JP2019001774A - Gelatin derivative, crosslinked gelatin hydrogel and porous body thereof, and methods for producing them - Google Patents

Abstract

To provide gelatin derivatives that is suitable for manufacturing a crosslinked gelatin hydrogel having a high crosslinking density and a high mechanical strength and a porous body thereof, to provide crosslinked gelatin hydrogel using the same and a porous body thereof, as well as to provide production methods thereof.SOLUTION: In the gelatin derivative in which the reactive group is introduced into the gelatin of the present invention, the amino group possessed by gelatin is bound to a methacryloyl group, and the hydroxy group and the carboxyl group possessed by gelatin are bound to a methacryloyl glyceryl ester group.SELECTED DRAWING: Figure 1

Description

  The present invention relates to living tissues such as skin and bone, cartilage, ligament, muscle, trachea, esophagus, nerve, blood vessel, bladder, heart, lung, kidney, pancreas, pancreas, liver, etc. damaged or lost due to a disease or accident -Gelatin derivatives, cross-linked gelatin hydrogels and porous bodies thereof that contain cells that differentiate and organize into their biological tissues / organs to repair organs, promote tissue regeneration, and production thereof It is about the method.

  Living bodies such as skin, bone, cartilage, ligament, muscle, trachea, esophagus, nerve, blood vessel, bladder, heart, lung, kidney, pancreas, pancreas, liver, etc. damaged or lost due to accidents or illness Conventionally, artificial organs and organ transplantation have been used to repair and treat tissues and organs. However, in the case of an artificial organ, there are problems such as insufficient function as compared with a natural organ and wear, loosening, or damage caused by an artificial object. In the case of living organ transplantation, in addition to the problem of lack of donors, when the donor is another person, there are many medical risks such as problems of rejection caused by immune responses and complications associated with the use of immunosuppressive agents. Due to the existence of such problems, the method of regenerating and treating living tissues and organs by the technique of living tissue engineering is ideal because it does not require a donor as compared with living organ transplantation. It is believed that.

  In the tissue engineering method, first, living cells are proliferated in vitro and seeded on a hydrogel used as a scaffold for living cells or tissues. This is cultured ex vivo, and after a living tissue is formed, it is transplanted in vivo. Alternatively, the living cells are mixed with a sol that becomes a hydrogel, and then injected into the defect site, gelled to induce regeneration of the living tissue in vivo. Therefore, hydrogels that induce and promote the formation of biological tissues play a very important role. The material forming this hydrogel is required to have a property of gelling after mixing with cells in a sol state when mixing with cells. In addition, biocompatibility as a property that does not affect the living body and bioabsorbability that is decomposed and absorbed while a new living tissue is formed are required.

  As a hydrogel for tissue regeneration, collagen gel, gelatin gel, hyaluronic acid gel, fibrin gel, alginic acid gel and the like are used. Techniques for producing hydrogels of these bioabsorbable polymers include temperature, pH, calcium concentration, antibody / antigen reaction, and photoreaction. For example, an aqueous collagen solution gels and forms a hydrogel when the body temperature reaches 37 ° C. in a sol state at a low temperature (eg, 4 ° C.). Alginic acid is a sol in the absence of calcium ions, and when calcium ions are added, it gels and forms a hydrogel. In the case of gelatin, a methacryloyl group capable of crosslinking polymerization is introduced into the side chain of the gelatin molecule and polymerized by exposure to ultraviolet rays in the presence of an initiator to form a hydrogel. However, the stability of these hydrogels and the mechanical strength of the hydrogel are not sufficient (for example, Non-Patent Document 1). In order to produce a Heidol gel porous body, a hydrogel having high strength is required.

  A pore-forming agent is used to introduce a porous structure into the hydrogel. Alginic acid gel particles and gelatin gel particles are used as a pore-forming agent. However, so far, high concentrations of calcium ions, organic solvents, edible oils, and the like have been used to produce alginate gel particles and gelatin gel particles (for example, Non-Patent Documents 2 and 3). However, since these production conditions are feared to reduce the viability of the encapsulated cells, a method for producing gel particles capable of minimizing the influence on the cells is desired. Moreover, since the hydrogel porous body has lower mechanical strength than the dense hydrogel, it is desirable that the crosslink density is high in order to improve the stability of the hydrogel.

Xiaomeng Li, Shangwu Chen, Jingchao Li, Xinlong Wang, Jing Zhang, Naoki Kawazoe, and Guoping Chen; 3D culture of chondrocytes in gelatin hydrogels with different stiffness.Polymers, 8, 269 (15 pages) (2016). Hongzhi Zhou and Hockin H. K. Xu; The fast release of stem cells from alginate-fibrin microbeads in injectable scaffolds for bone tissue engineering.Biomaterials. 32 (30): 7503-7513 (2011). Lei Zeng, Xiaofeng Chen, Qing Zhang, Feng Yu, Yuli Li, Yongchang Yao; Redifferentiation of dedifferentiated chondrocytes in a novel three-dimensional microcavitary hydrogel.J Biomed Mater Res A. 103 (5): 1693-702 (2015).

  However, in the case of a gelatin hydrogel prepared using gelatin having a methacryloyl group introduced as in Non-Patent Document 1, only the amino group of gelatin is reacted with anhydrous methacrylic acid to introduce a methacryloyl group. The crosslink density by cross-linking polymerization by irradiation is low, and the mechanical strength of the formed hydrogel is still low. In particular, in order to maintain the structure and shape of the formed hydrogel porous body in order to produce the hydrogel porous body, a bulk hydrogel with high mechanical strength is necessary.

  Moreover, in order to produce the unmodified gelatin gel particles used for producing the hydrogel porous body, a solvent or liquid that does not dissolve gelatin such as an organic solvent or oil has been used. In the case of using an organic solvent or oil, there is a concern that the remaining organic solvent or oil may adversely affect the cells. Furthermore, when cells are encapsulated in gelatin gel particles, a suspension of cells and an aqueous gelatin solution is dropped or added to an organic solvent or oil to prepare an emulsion. There are concerns about adverse effects on abilities. A method for producing gelatin gel particles encapsulating cells that does not affect cell activity is desirable.

  The present invention has been made in view of the circumstances as described above. A crosslinked gelatin hydrogel having a high crosslinking density and a high mechanical strength, a gelatin derivative preferable for producing a porous body thereof, and a crosslinked gelatin hydrogel using the same. And a porous body thereof, and a method for producing the same. Another object of the present invention is to provide a crosslinked gelatin hydrogel having a high crosslinking density, a high mechanical strength and encapsulating cells and a porous body thereof under mild and non-cytotoxic conditions. It is another object of the present invention to provide a method for producing a pore-forming agent having a porous structure under mild and non-cytotoxic conditions.

In the gelatin derivative according to the present invention in which a reactive group is introduced, the amino group of the gelatin is bonded to the methacryloyl group, and the hydroxy group and the carboxyl group of the gelatin are bonded to the methacryloyl glyceryl ester group. This solves the above problem.
The content of the methacryloyl group satisfies a range of 0.01 mmol to 10 mmol with respect to 1 g of the gelatin derivative, and the content of the methacryloyl glyceryl ester group is 0.01 mmol to 10 mmol with respect to 1 g of the gelatin derivative. The range may be satisfied.
The content of the methacryloyl group satisfies the range of 0.1 mmol to 1 mmol with respect to 1 g of the gelatin derivative, and the content of the methacryloyl glyceryl ester group is from 0.1 mmol to 1 mmol with respect to 1 g of the gelatin derivative. The range may be satisfied.
The gelatin may be at least one selected from the group of animal bones, animal skins, fish bones, fish skins and fish scales.
The method for producing the aforementioned gelatin derivative according to the present invention comprises a step of reacting gelatin with methacrylic anhydride to synthesize a gelatin primary modified product in which a methacryloyl group is bonded to an amino group of the gelatin, and the gelatin primary modification. Reacting the body with glycidyl methacrylate, and synthesizing the gelatin derivative, which is a gelatin secondary modified product in which a methacryloylglyceryl ester group is bonded to a hydroxy group and a carboxy group of the gelatin primary modified product, This solves the above problem.
In the step of synthesizing the gelatin primary modified product, a volume ratio of the methacrylic anhydride to the gelatin aqueous solution is 10000 to a gelatin aqueous solution having a gelatin concentration of 1 (w / v)% to 50 (w / v)%. : The methacrylic anhydride may be added so as to satisfy 1-1: 1.
The step of synthesizing the gelatin secondary modified product includes the step of synthesizing the gelatin primary modified product in an aqueous gelatin primary modified product solution having a gelatin primary modified product concentration of 0.1 (w / v)% or more and 40 (w / v)% or less. The methacrylic anhydride may be added so that the volume ratio of the glycidyl methacrylate to the aqueous solution satisfies 10,000: 1 to 1: 1.
The gelatin primary modified aqueous solution may have a pH in the range of 2 to 5, or 6.5 to 8.
The cross-linked gelatin hydrogel and porous body thereof according to the present invention contain a cross-linked product in which the above-described gelatin derivative is cross-linked, thereby solving the above-mentioned problems.
Crosslink density of the gelatin derivative may be a 0.02 mol / ^{m 3} or more 10000mol / ^{m 3} or less.
Cells may be encapsulated.
The cells are chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, adipocytes, neurons, vascular endothelial cells, smooth muscle cells, cardiomyocytes, epithelial cells, hepatocytes, pancreatic β cells, kidneys At least one may be selected from the group consisting of cells, bone marrow-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, neural stem cells, embryonic stem cells, and induced pluripotent stem cells.
The method for producing a cross-linked gelatin hydrogel according to the present invention cross-links using the above-mentioned gelatin derivative, thereby solving the above-mentioned problems.
You may include the process of preparing the aqueous solution containing the said gelatin derivative and a photoinitiator, and the process of irradiating the said aqueous solution with an ultraviolet-ray.
The method further includes a step of preparing a cell suspension by suspending cells in the aqueous solution obtained by the step of preparing the aqueous solution, and the step of irradiating the aqueous solution with ultraviolet rays comprises applying ultraviolet rays to the cell suspension. It may be irradiated.
The method for producing a crosslinked gelatin hydrogel porous body according to the present invention crosslinks using the gelatin derivative and gel particles described above, thereby solving the above-mentioned problems.
A step of preparing an aqueous solution containing the gelatin derivative and a photopolymerization initiator, a step of preparing a mixture obtained by adding gel particles made of gelatin hydrogel to the aqueous solution obtained by the step of preparing the aqueous solution, and the mixture. And irradiating with ultraviolet rays.
You may further include the process of suspending a cell in the said aqueous solution obtained by the process of preparing the said aqueous solution, and preparing a cell suspension.
The gel particles may contain cells.
The cell may be an animal cell derived from an animal.

