 at a wavelength of 405 nm was about 0.2, below 0.3.

[0091] Also, the final turbidity of hydrogel N-γ15 was higher than that of hydrogel N-γ5. From this, it was found that the amount of the fibrous structure in the hydrogel prepared by the method of N-γ changed greatly depending on the irradiation dose of gamma rays.

[0092] 〔Example 3: Observation of fibrous structure〕 (Observation by Second Harmonic Generation Light) The results of observing the second harmonic generation light (SHG) when excited at 950 nm using a multiphoton excitation microscope are shown in Fig. 5. Hydrogels G, G-γ15, and N-γ15 had fibrous structures. In particular, the fibrous structure of hydrogel N-γ15 had a high density. On the other hand, no fibrous structure was confirmed in hydrogel A-γ15.

[0093] (Denatured Collagen) To the four types of hydrogels prepared in Example 1, a denatured collagen detection reagent (BindCOL, biotin-conjugated, Funakoshi FDV-0035) dissolved at 1 μg / mL in 1% BSA / PBS was added, and the mixture was allowed to stand at room temperature for 60 minutes. The denatured collagen detection reagent is a reagent that binds to the part where the conventional triple helix of collagen is unraveled. Thereafter, Streptavidin-FITC (Invitrogen SA1001) dissolved at 0.8 μg / mL in 1% BSA / PBS was added, and the mixture was allowed to stand at room temperature for 60 minutes for staining.

[0094] The results of observing the stained hydrogels by a confocal microscope are shown in Fig. 6. Hydrogels G, G-γ15, and N-γ15 had little denatured collagen, but it was found that A-γ15 contained more denatured collagen compared to the other hydrogels.

[0095] [Example 4: Compressive Elastic Modulus of Hydrogel] (Compressive Elastic Modulus of Each Hydrogel) For the hydrogels G, G-γ15, N-γ15, and A-γ15 prepared in Example 1, a compression test was performed using a rheometer to determine the compressive elastic modulus. The compressive elastic modulus was calculated from the stress-strain curve from the surface of the hydrogel to 10% of the thickness. The measurement conditions are as follows.

[0096] Measuring instrument: Creep meter (Yamaden Co., Ltd., RE2-33005C) Compression speed: 50 μm / s Maximum test load: 2 N Temperature: Measured immediately after taking the hydrogel out of a 37 °C incubator into a room temperature environment (25 °C).

[0097] The results are shown in Fig. 7. The compression elastic moduli of hydrogels A-γ and N-γ exceeded 3 kPa. Furthermore, the compression elastic moduli of hydrogels A-γ15 and N-γ15 were higher than those of hydrogels A-γ5 and N-γ5, respectively.

[0098] The compression elastic modulus of hydrogel G was approximately 1.7 kPa, and there was no hydrogel with a compression elastic modulus of 3 kPa or more. The compression elastic modulus of hydrogel G-γ, which was obtained by irradiating hydrogel G with gamma rays, was also at the same level as that of hydrogel G. This indicates that hydrogel G is less affected by gamma ray irradiation in terms of the compression elastic modulus. This may be partly because, in hydrogel G-γ, the fibrous structure of collagen has been formed before gamma ray irradiation, restricting the movement of collagen molecules and making it difficult for the cross-linking effect due to gamma ray irradiation to appear.

[0099] Also, the compression elastic moduli of hydrogels G-γ5 and G-γ15 were at the same level. This indicates that the gamma ray dose has no effect on the compression elastic modulus of hydrogel G-γ.

[0100] (Relationship between the compression elastic modulus of hydrogel N-γ and the gamma ray dose) Regarding hydrogel N-γ, the pressure elastic moduli were measured for a plurality of hydrogels prepared by changing the gamma ray irradiation dose. The maximum gamma ray irradiation dose was 80 kGy.

[0101] The results are shown in Fig. 8. The compression elastic modulus increased according to the gamma ray irradiation dose. When the gamma ray irradiation dose was 80 kGy, the compression elastic modulus was 30 kPa.

[0102] (Effect of the concentration of acidic collagen solution on the compression elastic modulus in hydrogel G-γ) A hydrogel was prepared in the same manner as hydrogel G or hydrogel G-γ15, except that the concentration of the acidic collagen solution was 4.5 mg / ml. The hydrogels were designated as hydrogel G’ and hydrogel G-γ15’, respectively. When the compressive elastic moduli of hydrogel G’ and hydrogel G-γ15’ were measured by the same measurement method as above and the average values were calculated, they were 2.5 kPa and 2.8 kPa, respectively, and no hydrogel having a compressive elastic modulus of 3.0 kPa or more was obtained.

[0103] When the concentration of the acidic collagen solution was made higher than 4.5 mg / ml, the acidic collagen solution increased in viscosity and became honey-like. Therefore, it was impossible to mix with the neutralizing solution and perform pipetting operations, and no hydrogel could be prepared.

