Material for gel formation, gel composition, and method for producing the same

Abstract

To provide a material that can form a gel composition having enhanced fracture stress and toughness, and exhibiting biocompatibility.SOLUTION: A material for gel formation comprising a collagen component, a crosslinking agent for the collagen component, a precursor capable of forming a biocompatible gel through a polymerization reaction, and a polymerization initiator for initiating polymerization of the precursor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention also relates to a gel-forming material, a gel composition, and a method for producing the same. [Background technology]

[0002] Gels of extracellular matrix components such as collagen are biocompatible and are commonly used as scaffold materials for cell culture. For example, Patent Document 1 discloses a gel composition containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, and ions of a metal element. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2021 / 261355 Summary of the Invention [Problem to be solved by the invention]

[0004] Biocompatible gel compositions have room for improvement in terms of breaking stress and toughness.

[0005] An object of the present invention is to provide a biocompatible gel composition with improved breaking stress and toughness, as well as a material from which the gel composition can be formed. [Means for solving the problem]

[0006] The present disclosure includes the following inventions. [1] A gel-forming material comprising a collagen component, a cross-linking agent for the collagen component, a precursor capable of forming a biocompatible gel by polymerization, and a polymerization initiator that initiates polymerization of the precursor. [2] The gel-forming material according to [1], wherein the crosslinking agent is an ion of a metal element. [3] The gel-forming material according to [2], wherein the metal element is platinum. [4] The gel-forming material according to any one of [1] to [3], wherein the precursor is fibrinogen and the polymerization initiator is thrombin. [5] A gel composition comprising a crosslinked collagen component and a biocompatible polymer, wherein the biocompatible polymer is a reaction product of a precursor capable of forming a biocompatible gel by polymerization reaction and a polymerization initiator that initiates polymerization of the precursor. [6] The gel composition according to [5], wherein the crosslinked material is a crosslinked material formed by ions of a metal element. [7] The gel composition according to [6], wherein the metal element is platinum. [8] The gel composition according to any one of [5] to [7], wherein the precursor is fibrinogen and the polymerization initiator is thrombin. [9] A method for producing a gel composition, comprising the steps of: preparing a mixture containing a precursor capable of forming a biocompatible gel by polymerization, a collagen component, and an aqueous medium; and mixing a crosslinker for the collagen component and a polymerization initiator that initiates polymerization of the precursor into the mixture to obtain a gel composition.

[10] The method for producing a gel composition according to [9], wherein the crosslinking agent is an ion of a metal element.

[11] The method for producing a gel composition according to

[10] , wherein the metal element is platinum.

[12] The method for producing a gel composition according to any one of [9] to

[11] , wherein the precursor is fibrinogen and the polymerization initiator is thrombin. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a gel composition having improved breaking stress and toughness and biocompatibility, as well as a material capable of forming the gel composition. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows an example of a stress-strain curve in a compression test. [Figure 2] FIG. 2 is a graph showing the evaluation results of the gel compositions of Example 1 and Comparative Examples 1-1 and 1-2, where (A) shows the evaluation results of elongation at break, (B) shows the evaluation results of stress at break, and (C) shows the evaluation results of toughness. [Figure 3] FIG. 3 is a graph showing the evaluation results of the gel compositions of Comparative Examples 2-1 to 2-3, where (A) shows the evaluation results of elongation at break, (B) shows the evaluation results of stress at break, and (C) shows the evaluation results of toughness. [Figure 4] FIG. 4 is a graph showing the evaluation results of the gel compositions of Comparative Examples 3-1 to 3-3, where (A) shows the evaluation results of elongation at break, (B) shows the evaluation results of stress at break, and (C) shows the evaluation results of toughness. [Figure 5] FIG. 5 is a graph showing the relationship between the ratio of the mass of fibrin to the mass of the collagen component and the breaking point. [Figure 6] FIG. 6 is a graph showing the evaluation results of the water content of the gel composition. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0010] [Gel-forming material] The gel-forming material according to this embodiment contains collagen, a collagen cross-linking agent, a precursor capable of forming a biocompatible gel through a polymerization reaction, and a polymerization initiator that initiates polymerization of the precursor. The gel-forming material is a material that forms a gel composition by mixing all of the above components (collagen, cross-linking agent, precursor, and polymerization initiator).

