JP7050296B2 - Gelatin derivatives, crosslinked gelatin hydrogels and their porous bodies, and methods for producing them. - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Published on June 15, 2017 Website of Journal of Chemistry B, a journal published by The Royal Society of Chemistry <http: // pubs. rsc. org / en / content / articlelanding / 2017 / tb / c7tb01350g #! divAbstract>, <http: // pubs. rsc. org / en / content / articlepdf / 2017 / tb / c7tb01350g> DOI 10.1039 / C7TB01350G

The present invention relates to living tissues such as skin and bones, cartilage, ligaments, muscles, trachea, esophagus, nerves, blood vessels, bladder, heart, lungs, kidneys, pancreas, pancreas, and liver that have been damaged or lost due to diseases or accidents. -Gelatin derivatives, cross-linked gelatin hydrogels and their porous bodies, and their production, which enclose cells that differentiate and organize into those biological tissues / organs and promote tissue regeneration in order to repair the organs. It's about the method.

Living organisms such as skin, bones, cartilage, ligaments, muscles, trachea, esophagus, nerves, blood vessels, bladder, heart, lungs, kidneys, pancreas, pancreas, and liver that have been damaged or lost due to accidents or illnesses. Traditionally, artificial organs and organ transplants have been used to repair and treat tissues and organs. However, in the case of artificial organs, there are problems that the functions are not sufficient as compared with natural organs, and that the artificial organs are worn, loosened, and damaged. In the case of living organ transplantation, in addition to the problem of a shortage of donors, when the donor is another person, there are considerable medical risks such as the problem of rejection caused by an immune response 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 method of biological tissue engineering is now ideal because it does not require a donor as compared with living organ transplantation. It is believed that.

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

As the hydrogel for tissue regeneration, collagen gel, gelatin gel, hyaluronic acid gel, fibrin gel, alginate gel and the like are used. Examples of 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 at a low temperature (for example, 4 ° C.) in a sol state and reaches a body temperature of 37 ° C. to form a hydrogel. Alginic acid is a sol in the absence of calcium ions, and when calcium ions are added, it gels to form a hydrogel. Further, in the case of gelatin, a methacryloyl group capable of crosslink 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 hydrogels are not sufficient (for example, Non-Patent Document 1). In order to produce a hydrel gel porous body, a hydrogel having high strength is required.

In addition, 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 pore-forming agents. However, until now, high-concentration calcium ions, organic solvents, edible oils, and the like have been used to produce alginic acid gel particles and gelatin gel particles (for example, Non-Patent Documents 2 and 3). However, since there is a concern that these production conditions may reduce the viability of the contained cells, a method for producing gel particles that can minimize the effect on the cells is desired. Further, 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 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, since the methacryloyl group was introduced by reacting only the amino group of gelatin with anhydrous metamethacrylic acid, ultraviolet rays were introduced. Cross-linking by irradiation The cross-linking density by polymerization is low, and the mechanical strength of the formed hydrogel is still low. In particular, in order to produce a hydrogel porous body, a bulk hydrogel having high mechanical strength is required in order to maintain the structure and shape of the formed hydrogel porous body.

Further, in order to prepare the unmodified gelatin gel particles used for producing the hydrogel porous body, a solvent or liquid in which gelatin is not dissolved such as an organic solvent or oil has been used. When an organic solvent or oil is used, there is a concern that the residual organic solvent or oil may adversely affect the cells. Further, when the cells are encapsulated in the gelatin gel particles, the suspension of the cells and the gelatin aqueous solution is dropped or added to an organic solvent or oil to prepare an emulsion, and the cells are purchased with an organic solvent or oil. There is concern that it will adversely affect abilities. A method for producing gelatin gel particles containing cells that does not affect the activity of cells is desirable.

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

In the gelatin derivative in which the reactive group is introduced into the gelatin according to the present invention, 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 the range of 0.01 mmol or more and 10 mmol or less with respect to 1 g of the gelatin derivative, and the content of the methacryloyl glyceryl ester group is 0.01 mmol or more and 10 mmol or less with respect to 1 g of the gelatin derivative. The range may be met.
The content of the methacryloyl group satisfies the range of 0.1 mmol or more and 1 mmol or less with respect to 1 g of the gelatin derivative, and the content of the methacryloyl glyceryl ester group is 0.1 mmol or more and 1 mmol or less with respect to 1 g of the gelatin derivative. The range may be met.
The gelatin may be selected from at least one group of animal bones, animal skins, fish bones, fish skins and fish scales.
The method for producing the above-mentioned gelatin derivative according to the present invention comprises a step of reacting gelatin with a methacrylic acid 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. It comprises a step of reacting a body with glycidyl methacrylate to synthesize the gelatin derivative which is a gelatin secondary modified product in which a methacryloyl glyceryl 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 primary modified gelatin, the volume ratio of the methacrylic anhydride to the aqueous gelatin solution is 10,000 to the aqueous solution of gelatin having a gelatin concentration of 1 (w / v)% or more and 50 (w / v)% or less. The methacrylic anhydride may be added so as to satisfy 1: 1 to 1: 1.
In the step of synthesizing the gelatin secondary modified product, the gelatin primary modified product is added to an aqueous solution of the gelatin primary modified product having a gelatin primary modified product concentration of 0.1 (w / v)% or more and 40 (w / v)% or less. The methacrylic acid anhydride may be added so that the volume ratio of the glycidyl methacrylate to the aqueous solution satisfies 10000: 1 to 1: 1.
The gelatin primary modified aqueous solution may have a pH in the range of 2 or more and 5 or less, or 6.5 or more and 8 or less.
The crosslinked gelatin hydrogel and its porous body according to the present invention contain a crosslinked body in which the above-mentioned gelatin derivative is crosslinked, thereby solving the above-mentioned problems.
The crosslink density of the gelatin derivative may be in the range of 0.02 mol / m ³ or more and 10,000 mol / m ³ or less.
It may contain cells.
The cells include cartilage cells, osteoblasts, fibroblasts, myoblasts, ligament cells, fat cells, nerve cells, vascular endothelial cells, smooth muscle cells, myocardial cells, epithelial cells, hepatocytes, pancreatic β cells, and kidneys. At least one may be selected from the group consisting of cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, nerve stem cells, embryonic stem cells, and artificial pluripotent stem cells.
The method for producing a crosslinked gelatin hydrogel according to the present invention solves the above-mentioned problems by cross-linking using the above-mentioned gelatin derivative.
A step of preparing an aqueous solution containing the gelatin derivative and a photopolymerization initiator and a step of irradiating the aqueous solution with ultraviolet rays may be included.
The step of suspending cells in the aqueous solution obtained by the step of preparing the aqueous solution to prepare a cell suspension is further included, and the step of irradiating the aqueous solution with ultraviolet rays irradiates the cell suspension with ultraviolet rays. It may be irradiated.
The method for producing a crosslinked gelatin hydrogel porous body according to the present invention solves the above-mentioned problems by cross-linking using the above-mentioned gelatin derivative and gel particles.
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. May include the step of irradiating the polymer with ultraviolet rays.
A step of suspending cells in the aqueous solution obtained by the step of preparing the aqueous solution to prepare a cell suspension may be further included.
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 the reactive group capable of cross-linking polymerization by ultraviolet irradiation is introduced to the maximum extent. ing. Since the crosslinked gelatin hydrogel of the present invention and its porous body are produced using the gelatin derivative of the present invention, the crosslink density is high. As a result, the mechanical strength is high and the shape stability is excellent. Moreover, since cells can be seeded in both bulk and pores of hydrogel, more efficient tissue regeneration is possible.

