JP2007297360A - Hydrogel, production method thereof and use thereof - Google Patents

Abstract

Provided is a polymer material imparted with cell affinity in a gel based on polysaccharides such as hyaluronic acid and having excellent hydrophilicity, and particularly suitable for use as a cell culture carrier. thing.
A hydrogel comprising a polysaccharide cross-linked product having a phenolic hydroxyl group introduced therein and fibrillated collagen. As the polysaccharide, hyaluronic acid is preferable, and tyramine is preferably condensed to introduce a phenolic hydroxyl group. As the fibrillated collagen, those derived from aquatic animals are preferable, and those derived from cocoons are more preferable.
[Selection] Figure 3

Description

  The present invention relates to a hydrogel containing a cross-linked polysaccharide such as hyaluronic acid and a fibrillated collagen, a method for producing the hydrogel, and a cell culture carrier containing the hydrogel.

Hyaluronic acid (HA) is a component of the extracellular matrix, and is a glycosaminoglycan that is abundant in joints, vitreous, skin and the like. HA has various functions such as high water retention and viscosity, various physiological activities, water retention in the body, lubrication / buffering action, promotion of wound healing, etc. HA has been studied for application to biomaterials. .
For example, in Patent Document 1, a polymerizable compound having a phenolic hydroxyl group such as tyramine is bound to an acidic polysaccharide such as HA using a condensing agent such as 1,1-carbonyldiimidazole and then treated with an enzyme. It has been proposed to produce a polysaccharide hydrogel by polymerization and curing.

Japanese Patent Laying-Open No. 2005-200944

  However, since HA has low cell adhesiveness, it is necessary to impart cell adhesiveness when considering application to cell culture carriers. Therefore, an object of the present invention is a polymer material imparted with cell affinity in an HA-based gel with excellent hydrophilicity, and particularly provides a polymer material that can be suitably used for applications such as cell culture carriers. There is to do.

As a result of studying imparting cell affinity to a polysaccharide gel such as HA, the present inventors have found that a polymer material obtained by complexing fibrotic collagen when the polysaccharide is cross-linked and gelled can achieve the above object. The present invention was completed. Collagen is a protein that is abundant in skin, ligaments, bones, and the like, and is widely applied with biocompatibility and various physiological activities.
In addition, animal collagen such as cattle and pigs has been used as a raw material for collagen, but since the BSE (bovine spongiform encephalopathy) problem has occurred, the existence of various pathogens typified by abnormal prions cannot be denied. As pointed out, in recent years, highly safe aquatic organism-derived collagen (such as marine collagen) has attracted attention, and there are cases where it is appropriate to use aquatic organism-derived collagen. However, since the denaturation temperature of aquatic organism-derived collagen is lower than that of animal collagen, there is a problem that when used as a biomaterial, thermal stability, strength, etc. are inferior under physiological conditions. In the present invention, such a problem can also be solved.
That is, this invention provides uses, such as a hydrogel containing the following hyaluronic acid and collagen, its manufacturing method, and a cell culture carrier containing the hydrogel.

[1] A hydrogel comprising a polysaccharide cross-linked product having a phenolic hydroxyl group introduced therein and fibrillated collagen.
[2] A polysaccharide into which a phenol group is introduced is a polysaccharide and the following general formula (1)
(In the formula, R represents a hydroxyl group, —NHR ¹ group, or carboxyl group; R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; a is an integer of 1 to 10; ^{1 to} A ⁵ each independently represents a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 6 carbon atoms, wherein one or two of A ^{1 to} A ⁵ are a hydroxyl group, and two are hydroxyl groups. Is assumed that both hydroxyl groups are in the ortho-position or para-position.)
2. The hydrogel as described in 1 above, which is a condensate with a phenol compound represented by formula (1).
[3] The hydrogel as described in 2 above, wherein R is a hydroxyl group, a primary amino group or a secondary amino group.
[4] The hydrogel according to any one of 1 to 3, wherein the condensation ratio of the polysaccharide and the phenol compound represented by the general formula (1) is 1: 0.003 to 0.1 (mass ratio).
[5] The hydrogel as described in any one of 1 to 4 above, wherein the polysaccharide is hyaluronic acid, alginic acid or a derivative thereof.
[6] The hydrogel as described in 2 above, wherein the phenol compound represented by the general formula (1) is tyramine.
[7] The hydrogel according to any one of 1 to 6, wherein the fibrillated collagen is derived from an aquatic animal.
[8] The hydrogel as described in 7 above, wherein the aquatic animal is a rabbit.
[9] The hydrogel as described in any one of 1 to 8 above, wherein the mass ratio of the cross-linked polysaccharide having a phenol group introduced to the fibrillated collagen is 1: 0.1-5.
[10] A method for producing a hydrogel, characterized in that a polysaccharide into which a phenol group is introduced and collagen containing non-fibrotic collagen are subjected to crosslinking and fibrosis in the presence of an enzyme to form a gel.
[11] The production method according to 10 above, wherein the enzyme is selected from laccase, tyrosinase, catalase and peroxidase.
[12] A cell culture carrier comprising the hydrogel according to any one of 1 to 9 above.