[0011] <Collagen ingredients> Examples of collagen components include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specific examples include type I collagen, type II collagen, and type III collagen. Examples of non-fibrous collagen include type IV collagen. The collagen component is preferably fibrous collagen, and more preferably type I collagen. Commercially available collagen may be used as the fibrous collagen, and a specific example thereof is type I collagen derived from pig skin manufactured by Nippon Meat Packers, Ltd.

[0012] Examples of animal species from which the collagen component is derived include, but are not limited to, humans, pigs, cows, etc. The collagen component may be derived from a single type of animal, or may be derived from multiple types of animals in combination.

[0013] The collagen component may include a fragmented collagen component. The fragmented collagen component can be obtained by fragmenting the collagen component. "Fragmentation" means breaking down an aggregate of collagen molecules into smaller sizes. Fragmentation may be performed under conditions that cleave the bonds within the collagen molecules, or under conditions that do not cleave the bonds within the collagen molecules. The fragmented collagen component may include a defibrated collagen component (defibrated collagen component), which is a collagen component defibrated by the application of physical force. Defibration is one form of fragmentation, and is performed, for example, under conditions that do not cleave the bonds within the collagen molecules.

[0014] The method for fragmenting the collagen component is not particularly limited. For example, the collagen component may be defibrated by applying a physical force using an ultrasonic homogenizer, an agitation homogenizer, a high-pressure homogenizer, or the like. When using an agitation homogenizer, the collagen component may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, by adjusting the homogenization time, number of times, etc., it is also possible to obtain millimeter-sized or nanometer-sized defibrated collagen components. Defibrated collagen components can also be obtained by defibrating the collagen component through repeated freezing and thawing.

[0015] The fragmented collagen component may contain at least a defibrated collagen component. Alternatively, the fragmented collagen component may consist solely of a defibrated collagen component. In other words, the fragmented collagen component may be a defibrated collagen component.

[0016] The defibrated collagen component preferably maintains the triple helix structure derived from collagen. The defibrated collagen component may be a component that partially maintains the triple helix structure derived from collagen.

[0017] The shape of the fragmented collagen component can be, for example, fibrous. Fibrous collagen components include thin threads (microfibrils) formed by the aggregation of multiple thread-like collagen molecules, threads formed by the further aggregation of microfibrils, and defibrillated versions of these threads. In fibrous collagen components, the RGD sequence is preserved without being destroyed. The RGD sequence is a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). The presence of the RGD sequence further promotes cell adhesion, making the material more suitable as a scaffold for cell culture, for example.

[0018] The average length of the fragmented collagen components may be 100 nm or more and 400 μm or less, or 100 nm or more and 200 μm or less. In one embodiment, the average length of the fragmented collagen components may be 5 μm or more and 400 μm or less, 10 μm or more and 400 μm or less, 22 μm or more and 400 μm or less, or 100 μm or more and 400 μm or less. In another embodiment, the average length of the fragmented collagen components may be 100 μm or less, 50 μm or less, 30 μm or less, 15 μm or less, 10 μm or less, 1 μm or less, or 100 nm or more. Of the entire fragmented collagen components, it is preferable that the average length of the majority of the fragmented collagen components is within the above numerical range. Specifically, it is preferable that the average length of 95% of the entire fragmented collagen components is within the above numerical range. The fragmented collagen components are more preferably defibrated collagen components whose average length is within the above numerical range.