Since the above-mentioned gelatin derivative is used in the method for producing a crosslinked gelatin hydrogel and a porous body thereof of the present invention, a crosslinked gelatin hydrogel having a high crosslinking density, high mechanical strength, and excellent shape stability and a porous body thereof are provided. can. Further, under mild and non-cytotoxic conditions, a crosslinked gelatin hydrogel having a high crosslink density containing cells, a high mechanical strength, and excellent shape stability and a porous body thereof can be easily produced.

The figure which shows typically the gelatin derivative of this invention. Flow chart for producing the gelatin derivative of the present invention A flowchart showing a procedure of a crosslinked gelatin hydrogel obtained by using the gelatin derivative of the present invention and a porous body thereof. The figure which shows the synthesis step (a) of the gelatin primary modification and the synthesis step (b) of the gelatin secondary modification. The figure which shows the ¹ H NMR spectrum of the gelatin primary modifier. The figure which shows the ¹ H NMR spectrum of the gelatin secondary modifier. Relationship between the storage elastic modulus (G') and the temperature loss elastic modulus (G ") of a 10 (w / v%) gelatin secondary modified aqueous solution. The figure which shows the fluorescence microscope image of the life-and-death staining of the cross-linked gelatin hydrogel containing the chondrocyte according to 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 body according to Example 4. The figure which shows the fluorescence microscope image of the life-and-death staining of a cross-linked gelatin hydrogel porous body containing a chondrocyte according to Example 4. Photograph showing the appearance of cartilage tissue regenerated in the crosslinked gelatin hydrogel porous body 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 body according to Example 5. The figure which shows the fluorescence microscope image of the life-and-death staining of a cross-linked gelatin hydrogel porous body containing a chondrocyte according to Example 5. Photograph showing the appearance of cartilage tissue regenerated with the crosslinked gelatin hydrogel porous body according to Example 5. Photograph showing the appearance of the crosslinked gelatin hydrogel porous body according to Example 6. The figure which shows the fluorescence microscope image of the life-and-death staining of the cross-linked gelatin hydrogel porous body containing the chondrocyte according to Example 6. Photograph showing the appearance of cartilage tissue regenerated in the crosslinked gelatin hydrogel porous body according to Example 6.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(Embodiment 1)
In the first embodiment, a gelatin derivative which is preferable for producing a crosslinked gelatin hydrogel having a high crosslink density and a 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. Both the methacryloyl group 120 and the methacryloyl glyceryl ester group 130 are reactive groups capable of crosslink polymerization, and if a crosslinked gelatin hydrogel is produced using the gelatin derivative 100 of the present invention, high crosslink density and high dynamics can be obtained. It is possible to provide a crosslinked gelatin hydrogel that achieves the desired strength and has excellent shape stability. Further, if a pore-forming agent is used in the production of the crosslinked gelatin hydrogel, it is possible to provide a crosslinked gelatin hydrogel porous body which achieves high crosslink density and high mechanical strength and has excellent shape stability. Further, if cells are encapsulated at the time of producing the crosslinked gelatin hydrogel, and / or if a pore-forming agent containing cells is used, a crosslinked gelatin hydrogel containing cells and a porous body thereof can be provided.

The gelatin derivative 100 of the present invention preferably satisfies the range in which the content of the methacryloyl group 120 with respect to 1 g of the gelatin derivative is 0.01 mmol or more and 10 mmol or less, and the content of the methacryloyl glyceryl ester group 130 with respect to 1 g of the gelatin derivative is 0.01 mmol. It satisfies the range of 10 mmol or less. Thereby, the cross-linking strength and the mechanical strength can be improved.

More preferably, the gelatin derivative 100 of the present invention satisfies the range in which the content of the methacryloyl group 120 with respect to 1 g of the gelatin derivative is 0.1 mmol or more and 1 mmol or less, and the content of the methacryloyl glyceryl ester group 130 with respect to 1 g of the gelatin derivative is 0. It satisfies the range of 1 mmol or more and 1 mmol or less. Thereby, the cross-linking strength and the mechanical strength can be surely improved.

The above gelatin 110 is derived from any one material selected from a group of tissues such as bones and skins of animals such as pigs, cows, sheep and chickens, fish bones, fish skins and fish scales, or a plurality of materials. It is gelatin. The molecular weight of the gelatin molecule is preferably in the range of 100 daltons or more and 10,000,000 daltons or less, and if it is in this range, it dissolves in water and forms a hydrogel. The molecular weight of the gelatin molecule is more preferably in the range of 500 daltons or more and 1,000,000 daltons or less. This is particularly advantageous for the formation of hydrogels having a stable shape and diffusivity of nutrients and waste products.

The presence of the methacryloyl group 120 bonded to the amino gas 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. Further, the content of the methacryloyl group and the methacryloyl glyceryl ester group can be calculated from the integrated value of the NMR signal.

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

The method for producing the gelatin derivative 100 of the present invention will be described with reference to FIG. Hereinafter, for the sake of clarity, the gelatin derivative will be referred to as a secondary gelatin modified product. This is because, as will be described later, the gelatin derivative of the present invention is obtained by modifying (introducing) a reactive group in gelatin in two steps.