  Since the hydrogel of the present invention is complexed with a fibrillated collagen in a polysaccharide cross-linked product such as hyaluronic acid, and is excellent in cell proliferation based on hyaluronic acid and cell adhesion based on collagen, It is useful as a biomaterial such as a culture carrier. Further, by using an aquatic organism-derived collagen as a collagen, it can be used as a highly safe material without worrying about zoonotic diseases such as BSE.

  In the present invention, the cross-linked polysaccharide having a phenolic hydroxyl group introduced therein includes those formed in a cross-linked state at the phenol group portion of the polysaccharide having a phenolic hydroxyl group introduced.

  The polysaccharide used in the present invention is not particularly limited as long as it is condensed with a phenol compound to be described later and crosslinks by the action of an enzyme, but a functional group for introducing a phenolic hydroxyl group (for example, a carboxylic acid group or an ester thereof). And the like are preferred. When the hydrogel of the present invention is used as a biomaterial such as a cell culture carrier, the polysaccharide is preferably hyaluronic acid, alginic acid or a derivative thereof, more preferably hyaluronic acid. Examples of the derivative include various modified products, and the site to be modified and the modifying group are not particularly limited as long as the effects of the present invention are not significantly impaired.

Such a phenolic introduction into a polysaccharide can be obtained by subjecting a compound having a functional group for condensing with a polysaccharide and a phenolic hydroxyl group to a condensation reaction with the polysaccharide. As a compound having a functional group for condensing with a polysaccharide and a phenolic hydroxyl group, for example, the following general formula (1)
(In the formula, R represents a hydroxyl group, —NHR ¹ group, or carboxyl group; R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; a is an integer of 1 to 10; ^{1 to} A ⁵ each independently represents a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 6 carbon atoms, wherein one or two of A ^{1 to} A ⁵ are a hydroxyl group, and two are hydroxyl groups. Is assumed that both hydroxyl groups are in the ortho-position or para-position.)
The phenol compound shown by these is mentioned.

  Here, as a C1-C6 alkyl group, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and these isomer groups are represented, As a C1-C6 alkoxy group, Methoxy group, ethoxy group, propoxy group, butoxy group, pentyloxy group, hexyloxy group and isomers thereof.

R is preferably a hydroxyl group or —NHR ¹ group, and a is preferably an integer of 1 to 6. A ^{1 to} A ⁵ are preferably a group other than a hydroxyl group, more preferably a hydroxyl group, and the other is a hydrogen atom.

  As the phenol compound represented by the general formula (1), tyramine and homovanillic acid derivatives are preferable as those having one phenolic hydroxyl group, and those having two phenolic hydroxyl groups include dopamine, noradrenaline or adrenaline. Catecholamine derivatives are mentioned. Of these, tyramine derivatives are preferred.