[0019] The average diameter of the fragmented collagen component may be 10 nm to 30 μm, 30 nm to 30 μm, 50 nm to 30 μm, 100 nm to 30 μm, 1 μm to 30 μm, 2 μm to 30 μm, 3 μm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. The fragmented collagen component is more preferably a defibrated collagen component having an average diameter within the above range.

[0020] The average length and average diameter of the fragmented collagen components can be determined by measuring individual collagen components using an optical microscope and analyzing the images. In this specification, "average length" refers to the average length of the measured sample in the longitudinal direction, and "average diameter" refers to the average length of the measured sample in the direction perpendicular to the longitudinal direction.

[0021] <Crosslinking agent> The crosslinking agent may be, for example, one capable of crosslinking carboxyl groups with each other, one capable of crosslinking amino groups with each other, or one capable of crosslinking carboxyl groups with amino groups. Examples of the crosslinking agent include metal ions, aldehyde crosslinking agents (e.g., glutaraldehyde), methacrylamide, etc. The crosslinking of collagen molecules may be due to the formation of hydrogen bonds between the carboxyl groups of the collagen molecules and the metal ions.

[0022] The metal element is preferably at least one metal element selected from the group consisting of transition metal elements and base metal elements. Specific examples of the at least one metal element selected from the group consisting of transition metal elements and base metal elements include titanium (Ti), copper (Cu), zinc (Zn), palladium (Pd), platinum (Pt), and gold (Au). Specific examples of ions of at least one metal element selected from the group consisting of transition metal elements and base metal elements include Ti. + , Ti 2+ , Ti 3+ , Ti 4+ , Cu + , Cu 2+ , Zn + , Zn 2+ , Pd 2+ , Pd 4+ , Pt 2+ , Pt 4+ , Au + , Au 2+ , Au 4+ etc.

[0023] The metal element is more preferably at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table. Specific examples of the at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table include copper (Cu), zinc (Zn), palladium (Pd), platinum (Pt), and gold (Au). Specific examples of ions of at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table include Cu.2+ , Zn 2+ , Pd 2+ , Pd 4+ , Pt 2+ , Pt 4+ , Au + , Au 2+ , Au 4+ etc.

[0024] From the viewpoint of being non-cytotoxic or low-cytotoxic and safe for living organisms, the metal element ions are preferably ions of metal elements selected from titanium (Ti), copper (Cu), zinc (Zn), platinum (Pt), and gold (Au). If the gel composition is safe for living organisms, it can be suitably used, for example, as a scaffold material for cell culture when forming artificial tissue.

[0025] <Precursor and polymerization initiator> The precursor is a substance capable of forming a biocompatible gel by polymerization, which gives a polymer with a three-dimensional network structure by reaction with a polymerization initiator.

[0026] The gel-forming material according to this embodiment can form a gel composition having a network structure (first network) derived from a crosslinked collagen component and a network structure (second network) derived from a reaction product of a precursor. Because the gel composition has both a first and a second network, it can also be called a double-network gel (DN gel). The gel-forming material according to this embodiment can provide a gel composition with improved breaking stress and toughness because, for example, the second network is formed by a three-dimensional network structure formed by a polymerization reaction.

[0027] An example of a combination of a precursor and a polymerization initiator is a combination of fibrinogen and thrombin. Fibrinogen reacts with thrombin to form fibrin (a polymer) through a polymerization reaction.

[0028] <Amount of precursor used> The ratio of the mass of the precursor to the mass of the collagen component (precursor / collagen component) is preferably 10 or less, and may be, for example, 8.0 or less, 6.0 or less, 4.0 or less, or 2.5 or less, since this further improves the breaking stress of the gel composition. The ratio of the mass of the precursor to the mass of the collagen component may be 2.0 or more, 3.0 or more, 4.0 or more, or 5.0 or more. The ratio of the mass of the precursor to the mass of the collagen component may be 2.0 or more and 10 or less, or 2.0 to 20, since this further improves the breaking stress and toughness of the gel composition.