  In the gelatin derivative of the present invention, the amino group is bonded to the methacryloyl group, the hydroxy group and the carboxyl group of the gelatin are bonded to the methacryloyl glyceryl ester group, and a reactive group capable of crosslinking polymerization by ultraviolet irradiation is introduced to the maximum. ing. Since the crosslinked gelatin hydrogel and porous body thereof of the present invention are produced using the gelatin derivative of the present invention, the crosslinking density is high. As a result, the mechanical strength is high and the shape stability is excellent. In addition, since cells can be seeded in the bulk or pores of the hydrogel, more efficient tissue regeneration is possible.

  The method for producing a crosslinked gelatin hydrogel and porous body thereof according to the present invention uses the above-described gelatin derivative, and therefore provides a crosslinked gelatin hydrogel having high crosslinking density, high mechanical strength, and excellent shape stability, and a porous body thereof. it can. In addition, a crosslinked gelatin hydrogel having a high crosslinking density, high mechanical strength and excellent shape stability and a porous body thereof can be easily produced under mild and non-cytotoxic conditions.

The figure which shows typically the gelatin derivative of this invention Flow chart for producing the gelatin derivative of the present invention Flow chart showing the procedure of a crosslinked gelatin hydrogel obtained using the gelatin derivative of the present invention and a porous body thereof The figure which shows the synthetic | combination process (a) of a gelatin primary modified body, and the synthetic | combination process (b) of a gelatin secondary modified body It shows a ¹ H NMR spectrum of the gelatin primary modifications It shows a ¹ H NMR spectrum of the gelatin secondary modifications Relationship between storage elastic modulus (G ′) and loss elastic modulus (G ″) of 10% (w / v%) gelatin secondary modified aqueous solution The figure which shows the fluorescence microscope image of the life-and-death dyeing | staining of the cell in the bridge | crosslinking gelatin hydrogel which included the chondrocyte by Example 3 Optical micrograph showing the appearance of rectangular parallelepiped particles made of gelatin hydrogel Photograph showing the appearance of the crosslinked gelatin hydrogel porous material according to Example 4 The figure which shows the fluorescence microscope image of the life-and-death dyeing | staining of the cell in the bridge | crosslinking gelatin hydrogel porous body which included the chondrocyte by Example 4 Photograph showing the appearance of cartilage tissue regenerated with a crosslinked gelatin hydrogel porous material according to Example 4 Optical micrograph showing the appearance of rectangular parallelepiped particles made of gelatin hydrogel Photograph showing the appearance of the crosslinked gelatin hydrogel porous material according to Example 5 The figure which shows the fluorescence microscope image of the life-and-death dyeing | staining of the cell in the bridge | crosslinking gelatin hydrogel porous body which included the chondrocyte by Example 5 Photograph showing the appearance of cartilage tissue regenerated with a crosslinked gelatin hydrogel porous material according to Example 5 Photograph showing the appearance of the crosslinked gelatin hydrogel porous material according to Example 6 The figure which shows the fluorescence microscope image of the life-and-death dyeing | staining of the cell in the bridge | crosslinking gelatin hydrogel porous body which included the chondrocyte by Example 6 Photograph showing the appearance of cartilage tissue regenerated with a crosslinked gelatin hydrogel porous material according to Example 6

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(Embodiment 1)
In Embodiment 1, a gelatin derivative that is preferable for producing a crosslinked gelatin hydrogel having a high crosslinking density and high mechanical strength or a porous body thereof will be described.

  FIG. 1 is a diagram schematically showing a gelatin derivative of the present invention.

  In the gelatin derivative 100 of the present invention, in the gelatin 110, the amino group of the gelatin is bonded to the methacryloyl group 120, and the hydroxy group and the carboxyl group of the gelatin are bonded to the methacryloyl glyceryl ester group 130. The methacryloyl group 120 and the methacryloyl glyceryl ester group 130 are both reactive groups capable of cross-linking polymerization. If a cross-linked gelatin hydrogel is produced using the gelatin derivative 100 of the present invention, a high cross-linking density and a high mechanical strength are obtained. It is possible to provide a crosslinked gelatin hydrogel that achieves the desired strength and is excellent in shape stability. Further, if a pore-forming agent is used during the production of the crosslinked gelatin hydrogel, a crosslinked gelatin hydrogel porous body that achieves a high crosslinking density and a high mechanical strength and is excellent in shape stability can be provided. Moreover, if cells are encapsulated during the production of the crosslinked gelatin hydrogel and / or a pore-forming agent that encapsulates cells is used, a crosslinked gelatin hydrogel that encapsulates cells and a porous body thereof can be provided.

  In the gelatin derivative 100 of the present invention, the content of the methacryloyl group 120 with respect to 1 g of the gelatin derivative preferably satisfies the range of 0.01 mmol to 10 mmol, and the content of the methacryloyl glyceryl ester group 130 with respect to 1 g of the gelatin derivative is 0.01 mmol. The range of 10 mmol or less is satisfied. Thereby, bridge | crosslinking strength and mechanical strength can be improved.

  In the gelatin derivative 100 of the present invention, more preferably, the content of the methacryloyl group 120 relative to 1 g of the gelatin derivative satisfies the range of 0.1 mmol to 1 mmol, and the content of the methacryloyl glyceryl ester group 130 relative to 1 g of the gelatin derivative is 0.00. The range of 1 mmol or more and 1 mmol or less is satisfied. Thereby, bridge | crosslinking strength and mechanical strength can be improved reliably.

  The gelatin 110 is derived from any one material selected from a group of bones and skins of animals such as pigs, cows, sheep, chickens, fish bones, fish skins, fish scales, or a plurality of materials. Gelatin. The molecular weight of the gelatin molecule is preferably in the range of 100 daltons to 10,000,000 daltons, and in this range, it dissolves in water and forms a hydrogel. The molecular weight of the gelatin molecule is more desirably in the range of 500 daltons to 1,000,000 daltons. This is particularly advantageous for the formation of hydrogels with a stable shape, diffusibility such as nutrients and wastes.

  The presence of the methacryloyl group 120 bonded to the amino group and the methacryloyl glyceryl ester group 130 bonded to the hydroxy group and the carboxyl group can be easily confirmed by nuclear magnetic resonance (NMR) spectroscopy. Moreover, the content rate of a methacryloyl group and a methacryloyl glyceryl ester group is computable with the integrated value of a NMR signal.

  FIG. 2 is a flowchart for producing the gelatin derivative of the present invention.

  The manufacturing method of the gelatin derivative 100 of this invention is demonstrated using FIG. Hereinafter, for ease of understanding, the gelatin derivative is referred to as a gelatin secondary modification. This is because, as will be described later, the gelatin derivative of the present invention can be obtained by modifying (introducing) a reactive group in gelatin in two stages.

  The method for producing a gelatin secondary modified product of the present invention comprises a step of reacting gelatin with methacrylic anhydride to synthesize a gelatin primary modified product in which a methacryloyl group is bonded to an amino group of gelatin (step S210 in FIG. 2). And a step of synthesizing a gelatin secondary modified product in which a gelatin primary modified product is reacted with glycidyl methacrylate to bind a hydroxy group and a carboxyl group of the gelatin primary modified product to a methacryloyl glyceryl ester group (step of FIG. 2). S220) is included.

  The step of synthesizing the primary gelatin modification will be described in detail. First, gelatin is dissolved in a heated aqueous solution to prepare an aqueous gelatin solution. The aqueous solution in which gelatin is dissolved is phosphate buffered saline having a pH of 4 to 11, or HEPES buffer, saline and pure water, but phosphate buffered saline (PBS) having a pH of 6 to 8 is used. desirable. The temperature of the heated aqueous solution is a temperature at which gelatin is dissolved, and is 30 ° C. to 80 ° C., but a desirable temperature is 35 ° C. to 55 ° C. The concentration of the gelatin aqueous solution obtained by dissolving gelatin in a warmed aqueous solution is 1 to 50 (w / v)%, and a desirable concentration is 5 to 20 (w / v)%. Within this range, uniform stirring is possible, which is advantageous for uniform modification reaction.