  There is no particular limitation on the condensation reaction conditions between the polysaccharide and the phenol compound represented by the general formula (1), and the reaction can be carried out by a conventionally known method. Examples of the condensing agent used for condensation include 1,1-carbonyldiimidazole, dicyclohexylcarbodiimide, 3- (3-dimethylaminopropyl) -1-ethylcarbodiimide / hydrochloride, and the like. Examples of the solvent to be used include dimethylformamide and dimethylsulfoxide, and dimethylformamide is particularly preferable. This is because polysaccharides such as hyaluronic acid are well dispersed.

  Further, the blending ratio (condensation ratio) of the phenol compound represented by the general formula (1) with respect to the polysaccharide is preferably 0.003 to 0.1 (mass ratio) with respect to the polysaccharide 1, and more preferably 0.01 to 0.05 (mass ratio). If the amount of the phenol compound is too small, crosslinking is not sufficiently performed and the strength becomes too low. If the amount of the phenol compound is too large, the resulting crosslinked gel becomes too hard and brittle.

The polysaccharide introduced with a phenolic hydroxyl group used in the present invention is preferably the following repeating unit:
The thing containing is mentioned.

The fibrotic collagen contained in the hydrogel of the present invention is formed by fibrosis of collagen having fibrotic ability. Although it does not specifically limit as collagen which has a fibrosis ability, From a viewpoint of industrial utilization, type I collagen with a high yield or collagen which has it as a main component is preferable.
Further, the molecular structure of the collagen having fibrotic ability is not particularly limited. It has been reported that non-helical regions (telopeptides) present at both ends of the collagen molecule have antigenicity, and the collagen used in the present invention may or may not be removed from the telopeptide. .

  The collagen having fibrosis used in the present invention is not particularly limited with respect to its denaturation. It is known that even once denatured collagen, the collagen helical structure is partially restored and the fibrosis ability is restored. In order to achieve the present invention, the spiral rate (%) is preferably 50 or more from the viewpoint of fibrosis ability. The spiral rate (%) is synonymous with the spiral recovery rate (%) described in Journal of Food Chemistry 60, p.1233 (1995). That is, it shows the helical recovery rate (%) obtained from the specific optical rotation measured with the polarimeter.

  The origin of the collagen having fibrosis used in the present invention is not particularly limited. Collagen derived from vertebrate dermis is preferably used from the viewpoint of the amount of resources and collagen yield. Among these, collagen derived from aquatic organisms that have a lower possibility of holding pathogens such as BSE than livestock is preferable. Specifically, fish dermis collagen such as scab, shark skin, tuna skin, cod skin, and flounder skin is particularly preferable.

  The collagen fiber in the present invention means a thread-like structure as shown in a scanning electron micrograph in the literature (Journal of Agricultural Food Chemistry, 48, p.2028-2032 (2000)).

  The hydrogel of the present invention is produced by cross-linking polysaccharides having phenolic hydroxyl groups introduced and collagen fibrosis. The reaction is preferably carried out in one pot.

An enzyme can be used to crosslink the polysaccharide.
Examples of the enzyme include laccase, tyrosinase, catalase, and peroxidase. Laccase and tyrosinase are excellent in that there is no possibility that the function-expressing substance is denatured during gelation because there is no need to add any other chemicals that do not constitute gel components. Peroxidase can be instantly gelled when used in combination with an oxidizing agent such as hydrogen peroxide, and is suitable as an instantaneous adhesive or hemostatic agent at medical sites. These enzymes can be used alone or in combination of two or more.

The origin of the enzyme is not particularly limited, but includes the following.
Examples of the origins of copper enzymes such as laccase and tyrosinase (phenol oxidase) include urushi, mushrooms (Tuchikaburi, mushrooms), and molds (Polyporus vericolor). The origins of hydroperoxidases such as catalase and peroxidase are, for example, bovine liver, horse blood cells, human blood cells, M. lysodeikticus, horseradish, soybean, radish, turnip, thyroid, milk, intestine, leukocytes, erythrocytes, yeast, Caldariomyces Examples include fumago and Steptococcus faecalis.
Among these, tyrosinase is preferably derived from mushrooms and peroxidase is preferably derived from horseradish.