[0029] [Gel Composition] The gel composition according to this embodiment contains a crosslinked collagen component and a biocompatible polymer.

[0030] <Cross-linked collagen components> A crosslinked collagen component is a component in which collagen molecules are crosslinked by a crosslinking agent. The crosslinked collagen component is formed by contacting the collagen component with the crosslinking agent. The crosslinking agent can be any of the above-mentioned crosslinking agents.

[0031] The content of the collagen component may be, for example, 0.1% by mass or more, 0.15% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 2.0% by mass or more, or 3.0% by mass or more, based on the total amount of the gel composition. The upper limit of the content of the collagen component in the gel composition is not particularly limited as long as it can be prepared, but may be, for example, 10.0% by mass or less, or 5.0% by mass or less, based on the total amount of the gel composition.

[0032] The content of the crosslinking agent may be 0.41 mg or less and 0.020 mg or more per 1 g of the collagen component.

[0033] The concentration of the crosslinking agent may be 0.1 mM or more and 1.0 mM or less based on the total amount of the gel composition.

[0034] <Biocompatible polymers> The biocompatible polymer is a polymerized product formed by the reaction of a precursor with a polymerization initiator. Examples of such biocompatible polymers include fibrin. The gel composition may or may not further contain other biocompatible polymers (e.g., alginate).

[0035] The amount of precursor used may be, for example, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.15% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 2.0% by mass or more, or 3.0% by mass or more, based on the total amount of the gel composition. The upper limit of the amount of precursor used is not particularly limited as long as it can be prepared, but may be, for example, 10.0% by mass or less, or 5.0% by mass or less, based on the total amount of the gel composition.

[0036] When the precursor is fibrinogen, the polymerization initiator is preferably thrombin, and in this case, the amount of thrombin used may be, for example, 3.0 U / ml or less based on the total amount of the gel composition.

[0037] <Other ingredients> The gel composition may further contain other components in addition to the above components depending on the intended use of the gel composition, such as nutrients for the cells to be cultured, pH adjusters, etc., when used as a scaffold material for cell culture.

[0038] <Physical properties of gel composition> The gel composition has improved breaking stress and toughness. The breaking stress of the gel composition may be, for example, 1 kPa or more, 10 kPa or more, 100 kPa or more, or 150 kPa or more. The toughness of the gel composition may be, for example, 5000 J / m 3 More than 7000J / m 3 or more than 10,000 J / m 3 may be greater than or equal to 18000 J / m 3 The breaking stress and toughness can be measured by the method described in the examples below.

[0039] [Method for producing gel composition] The gel composition can be obtained, for example, by a method including a step of preparing a mixed solution containing a precursor, a collagen component, and an aqueous medium (mixing step), and a step of mixing a crosslinker for the collagen component and a polymerization initiator into the mixed solution to obtain a gel composition (gelling step).

[0040] In the mixing step, a mixed liquid containing a precursor, a collagen component, and an aqueous medium is prepared. The mixing step can be performed by a method of mixing a precursor-containing liquid containing a precursor and an aqueous medium with a collagen-containing liquid containing a collagen component and an aqueous medium, a method of mixing a precursor into a collagen-containing liquid, a method of mixing a collagen component into a precursor-containing liquid, or a method of mixing a collagen component into a precursor-containing liquid. The mixing step is preferably performed by mixing a precursor-containing liquid with a collagen-containing liquid.

[0041] As used herein, the term "aqueous medium" refers to a liquid containing water as an essential component. Examples of aqueous media include physiological saline solutions such as phosphate-buffered saline (PBS) and liquid media such as Dulbecco's Modified Eagle's Medium (DMEM).

[0042] The temperature (mixing temperature) at which the precursor and collagen component are mixed is not particularly limited, but from the viewpoint of avoiding excessive reaction, it is preferable to carry out the mixture at, for example, 10° C. or less, and more preferably 5° C. or less. The lower limit of the temperature is not particularly limited, but it is usually −3° C. or more, and preferably 0° C. or more.