  Next, while stirring the aqueous gelatin solution, methacrylic anhydride is added, and the reaction is carried out for several hours in the dark while maintaining the temperature at which the reaction product and the product (here, the primary modified product of gelatin) are dissolved. Here, the addition rate of the methacrylic anhydride is preferably 0.01 to 20.0 mL / min. Within this range, it is advantageous for keeping the reaction temperature constant. A more desirable addition rate is 0.1 to 5.0 mL / min. The volume charge ratio of methacrylic anhydride added to the gelatin aqueous solution is preferably 10,000: 1 to 1: 1. Within this range, the amino group of gelatin can be modified. A more desirable charging ratio is 100: 1 to 2: 1. The reaction temperature is from 30 ° C to 80 ° C, but the desired temperature is from 35 ° C to 55 ° C. The reaction time is 10 minutes to 96 hours, but the desired reaction time is 1 hour to 48 hours.

  Next, the reaction product is diluted with pure water at a temperature where the reaction product and the product are dissolved, and dialyzed in pure water using a dialysis membrane while maintaining the temperature at which the reaction product and the product are dissolved. Thereby, salts and unreacted methacrylic anhydride are removed, and the synthesized gelatin primary modified product is purified. Here, the pure water for dilution may be ultrapure water. The temperature of pure water for dilution is maintained at a temperature at which the reactant and product can be maintained as a solution, preferably 30 ° C. to 80 ° C., but a desirable temperature is 35 ° C. to 60 ° C. The fractional molecular weight of the dialysis membrane used for purification of the product may be within a range where the synthesized primary gelatin modification product and the unreacted methacrylic anhydride and low molecular weight salts can be separated.

  Next, the purified gelatin primary modified aqueous solution may be freeze-dried to obtain a gelatin primary modified solid. The lyophilization time is not particularly limited as long as it can remove water from the frozen material, but illustratively it is 1 day to 1 month. The desired lyophilization time is 2 days to 2 weeks. In this way, a gelatin primary modified product in which a methacryloyl group is bonded to an amino group of gelatin is obtained.

  The process of synthesizing the secondary gelatin modification will be described in detail. First, the gelatin primary modification is dissolved in warm water to prepare an aqueous solution, and then the pH of the aqueous solution is adjusted. Any ion-exchanged water, distilled water, or ultrapure water can be used as hot water for dissolving the gelatin primary modified product. The water temperature is 25 ° C. to 80 ° C. at a temperature at which the gelatin primary modified product is dissolved, but a desirable temperature is 35 ° C. to 55 ° C. The concentration of the gelatin primary modified body solution in which the gelatin primary modified body is dissolved in warm water is 0.1 to 40 (w / v)%, and a desirable concentration is 0.2 to 20 (w / v)%. If it is this range, since it is fluid, it is advantageous to uniform modification reaction. Examples of the solution used for adjusting the pH of the aqueous gelatin primary modification solution include hydrochloric acid, nitric acid, and sulfuric acid, and hydrochloric acid is preferable. The concentration of hydrochloric acid, nitric acid and sulfuric acid for adjusting the pH is 0.1 to 5M, and preferably 0.2 to 2M. The pH of the aqueous solution is preferably adjusted to a range of 1.0 to 10. If it is this range, reaction with the glycidyl methacrylate mentioned later will arise. Desirable pH is in the range of 2-5 or 6.5-8.

  Next, glycidyl methacrylate is added to the gelatin primary modified aqueous solution, and the reaction is performed for several hours in the dark while maintaining the temperature at which the reaction product and the product (herein, gelatin secondary modified) are dissolved. The addition rate of the glycidyl methacrylate is preferably 0.01 to 20.0 mL / min. Within this range, it is advantageous to keep the reaction temperature constant. A more desirable addition rate is 0.1 to 5.0 mL / min. The volume charge ratio of glycidyl methacrylate added to the gelatin primary modified aqueous solution is preferably 10,000: 1 to 1: 1. Within this range, modification of the hydroxyl group and carboxyl group of gelatin becomes possible. A more desirable charging ratio is 250: 1 to 3: 1. The reaction temperature is 30 ° C to 80 ° C, but a desirable temperature is 35 ° C to 60 ° C. The reaction time is 1 hour to 96 hours, but the desired reaction time is 2 hours to 48 hours.

  Next, the reaction solution is diluted with water at a temperature at which the reactant and product are dissolved, and dialyzed in pure water while maintaining a temperature at which the reactant and product are dissolved using a dialysis membrane. As a result, salts, unreacted glycidyl methacrylate, and unreacted gelatin primary modified product are removed, and the synthesized gelatin secondary modified product is purified. Here, any of ion-exchanged water, distilled water, and ultrapure water can be used as the dilution water. The temperature of the water for dilution is maintained at a temperature at which the reactant and product can be maintained in solution, preferably 30 ° C. to 80 ° C., but the desired temperature is 35 ° C. to 60 ° C. The fractional molecular weight of the dialysis membrane used for the purification of the product may be within a range in which the synthesized gelatin secondary modified product and unreacted glycidyl methacrylate, unreacted gelatin primary modified product and low molecular weight salts can be separated.

  Subsequently, the purified gelatin secondary modified aqueous solution may be freeze-dried to obtain a gelatin secondary modified solid. The lyophilization time is not particularly limited as long as it can remove water from the frozen material, but illustratively it is 1 day to 1 month. The desired lyophilization time is 2 days to 2 weeks. In this way, a gelatin secondary modified product in which a methacryloyl group and a methacryloyl glyceryl ester group are bonded to the hydroxy group and carboxyl group of the gelatin primary modified product is obtained.

(Embodiment 2)
In the second embodiment, a method for producing a pore-forming agent used for forming a porous structure in a hydrogel will be described.

(1) Method for Producing Gel Particles Not Encapsulating Cells A method for producing gel particles that function as a pore-forming agent comprises a step of producing a gelatin hydrogel at a low temperature, and the obtained gelatin hydrogel is converted into gelatin hydrogel particles ( For example, it includes a step of cutting into cubic particles).

  The process for producing the gelatin hydrogel will be described in detail. Gelatin is dissolved in warm water at a temperature at which gelatin dissolves, phosphate buffered saline, HEPES buffer, saline, a medium for cell culture, or a mixed solution of phosphate buffered saline and a medium to prepare an aqueous gelatin solution. The temperature of the gelatin aqueous solution is 37 ° C. to 80 ° C., and a desirable temperature is 40 ° C. to 60 ° C. Here too, gelatin is derived from any material selected from the group of bones and skins of animals such as pigs, cattle, sheep and chickens, fish bones, fish skins and fish scales, or a plurality of materials. Gelatin. The molecular weight of the gelatin molecule is preferably in the range of 100 daltons to 10,000,000 daltons, and in this range, it dissolves in water and forms a hydrogel. The molecular weight of the gelatin molecule is more desirably in the range of 500 daltons to 1,000,000 daltons. This is particularly advantageous for the formation of hydrogels with a stable shape, diffusibility such as nutrients and wastes.

  The above medium is a medium used for cell culture, and may be one kind or several kinds of mixed medium such as Dulbecco's modified Eagle medium and Eagle minimum essential medium. The medium may or may not contain serum. When serum is included, the concentration of serum is 0.1-50%, with a desirable concentration of 0.5-20%. In the case of the above mixed solution of phosphate buffered saline and culture medium, the volume mixing ratio is 50: 1 to 1:50, and the desirable volume mixing ratio is 10: 1 to 1:10. The concentration of the gelatin aqueous solution is preferably 0.1 to 50 (w / v)%. Within this range, a hydrogel can be formed. A more desirable concentration is 0.5 to 20 (w / v)%.

  Next, the gelatin aqueous solution is sterilized with a filter membrane for filter sterilization. The pore diameter of the filter membrane for filtration sterilization of the gelatin aqueous solution is 0.1 to 0.5 μm, and the desirable pore diameter of the filter membrane is 0.1 to 0.3 μm.

  The gelatin aqueous solution sterilized by filtration is gelled at a low temperature to form a gelatin hydrogel. The temperature for gelatinizing the gelatin aqueous solution is not particularly limited as long as the gelatin aqueous solution can be gelled, but it is preferably a low temperature in the range of 0.5 ° C to 30 ° C. This makes it possible to form a hydrogel. A desirable temperature is 3 ° C to 15 ° C. In this way, a gelatin hydrogel is produced. In the present embodiment, low temperature intends the above range.

  Next, the step of cutting the gelatin hydrogel will be described in detail. The formed gelatin hydrogel is thinly cut to obtain a thin gelatin hydrogel sheet. Alternatively, an aqueous gelatin solution is pipetted into a frame space having a low frame space and a wide frame space, and gelled at a low temperature to form a thin gelatin hydrogel sheet having the same height as the frame space. Here, the thickness of the thin gelatin hydrogel sheet is 20 μm to 2000 μm, and preferably 50 μm to 1000 μm. In order to cut gelatin hydrogel thinly, a cutter or a thread may be used. A silicone rubber frame, a glass frame, a plastic frame, or the like can be used as a frame space when a thin gelatin hydrogel sheet is formed in the frame space.

  Next, a thin gelatin hydrogel sheet is chopped to produce gelatin hydrogel cubic particles. In order to chop the formed gelatin hydrogel sheet, a cutter, thread or mesh may be used. Nylon mesh or stainless steel mesh can be used as the mesh. The mesh size of the mesh may be 20 μm × 20 μm to 2000 μm × 2000 μm, but the desired mesh size is 50 μm × 50 μm to 1000 μm × 1000 μm. The length of one side of the above-mentioned gelatin hydrogel cubic particles may be 20 μm to 2000 μm, but the desired mesh size is 50 μm to 1000 μm. In this way, cubic particles made of gelatin hydrogel are produced. The cubic particles thus obtained are made of unmodified gelatin (also referred to as pure gelatin) without intentionally modifying the gelatin used as a raw material.