  The hydrogel of the present invention contains, for example, a collagen solution and a salt necessary for fibrosis of collagen in a buffer solution in which a phenolic hydroxyl group-introduced polysaccharide and enzyme are mixed, and preferably contains an oxidizing agent such as hydrogen peroxide. And can be prepared by reacting. As a result, a tissue including a hybrid composite structure in which fibrillated collagen is inserted into a cross-linked polysaccharide having a phenolic hydroxyl group introduced therein is formed.

  The pH of the collagen solution varies depending on the method for producing the collagen raw material. Collagen is mainly divided into an acid-solubilized collagen extracted with an acidic aqueous solution or an acidic aqueous solution containing a proteolytic enzyme, and an alkali-solubilized collagen extracted with an alkaline aqueous solution. When the collagen solution is an acid-solubilized collagen solution, the pH is preferably between 2.0 and 6.0. When the pH is lower than 2.0, the collagen molecules may be hydrolyzed, which is not preferable. If the pH is higher than 6.0, collagen may not be sufficiently solubilized, which is not preferable. On the other hand, when the collagen solution is an alkali-solubilized collagen solution, the pH is preferably between 5.5 and 10. When the pH is lower than 5.5, the collagen may not be sufficiently solubilized, which is not preferable. When the pH is higher than 10, the collagen molecule may undergo hydrolysis, which is not preferable.

  As the solvent for the collagen solution, in the case of an acidic solvent, water that is safe from the end use and widely used for industrial use, or an aqueous solution of hydrochloric acid, acetic acid, citric acid, fumaric acid, or the like is desirable. In the case of neutral to alkaline, water or an aqueous solution of phosphate, acetate, Tris or the like is desirable for the same reason as described above.

  The solute concentrations of the acidic solvent and the neutral to alkaline solvent are not particularly limited as long as the pH at which the collagen used is solubilized can be imparted to the solvent. However, if the solute concentration is too high, depending on the solute, the pH in the target range cannot be imparted, collagen fibrosis is inhibited, or physical properties such as cell adhesion of the resulting gel may be inhibited. Preferably it is 1.0M or less, More preferably, it is 0.50M or less.

  Various functional substances can be added to the collagen solution so as to further enhance the function of the collagen gel as long as the effect of the present invention of obtaining a highly heat-stable collagen gel is not inhibited. Specific examples include functional proteins such as cell growth factors, and functional polysaccharides such as hyaluronic acid, chondroitin sulfate, polylactic acid, β1-3 glucan, chitin, and chitosan.

  The collagen concentration of the collagen solution is preferably in the range of 0.01 to 3.0 (w / v)% from the viewpoint of collagen solubility, solution viscosity, or gel physical properties. When the concentration is lower than 0.01 (w / v)%, the gel strength may be insufficient, which is not preferable. When the concentration is higher than 3.0 (w / v)%, the viscosity of the collagen solution is too high, and it may be difficult to produce a gel. Preferably it is the range of 0.05-2.0 (w / v)%.

  The solvent for causing collagen fibrosis is not particularly limited. However, considering the end use of cell carriers or medical materials, salts having a buffering capacity such as phosphates, acetates, carbonates, Tris and the like that are not or low in cytotoxicity and are widely used for industrial use. It is preferable to use an aqueous solution. The pH suitable for collagen fibrillation varies depending on the type of collagen, but it is often in the range of pH 5 to 9, and a phosphate having a high buffering capacity in that range is particularly preferably used.

  The operation of mixing the collagen solution and the solution for causing fibrosis is performed while keeping the solution temperature at a temperature that does not greatly exceed the denaturation temperature. It is preferably the denaturation temperature of the collagen used + 5 ° C. or lower, more preferably the collagen denaturation temperature used or lower.

  The above-mentioned collagen denaturation temperature is a value determined from a change in optical rotation of a collagen solution when the collagen solution is heated stepwise as described in Journal of Food Chemistry 60, p.1233 (1995). .

  The amount of the fibrillated collagen in the hydrogel of the present invention is preferably 0.1 to 5 (mass ratio), more preferably 0.3 to 2 (mass ratio) with respect to the polysaccharide cross-linked product 1 having a phenol group introduced therein. ). When the amount of fibrotic collagen is large, the physical properties of the gel are lowered, and when the amount of fibrotic collagen is small, the cell proliferation action on the gel is lowered.