[0043] In the gelation process, a crosslinking agent and a polymerization initiator are mixed into a mixed solution to obtain a gel composition. In the gelation process, the collagen component and the crosslinking agent come into contact with each other to form a crosslinked collagen component, and a biocompatible polymer is formed by a polymerization reaction between a precursor substance and the polymerization initiator.

[0044] The gelling step may be carried out, for example, by mixing a solution containing a crosslinking agent and an aqueous medium and a solution containing a polymerization initiator and an aqueous medium into a mixed solution.

[0045] The solution containing the crosslinking agent may be a solution containing ions of a metal element. The source of the metal element ions is not particularly limited, and may be, for example, an inorganic salt or an organic salt.

[0046] The gelation step may include mixing the crosslinker and the polymerization initiator and then incubating the mixture. The temperature during incubation may be, for example, 37° C. The incubation time can be appropriately set depending on the type of materials used, and may be, for example, 1 to 24 hours.

[0047] [Method of using the gel composition] The gel composition according to this embodiment is suitably used, for example, as a scaffold material for forming a cell structure (three-dimensional tissue). [Example]

[0048] The present invention will be explained in more detail below based on test examples, but the present invention is not limited to the following test examples.

[0049] 〔reagent〕 Fibrinogen (Sigma-Aldrich F8630-5G Fibrinogen from Bovine Plasma, Type IS (30 mg / mL)) Thrombin (Sigma-Aldrich T4648-10KU Thrombin from Bovine Plasma, 1000U, 104 Units / mg, 10KU / 96.2mg) Phosphate-buffered saline (PBS) Collagen (NMP Collagen PS, manufactured by Nippi Corporation, lot. PSBUL03S) Potassium tetrachloroplatinate(II) (MW: 415.09) (Sigma-Aldrich, 206075-1G, MKCK0067) Sodium alginate (Sigma-Aldrich, CAS: 42000501-10G, Lot BP-2110-27) Calcium sulfate dihydrate (CAS: 034-00925) N,N-Dimethylacrylamide (DMA) (CAS: 2680-03-7) N,N'-methylenebisacrylamide (BIS) (CAS: 110-26-9) N,N,N',N'-Tetramethylethylenediamine (TEMED) (CAS: 110-18-9) Ammonium persulfate (APS)

[0050] [Production of gel composition] Example 1 A gel composition containing a crosslinked collagen component and fibrin (Col / Fib DN gel) was prepared according to the following procedure.

[0051] [Experimental Procedure] (Preparation of fibrinogen solution) 360 mg of fibrinogen was weighed into a 15 mL centrifuge tube. The fibrinogen was added to 6 mL of PBS that had been heated to 37°C. The PBS containing fibrinogen was stirred with a rotator for about 1 hour to disperse the fibrinogen, yielding a fibrinogen solution with a fibrinogen concentration of 6% by mass.

[0052] (Preparation of thrombin solution) 9.6 mg of thrombin was weighed into a 1.5 mL microtube. 1 mL of PBS was added to the microtube containing thrombin to obtain a thrombin solution with a thrombin concentration of 1000 U / mL. The thrombin solution was dispensed into 100 μL aliquots and stored at -20°C.

[0053] (Preparation of collagen-containing solution) Collagen was placed in an autoclave-sterilized glass dish in a clean bench using an autoclave-sterilized spatula and spread thinly. The glass dish containing the collagen was then UV-sterilized for 3 minutes. 50 mg of collagen was dispersed in 5 mL of PBS using a new autoclave-sterilized spatula to obtain a collagen dispersion with a collagen concentration of 10 mg / mL. The collagen dispersion was homogenized using a homogenizer for 5 minutes (Power 6) to obtain a collagen-containing solution. The collagen-containing solution was left to stand overnight in a refrigerator. After standing, the collagen-containing solution was centrifuged at 15,000 rpm for 5 minutes to remove bubbles. This yielded a collagen-containing solution (CNF) with a collagen concentration of 2% by mass.