(2) Method for Producing Gel Particles Encapsulating Cells A method for producing gel particles encapsulating cells functioning as a pore-forming agent comprises a step of producing a gelatin hydrogel encapsulating cells at a low temperature, and the obtained cells The method includes a step of cutting the encapsulated gelatin hydrogel into particles (for example, cubic particles). When using gelatin hydrogel cubic particles containing cells as a pore-forming agent, pores are formed inside the bulk hydrogel and cells remain in the formed pores.

  The process for producing a gelatin hydrogel encapsulating cells will be described in detail. The process until the gelatin aqueous solution is sterilized with the filter membrane is the same as the above-described process (1) for producing the gelatin hydrogel, and the description thereof is omitted. The filter-sterilized gelatin aqueous solution is lowered to the lowest possible temperature at which the gelatin aqueous solution does not gel. Here, the temperature which makes it as low as possible at the above-mentioned temperature which does not gelatinize is 37 degreeC. At this temperature, gelation can be prevented.

Next, the cells are suspended in an aqueous gelatin solution at a reduced temperature. The gelatin aqueous solution in which the cells are suspended is gelled at a low temperature to form a gelatin hydrogel containing the cells. The cell concentration of the cell suspension of the gelatin aqueous solution is preferably in the range of 10 ³ cells / mL to 10 ⁸ cells / mL. Within this range, uniform cell distribution and efficient proliferation are possible. A more desirable concentration is 10 ⁴ cells / mL to 5 × 10 ⁸ cells / mL. The temperature at which the cell suspension of the gelatin aqueous solution is gelled is 0.5 ° C. to 30 ° C., but the desired temperature is 3 ° C. to 15 ° C. In this way, a gelatin hydrogel containing cells is produced.

  As the above-mentioned cells, cells derived from humans or other animals can be used. Chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, adipocytes, nerve cells, vascular endothelial cells, smooth muscle cells, cardiomyocytes, epithelial cells, hepatocytes, pancreatic β cells, kidneys collected from tissues Cells such as cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells can be encapsulated in gelatin hydrogel.

  Next, a step of cutting the gelatin hydrogel encapsulating the cells is performed, but since it is the same as the step of cutting the gelatin hydrogel described in (1) above, description thereof is omitted. In this way, cubic particles made of gelatin hydrogel containing cells are produced.

  The cubic particles made of gelatin hydrogel not encapsulating / encapsulating cells thus obtained function as a granulating agent. Therefore, when used together with the gelatin derivative of the present invention described in Embodiment 1, cells are encapsulated. A crosslinked gelatin hydrogel porous body which is not / encapsulated can be provided.

  Here, the gelatin hydrogel that does not encapsulate / encapsulate cells has been described as being cut into cubic particles, but the shape after cutting is not limited to a cube. For example, the shape of the particles is arbitrarily set according to a desired porous structure such as a rectangular parallelepiped, a cylinder, or a sphere. Also in this case, the gelatin hydrogel is cut so that the major axis of the particles is 20 μm to 2000 μm.

(Embodiment 3)
In Embodiment 3, the gelatin derivative (gelatin secondary modified product) described in Embodiment 1 and the gel particles described in Embodiment 2 (including / not including cells) are used for cross-linking as necessary. The gelatin hydrogel and its porous body, and the production method thereof will be described.

  FIG. 3 is a flowchart showing a procedure for a crosslinked gelatin hydrogel obtained by using the gelatin derivative of the present invention and a porous body thereof.

  By using the gelatin derivative 100 of the present invention (the gelatin derivative 100 described in Embodiment 1), a crosslinked gelatin hydrogel 310 that does not encapsulate cells, a crosslinked gelatin hydrogel 320 that encapsulates cells, and a crosslinked gelatin hydrogel that does not encapsulate cells. A gel porous body 330 and a crosslinked gelatin hydrogel porous body 340 enclosing cells are obtained.

Any cross-linked gelatin hydrogel and its porous bodies 310, 320, 330, and 340 have high cross-linking density and high mechanical strength, and thus have excellent shape stability. Illustratively, the crosslinking density of the crosslinked gelatin hydrogel and the porous body 310, 320 has a 0.02 mol / ^{m 3} or more 10000mol / ^{m 3} or less. When the crosslink density is within this range, the shape stability is excellent. More preferably, the crosslink density, has a 0.1 mol / ^{m 3} or more 4000mol / ^{m 3} or less. The crosslinked gelatin hydrogel and its porous bodies 310, 320, 330, and 340 have a storage elastic modulus in the range of 0.1 kPa to 10 kPa, and have high mechanical strength. The crosslinking density n can be calculated based on, for example, n = E ′ / 4RT (where E ′ is a dynamic storage elastic modulus, R is a gas constant, and T is a temperature).

  According to FIG. 3, cells can be seeded in the bulk of cross-linked gelatin hydrogel, in the pores of hydrogel, or both by using gelatin derivative 100 of the present invention, enabling more efficient tissue regeneration. To. The crosslinked gelatin hydrogel 320 containing cells and the porous body 340 thereof may be cut to an appropriate size to form a transplant, or cultured in a culture medium or transplanted to an animal to regenerate a tissue. May be.

Next, each manufacturing method of crosslinked gelatin hydrogel and its porous body 310,320,330,340 is demonstrated.
(1) Method for producing cross-linked gelatin hydrogel 310 without encapsulating cells (Procedure A in FIG. 3)
The method for producing the crosslinked gelatin hydrogel 310 includes a step of preparing an aqueous solution containing the gelatin secondary modified product (gelatin derivative) 100 of the present invention and a photopolymerization initiator, and a step of irradiating the aqueous solution with ultraviolet rays. It is characterized by.

  The step of preparing an aqueous solution of gelatin secondary modified product and photopolymerization initiator will be described in detail. The aqueous solution is maintained at, for example, 23 ° C. or higher and 37 ° C. or lower, preferably 25 ° C. or higher and 30 ° C. or lower. As the solvent, pure water, phosphate buffered saline, HEPES buffer, saline, cell culture medium, or a mixed solution of phosphate buffered saline and medium can be used. The above medium is a medium used for cell culture, and may be one kind or several kinds of mixed medium such as Dulbecco's modified Eagle medium and Eagle minimum essential medium. The medium may or may not contain serum. The serum concentration is 0.1-50%, and the desired concentration is 0.5-20%. The volume mixing ratio of the mixed solution of the above phosphate buffered saline and the medium is 50: 1 to 1:50, preferably 10: 1 to 1:10.

  The concentration of the aqueous solution of the gelatin secondary modified product is 0.1 to 50 (w / v)% in terms of the weight of the gelatin secondary modified product and the concentration of the volume of the solution. This promotes gelation. A desirable concentration is 0.5 to 30 (w / v)%.

  The above photopolymerization initiator is illustratively 2-hydroxy-1- (4- (hydroxyethoxy) phenyl) -2-methyl 1-propanone, ammonium persulfate and N, N, N ′, N ′. -A liquid mixture containing tetramethylethylenediamine or a liquid mixture containing potassium persulfate and N, N, N ', N'-tetramethylethylenediamine, preferably 2-hydroxy-1- (4- (hydroxy Ethoxy) phenyl) -2-methyl l-1-propanone. The concentration of the photopolymerization initiator is 0.01 to 10 (w / v)%, preferably 0.05 to 2 (w / v)% in terms of the weight of the photopolymerization initiator to the volume of the solution. . This promotes polymerization.

  Next, the aqueous solution containing the gelatin secondary modified product and the photopolymerization initiator is sterilized with a filter membrane for filter sterilization. The pore diameter of the filter membrane for filtration sterilization of the gelatin aqueous solution is 0.1 to 0.5 μm, and the desirable pore diameter of the filter membrane is 0.1 to 0.3 μm.

  Next, the process of irradiating this aqueous solution with ultraviolet light will be described in detail. The prepared aqueous solution is maintained at, for example, 23 ° C. or higher and 37 ° C. or lower, preferably 25 ° C. or higher and 30 ° C. or lower, and irradiated with ultraviolet rays. Thereby, aqueous solution is exposed, a crosslinking polymerization reaction starts and it gelatinizes. Here, the irradiation condition of ultraviolet rays is preferably 10 seconds to 30 minutes, and desirably 20 seconds to 10 minutes. In the case of producing a cross-linked gelatin hydrogel 310 having a constant thickness, the gelatin secondary modification product and the photopolymerization initiator are formed in a space formed between two quartz cover glasses separated by a space frame having a constant thickness. It is only necessary to inject an aqueous solution and irradiate with ultraviolet rays.

  In this way, a crosslinked gelatin hydrogel 310 having no cells is obtained. Alternatively, the cross-linked gelatin hydrogel 310 may be cut to a required size, and the cells may be appropriately seeded. To cut to a certain size, a cutter, thread or mesh may be used.

(2) Method for producing crosslinked gelatin hydrogel 320 containing cells (Procedure B in FIG. 3)
The method for producing the crosslinked gelatin hydrogel 320 includes a step of preparing an aqueous solution containing the gelatin secondary modified product (gelatin derivative) 100 of the present invention and a photopolymerization initiator, and a cell suspension in which cells are suspended. It includes a step of preparing and a step of irradiating the cell suspension with ultraviolet rays.