[0104] From the above, it was found that it was difficult to prepare a hydrogel having a compressive elastic modulus of 3.0 kPa or more by the production method of hydrogel G-γ.

[0105] 〔Example 5: Collagen Concentration in Hydrogel〕 (Collagen Concentration in Each Hydrogel) The concentration of type I collagen (the ratio of type I collagen constituting the gel) contained in the hydrogel prepared in Example 1 was calculated from the ratio of the dry weight to the swollen weight.

[0106] The results are shown in Fig. 9. The content of type I collagen relative to the total amount of each of hydrogel A-γ and hydrogel N-γ exceeded 1.3% by mass. Furthermore, hydrogel A-γ15 and hydrogel N-γ15 each had a higher content of type I collagen than hydrogel A-γ5 and hydrogel N-γ5, respectively.

[0107] The content of type I collagen in hydrogel G was approximately 1% by mass, and there was no hydrogel with 1.3% by mass or more. The hydrogel G-γ obtained by irradiating hydrogel G with gamma rays also had a type I collagen concentration comparable to that of hydrogel G. This indicates that irradiating hydrogel G with gamma rays has little effect on the concentration of type I collagen in the hydrogel. This may be due to the fact that in hydrogel G-γ, the fibrous structure of collagen has been formed before gamma ray irradiation, so the movement of collagen molecules is restricted and the effect of cross-linking by gamma ray irradiation is less likely to appear.

[0108] Also, the type I collagen concentrations of hydrogels G-γ5 and G-γ15 were comparable. This indicates that the dose of gamma rays has no effect on the type I collagen concentration of hydrogel G-γ.

[0109] (Relationship between the type I collagen concentration of hydrogel N-γ and the gamma ray dose) Regarding hydrogel N-γ, the type I collagen concentrations were calculated for a plurality of hydrogels prepared by changing the irradiation dose of gamma rays. The maximum irradiation dose of gamma rays was 80 kGy.

[0110] The results are shown in Fig. 10. The type I collagen concentration increased according to the irradiation dose of gamma rays. When the irradiation dose of gamma rays was 80 kGy, the type I collagen concentration was approximately 5% by mass.

[0111] [Example 6: Cell culture] Hydrogels G and G-γ15 were prepared by dispensing 1.4 mL of neutralized collagen solution into 35-mm dishes. Hydrogels N-γ15 and A-γ15 were prepared by dispensing 2 mL of neutralized collagen solution into 12-well microplates and then transferred to 35-mm dishes after preparation. 2 mL of medium was injected into each of them, and after standing in a 37 °C incubator for 30 to 60 minutes, they were used for cell culture experiments.

[0112] 3T3 cells, which are fibroblasts derived from mouse fetal skin, at 22 cells / mm 2Seeded on various hydrogels at the concentration of , and cultured for 2 days. Then, it was fixed with 4% paraformaldehyde, and actin filaments were stained with Phalloidin-FITC.

[0113] The results of observing the stained samples with a confocal microscope are shown in Fig. 11. It can be seen that in hydrogel N-γ15, cells adhered and grew well compared to hydrogels A-γ15 and G. In addition, in the case of hydrogel G-γ15, the same results as those of hydrogel G were obtained.

Industrial Applicability

[0114] The present invention can be used for cell culture substrates and the like.

Claims

1. having a compression elastic modulus of 3 kPa or more and 500 kPa or less, containing collagen that has been fibrillated, and having a radiation crosslinked structure that crosslinks the collagens, a hydrogel.

2. The hydrogel according to claim 1, having an optical density at a wavelength of 405 nm of 0.3 or more.

3. The hydrogel according to claim 1, containing 1.5% by mass or more and 30% by mass or less of the collagen with respect to the hydrogel.

4. The hydrogel according to claim 1, having a compression elastic modulus of 5 kPa or more.

5. The hydrogel according to claim 1, wherein the collagen contains type I collagen.

6. The hydrogel according to any one of claims 1, which is a material for cell culture.

7. A method for producing a cell culture, comprising culturing cells by bringing the cells into contact with the hydrogel according to any one of claims 1 to 6.

8. A neutralization pH adjustment step of adjusting a collagen solution containing 0.1 to 70% by mass of collagen molecules to a pH greater than 5 and less than 8 to obtain a neutralized collagen solution; an irradiation step of irradiating the neutralized collagen solution with radiation having a dose of 1 to 1000 kGy; a heating step of heating the irradiated neutralized collagen solution to 30°C or more and 45°C or less to fibrillate the collagen molecules in the neutralized collagen solution; A method for producing a hydrogel, comprising.

9. In the neutralization pH adjustment step, the pH is adjusted using at least one of a basic solution, buffered physiological saline, a medium, and water. The method for producing a hydrogel according to claim 8.

10. The method for producing a hydrogel according to claim 8, wherein the dose in the irradiation step is 1 to 200 kGy.

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