The method for producing a secondary gelatin modifier of the present invention is a step of reacting gelatin with a methacrylate anhydride to synthesize a primary gelatin modifier in which a methacryloyl group is bonded to an amino group of gelatin (step S210 in FIG. 2). , And a step of reacting a gelatin primary modified product with glycidyl methacrylate to synthesize a gelatin secondary modified product in which a methacryloyl glyceryl ester group is bonded to a hydroxy group and a carboxyl group of the gelatin primary modified product (step in FIG. 2). It is characterized by including S220).

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

Then, while stirring the gelatin aqueous solution, methacrylic anhydride is added, and the reaction product is reacted in a dark place for several hours at a temperature at which the product (here, the primary modified gelatin) is dissolved. Here, the addition rate of the methacrylic anhydride 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 0.1-5.0 mL / min. The volume charging ratio of the methacrylic anhydride added to the aqueous gelatin solution is preferably 10000: 1 to 1: 1. Within this range, it is possible to modify the amino group of gelatin. A more desirable charging ratio is 100: 1 to 2: 1. The reaction temperature is 30 ° C to 80 ° C, but the desired temperature is 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.

Then, the reaction product is diluted with pure water at a temperature at which the reaction product and the product are dissolved, and dialyzed in pure water while maintaining the temperature at which the reaction product and the product are dissolved using a dialysis membrane. As a result, 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 the pure water for dilution is maintained at a temperature at which the reaction product and the product can be maintained as a solution, preferably 30 ° C. to 80 ° C., but a desirable temperature is 35 ° C. to 60 ° C. The molecular weight cut-off of the dialysis membrane used for purifying the product may be as long as the synthesized gelatin primary modifier can be separated from the unreacted methacrylic anhydride and small molecules of salts.

Then, the purified aqueous solution of the primary gelatin modifier may be freeze-dried to obtain a solid substance of the primary gelatin modifier. The freeze-drying time is not particularly limited as long as it can remove water from the frozen product, but is exemplifiedly 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 contained in gelatin can be obtained.

The process of synthesizing the secondary gelatin modified product will be described in detail. First, the primary gelatin modifier is dissolved in warm water to prepare an aqueous solution, and then the pH of the aqueous solution is adjusted. Ion-exchanged water, distilled water, or ultrapure water can be used as the warm water for dissolving the gelatin primary modifier. The water temperature is 25 ° C to 80 ° C, which is the temperature at which the gelatin primary modifier is melted, but the desirable temperature is 35 ° C to 55 ° C. The concentration of the gelatin primary modified aqueous solution obtained by dissolving the gelatin primary modified in warm water is 0.1 to 40 (w / v)%, and the desirable concentration is 0.2 to 20 (w / v)%. Within this range, the fluidity is advantageous for a uniform modification reaction. Hydrochloric acid, nitric acid, and sulfuric acid are examples of the solution used for adjusting the pH of the gelatin primary modified aqueous solution, but hydrochloric acid is preferable. The concentrations of hydrochloric acid, nitric acid and sulfuric acid for pH adjustment are 0.1 to 5 M, preferably 0.2 to 2 M. The pH of the aqueous solution is preferably adjusted to the range of 1.0-10. Within this range, a reaction with glycidyl methacrylate, which will be described later, occurs. The desirable pH is in the range of 2 to 5 or 6.5 to 8.

Next, glycidyl methacrylate is added to the aqueous solution of the primary modified gelatin, and the reaction is reacted in a dark place for several hours at a temperature at which the reaction product and the product (here, the secondary modified gelatin) are dissolved. The addition rate of the above-mentioned 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 0.1-5.0 mL / min. The volume charging ratio of glycidyl methacrylate to be added to the gelatin primary modified aqueous solution is preferably 10000: 1 to 1: 1. This range allows modification of the hydroxyl and carboxyl groups of gelatin. A more desirable charging ratio is 250: 1 to 3: 1. The reaction temperature is 30 ° C to 80 ° C, but the desired 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.

Then, the reaction solution is diluted with water at a temperature at which the reaction product and the product are dissolved, and dialyzed in pure water while maintaining the temperature at which the reaction product and the 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 water for dilution. The temperature of the water for dilution is maintained at a temperature at which the reaction product and the product can be maintained as a solution, preferably 30 ° C. to 80 ° C., but a desirable temperature is 35 ° C. to 60 ° C. The molecular weight cut-off of the dialysis membrane used for purifying the product may be within the range in which the synthesized gelatin secondary modified product can be separated from the unreacted glycidyl methacrylate, the unreacted gelatin primary modified product, and small molecules of salts.

Then, the purified aqueous solution of the secondary gelatin modification may be freeze-dried to obtain a solid substance of the secondary gelatin modification. The freeze-drying time is not particularly limited as long as it can remove water from the frozen product, but is exemplifiedly 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 the carboxyl group of the gelatin primary modified product can be 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 that do not contain cells The method for producing gel particles that function as a pore-forming agent is a step of producing gelatin hydrogel at a low temperature, and the obtained gelatin hydrogel is used as gelatin hydrogel particles (1). For example, it is characterized by including a step of cutting into (cubic particles).

The process of producing the gelatin hydrogel will be described in detail. The gelatin is dissolved in warm water at a temperature at which the gelatin dissolves, phosphate buffered saline, HEPES buffer, saline, a medium for cell culture, or a mixed solution of phosphate buffered saline and the medium to prepare an aqueous gelatin solution. The temperature of the above gelatin aqueous solution is 37 ° C to 80 ° C, and the desirable temperature is 40 ° C to 60 ° C. Again, gelatin is derived from tissues such as bone and skin of animals such as pigs, cows, sheep and chickens, any one material selected from the group of fish bones, fish skins and fish scales, or multiple materials. It is gelatin. The molecular weight of the gelatin molecule is preferably in the range of 100 daltons or more and 10,000,000 daltons or less, and if it is in this range, it dissolves in water and forms a hydrogel. The molecular weight of the gelatin molecule is more preferably in the range of 500 daltons or more and 1,000,000 daltons or less. This is particularly advantageous for the formation of hydrogels having a stable shape and diffusivity of nutrients and waste products.