  Since the step of preparing the aqueous solution of the gelatin secondary modified product and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) described above, description thereof is omitted. In the step of preparing a cell suspension in which cells are suspended in the obtained aqueous solution, the aqueous solution of gelatin secondary modified product and photopolymerization initiator is sterilized with a filter membrane for filter sterilization, and then the temperature of the aqueous solution is adjusted. The temperature is kept as low as possible, for example, 23 ° C. or higher and 37 ° C. or lower, preferably 25 ° C. or higher and 30 ° C. or lower. This prevents gelation of the aqueous solution. Cells are added to an aqueous solution of a gelatin secondary modified product and a photopolymerization initiator at a low temperature to prepare a cell suspension.

The cell concentration of the cell suspension is preferably in the range of 10 ³ cells / mL to 10 ⁸ cells / mL. Within this range, uniform cell distribution and efficient proliferation are possible. A more desirable concentration is 10 ⁴ cells / mL to 5 × 10 ⁸ cells / mL.

  As the above-mentioned cells, cells derived from humans or other animals can be used. Chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, adipocytes, nerve cells, vascular endothelial cells, smooth muscle cells, cardiomyocytes, epithelial cells, hepatocytes, pancreatic β cells, kidneys collected from tissues Cells such as cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells can be encapsulated in gelatin hydrogel.

  Subsequently, this cell suspension is irradiated with ultraviolet rays to carry out cross-linking polymerization. In addition, since the process of irradiating this cell suspension with ultraviolet rays is the same as the process of irradiating the aqueous solution (1) with ultraviolet rays, the description thereof is omitted. In this way, a crosslinked gelatin hydrogel 320 containing cells is obtained. Again, in the case of producing a cross-linked gelatin hydrogel 320 having a constant thickness, the cell suspension is injected into a space formed between two quartz cover glasses separated by a space frame having a constant thickness. Irradiation with ultraviolet rays is sufficient.

(3) Method for producing crosslinked gelatin hydrogel porous body 330 that does not encapsulate cells (procedure C in FIG. 3)
The method for producing the crosslinked gelatin hydrogel porous body 330 includes a step of preparing an aqueous solution containing the gelatin secondary modified product (gelatin derivative) 100 of the present invention and a photopolymerization initiator, and gel particles (described in Embodiment 2). And a step of preparing a mixture to which cubic particles made of gelatin hydrogel not encapsulating cells) are added, and a step of irradiating the mixture with ultraviolet rays.

  Since the step of preparing the aqueous solution of the gelatin secondary modified product and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) described above, description thereof is omitted. In the step of preparing a mixture in which gel particles are added to the obtained aqueous solution, the aqueous solution of the gelatin secondary modified product and the photopolymerization initiator is sterilized with a filter membrane for filter sterilization, and then the temperature of the aqueous solution is lowered as low as possible. And 23 ° C. or higher and 37 ° C. or lower, preferably 25 ° C. or higher and 30 ° C. or lower. This prevents gelation of the aqueous solution. Gel particles are added to an aqueous solution of a gelatin secondary modified product and a photopolymerization initiator at a low temperature to prepare a mixture. The mixing ratio of the gelatin secondary modified product, the photopolymerization initiator aqueous solution, and the gel particles is such that the ratio of the volume of the aqueous solution to the amount of the gel particles is 1: 5 (v / w) to 50: 1 (v / w). w) is mixed to satisfy. Thereby, a crosslinked gelatin hydrogel porous body having a desired porous structure is obtained. The mixing ratio is desirably 1: 2 (v / w) to 5: 1 (v / w).

  Next, the mixture is irradiated with ultraviolet rays to carry out cross-linking polymerization. The step of irradiating the mixture with ultraviolet rays is the same as the step of irradiating the aqueous solution (1) with ultraviolet rays, but the temperature is higher than the gelation temperature of the gelatin derivative of the present invention. The gelatin derivative of the present invention is subjected to cross-linking polymerization while maintaining the temperature (25 ° C. in the examples) so as to be lower than the gelation temperature of). After the cross-linking polymerization, if the product is set to a sol-gel transition point (37 ° C.) or higher of gel particles (pure gelatin), hydrogen bonds between pure gelatin molecules are broken and only the gel particles are dissolved, so that a porous structure is formed. Is done. In this way, a crosslinked gelatin hydrogel porous body 330 that does not encapsulate cells is obtained. Again, in the case of producing a crosslinked gelatin hydrogel porous body 330 having a constant thickness, the mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a constant thickness, What is necessary is just to irradiate with an ultraviolet-ray.

(4) Method for Producing Crosslinked Gelatin Hydrogel Porous Material 340 Encapsulating Cells Crosslinked gelatin hydrogel porous material 340 is produced by three different procedures DF as shown in FIG.

(A) A method for manufacturing the procedure D in FIG. 3 will be described.
The method for producing the crosslinked gelatin hydrogel porous body 340 includes a step of preparing an aqueous solution containing the gelatin secondary modified product (gelatin derivative) 100 of the present invention and a photopolymerization initiator, and a cell suspension in which cells are suspended. A step of preparing a liquid, a step of preparing a mixture in which gel particles (cubic particles made of gelatin hydrogel not containing cells described in Embodiment 2) are added, and a step of irradiating the mixture with ultraviolet rays It is characterized by including.

  Here, the step of preparing the aqueous solution containing the gelatin secondary modified product and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) described above. The step of preparing the cell suspension in which the cells are suspended in the obtained aqueous solution is the same as the step of preparing the cell suspension in (2) above. The step of preparing the mixture in which the gel particles are added to the cell suspension is the same as the step of preparing the mixture of (3) described above. The step of irradiating the obtained mixture with ultraviolet rays is the same as the step of irradiating the mixture (3) with ultraviolet rays. Therefore, the description of the above steps is omitted.

  By such a combination of steps, a crosslinked gelatin hydrogel porous body 340 enclosing cells is obtained. Here again, in the case of producing a crosslinked gelatin hydrogel porous body 340 having a constant thickness, the mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a constant thickness. Then, it may be irradiated with ultraviolet rays.

(B) A method for manufacturing the procedure E in FIG. 3 will be described.
The method for producing the crosslinked gelatin hydrogel porous body 340 includes a step of preparing an aqueous solution containing the gelatin secondary modified product (gelatin derivative) 100 of the present invention and a photopolymerization initiator, and gel particles (described in Embodiment 2). And a step of preparing a mixture to which cubic particles made of gelatin hydrogel encapsulating the cells) are added, and a step of irradiating the mixture with ultraviolet rays.

Here, the step of preparing the aqueous solution containing the gelatin secondary modified product and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) described above. The step of preparing the mixture obtained by adding gel particles to the obtained aqueous solution is the same as the step of preparing the mixture of (3) above, but the cell concentration contained in the gel particles enclosing the cells is The concentration is preferably in the range of 10 ³ cells / mL to 10 ⁸ cells / mL. A more desirable concentration is 10 ⁴ cells / mL to 5 × 10 ⁸ cells / mL. The step of irradiating the obtained mixture with ultraviolet rays is the same as the step of irradiating the mixture (3) with ultraviolet rays. Therefore, the description of the above steps is omitted.

  By such a combination of steps, a crosslinked gelatin hydrogel porous body 340 enclosing cells is obtained. Here again, in the case of producing a crosslinked gelatin hydrogel porous body 340 having a constant thickness, the mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a constant thickness. Then, it may be irradiated with ultraviolet rays.

(C) A method for manufacturing the procedure F of FIG. 3 will be described.
The method for producing the crosslinked gelatin hydrogel porous body 340 includes a step of preparing an aqueous solution containing the gelatin secondary modified product (gelatin derivative) 100 of the present invention and a photopolymerization initiator, and a cell suspension in which cells are suspended. A step of preparing a liquid, a step of preparing a mixture in which gel particles (cubic particles made of gelatin hydrogel encapsulating cells described in Embodiment 2) are added, and a step of irradiating the mixture with ultraviolet rays It is characterized by including.

  Here, the step of preparing the aqueous solution containing the gelatin secondary modified product and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) described above. The step of preparing the cell suspension in which the cells are suspended in the obtained aqueous solution is the same as the step of preparing the cell suspension in (2) above. The step of preparing the mixture in which the gel particles are added to the cell suspension is the same as the step of preparing the mixture of (3) described above. The step of irradiating the obtained mixture with ultraviolet rays is the same as the step of irradiating the aqueous solution (1) with ultraviolet rays. Therefore, the description of the above steps is omitted.

  By such a combination of steps, a crosslinked gelatin hydrogel porous body 340 enclosing cells is obtained. Here again, in the case of producing a crosslinked gelatin hydrogel porous body 340 having a constant thickness, the mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a constant thickness. Then, it may be irradiated with ultraviolet rays.

  EXAMPLES Hereinafter, although this invention is explained in full detail using a specific Example, this invention is not limited to these Examples at all.

<Example 1>
In Example 1, using pig skin-derived gelatin, an amino group was bonded to a methacryloyl group (120 in FIG. 1), and a hydroxy group and a carboxyl group of gelatin were bonded to a methacryloyl glyceryl ester group (130 in FIG. 1). The gelatin derivative of the present invention (hereinafter simply referred to as gelatin secondary modified product) was synthesized.

  FIG. 4 is a diagram showing a step (a) for synthesizing a gelatin primary modified product and a step (b) for synthesizing a gelatin secondary modified product.