  The hydrogel of the present invention obtained by the above method has excellent mechanical strength with no decrease in strength due to the collagen portion, and at the same time, excellent thermal stability. Therefore, even when aquatic organism-derived collagen having a low denaturation temperature is used, a hydrogel having sufficient mechanical strength and thermal stability for application to cell carriers such as materials in regenerative medicine or medical materials can be obtained. .

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples.

Example 1: Synthesis of phenol group-containing hyaluronic acid derivative 100 mg (0.23 mmol) of hyaluronic acid (HA) was dissolved in 40 mL of deionized water, and 4.0 mg (0.029 mmol) of tyramine, 27 mg of N-hydroxysuccinimide (NHS) ( 0.23 mmol) was added and dissolved. Furthermore, 44 mg (0.23 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added and stirred at room temperature for 24 hours. Thereafter, the reaction solution was concentrated under reduced pressure, and the resulting concentrated solution was poured into a dialysis membrane (cut-off molecular weight 2000), followed by dialysis in water for 2 days (changing water 5 times). After the dialysis membrane, 1.0 g of a cation exchange resin (Amberlite IR120BH) was added to the reaction solution, and after stirring for 12 hours, the cation exchange resin was removed by filtration. The obtained reaction solution was dropped into a large excess of ethanol to perform reprecipitation, and the precipitated product was collected by centrifugation. A phenol group-containing hyaluronic acid derivative (HA derivative) was obtained by washing and drying under reduced pressure.

Example 2: Synthesis of gel The HA derivative obtained above was dissolved in a phosphate buffer (PBS) to prepare a 2.0% (w / v) HA solution, which was used for the production of each gel. As a phosphate buffer, PBS-1 does not contain NaCl as a 0.10M phosphate buffer (pH 7.0), and PBS-2 includes NaCl = 105 mM and sodium phosphate = 52.5 mM phosphate buffer (pH 6.8). ) Was used.
A collagen solution was prepared as follows.
The skin of a salmon (white salmon, scientific name: Oncorhynchus Keta) from which scales and body were removed with a scalpel was cut into approximately 3 cm squares. This was repeated three times with an equal volume mixed solvent of chloroform / methanol, degreased, washed twice with methanol to remove chloroform, and then washed three times with water to remove methanol. All subsequent steps were performed at 4 ° C.
130 g of the obtained defatted husk was immersed in 5 L of 0.5 M acetic acid at 4 ° C. and allowed to stand for 4 days. The swollen scab was filtered with medical gauze, and the filtrate was centrifuged at 10,000 × g for 30 minutes to precipitate insoluble matter, and 1.5 L of supernatant was collected. The supernatant was mixed with 50 mg of pepsin powder and gently stirred for 2 days. Sodium chloride was added to the obtained collagen solution so as to have a final concentration of 5%, and after gently stirring with a glass rod for 1 minute, it was allowed to stand for 24 hours. The white insoluble matter produced by salting out was centrifuged (same conditions as above) to recover the precipitate, and the precipitate was added to 2 L of 0.5 M acetic acid and dissolved by gently stirring. It took 3 days to dissolve. This operation was repeated once to obtain a colorless and transparent collagen solution. This collagen solution was dialyzed against deionized water using a cellulose tube. Deionized water was repeatedly exchanged until the pH of the dialyzed external solution was neutral, and the resulting neutral collagen solution was lyophilized to obtain white sponge-like collagen.
This sponge-like collagen was dried under reduced pressure using a desiccator containing silica gel, and added to pH 3.0 dilute hydrochloric acid preliminarily cooled to 4 ° C. so as to be 0.50 (w / v)% using the exact balance value, and gently stirred. And completely dissolved. This was used as a collagen solution.
Each gel was synthesized in a 24 well plate.
(1) Synthesis of hyaluronic acid gel Add 334 μL of PBS to 125 μL of 2.0% HA solution (PBS-1), 10 μL of 10 units / mL horseradish peroxidase (HRP) solution (solvent: PBS-1), and stir well did. Thereafter, 31 μL of a 0.06% (v / v) aqueous hydrogen peroxide solution was added and stirred for 1 to 2 minutes to obtain a hyaluronic acid gel.
(2) Synthesis of hyaluronic acid-non-fibrotic collagen hybrid gel To 125 μL of 2.0% HA solution (PBS-1), 334 μL of collagen solution and 10 μL of 10 units / mL HRP solution (solvent: PBS-1) were added. Stir well. Thereafter, 31 μL of a 0.06% (v / v) aqueous hydrogen peroxide solution was added and stirred for 1 to 2 minutes to obtain a hyaluronic acid-non-fibrotic collagen hybrid gel.
(3) Synthesis of hyaluronic acid-fibrotic collagen hybrid gel To 125 μL of 2.0% HA solution (PBS-2), 10 μL of 10 units / mL HRP solution (solvent: PBS-2) was added and stirred well. Then, 334 μL of collagen solution and 31 μL of 0.06% (v / v) hydrogen peroxide aqueous solution were quickly added and stirred for 1 to 2 minutes to obtain a hyaluronic acid-fibrotic collagen hybrid gel. The final concentrations of NaCl and sodium phosphate in the gel are 35 mM and 17.5 mM, respectively.