[0054] (Preparation of crosslinker solution) A platinum solution (concentration 12.5 mM, 5.18 mg / mL) was prepared from potassium tetrachloroplatinate(II) and PBS using a plastic spatula.

[0055] (Preparation of gel composition) 54 μL of ice-cold PBS was added to 200 μL of collagen-containing solution with a collagen concentration of 2% by mass and stirred. Then, 333 μL of fibrinogen solution with a fibrinogen concentration of 6% by mass was added to the collagen-containing solution and pipetted. 80 μL of platinum solution was added to the mixed solution after pipetting. 333 μL of 10 U / mL thrombin solution, prepared by diluting 1000 U / mL thrombin solution 100-fold, was added to the mixed solution immediately after the platinum solution was added and pipetted. The resulting mixed solution was placed in a mold and incubated at 37°C for 24 hours. This procedure yielded a cylindrical gel composition (total volume 1000 μL) with a diameter of 8 mm and a height of 4 mm. This resulted in a gel composition with a fibrinogen:collagen (mass ratio) of 5:1 (2.0% by mass:0.4% by mass), a thrombin concentration of 3 U / mL, and a Pt concentration of 1 mM.

[0056] Gel compositions were prepared in the same manner as above, except that the fibrinogen:collagen (mass ratio) was changed to 20:1 (2.0% by mass:0.1% by mass), 10:1 (2.0% by mass:0.2% by mass), or 2:1 (2.0% by mass:1.0% by mass).

[0057] Comparative Example 1-1 A gel composition containing crosslinked collagen but not containing fibrin (Collagen SN gel) was prepared by the following procedure.

[0058] 200 μl of collagen-containing solution was added to 720 μl of PBS and pipetted. 80 μl of platinum solution was added to the pipetted solution. The resulting mixed solution was placed in a mold and incubated at 37°C for 24 hours to obtain a gel composition. Gel compositions with different concentrations were also prepared in the same manner.

[0059] Comparative Example 1-2 A gel composition (Fibrin SN gel) containing fibrin but not containing a crosslinked collagen component was prepared by the following procedure.

[0060] 333 μL of fibrinogen solution was added to 253 μL of PBS and pipetted. 80 μL of platinum solution was added to the pipetted solution. 333 μL of thrombin solution with a thrombin concentration of 10 U / mL, which had been prepared by diluting 1000 U / mL thrombin solution 100-fold, was added to the mixture after the addition of the platinum solution and pipetted. The resulting mixture was placed in a mold and incubated at 37°C for 24 hours to obtain a gel composition.

[0061] Comparative Example 2-1 A gel composition containing a crosslinked collagen component and calcium alginate (Col / Alg DN gel) was prepared according to the following procedure.

[0062] (Preparation of collagen-containing solution and platinum solution) The collagen-containing liquid and platinum solution were prepared in the same manner as in Example 1 and used.

[0063] (Preparation of alginate solution) 300 mg of sodium alginate was dispersed in 5 mL of cooled PBS, and if the sodium alginate did not dissolve, the solution was heated to prepare an alginate solution with an alginate concentration of 6% by mass.

[0064] (Preparation of calcium solution) A calcium slurry with a calcium ion concentration of 1.22M was prepared by adding 20mL of PBS to 4.2g of CaSO4·2H2O and stirring vigorously. 400µL of the calcium slurry was taken while stirring with a stirrer and added to 600µL of PBS to prepare a calcium solution with a calcium ion concentration of 488mM. The calcium solution was pipetted thoroughly before use.