  According to step S210 shown in FIG. 2, a gelatin primary modified product (denoted as GelMA in FIG. 4 (a)) was synthesized based on the synthesis procedure of FIG. 4 (a). Specifically, 5 g of porcine skin-derived gelatin (molecular weight: 50,000 to 100,000 daltons) was dissolved in PBS while stirring at 50 ° C. to obtain a 10 (w / v)% gelatin solution. Next, 5 mL of methacrylic anhydride was added to the gelatin solution at 0.5 mL / min while stirring at 50 ° C., and reacted for 3 hours in the dark. Here, the volume charge ratio of methacrylic anhydride to the gelatin aqueous solution was 10: 1. The reaction product is diluted 5-fold in PBS at 50 ° C. and dialyzed for 7 days in ultrapure water at 40 ° C. using a dialysis membrane (fractional molecular weight: 12,000 to 14,000 daltons), so The reaction methacrylic anhydride was removed and the reaction product was purified.

The molecular structure of the reaction product was analyzed by ¹ H NMR. ¹ H NMR spectra were measured using an NMR spectrometer with a frequency of 300 MHz and a single axial gradient inverse probe. Before measuring, the reaction product, 40 ° C. to heavy water 1mL containing internal standard 0.05 (w / v)% 3- ( trimethylsilyl) _{-2,2,3,3-d 4} acid sodium salt propionate Melted in The ¹ H NMR spectrum of the reaction product is shown in FIG.

FIG. 5 is a diagram showing a ¹ H NMR spectrum of a gelatin primary modified product.

  The signals at 5.4 ppm and 5.7 ppm in FIG. 5 were attributed to the vinyl proton of the methacrylate group. The sum of the signal intensities of 5.4 ppm and 5.7 ppm was 0.92. From this, it was confirmed that the obtained reaction product was a gelatin primary modified product in which a methacryloyl group was bonded to an amino group.

  The obtained gelatin primary modified product was freeze-dried for 7 days to obtain a solid.

  According to step S220 shown in FIG. 2, a gelatin secondary modification (indicated as GelMAGMA in FIG. 4 (b)) was synthesized based on the synthesis procedure of FIG. 4 (b). Specifically, the gelatin primary modification obtained above was reacted with glycidyl methacrylate. First, 2.5 g of gelatin primary modified product was dissolved in ultrapure water to prepare a 2 (w / v)% solution, and then the pH was adjusted to 3.5 with 1M HCl. Next, 5 mL of glycidyl methacrylate was added to the gelatin primary modified aqueous solution at 0.5 mL / min and reacted at 50 ° C. for 24 hours. Here, the volume charge ratio of glycidyl methacrylate to the gelatin primary modified aqueous solution was 25: 1.

  The obtained reaction product was diluted 5-fold in PBS at 50 ° C. and dialyzed for 7 days in ultrapure water at 40 ° C. using a dialysis membrane (fraction molecular weight: 12,000 to 14,000 daltons). Unreacted glycidyl methacrylate was removed and the reaction product was purified.

The molecular structure of the reaction product was analyzed by ¹ H NMR. ¹ H-NMR spectra were measured using an NMR spectrometer having a frequency of 300 MHz and a single axial gradient inverse probe. Before measuring, the reaction product, 40 ° C. to heavy water 1mL containing internal standard 0.05 (w / v)% 3- ( trimethylsilyl) _{-2,2,3,3-d 4} acid sodium salt propionate Melted in The ¹ H NMR spectrum of the reaction product is shown in FIG.

FIG. 6 is a diagram showing a ¹ H NMR spectrum of a gelatin secondary modified product.

  The signals of 5.4 ppm and 5.7 ppm in FIG. 6 are the vinyl protons of the methacrylate group derived from methacrylic anhydride, and the signals of 5.8 ppm and 6.2 ppm are the vinyl protons of the methacrylate group derived from glycidyl methacrylate. Assigned. Thus, the obtained reaction product is a gelatin secondary modified product (gelatin derivative of the present invention) in which an amino group is bonded to a methacryloyl group and a hydroxy group and a carboxyl group of gelatin are bonded to a methacryloyl glyceryl ester group. I confirmed that there was.

  The sum of the signal intensities of 5.4 ppm, 5.7 ppm, 5.8 ppm and 6.2 ppm was 1.76. The sum of the signal intensities of protons derived from the vinyl group in the methacrylate group of the gelatin secondary modified product is about twice the value of the vinyl proton in the methacrylate group of the gelatin primary modified product of Example 1, and more photoreactions occur. It was found that a functional methacrylate group was introduced. From the NMR spectrum, the contents of methacrylate group and methacryloyl glyceryl ester group were 0.35 mmol and 0.70 mmol, respectively, in terms of 1 g of gelatin secondary modified product.

  The purified gelatin secondary modified product was lyophilized for 7 days to obtain a solid. In the following examples, the obtained solid was used.

  Next, the rheological properties of the gelatin secondary modified aqueous solution were measured with a rheometer, and the sol-gel transition temperature was measured. The gelatin secondary modification was dissolved in PBS at 50 ° C. to prepare a 10 (w / v%) aqueous solution, and 300 μL was used for the measurement. Using a parallel plate measurement geometry with a diameter of 50 mm, temperature sweep (37 ° C to 20 ° C, cooling rate 0.15 ° C / min) with constant amplitude (γ = 5%) and frequency (1 Hz), storage modulus (G ′) and loss elastic modulus (G ″) were measured. FIG. 7 shows the temperature dependence of the storage elastic modulus (G ′) and loss elastic modulus (G ″) of the gelatin secondary modified aqueous solution.

  FIG. 7 is a graph showing the relationship between the storage elastic modulus (G ′) and the loss elastic modulus (G ″) of a 10 (w / v%) gelatin secondary modified aqueous solution.

  The transition temperature from the sol to the gel was determined from the intersection of the storage elastic modulus (G ′) and the loss elastic modulus (G ″). The transition temperature from the sol to the gel of the aqueous gelatin secondary modified product was 22.7 ° C. The transition temperature from the sol to the gel of the gelatin secondary modified aqueous solution was lower than room temperature, and the gelatin secondary modified aqueous solution did not gel at room temperature, so that it was easy to handle.

<Example 2>
In Example 2, a crosslinked gelatin hydrogel (310 in FIG. 3) was produced based on the procedure A of FIG. 3 using the gelatin secondary modified product synthesized in Example 1.

  Specifically, gelatin secondary modified product (10%, w / v) and photopolymerization initiator (2-hydroxy-1- (4- (hydroxyethoxy) phenyl) -2-methyl-1-propanone) (0 0.5%, w / v) was dissolved in a PBS solution at 25 ° C. to prepare an aqueous solution. This aqueous solution was sterilized with a filter membrane (pore size: 0.22 μm) for filtration sterilization. After sterilization, these aqueous solutions are pipetted between two quartz cover glasses separated by a 1.5 mm thick silicone rubber frame, kept at 25 ° C., and irradiated with ultraviolet rays for 5 minutes to crosslink. A gelatin hydrogel was prepared.

The obtained crosslinked gelatin hydrogel was cut into a disk shape having a diameter of 10 mm and used for a rheological test. The storage modulus was measured with a rheometer equipped with a 10 mm parallel plate. The temperature was set at 37 ° C. and the sample was allowed to equilibrate for 3 minutes before starting the test. The storage elastic modulus was measured by applying an oscillating shear deformation under a constant frequency (1 Hz) and a constant longitudinal strain (5%). Three samples were tested to calculate the mean and standard deviation. As a result, it was found that the storage elastic modulus was 3295.6 ± 276.8 Pa and had high mechanical strength. When the crosslinking density was calculated, it was 70 mol / m ³ .

<Example 3>
In Example 3, using the gelatin secondary modified product synthesized in Example 1, a crosslinked gelatin hydrogel (320 in FIG. 3) containing chondrocytes was produced based on the procedure B of FIG.

First, primary chondrocytes isolated from calf knee joint cartilage were cultured at 37 ° C. and 5% CO ₂ . Culture includes 10% fetal bovine serum, 4500 mg / L glucose, 4 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.1 mM non-essential amino acid, 0.4 mM proline, 1 mM pyruvate sodium salt, 50 μg / Dulbecco's modified Eagle's medium (DMEM), 75 cm ² tissue culture flask supplemented with mL ascorbic acid was used. The medium was changed every 3 days. It was used for experiments in which chondrocytes that had been subcultured once were encapsulated in hydrogel. Chondrocytes that reached confluence were detached with a trypsin / EDTA solution, the cells were collected by centrifugation, and the number of cells was counted with a hemocytometer.

Next, an aqueous solution of a gelatin secondary modified product (10%, w / v) and a photopolymerization initiator (0.5%, w / v) was prepared in the same procedure as in Example 2, and this was prepared. The chondrocytes were suspended, and a cell suspension of chondrocytes containing a gelatin secondary modified product and a photopolymerization initiator was prepared. Here, the cell concentration of the cell suspension was 2 × 10 ⁷ cells / mL. The cell suspension was kept at 25 ° C. The cell suspension was pipetted into a space formed by sandwiching a 1.5 mm thick silicone rubber frame between two quartz cover glasses. Next, the cell suspension was kept at 25 ° C. and irradiated with ultraviolet rays for 5 minutes to prepare a crosslinked gelatin hydrogel encapsulating chondrocytes. Further, it was cut out into a disk shape having a diameter of 10 mm and cultured in a T-flask for 2 weeks while shaking at a speed of 60 rpm. The medium was changed every 2 days.

  Viable cells in the crosslinked gelatin hydrogel were stained with calcein-AM and dead cells were stained with propidium iodide to evaluate the viability of chondrocytes. Immediately after the preparation of the crosslinked gelatin hydrogel and after culturing for 2 weeks, the disk-shaped crosslinked gelatin hydrogel disk containing the cells was washed three times with PBS, and contained no calcein-AM (2 μM) and propidium iodide (4 μM). Incubated in serum medium at 37 ° C. for 15 minutes. The stained cells were observed with a confocal laser microscope. The results are shown in FIG.