Example 3: Cell proliferation experiment (1) Cell culture The operation of adding 2.0 mL of PBS to a 24 well plate containing gel and allowing it to stand for several hours was performed three times to remove HRP and H ₂ O ₂ . During this operation, the gel was irradiated with UV to sterilize the gel. This gel was seeded with 3T3 cells at a density of 1.0 × 10 ⁴ cells / mL. The medium used was Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum (FBS) and penicillin-streptomycin added to a final concentration of 10% and 1 units / mL, respectively. The medium was changed every 3 days of culture.

(2) Observation of gel surface by SEM After 1, 3, 5, and 7 days of culture, the medium in the well was removed. 1 mL of 2.5% glutaraldehyde was added thereto, and the cells on the gel surface were immobilized by incubating for 1 hour in a bioshaker at a rotation speed of 200 rpm. Thereafter, glutaraldehyde was removed and washed with PBS three times. The gel in which the washed gel is immersed in 30%, 50%, 70%, 90%, and 100% ethanol aqueous solutions for 15 minutes in order to gradually replace the water in the gel with ethanol and completely replace with ethanol. Was immersed in t-butanol and replaced with t-butanol, and then the gel was freeze-dried to obtain a sponge. Gold was deposited on the sponge surface, and the surface was observed with SEM.
The results are shown in FIGS. In the case of a hyaluronic acid gel containing no collagen, the cells did not adhere to the gel at all as shown in FIG. 1 and formed aggregates. In the case of collagen that contains collagen but is not fibrotic, as shown in FIG. 2, the cells are only slightly adhered to the gel surface after 1 day of culture, and small cell aggregates after 3 days. It was observed. Although the aggregates of cells increased with the passage of the culture days, the amount of cells adhered to the gel surface was small. On the other hand, in the gel of the present invention, as shown in FIG. 3, many cells adhere to the gel surface after 1 day of culture, and after 3 days, the cells adhered to the gel surface grow and Was forming. After 5 days, the cells covered the gel over a wide area, and after 7 days, almost the entire surface of the gel was covered and became confluent.

(3) Measurement of the number of cells The number of cells grown on each gel was measured. The measurement was performed using a calibration curve prepared in advance. A calibration curve was prepared according to the following.
0 cells / mL, 1 × 10 ³ cells / mL, 5 × 10 ³ cells / mL, 1 × 10 ⁴ cells / mL, 2.5 × 10 ⁴ cells / mL, 5 × 10 ⁴ cells / mL, 7.5 × ^{10 4 cells / mL, 1 ×} 10 5 cells / mL, 2.5 × 10 5 cells / mL, 5 × 10 5 cells / mL, 1 × 10 of ⁶ cells / mL of each cell suspension in 24 well plate After adding 1 mL at a time, the cells were incubated in a CO ₂ incubator (temperature: 37 ° C., CO ₂ concentration: 5%) for 1 hour to allow the cells to adhere to the wells. After the medium of each well was changed, 100 μL of WST-1 (tetrazolium salt) was added and incubated in a CO ₂ incubator for 3 hours. Thereafter, the reaction is stopped by adding 100 μL of 100 mM hydrochloric acid, 1 mL of the reaction solution is taken to a newly prepared 24 well plate, 450 nm light is irradiated to the reaction solution in the well with a plate reader, and the absorbance is measured. did. A calibration curve was created using the obtained data (horizontal axis: number of cells, vertical axis: absorbance).