The above medium is a medium used for cell culture, and may be one or several mixed media such as Dulbecco's modified Eagle's medium and Eagle's minimum essential medium. The medium may or may not contain serum. When serum is included, the serum concentration is 0.1-50% and the desired concentration is 0.5-20%. In the case of the above-mentioned mixed solution of phosphate buffered saline and the medium, the volume mixing ratio is 50: 1 to 1:50, and the desired volume mixing ratio is 10: 1 to 1:10. The concentration of the above gelatin aqueous solution is preferably 0.1 to 50 (w / v)%. Within this range, hydrogels 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 filtration sterilization. The pore size of the filter membrane for filtration sterilization of the above gelatin aqueous solution is 0.1 to 0.5 μm, and the pore size of the desired filter membrane is 0.1 to 0.3 μm.

The filtered sterilized gelatin aqueous solution is gelled at a low temperature to form a gelatin hydrogel. The temperature at which the above-mentioned gelatin aqueous solution is gelled is not particularly limited as long as the gelatin aqueous solution can be gelled, but is preferably a low temperature in the range of 0.5 ° C. to 30 ° C. This allows the formation of hydrogels. The desirable temperature is 3 ° C to 15 ° C. In this way, gelatin hydrogel is produced. In the present embodiment, the low temperature is intended in the above range.

Next, the process of cutting the gelatin hydrogel will be described in detail. The formed gelatin hydrogel is sliced thinly to obtain a thin gelatin hydrogel sheet. Alternatively, an aqueous gelatin solution is pipetted into a frame space with a low frame space and a large 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, preferably 50 μm to 1000 μm. A cutter or thread can be used to cut the gelatin hydrogel into thin slices. A silicone rubber frame, a glass frame, a plastic frame, or the like can be used as the frame space when forming a thin gelatin hydrogel sheet in the above frame space.

Next, a thin sheet of gelatin hydrogel is shredded to prepare cubic particles of gelatin hydrogel. To shred the formed gelatin hydrogel sheet, a cutter, thread or mesh may be used. Nylon mesh or stainless 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 desirable mesh size is 50 μm × 50 μm to 1000 μm × 1000 μm. The length of one side of the cube particles of the above gelatin hydrogel may be 20 μm to 2000 μm, but the desirable mesh size is 50 μm to 1000 μm. In this way, cubic particles made of gelatin hydrogel are produced. The cube particles thus obtained are made of unmodified gelatin (also referred to as pure gelatin) in which the gelatin used as a raw material is not intentionally modified.

(2) Method for producing gel particles containing cells The method for producing gel particles containing cells that function as a pore-forming agent is a step of producing a gelatin hydrogel containing cells at a low temperature, and the obtained cells. It is characterized by including a step of cutting the encapsulated gelatin hydrogel into particles (for example, cubic particles). When the cube particles of gelatin hydrogel containing cells are used as a pore-forming agent, pores are formed inside the bulk hydrogel and cells remain in the formed pores.

The process of producing a gelatin hydrogel containing cells will be described in detail. Since the step of sterilizing the gelatin aqueous solution with the filter membrane is the same as the step of producing the gelatin hydrogel of (1) above, the description thereof will be omitted. The filtered sterilized gelatin aqueous solution is lowered to the lowest possible temperature at a temperature at which the gelatin aqueous solution does not gel. Here, the temperature as low as possible at the above-mentioned non-gelling temperature is 37 ° C. At this temperature, gelation can be prevented.

The cells are then suspended in a cold gelatin solution. 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 above gelatin aqueous solution is preferably in the range of 10 ³ cells / mL to ¹⁰⁸ cells / mL. Within this range, uniform cell distribution and efficient growth are possible. More desirable concentrations are 10 ⁴ cells / mL to 5 × 10 ⁸ cells / mL. The temperature at which the cell suspension of the above gelatin aqueous solution is gelled is 0.5 ° C to 30 ° C, but the desirable temperature is 3 ° C to 15 ° C. In this way, a gelatin hydrogel containing cells is produced.

As the above cells, cells derived from humans or other animals can be used. Chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, fat cells, nerve cells, vascular endothelial cells, smooth muscle cells, myocardial cells, 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, nerve stem cells, embryonic stem cells, and artificial pluripotent stem cells can be encapsulated in gelatin hydrogel.

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

The cube particles made of gelatin hydrogel that do not / contain cells thus obtained function as a granulator, and therefore, when used together with the gelatin derivative of the present invention described in the first embodiment, they contain cells. It is possible to provide a crosslinked gelatin hydrogel porous body which does not / contains.

Although it has been described here that the gelatin hydrogel that does not contain / contains cells is cut into cube particles, the shape after cutting is not limited to the 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. In this case as well, the gelatin hydrogel is cut so that the major axis of the particles is 20 μm to 2000 μm.

(Embodiment 3)
In the third embodiment, the gelatin derivative (gelatin secondary modified product) described in the first embodiment and, if necessary, the gel particles (encapsulating / not encapsulating cells) described in the second embodiment are used for cross-linking. Gelatin hydrogel and its porous body, and a method for producing them will be described.

FIG. 3 is a flowchart showing a procedure of 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 the first embodiment), a cross-linked gelatin hydrogel 310 that does not contain cells, a cross-linked gelatin hydrogel 320 that contains cells, and a cross-linked gelatin hydro that does not contain cells are used. A gel porous body 330 and a crosslinked gelatin hydrogel porous body 340 containing cells can be obtained.

All of the crosslinked gelatin hydrogels and their porous bodies 310, 320, 330, and 340 have high crosslink density and high mechanical strength, and thus are excellent in shape stability. Illustratively, the crosslinked gelatin hydrogel and its porous bodies 310, 320, 330, 340 have a crosslinked density in the range of 0.02 mol / m ³ or more and 10,000 mol / m ³ or less. If the crosslink density is in this range, the shape stability is excellent. More preferably, the crosslink density has a range of 0.1 mol / m ³ or more and 4000 mol / m ³ or less. Further, 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 or more and 10 kPa or less, and have high mechanical strength. The crosslink 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, by using the gelatin derivative 100 of the present invention, cells can be seeded in the bulk of the crosslinked gelatin hydrogel, in the pores of the hydrogel, or both, so that more efficient tissue regeneration is possible. To. The cross-linked gelatin hydrogel 320 or its porous body 340 containing cells may be cut into an appropriate size and used as a transplant, or the tissue may be regenerated by culturing in a medium or by transplanting into an animal. You may.