  FIG. 8 is a diagram showing a fluorescence microscopic image of cell viability staining in a crosslinked gelatin hydrogel encapsulating chondrocytes according to Example 3.

  FIG. 8 (a) shows a fluorescence microscopic image of life-and-death staining of cells immediately before culturing and (b) after 2 weeks of culturing. FIG. 8 is shown in grayscale, but the brightly shown areas indicate live cells and dead cells are indicated by arrows.

  According to FIG. 8, a few dead cells were detected immediately after encapsulating the cells, that is, immediately before the culture, but not after two weeks of culture. Some chondrocytes aggregated to form small aggregates. From these results, there was no obvious effect on the viability of the cells even when the gelatin secondary modified product was subjected to cross-linking polymerization in the state of encapsulating the cells. The obtained cross-linked gelatin hydrogel has high cytocompatibility, indicating that this method is suitable for preparing a cross-linked gelatin hydrogel encapsulating chondrocytes.

<Example 4>
In Example 4, using the gelatin secondary modified product synthesized in Example 1, a crosslinked gelatin hydrogel (340 in FIG. 3) containing chondrocytes was produced based on Procedure D in FIG. In Example 4, hydrogel cuboid particles made of pure unmodified gelatin that did not encapsulate cells were used as a pore-forming agent for a crosslinked gelatin hydrogel porous material.

  Unmodified gelatin (pure gelatin) undergoes a gel-sol transition at 37 ° C. Hydrogel cuboid particles made of unmodified gelatin were prepared from an aqueous solution of unmodified gelatin. Unmodified gelatin was dissolved in a mixed solution of PBS and DMEM (volume ratio 1: 1) to prepare a 5 (w / v)% gelatin solution. This gelatin solution was sterilized by filtration through a filter membrane having a pore size of 0.22 μm. The gelatin solution was injected into the space of a silicone rubber frame having a frame space of 100 mm × 20 mm × 300 μm with a pipette and gelled at 4 ° C. The formed unmodified gelatin hydrogel was pressed into a nylon mesh having a mesh size of 250 μm × 250 μm to cut into hydrogel cuboid particles of unmodified gelatin having a size of 250 × 250 × 300 μm. The state after cutting was observed with an optical microscope. The observation results are shown in FIG.

  FIG. 9 is an optical micrograph showing the state of rectangular parallelepiped particles made of gelatin hydrogel.

  According to FIG. 9, it was found that the rectangular parallelepiped particles have 250 × 250 × 300 μm.

  Next, an aqueous solution of gelatin secondary modified product (10%, w / v) and photopolymerization initiator (0.5%, w / v) was prepared in the same procedure as in Example 3, and prepared in this. The chondrocytes were suspended, and a cell suspension of chondrocytes containing a gelatin secondary modified product and a photopolymerization initiator was prepared. A mixture was prepared by adding rectangular parallelepiped particles made of gelatin hydrogel to a cell suspension of chondrocytes. Here, the cell suspension and the hydrogel cuboid particles were mixed at 2: 1 (v / w), and the mixture was maintained at 25 ° C.

  The mixture was pipetted between two quartz cover glasses separated by a 1.5 mm thick silicone rubber frame, kept at 25 ° C., and irradiated with ultraviolet rays for 5 minutes. As a result, the gelatin hydrogel was crosslinked and polymerized. The product was then held at 37 ° C. to dissolve the gel particles. A crosslinked gelatin hydrogel porous body containing cells in the crosslinked bulk hydrogel and no cells in the cuboid particles was prepared. This crosslinked gelatin hydrogel porous body was cut into a disk shape having a diameter of 6 mm. The appearance of the crosslinked gelatin hydrogel porous material was observed. The observation results are shown in FIG.

  FIG. 10 is a photograph showing the appearance of the crosslinked gelatin hydrogel porous material according to Example 4.

  According to FIG. 10, the obtained crosslinked gelatin hydrogel porous body was transparent and uniform immediately before the culture. Subsequently, the cells were cultured for 4 weeks in a cell culture T-flask while shaking at a speed of 60 rpm. The medium was changed every two days. All steps were performed under aseptic conditions.

  Viability of chondrocytes in the crosslinked gelatin hydrogel porous body was evaluated by the same procedure as in Example 3, and the stained cells were observed with a confocal laser microscope. The observation results are shown in FIG.

  FIG. 11 is a view showing a fluorescence microscopic image of life-and-death staining of cells in a crosslinked gelatin hydrogel porous body encapsulating chondrocytes according to Example 4.

  FIG. 11 (a) shows a fluorescence microscopic image of life-and-death staining of the cells immediately before culturing and (b) after culturing for 4 weeks. According to FIG. 11, a few dead cells (indicated by arrows in the figure) were detected immediately after encapsulating the cells, that is, immediately before the culture, but no dead cells were detected after 4 weeks of culture. In particular, chondrocytes were observed only in the bulk portion immediately after the preparation of the crosslinked gelatin hydrogel porous material, but were also observed in the pores after 4 weeks of culture. Moreover, according to FIG.11 (b), it was confirmed that the chondrocyte has aggregated.

  Further, the crosslinked gelatin hydrogel porous body of Example 4 was cultured in vitro for 1 day, and then implanted subcutaneously in the back of nude mice for 4 weeks to regenerate cartilage tissue. The state after reproduction is shown in FIG.

  FIG. 12 is a photograph showing the appearance of cartilage tissue regenerated with a crosslinked gelatin hydrogel porous material according to Example 4.

  As can be seen from a comparison between FIG. 10 and FIG. 12, a glossy cartilage tissue was regenerated as a whole.

<Example 5>
In Example 5, using the gelatin secondary modified product synthesized in Example 1, a crosslinked gelatin hydrogel (340 in FIG. 3) including chondrocytes was produced based on the procedure E in FIG. In Example 5, hydrogel cuboid particles made of pure unmodified gelatin containing cells were used as a pore-forming agent for a crosslinked gelatin hydrogel porous material.

In the same manner as in Example 4, unmodified gelatin was dissolved in a mixed solution of PBS and DMEM at a volume ratio of 1: 1 to prepare a 5 (w / v)% gelatin solution. This gelatin solution was sterilized by filtration through a filter membrane having a pore size of 0.22 μm. Chondrocytes were suspended in this gelatin solution to prepare a cell suspension. The cell concentration of the cell suspension was 4 × 10 ⁷ cells / mL. The cell suspension of gelatin solution was pipetted into a 100 mm × 20 mm × 300 μm silicone rubber frame space and gelled at 4 ° C. By pressing the formed non-modified gelatin hydrogel encapsulating chondrocytes against a nylon mesh having a mesh size of 250 μm × 250 μm, the hydrogel cuboid particles of unmodified gelatin encapsulating chondrocytes having a size of 250 × 250 × 300 μm are formed. Cut and processed. The state after cutting was observed with an optical microscope. The observation results are shown in FIG.

  FIG. 13 is an optical micrograph showing the state of rectangular parallelepiped particles made of gelatin hydrogel.

  According to FIG. 13, it was found that the rectangular parallelepiped particles have 250 × 250 × 300 μm.

  Next, an aqueous solution containing a gelatin secondary modified product and a photopolymerization initiator is prepared by the same procedure as in Example 2, and chondrocyte-encapsulating hydrogel cuboid particles are added thereto by the same procedure as in Example 4. The mixture was prepared (mixing ratio was 2: 1 (v / w)) and kept at 25 ° C.

  The mixture was pipetted between two quartz cover glasses separated by a 1.5 mm thick silicone rubber frame, kept at 25 ° C., and irradiated with ultraviolet rays for 5 minutes. As a result, the gelatin hydrogel was crosslinked and polymerized. The product was then held at 37 ° C. to dissolve the gel particles. In this way, a crosslinked gelatin hydrogel porous body containing no cells in the crosslinked bulk hydrogel and cells in the hydrogel cuboid particles was prepared. This gelatin hydrogel was cut into a disk having a diameter of 6 mm. The appearance of the crosslinked gelatin hydrogel porous material was observed. The observation results are shown in FIG.

  FIG. 14 is a photograph showing the appearance of the crosslinked gelatin hydrogel porous material according to Example 5.

  According to FIG. 14, the obtained crosslinked gelatin hydrogel porous body was transparent and uniform immediately before the culture. Next, cell culture is performed under the same conditions as in Example 4, the viability of the chondrocytes in the crosslinked gelatin hydrogel porous body is evaluated by the same procedure as in Example 3, and the stained cells are observed with a confocal laser microscope. did. The observation results are shown in FIG.

  FIG. 15 is a diagram showing a fluorescence microscopic image of cell viability staining in a crosslinked gelatin hydrogel porous body encapsulating chondrocytes according to Example 5.

  FIG. 15 (a) shows a fluorescence microscopic image of life-and-death staining of cells immediately before culturing and (b) after culturing for 4 weeks. According to FIG. 15, a few dead cells (indicated by arrows in the figure) were detected immediately after encapsulating the cells, that is, immediately before the culture, but no dead cells were detected after 4 weeks of culture. In particular, chondrocytes were observed only in the pores of the crosslinked gelatin hydrogel porous material immediately after the porous gelatin hydrogel porous material was prepared and after 4 weeks of culture. Moreover, according to FIG.15 (b), it was confirmed that the chondrocyte has aggregated.