The method for measuring the number of cells on each gel is as follows.
After replacing the medium in each gel, 100μL added WST-1, CO ₂ incubator (temperature: 37 °, CO ₂ concentration: 5%) were incubated for 3 hours in a. After 3 hours, the reaction was stopped by adding 100 μL of 100 mM hydrochloric acid, and 1 mL each of the reaction solution was taken into a newly prepared 24 well plate, and the reaction solution in the well was irradiated with 450 nm light by a plate reader to absorb the absorbance. Was measured, and the relative cell number was determined based on a calibration curve prepared in advance.

The results are shown in FIG. In the case of the hyaluronic acid gel not containing collagen and the gel containing non-fibrotic collagen, it can be seen that the cells hardly grew. On the other hand, in the gel of the present invention, remarkable cell proliferation was observed, and after 7 days of culture, it was about 120 times that at the time of seeding.
From the above results, it can be seen that the hydrogel of the present invention containing fibrotic collagen can be an excellent cell culture carrier having high cell affinity.

The microscope picture which shows the state after culture | cultivating with hyaluronic acid gel which does not contain collagen, and culture | cultivation 1,3,5,7 days. The photomicrograph which shows the state after culture | cultivating the cell culture with the hyaluronic acid gel containing a non-fibrotic collagen and culture | cultivating 1,3,5,7 days. The photomicrograph which shows the state after culture | cultivation by the hyaluronic acid gel containing a fibrosis collagen and culture | cultivation 1, 3, 5, and 7 days. The graph which shows the proliferation number of the cell using each gel.

Claims (12)

  A hydrogel comprising a polysaccharide cross-linked product having a phenolic hydroxyl group introduced therein and fibrotic collagen. A polysaccharide into which a phenol group has been introduced is a polysaccharide and the following general formula (1)
(In the formula, R represents a hydroxyl group, —NHR ¹ group, or carboxyl group; R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; a is an integer of 1 to 10; ^{1 to} A ⁵ each independently represents a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 6 carbon atoms, wherein one or two of A ^{1 to} A ⁵ are a hydroxyl group, and two are hydroxyl groups. Is assumed that both hydroxyl groups are in the ortho-position or para-position.)
The hydrogel according to claim 1, which is a condensate with a phenol compound represented by the formula:   The hydrogel according to claim 2, wherein R is a hydroxyl group, a primary amino group, or a secondary amino group.   The hydrogel according to any one of claims 1 to 3, wherein the condensation ratio of the polysaccharide and the phenol compound represented by the general formula (1) is 1: 0.003 to 0.1 (mass ratio).   The hydrogel according to any one of claims 1 to 4, wherein the polysaccharide is hyaluronic acid, alginic acid or a derivative thereof.   The hydrogel according to claim 2, wherein the phenol compound represented by the general formula (1) is tyramine.   The hydrogel according to any one of claims 1 to 6, wherein the fibrillated collagen is derived from an aquatic animal.   The hydrogel according to claim 7, wherein the aquatic animal is a rabbit.   The hydrogel according to any one of claims 1 to 8, wherein the mass ratio of the cross-linked polysaccharide having a phenol group introduced to the fibrillated collagen is 1: 0.1-5.   A method for producing a hydrogel, characterized in that a polysaccharide having a phenol group introduced therein and collagen containing non-fibrotic collagen are subjected to cross-linking and fibrosis in the presence of an enzyme to form a gel.   The production method according to claim 10, wherein the enzyme is selected from laccase, tyrosinase, catalase and peroxidase. A cell culture carrier comprising the hydrogel according to claim 1.