[0065] (Preparation of gel composition) 387 μL of chilled PBS was added to the microtube, and 200 μL of collagen-containing solution was added and pipetted. 80 μL of platinum solution and 50 μL of calcium solution were added to the mixed solution and pipetted. As soon as the cloudy Ca solution was dispersed throughout, 333 μL of alginate solution was added and pipetted. The resulting mixed solution was placed in a mold and incubated at 37°C for 24 hours. Through these operations, a gel composition was obtained with an alginate:collagen (mass ratio) of 5:1 (2.0% by mass:0.4% by mass), a Ca concentration of 24.4 mM, and a Pt concentration of 1 mM.

[0066] Gel compositions were prepared in the same manner as above, except that the alginate:collagen (mass ratio) was changed to 20:1 (2.0% by mass:0.1% by mass), 10:1 (2.0% by mass:0.2% by mass), or 2:1 (2.0% by mass:1.0% by mass).

[0067] Comparative Example 2-2 The gel composition containing a crosslinked collagen component but not containing calcium alginate (Collagen SN gel) was the same as that used in Comparative Example 1-1.

[0068] Comparative Example 2-3 A gel composition (Alginate SN gel) containing calcium alginate but not containing a crosslinked collagen component was prepared by the following procedure.

[0069] (Preparation of gel composition) 537 μL of chilled PBS was added to the microtube, and 333 μL of alginate solution was added and pipetted. 80 μL of platinum solution and 50 μL of calcium solution were then quickly added to the alginate solution and pipetted. The resulting mixture was placed in a mold and incubated at 37°C for 24 hours to obtain a gel composition.

[0070] Comparative Example 3-1 A gel composition containing a crosslinked collagen component and a DMA polymer (Col / DMA DN gel) was prepared by the following procedure.

[0071] (Preparation of collagen-containing solution and platinum solution) The collagen-containing liquid and platinum solution were prepared in the same manner as in Example 1 and used.

[0072] (Removal of polymerization inhibitors from DMA) DMA was mixed with a polymerization inhibitor remover (alumina oxide), which was then removed using a syringe filter or centrifugation.

[0073] (Preparation of gel composition) 62 μL of DMA and 0.9 mg of BIS were weighed into a sample tube, and 600 μL of collagen-containing solution and 293 μL of PBS were added. The DMA and BIS were dissolved by pipetting. 24 mg of APS was weighed into a black microtube and dissolved in 200 μL of PBS. After adding APS to the sample tube containing the mixed solution, the tube was gently shaken and then capped with a septum. The resulting mixture was placed in an ice bath and nitrogen bubbled for 30 minutes to remove oxygen from the system. The septum was removed, and 10-fold diluted TEMED solution (22.5 mL of TEMED + 202.5 mL of PBS) and 160 μL of platinum solution were simultaneously added at room temperature and quickly poured into a mold. The mixture was then incubated at 37°C for 24 hours. This procedure yielded a gel composition with a DMA:collagen (mass ratio) of 5:1 (4.0% by mass:0.8% by mass), a BIS concentration of 1.0 mol%, and a Pt concentration of 1 mM.

[0074] Gel compositions were prepared in the same manner as above, except that the DMA:collagen (mass ratio) was changed to 20:1 (4.0% by mass:0.2% by mass), 10:1 (4.0% by mass:0.4% by mass), or 4:1 (4.0% by mass:1.0% by mass).

[0075] Comparative Example 3-2 The gel composition (Collagen SN gel) containing a crosslinked collagen component but not containing a DMA polymer was the same as that used in Comparative Example 1-1.

[0076] Comparative Example 3-3 A gel composition (DMA SN gel) containing a DMA polymer but not a crosslinked collagen component was prepared in the same manner as in Comparative Example 3-1, except that no collagen-containing liquid was added (600 μL of PBS was used instead of 600 μL of collagen-containing liquid).

[0077] [Evaluation of Gel Composition] <Evaluation of breaking elongation, breaking stress and toughness> A compression test was carried out using the gel composition under the following measurement conditions. [Measurement conditions] Load cell 20N Approaching speed: 1.0 mm / min Jig: 15mm diameter, flat plate type Measurement began when the force reached 0.005N.