Next, methods for producing the crosslinked gelatin hydrogel and its porous bodies 310, 320, 330, and 340 will be described.
(1) Method for producing crosslinked gelatin hydrogel 310 that does not encapsulate 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 steps of preparing an aqueous solution of the gelatin secondary modifier and the photopolymerization initiator will be described in detail. The aqueous solution is kept 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, a medium for cell culture, or a mixed solution of phosphate buffered saline and a medium can be used. The above-mentioned medium is a medium used for cell culture, and may be one or several mixed media such as Dulbecco's modified Eagle's medium and Eagle's 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 above-mentioned mixed solution of 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 modifier is 0.1 to 50 (w / v)% in terms of the concentration of the weight of the gelatin secondary modifier to the volume of the solution. This promotes gelation. The desired concentration is 0.5-30 (w / v)%.

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

Then, the aqueous solution containing the gelatin secondary modifier and the photopolymerization initiator is sterilized with a filter membrane for filtration sterilization. The pore size of the filter membrane for filtration sterilization of the above gelatin aqueous solution is 0.1 to 0.5 μm, and the pore size of the desired filter membrane is 0.1 to 0.3 μm.

Next, the step of irradiating this aqueous solution with ultraviolet rays will be described in detail. The prepared aqueous solution is kept 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. As a result, the aqueous solution is exposed, the cross-linking polymerization reaction is started, and gelation occurs. Here, the irradiation condition of ultraviolet rays is preferably 10 seconds to 30 minutes, and preferably 20 seconds to 10 minutes. In the case of producing a crosslinked gelatin hydrogel 310 having a constant thickness, the gelatin secondary modifier and the photopolymerization initiator are formed in a space formed between two quartz cover glasses separated by a space frame having a constant thickness. The aqueous solution of the above may be injected and irradiated with ultraviolet rays.

In this way, a cell-free crosslinked gelatin hydrogel 310 is obtained. Alternatively, the crosslinked gelatin hydrogel 310 may be cut to a required size to allow cells to 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 is 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 is characterized by including 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 modifier and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) above, the description thereof will be omitted. In the step of preparing a cell suspension in which cells are suspended in the obtained aqueous solution, the aqueous solution of the gelatin secondary modifier and the photopolymerization initiator is sterilized with a filter membrane for filtration sterilization, and then the temperature of the aqueous solution is adjusted. Keep 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 low-temperature gelatin secondary modifier and a photopolymerization initiator to prepare a cell suspension.

The cell concentration of the above cell suspension is preferably in the range of 10 ³ cells / mL to ¹⁰⁸ cells / mL. Within this range, uniform cell distribution and efficient growth are possible. More desirable concentrations are 10 ⁴ cells / mL to 5 × 10 ⁸ cells / mL.

As the above cells, cells derived from humans or other animals can be used. Chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, fat cells, nerve cells, vascular endothelial cells, smooth muscle cells, myocardial cells, 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, nerve stem cells, embryonic stem cells, and artificial pluripotent stem cells can be encapsulated in gelatin hydrogel.

Next, the cell suspension is irradiated with ultraviolet rays to carry out cross-linking polymerization. Since the step of irradiating the cell suspension with ultraviolet rays is the same as the step of irradiating the aqueous solution of (1) above with ultraviolet rays, the description thereof will be omitted. In this way, a crosslinked gelatin hydrogel 320 containing cells is obtained. Again, in the case of producing a crosslinked gelatin hydrogel 320 having a certain thickness, the cell suspension is injected into the space created between two quartz cover glasses separated by a space frame having a certain thickness. , Ultraviolet rays may be irradiated.

(3) Method for Producing Crosslinked Gelatin Hydrogel Porous 330 that Does Not Enclose Cells (Procedure C in FIG. 3)
The method for producing the crosslinked gelatin hydrogel porous body 330 is 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). It is characterized by including a step of preparing a mixture (cubic particles made of gelatin hydrogel) which does not encapsulate the cells, and a step of irradiating the mixture with ultraviolet rays.

Since the step of preparing the aqueous solution of the gelatin secondary modifier and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) above, the description thereof will be 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 modifier and the photopolymerization initiator is sterilized with a filter membrane for filtration sterilization, and then the temperature of the aqueous solution is lowered as much 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. Gel particles are added to an aqueous solution of a low-temperature gelatin secondary modifier and a photopolymerization initiator to prepare a mixture. The mixing ratio of the above-mentioned secondary gelatin modifier, the aqueous solution of the photopolymerization initiator 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 /). It is mixed so as to satisfy w). As a result, a crosslinked gelatin hydrogel porous body having a desired porous structure can be obtained. The mixing ratio is preferably 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 this mixture with ultraviolet rays is the same as the step of irradiating the aqueous solution of (1) above with ultraviolet rays, but the gel particles (that is, pure gelatin) are higher than the gelation temperature of the gelatin derivative of the present invention. ) Is kept at a temperature (25 ° C. in the example) so as to be lower than the gelation temperature, and the gelatin derivative of the present invention is crosslinked and polymerized. After cross-linking polymerization, when the product is above the sol-gel transition point (37 ° C) of the gel particles (pure gelatin), the hydrogen bonds between the pure gelatin molecules are broken and only the gel particles are dissolved, so that a porous structure is formed. Will be 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 of a certain thickness, the above mixture is injected into a space formed between two quartz cover glasses separated by a space frame of a certain thickness. It suffices to irradiate with ultraviolet rays.

(4) Method for producing cross-linked gelatin hydrogel porous body 340 containing cells The cross-linked gelatin hydrogel porous body 340 is produced by three different procedures D to F as shown in FIG.

(A) The manufacturing method of the procedure D of FIG. 3 will be described.
The method for producing the crosslinked gelatin hydrogel porous body 340 is 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 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 the second embodiment) 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 modifier and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) 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 of (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) above. The step of irradiating the obtained mixture with ultraviolet rays is the same as the step of irradiating the mixture of the above-mentioned (3) with ultraviolet rays. Therefore, the description of the above steps will be omitted.

By combining such steps, a crosslinked gelatin hydrogel porous body 340 containing cells can be obtained. Again, in the case of producing a crosslinked gelatin hydrogel porous body 340 having a certain thickness, the above mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a certain thickness. Then, it is sufficient to irradiate with ultraviolet rays.