  Further, in the same manner as in Example 4, the crosslinked gelatin hydrogel porous material of Example 5 was cultured in vitro for 1 day and then implanted subcutaneously in the back of nude mice for 4 weeks to regenerate cartilage tissue. The state after reproduction is shown in FIG.

  FIG. 16 is a photograph showing the appearance of a cartilage tissue regenerated with a crosslinked gelatin hydrogel porous material according to Example 5.

  As can be seen from a comparison between FIG. 14 and FIG. 16, a glossy cartilage tissue was regenerated as a whole.

<Example 6>
In Example 6, using the gelatin secondary modified product synthesized in Example 1, a crosslinked gelatin hydrogel (340 in FIG. 3) containing chondrocytes was produced based on the procedure F in FIG. In Example 6, as in Example 5, hydrogel cuboid particles made of pure unmodified gelatin containing cells were used as a pore-forming agent for a crosslinked gelatin hydrogel porous material.

In the same procedure as in Example 3, an aqueous solution of gelatin secondary modified product (10%, w / v) and photopolymerization initiator (0.5%, w / v) was prepared, and chondrocytes prepared thereby Was suspended, and a cell suspension of chondrocytes containing a gelatin secondary modified product and a photopolymerization initiator was prepared. Here, the cell concentration of the cell suspension was 1.34 × 10 ⁷ cells / mL. Next, in the same procedure as in Example 5, a mixture to which chondrocyte-encapsulating hydrogel cuboid particles were added was prepared (mixing ratio was 2: 1 (v / w)) and maintained at 25 ° C. Here, the cell concentration of the cell suspension for producing the rectangular parallelepiped particles was 1.33 × 10 ⁷ cells / mL.

  The mixture was pipetted between two quartz cover glasses separated by a 1.5 mm thick silicone rubber frame, kept at 25 ° C., and irradiated with ultraviolet rays for 5 minutes. As a result, the gelatin hydrogel was crosslinked and polymerized. The product was then held at 37 ° C. to dissolve the gel particles. In this way, a crosslinked gelatin hydrogel porous body containing cells in both the crosslinked bulk hydrogel and the hydrogel cuboid particles was prepared. This gelatin hydrogel was cut into a disk having a diameter of 6 mm. The appearance of the crosslinked gelatin hydrogel porous material was observed. The observation results are shown in FIG.

  FIG. 17 is a photograph showing the appearance of a crosslinked gelatin hydrogel porous material according to Example 6.

  According to FIG. 17, the obtained crosslinked gelatin hydrogel porous body was transparent and uniform immediately before the culture. Next, cell culture is performed under the same conditions as in Example 4, the viability of the chondrocytes in the crosslinked gelatin hydrogel porous body is evaluated by the same procedure as in Example 3, and the stained cells are observed with a confocal laser microscope. did. The observation results are shown in FIG.

  FIG. 18 is a fluorescence microscopic image of cell viability staining in a crosslinked gelatin hydrogel porous body encapsulating chondrocytes according to Example 6.

  FIG. 18 (a) shows a fluorescence microscopic image of life-and-death staining of cells immediately before culturing and (b) after culturing for 4 weeks. According to FIG. 18, a few dead cells (indicated by arrows in the figure) were detected immediately after encapsulating the cells, that is, immediately before the culture, but no dead cells were detected after 4 weeks of culture. In particular, immediately after the preparation of the crosslinked gelatin hydrogel porous body, the chondrocytes were observed both in the bulk region and the pores of the hydrogel porous body and were uniformly distributed. Cells aggregated.

  Further, in the same manner as in Example 4, the crosslinked gelatin hydrogel porous material of Example 6 was cultured in vitro for 1 day and then implanted subcutaneously in the back of nude mice for 4 weeks to regenerate cartilage tissue. The state after reproduction is shown in FIG.

  FIG. 19 is a photograph showing the appearance of cartilage tissue regenerated with a crosslinked gelatin hydrogel porous material according to Example 6.

  As can be seen from the comparison between FIG. 17 and FIG. 19, a glossy cartilage tissue was regenerated as a whole.

  The gelatin derivative (gelatin secondary modified product) of the present invention has a reactivity in which an amino group is bonded to a methacryloyl group and a hydroxy group and a carboxyl group of gelatin are bonded to a methacryloyl glyceryl ester group, which can be crosslinked by ultraviolet irradiation. The group is introduced to the maximum. Since the crosslinked gelatin hydrogel and porous body thereof of the present invention are produced using the gelatin derivative of the present invention, the mechanical strength is high and the shape stability is excellent. Thereby, in addition to the use as an injectable gel to a load site | part, it can be used also for the tissue reproduction | regeneration by a biological tissue engineering method. Moreover, since cells can be seeded in both the bulk and pores of the hydrogel, more efficient tissue regeneration becomes possible, and shortening of the healing period can be expected. Therefore, the crosslinked gelatin hydrogel of the present invention and the porous body thereof are extremely useful for treating tissue defects.

100 Gelatin derivative 110 Gelatin 120 Methacryloyl group 130 Methacryloyl glyceryl ester group

Claims (20)

A gelatin derivative having a reactive group introduced into gelatin,
The amino group of the gelatin is bonded to the methacryloyl group;
A gelatin derivative in which a hydroxy group and a carboxyl group of the gelatin are bonded to a methacryloyl glyceryl ester group. The content of the methacryloyl group satisfies a range of 0.01 mmol to 10 mmol with respect to 1 g of the gelatin derivative,
The gelatin derivative according to claim 1, wherein a content of the methacryloyl glyceryl ester group satisfies a range of 0.01 mmol to 10 mmol with respect to 1 g of the gelatin derivative. The content of the methacryloyl group satisfies the range of 0.1 mmol to 1 mmol with respect to 1 g of the gelatin derivative,
The gelatin derivative according to claim 2, wherein a content of the methacryloyl glyceryl ester group satisfies a range of 0.1 mmol or more and 1 mmol or less with respect to 1 g of the gelatin derivative.   The gelatin derivative according to claim 1, wherein the gelatin is at least one selected from the group of animal bones, animal skins, fish bones, fish skins and fish scales. A method for producing the gelatin derivative according to claim 1,
A step of reacting gelatin with methacrylic anhydride and synthesizing a primary modified gelatin having a methacryloyl group bonded to an amino group of the gelatin;
Reacting the gelatin primary modification product with glycidyl methacrylate and synthesizing the gelatin derivative, which is a gelatin secondary modification product in which a methacryloylglyceryl ester group is bonded to a hydroxy group and a carboxy group of the gelatin primary modification product; Including the method.   In the step of synthesizing the gelatin primary modified product, a volume ratio of the methacrylic anhydride to the gelatin aqueous solution is 10000 to a gelatin aqueous solution having a gelatin concentration of 1 (w / v)% to 50 (w / v)%. The method according to claim 5, wherein the methacrylic anhydride is added so as to satisfy 1: 1 to 1: 1.   The step of synthesizing the gelatin secondary modified product includes the step of synthesizing the gelatin primary modified product in an aqueous gelatin primary modified product solution having a gelatin primary modified product concentration of 0.1 (w / v)% or more and 40 (w / v)% or less. The method according to claim 5, wherein the methacrylic anhydride is added so that a volume ratio of the glycidyl methacrylate to the aqueous solution satisfies 10,000: 1 to 1: 1.   The method according to claim 7, wherein the gelatin primary modified aqueous solution has a pH in the range of 2 to 5, or 6.5 to 8. A crosslinked gelatin hydrogel and a porous body thereof,
A cross-linked gelatin hydrogel containing the cross-linked product obtained by cross-linking the gelatin derivative according to claim 1 and a porous body thereof. Crosslink density of the gelatin derivatives have 0.02 mol / ^{m 3} or more 10000mol / ^{m 3} or less of the range, cross-linked gelatin hydrogel and a porous body according to claim 9.   The crosslinked gelatin hydrogel according to claim 9 and a porous body thereof, which encapsulate cells.   The cells are chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, adipocytes, neurons, vascular endothelial cells, smooth muscle cells, cardiomyocytes, epithelial cells, hepatocytes, pancreatic β cells, kidneys The crosslinked gelatin hydrogel according to claim 11, wherein at least one is selected from the group consisting of cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, neural stem cells, embryonic stem cells, and induced pluripotent stem cells. Gel and its porous body. A method for producing a crosslinked gelatin hydrogel comprising:
The method of bridge | crosslinking using the gelatin derivative in any one of Claims 1-4. Preparing an aqueous solution containing the gelatin derivative and a photopolymerization initiator;
And irradiating the aqueous solution with ultraviolet light. Further comprising a step of suspending cells in the aqueous solution obtained by the step of preparing the aqueous solution to prepare a cell suspension;
The method according to claim 14, wherein the step of irradiating the aqueous solution with ultraviolet rays irradiates the cell suspension with ultraviolet rays. A method for producing a crosslinked gelatin hydrogel porous body,
The method of bridge | crosslinking using the gelatin derivative and gel particle in any one of Claims 1-4. Preparing an aqueous solution containing the gelatin derivative and a photopolymerization initiator;
Preparing a mixture obtained by adding gel particles made of gelatin hydrogel to the aqueous solution obtained by the step of preparing the aqueous solution;
And irradiating the mixture with ultraviolet light.   The method according to claim 17, further comprising a step of preparing a cell suspension by suspending cells in the aqueous solution obtained by the step of preparing the aqueous solution.   The method according to claim 17 or 18, wherein the gel particles enclose cells.   The method according to any one of claims 15, 18 and 19, wherein the cell is an animal cell derived from an animal.