[0078] [Mechanical evaluation] The physical properties were evaluated from the stress-strain curves of stress (vertical axis) and strain (horizontal axis) obtained in the compression test. The results are shown in Figures 2 to 4.

[0079] (Method of determining the breaking point) The point at which the stress began to decrease significantly was taken as the breaking point. In the case of gels with too little stress or that stretched easily (fibrin gels and DMA polymer gels), the measurement was stopped at 75%, the limit size at which the gel would not protrude from the jig. Figure 1 shows an example of a stress-strain curve. As shown in Figure 1, the stress (Stress) at the breaking point was taken as the breaking stress (Pa), and the elongation (Strain) was taken as the breaking elongation (%). The area of ​​the graph up to the breaking point divided by the volume of the gel was taken as the toughness (J / m 3 ) All calculations were performed using TRAPEZIUM X (SHIMADZU, Kyoto, Japan).

[0080] As shown in FIGS. 2 to 4, it was confirmed that the gel composition containing a crosslinked collagen component and fibrin is a biocompatible gel composition, and also has improved breaking stress and toughness.

[0081] Figure 5 shows the ratio of fibrin mass to collagen mass (concentration ratio of 2 nd to 1 st The relationship between the fibrin network and the breaking point is shown. It was confirmed that the breaking stress increased as the ratio of the fibrin mass to the collagen mass increased.

[0082] <Evaluation of moisture content> The water content of the gel composition prepared by the above method was measured. The gel composition was placed in a 60 mm dish whose mass had been measured, and the mass was measured. This gave the mass of the gel composition containing water (W w The gel composition was then freeze-dried. The mass of the freeze-dried gel was measured, and the weight of the container was subtracted from the mass to determine the mass of the gel without water (W d The moisture content was calculated using the following formula: Formula:W w / (W w +W d )×100(%)

[0083] Figure 5 shows the results of measuring the water content. As shown in Figure 5, the gel composition of Example 1 containing a crosslinked collagen component and fibrin (Col / Fib DN gel) showed a high water content of 95% or more. This confirmed that the gel composition of Example 1 can contain a culture medium and therefore can be applied to three-dimensional culture.

Claims

1. Collagen components and a cross-linking agent for the collagen component; a precursor capable of forming a biocompatible gel upon polymerization; a polymerization initiator that initiates polymerization of the precursor.

2. The gel-forming material according to claim 1 , wherein the crosslinking agent is an ion of a metal element.

3. The gel-forming material according to claim 2 , wherein the metal element is platinum.

4. 3. The gel-forming material according to claim 1, wherein the precursor is fibrinogen and the polymerization initiator is thrombin.

5. The composition comprises a crosslinked collagen component and a biocompatible polymer, A gel composition, wherein the biocompatible polymer is a reaction product of a precursor capable of forming a biocompatible gel by polymerization reaction and a polymerization initiator that initiates polymerization of the precursor.

6. The gel composition according to claim 5 , wherein the crosslinked material is a crosslinked material formed by ions of a metal element.

7. The gel composition of claim 6, wherein the metal element is platinum.

8. 7. The gel composition of claim 5, wherein the precursor is fibrinogen and the polymerization initiator is thrombin.

9. A step of preparing a mixture containing a precursor capable of forming a biocompatible gel by polymerization reaction, a collagen component, and an aqueous medium; a step of mixing a crosslinking agent for the collagen component and a polymerization initiator that initiates polymerization of the precursor substance into the mixed liquid to obtain a gel composition.

10. The method for producing a gel composition according to claim 9, wherein the crosslinking agent is an ion of a metal element.

11. The method for producing a gel composition according to claim 10, wherein the metal element is platinum.

12. The method for producing a gel composition according to claim 9 or 10, wherein the precursor is fibrinogen and the polymerization initiator is thrombin.

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