(B) The manufacturing method of the procedure E of FIG. 3 will be described.
The method for producing the crosslinked gelatin hydrogel porous body 340 is 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). It is characterized by including a step of preparing a mixture (cubic particles made of gelatin hydrogel) containing gelatin hydrogels, and a step of irradiating the mixture with ultraviolet rays.

Here, the step of preparing the aqueous solution containing the gelatin secondary modifier and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) above. The step of preparing a mixture in which gel particles are added 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 containing cells is the cell concentration. The concentration is preferably in the range of 10 ³ cells / mL to 10 ⁸ cells / mL. More desirable concentrations are 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 of the above-mentioned (3) with ultraviolet rays. Therefore, the description of the above steps will be omitted.

By combining such steps, a crosslinked gelatin hydrogel porous body 340 containing cells can be obtained. Again, in the case of producing a crosslinked gelatin hydrogel porous body 340 having a certain thickness, the above mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a certain thickness. Then, it is sufficient to irradiate with ultraviolet rays.

(C) The manufacturing method of the procedure F of FIG. 3 will be described.
The method for producing the crosslinked gelatin hydrogel porous body 340 is 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 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 containing the cells described in the second embodiment) 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 modifier and the photopolymerization initiator is the same as the step of preparing the aqueous solution of (1) 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 of (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) above. The step of irradiating the obtained mixture with ultraviolet rays is the same as the step of irradiating the aqueous solution of (1) above with ultraviolet rays. Therefore, the description of the above steps will be omitted.

By combining such steps, a crosslinked gelatin hydrogel porous body 340 containing cells can be obtained. Again, in the case of producing a crosslinked gelatin hydrogel porous body 340 having a certain thickness, the above mixture is injected into a space formed between two quartz cover glasses separated by a space frame having a certain thickness. Then, it is sufficient to irradiate with ultraviolet rays.

Hereinafter, the present invention will be described in detail with reference to specific examples, but the present invention is not limited to these examples.

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

FIG. 4 is a diagram showing a step of synthesizing a primary gelatin modifier (a) and a step of synthesizing a secondary gelatin modifier (b).

According to step S210 shown in FIG. 2, a gelatin primary modifier (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 with 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 with stirring at 50 ° C., and the mixture was reacted in the dark for 3 hours. Here, the volume-filling ratio of methacrylic anhydride to the aqueous gelatin solution was 10: 1. The reaction product was diluted 5-fold with PBS at 50 ° C. and dialyzed against ultrapure water at 40 ° C. using a dialysis membrane (fractional molecular weight of 12,000 to 14,000 daltons) for 7 days to obtain salts and non-salts. 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 1} H NMR spectra were measured using an NMR spectrometer with a frequency of 300 MHz and a single axial gradient reverse probe. Prior to measurement, the reaction product was placed in 1 mL of heavy water containing 0.05 (w / v)% 3- (trimethylsilyl) propionic acid-2,2,3,3- _d sodium tetraate salt of the internal standard at 40 ° C. Melted with. 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 5.4 ppm and 5.7 ppm signals in FIG. 5 were assigned to the vinyl protons 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 bound to an amino group.

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

According to step S220 shown in FIG. 2, a gelatin secondary modifier (denoted as GelMAGMA in FIG. 4 (b)) was synthesized based on the synthesis procedure of FIG. 4 (b). Specifically, the previously obtained gelatin primary modifier was reacted with glycidyl methacrylate. First, 2.5 g of the primary gelatin modifier 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 the reaction was carried out at 50 ° C. for 24 hours. Here, the volume-added ratio of glycidyl methacrylate to the aqueous solution of the primary modified gelatin was 25: 1.

The obtained reaction product was diluted 5-fold with PBS at 50 ° C. and dialyzed against ultrapure water at 40 ° C. using a dialysis membrane (molecular weight cut off of 12,000 to 14,000 daltons) for 7 days to obtain salts and the like. 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 The 1 H-NMR spectrum was measured using an NMR spectrometer having a frequency of 300 MHz and a single axial gradient reverse probe. Prior to measurement, the reaction product was placed in 1 mL of heavy water containing 0.05 (w / v)% 3- (trimethylsilyl) propionic acid-2,2,3,3- _d sodium tetraate salt of the internal standard at 40 ° C. Melted with. 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 secondary gelatin 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. Attributed. From this, 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 the vinyl group-derived protons in the methacrylate group of the gelatin secondary modifier is about twice the value of the vinyl protons in the methacrylate group of the gelatin primary modifier of Example 1, and more photoreactions occur. It was found that a sex methacrylate group was introduced. From the NMR spectrum, the contents of the methacrylate group and the methacryloylglyceryl ester group were 0.35 mmol and 0.70 mmol, respectively, in terms of 1 g of the gelatin secondary modified product.

The purified gelatin secondary modification was freeze-dried for 7 days to obtain a solid substance. In the following examples, the obtained solid matter 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 secondary gelatin 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) was performed with a constant amplitude (γ = 5%) and frequency (1 Hz), and the storage elastic modulus. (G') and the loss elastic modulus (G ″) were measured. The storage elastic modulus (G ′) and the loss elastic modulus (G ″) of the gelatin secondary modified aqueous solution show the temperature dependence in FIG. 7.

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

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 of the gelatin secondary modified aqueous solution from the sol to the gel was 22.7 ° C. The transition temperature of the sol-to-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 in FIG. 3 using the gelatin secondary modifier synthesized in Example 1.

Specifically, a secondary gelatin modifier (10%, w / v) and a photopolymerization initiator (2-hydroxy-1- (4- (hydroxyethoxy) phenyl) -2-methyl l-1-propanol) (0). An aqueous solution was prepared by dissolving 5.5%, w / v) in a PBS solution at 25 ° C. This aqueous solution was sterilized with a filter membrane for filtration sterilization (pore size: 0.22 μm). After sterilization, these aqueous solutions are pipetted into the gap between two quartz cover glasses separated by a 1.5 mm thick silicone rubber frame, kept at 25 ° C, and crosslinked by irradiating with ultraviolet rays for 5 minutes. A gelatin hydrogel was prepared.

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

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

First, primary chondrocytes isolated from calf knee articular cartilage were cultured at 37 ° C. and 5% CO ₂ . For culture, 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 sodium pyruvate, 50 μg / Dalvecco's modified Eagle's medium (DMEM) supplemented with mL ascorbic acid and a 75 cm ² tissue culture flask were used. The medium was changed every 3 days. It was used in an experiment in which chondrocytes that had been subcultured once were encapsulated in hydrogel. Chondrocytes that reached confluence were exfoliated with a trypsin / EDTA solution, the cells were collected by centrifugation, and the number of cells was counted with a blood cell calculator.

Next, an aqueous solution of the gelatin secondary modifier (10%, w / v) and the photopolymerization initiator (0.5%, w / v) was prepared and prepared in the same procedure as in Example 2. The chondrocytes were suspended to prepare a cell suspension of chondrocytes containing a gelatin secondary modifier and a photopolymerization initiator. Here, the cell concentration of the cell suspension was 2 × ¹⁰⁷ cells / mL. The cell suspension was kept at 25 ° C. The cell suspension was pipetted into a space created 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 containing 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 rate of 60 rpm. The medium was changed every 2 days.

Live cells in the cross-linked gelatin hydrogel were stained with calcein-AM, and dead cells were stained with propidium iodide to evaluate the viability of chondrocytes. Immediately after preparation of the cross-linked gelatin hydrogel and after culturing for 2 weeks, the disk-shaped cross-linked gelatin hydrogel disk containing cells was washed 3 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 scanning microscope. The results are shown in FIG.

FIG. 8 is a diagram showing a fluorescence microscope image of life-and-death staining of cells in a cross-linked 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 culturing for 2 weeks. FIG. 8 is shown in grayscale, where 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 encapsulation of the cells, that is, immediately before culturing, but not after culturing for 2 weeks. Some chondrocytes aggregated to form small aggregates. From these results, there was no clear effect on the viability of the cells even if the gelatin secondary modifier was cross-linked and polymerized with the cells encapsulated. Since the obtained cross-linked gelatin hydrogel has high cell compatibility, it was shown that this method is suitable for producing a cross-linked gelatin hydrogel containing chondrocytes.

<Example 4>
In Example 4, a crosslinked gelatin hydrogel (340 in FIG. 3) encapsulating chondrocytes was produced using the gelatin secondary modifier synthesized in Example 1 based on procedure D in FIG. In Example 4, hydrogel rectangular parallelepiped particles made of pure unmodified gelatin without encapsulating cells were used as a pore-forming agent for the crosslinked gelatin hydrogel porous body.

Unmodified gelatin (pure gelatin) undergoes a gel-sol transition at 37 ° C. Hydrogel rectangular parallelepiped 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 pipetted into the space of a silicone rubber frame having a frame space of 100 mm × 20 mm × 300 μm and gelled at 4 ° C. The formed unmodified gelatin hydrogel was pressed against a nylon mesh having a mesh size of 250 μm × 250 μm to cut into unmodified gelatin hydrogel rectangular parallelepiped particles 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 appearance of rectangular parallelepiped particles made of gelatin hydrogel.

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

Next, an aqueous solution of the gelatin secondary modifier (10%, w / v) and the photopolymerization initiator (0.5%, w / v) was prepared and prepared in the same procedure as in Example 3. The chondrocytes were suspended to prepare a cell suspension of chondrocytes containing a gelatin secondary modifier and a photopolymerization initiator. A mixture was prepared by adding rectangular particles consisting of gelatin hydrogel to a cell suspension of chondrocytes. Here, the cell suspension and the hydrogel rectangular parallelepiped particles were mixed so as to be 2: 1 (v / w), and the mixture was 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 light 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 cross-linked gelatin hydrogel porous body containing cells in the cross-linked bulk hydrogel and no cells in the rectangular 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 body was observed. The observation results are shown in FIG.

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

According to FIG. 10, the obtained crosslinked gelatin hydrogel porous body was transparent and uniform immediately before culturing. Then, the cells were cultured in a T-flask for 4 weeks while shaking at a rate of 60 rpm. The medium was changed every 2 days. All steps were performed aseptically.

The 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 scanning microscope. The observation results are shown in FIG.

FIG. 11 is a diagram showing a fluorescence microscope 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 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 encapsulation of cells, that is, immediately before culturing, but no dead cells were detected after culturing for 4 weeks. In particular, chondrocytes were observed only in the bulk portion immediately after preparation of the crosslinked gelatin hydrogel porous body, but were also observed in the pores after culturing for 4 weeks. Further, according to FIG. 11B, it was confirmed that the chondrocytes were aggregated.

Further, after culturing the crosslinked gelatin hydrogel porous body of Example 4 in vitro for 1 day, it was implanted subcutaneously under the back of a nude mouse 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 the crosslinked gelatin hydrogel porous body according to Example 4.

As can be seen from the comparison between FIGS. 10 and 12, a totally glossy cartilage tissue was regenerated.

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

Similar to Example 4, unmodified gelatin was dissolved in a mixed solution of PBS and DMEM in 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 × ¹⁰⁷ cells / mL. A cell suspension of gelatin solution was pipetted into a space of a 100 mm × 20 mm × 300 μm silicone rubber frame and gelled at 4 ° C. By pressing the formed unmodified gelatin hydrogel containing chondrocytes against a nylon mesh having a mesh size of 250 μm × 250 μm, hydrogel square particles of chondrocyte-encapsulated unmodified gelatin having a size of 250 × 250 × 300 μm are obtained. It was cut. 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 appearance of rectangular parallelepiped particles made of gelatin hydrogel.

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

Next, an aqueous solution containing a gelatin secondary modifier and a photopolymerization initiator was prepared by the same procedure as in Example 2, and chondrocyte-encapsulating hydrogel rectangular particles were 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 light 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 was prepared in which the crosslinked bulk hydrogel did not contain cells and the hydrogel rectangular particles contained cells. This gelatin hydrogel was cut into a disk having a diameter of 6 mm. The appearance of the crosslinked gelatin hydrogel porous body was observed. The observation results are shown in FIG.

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

According to FIG. 14, the obtained crosslinked gelatin hydrogel porous body was transparent and uniform immediately before culturing. Next, cell culture was performed under the same conditions as in Example 4, the 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 scanning microscope. bottom. The observation results are shown in FIG.

FIG. 15 is a diagram showing a fluorescence microscope image of life-and-death staining of cells 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 encapsulation of cells, that is, immediately before culturing, but no dead cells were detected after culturing for 4 weeks. In particular, chondrocytes were observed only in the pores of the crosslinked gelatin hydrogel porous body immediately after preparation of the crosslinked gelatin hydrogel porous body and after culturing for 4 weeks. Further, according to FIG. 15 (b), it was confirmed that the chondrocytes were aggregated.

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

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

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

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

A chondrocyte prepared by preparing an aqueous solution of a gelatin secondary modifier (10%, w / v) and a photopolymerization initiator (0.5%, w / v) in the same procedure as in Example 3. Was suspended to prepare a cell suspension of chondrocytes containing a gelatin secondary modifier and a photopolymerization initiator. Here, the cell concentration of the cell suspension was 1.34 × ¹⁰⁷ cells / mL. Then, in the same procedure as in Example 5, a mixture containing chondrocyte-encapsulating hydrogel rectangular parallelepiped particles was prepared (mixing ratio was 2: 1 (v / w)) and kept at 25 ° C. Here, the cell concentration of the cell suspension for producing rectangular parallelepiped particles was 1.33 × ¹⁰⁷ 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 light 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 rectangular 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 body was observed. The observation results are shown in FIG.

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

According to FIG. 17, the obtained crosslinked gelatin hydrogel porous body was transparent and uniform immediately before culturing. Next, cell culture was performed under the same conditions as in Example 4, the 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 scanning microscope. bottom. The observation results are shown in FIG.

FIG. 18 is a diagram showing a fluorescence microscope image of life-and-death staining of cells 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 encapsulation of cells, that is, immediately before culturing, but no dead cells were detected after culturing for 4 weeks. In particular, immediately after the cross-linked gelatin hydrogel porous body was prepared, chondrocytes were observed in both the bulk site and the pores of the hydrogel porous body and were uniformly distributed. The cells aggregated.

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

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

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

In the gelatin derivative (secondary modified gelatin) of the present invention, the amino group 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, and the reactivity can be crosslinked and polymerized by irradiation with ultraviolet rays. The group has been introduced to the maximum. Since the crosslinked gelatin hydrogel of the present invention and its porous body are produced by using the gelatin derivative of the present invention, they have high mechanical strength and excellent shape stability. As a result, in addition to being used as an injectable gel for a loaded site, it can also be used for tissue regeneration by a biotissue engineering method. Moreover, since cells can be seeded in both the bulk and the pores of the hydrogel, more efficient tissue regeneration is possible, and a shortening of the healing period can be expected. Therefore, the crosslinked gelatin hydrogel of the present invention and its porous body are extremely useful for treating tissue defects.

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

Claims (21)

A gelatin derivative in which a reactive group is introduced into gelatin.
The amino group of gelatin is bonded to the methacryloyl group,
A gelatin derivative in which the hydroxy group and the carboxyl group of the gelatin are bonded to the methacryloylglyceryl ester group. The content of the methacryloyl group satisfies the range of 0.01 mmol or more and 10 mmol or less with respect to 1 g of the gelatin derivative.
The gelatin derivative according to claim 1, wherein the content of the methacryloyl glyceryl ester group satisfies the range of 0.01 mmol or more and 10 mmol or less with respect to 1 g of the gelatin derivative. The content of the methacryloyl group satisfies the 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 2, wherein the content of the methacryloyl glyceryl ester group satisfies the 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 selected from the group of animal bone, animal skin, fish bone, fish skin and fish scale. The method for producing a gelatin derivative according to any one of claims 1 to 4.
A step of reacting gelatin with a methacrylic anhydride to synthesize a primary modified gelatin in which a methacryloyl group is bonded to an amino group of the gelatin.
A step of reacting the gelatin primary modified product with glycidyl methacrylate to synthesize the gelatin derivative which is a gelatin secondary modified product in which a methacryloyl glyceryl ester group is bonded to a hydroxy group and a carboxy group of the gelatin primary modified product. Inclusive, method. Cross-linked gelatin hydrogel,
A crosslinked gelatin hydrogel containing a crosslinked product in which the gelatin derivative according to any one of claims 1 to 4 is crosslinked. The crosslinked gelatin hydrogel according to claim 6, wherein the crosslinked density of the gelatin derivative has a range of 0.02 mol / m ³ or more and 10000 mol / m ³ or less. The crosslinked gelatin hydrogel according to claim 6, which comprises cells. The cells include cartilage cells, osteoblasts, fibroblasts, myoblasts, ligament cells, fat cells, nerve cells, vascular endothelial cells, smooth muscle cells, myocardial cells, epithelial cells, hepatocytes, pancreatic β cells, and kidneys. The crosslinked gelatin hydro of claim 8 , 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 artificial pluripotent stem cells. gel. A method for producing crosslinked gelatin hydrogels.
A method for crosslinking using the gelatin derivative according to any one of claims 1 to 4. The step of preparing an aqueous solution containing the gelatin derivative and the photopolymerization initiator, and
The method according to claim 10, comprising the step of irradiating the aqueous solution with ultraviolet rays. Further including the 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 11 , wherein the step of irradiating the aqueous solution with ultraviolet rays is to irradiate the cell suspension with ultraviolet rays. Cross-linked gelatin hydrogel porous body
A crosslinked gelatin hydrogel porous body containing a crosslinked body in which the gelatin derivative according to any one of claims 1 to 4 is crosslinked. The crosslinked gelatin hydrogel porous body according to claim 13, wherein the crosslinked density of the gelatin derivative has a range of 0.02 mol / m ³ or more and 10000 mol / m ³ or less. The crosslinked gelatin hydrogel porous body according to claim 13 , which encloses cells. The cells include cartilage cells, osteoblasts, fibroblasts, myoblasts, ligament cells, fat cells, nerve cells, vascular endothelial cells, smooth muscle cells, myocardial cells, epithelial cells, hepatocytes, pancreatic β cells, and kidneys. The crosslinked gelatin hydro of claim 15 , 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 artificial pluripotent stem cells. Gel porous body. A method for producing a crosslinked gelatin hydrogel porous body.
A method for crosslinking using the gelatin derivative and gel particles according to any one of claims 1 to 4. The step of preparing an aqueous solution containing the gelatin derivative and the photopolymerization initiator, and
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 a step of preparing the mixture.
17. The method of claim 17, comprising the step of irradiating the mixture with ultraviolet light. The method according to claim 18, further comprising the step of preparing a cell suspension by suspending cells in the aqueous solution obtained by the step of preparing the aqueous solution. 17. The method of claim 17 or 18, wherein the gel particles encapsulate cells. The method of claim 19 or 20, wherein the cell is an animal cell of animal origin.

Images