JP2000239304A - Cell culture substrate - Google Patents

Abstract

(57) [Summary] [PROBLEMS] An artificial skin or the like by culturing various cells inside and / or on the surface of a culture substrate without using any crosslinking agent or the like, and having excellent safety and biocompatibility. Provided is a cell culture substrate that can be used as a material for constructing an artificial organ and also used for producing a useful substance by cell culture. SOLUTION: The cell culture substrate comprises a gel formed of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cell culture substrate containing a gel formed of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution.

[0002]

2. Description of the Related Art Conventionally, in the treatment of skin damage due to burns, abrasions, etc., human skin transplantation or freeze-dried swine dermis or chitin has been used to cover the injured site and replace defective tissue. Wound dressings such as nonwovens have been used. However, the amount of skin transplants that can be collected is limited,
Wound dressings only cover the damage and do not replace the function of the skin.Therefore, more effective treatments include artificial skin made of a culture substrate that provides a place for the growth of skin cells at the damaged site. Artificial skin in which skin cells are cultured in advance on a culture substrate has been studied.

[0003] As a material for a culture substrate used for such artificial skin, it is desirable to use a material that can be selected from various forms according to damage in addition to high biocompatibility and bioabsorbability.
Collagen, chondroitin sulfate, hyaluronic acid and the like have been studied. Above all, hyaluronic acid is a substance originally present in the human body and has a long track record of use as an ophthalmic surgery adjuvant and a drug for joint injection, so its biocompatibility and bioabsorbability have been fully confirmed. It had been. However, since hyaluronic acid is water-soluble, its use as a culture substrate by itself has been limited.

Therefore, attempts have been made to combine with other materials or to chemically modify hyaluronic acid to make it hardly water-soluble. For example, (1) a water-insoluble medical material comprising an ionic complex of hyaluronic acid and a polymer compound having an amino group or an imino group (JP-A-6-731)
No. 03), (2) a water-insoluble biocompatible material by a condensation reaction between hyaluronic acid and a crosslinking agent di (or bi) hydrazine or di (or bi) hydrazide (Japanese Patent Laid-Open No. 9-5)
No. 9303), and (3) artificial skin for surgical dermatology comprising a complete or partial ester of hyaluronic acid and an aliphatic or araliphatic ester (Japanese Patent No. 2569012).

However, (1) does not form a carrier by itself, and (2) and (3) are substances whose chemical structures are different from those of the original hyaluronic acid.

[0006]

SUMMARY OF THE INVENTION In order to make the most of the inherent biocompatibility inherent in hyaluronic acid itself, it is necessary to use no chemical cross-linking agent or chemical modifier and to use a cationic A means for rendering hyaluronic acid itself poorly water-soluble without forming a complex with a polymer compound has not been developed so far. We have found for the first time to produce a poorly water-soluble hyaluronic acid gel composed of hyaluronic acid alone without using a crosslinking agent or the like by a simple method (PCT / JP98 / 03536). The present inventors have intensively studied the possibility of application of the present invention to a cell culture substrate, found its usefulness, and completed the present invention.

[0007]

That is, the present invention provides (1)
A cell culture substrate containing a gel formed of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution, (2) the following (a),
(A) a cell culture substrate comprising a gel formed of hyaluronic acid alone which satisfies the requirement of (b);
(B) Hyaluronic acid solubilized by treating a hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid is 50% or less in a neutral aqueous solution at 37 ° C. in 12 hours. (3) a gel formed solely of hyaluronic acid, which has a structure and partially contains a molecular weight fraction having a degree of branching of 0.5 or more in the solubilized hyaluronic acid, 1 selected from the group consisting of crushed, sponge, lump, fibrous, or tubular
(2) the cell culture substrate according to (2), which has a dissolution rate of 50% or less in 12 hours in a neutral aqueous solution at 37 ° C., and promotes acid hydrolysis of hyaluronic acid; Hyaluronic acid gel partially containing a molecular weight fraction having a degree of branching of 0.5 or more in hyaluronic acid solubilized by treating the hyaluronic acid gel under conditions, and cells containing hyaluronic acid that has not been gelated A culture substrate, (5) a sheet-shaped, film-shaped, crushed, sponge-shaped, lump-shaped, fibrous, or tube-shaped hyaluronic acid gel formed of hyaluronic acid alone, and a non-gelled hyaluronic acid. (6) The cell culture substrate according to any one of (1) to (5), wherein the cell culture substrate is for artificial skin.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The hyaluronic acid gel referred to in the present invention is a polymer having a three-dimensional network structure and a swollen body thereof. The three-dimensional network structure is formed by a cross-linked structure of hyaluronic acid.

One example is the pH of hyaluronic acid.
Freezing and then thawing an aqueous solution of 3.5 or less to obtain a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution in a sheet, film, crushed, sponge, lump, fibrous, or tubular form. Can be. This will be described more specifically below.

The hyaluronic acid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissues or manufactured by fermentation. The strain used in the fermentation method is a microorganism having hyaluronic acid-producing ability, such as Streptococcus sp.
No. 3-123392, Streptococcus equi FM-100 (Microtechnical Laboratories No. 9027), and Streptococcus equi FM-300 described in Japanese Patent Application Laid-Open No. Hei 2-234689 (Microtechnical Labs No. 2319) )), A mutant which stably produces hyaluronic acid in high yield is desirable. Those cultured and purified using the above mutant strains are used.

The molecular weight of the hyaluronic acid used in the present invention is preferably in the range of about 1 × 10 ⁵ to about 1 × 10 ⁷ daltons. In addition, as long as it has a molecular weight within the above range, those obtained by subjecting them to hydrolysis treatment or the like from those having a higher molecular weight can also be preferably used. It should be noted that the hyaluronic acid according to the present invention is used in a concept that also includes alkali metal salts thereof, for example, sodium, potassium and lithium salts.

[0012] The term "hyaluronic acid alone" as used in the present invention means that no chemical crosslinking agent or chemical modifier other than hyaluronic acid is used, and that it does not form a complex with a cationic polymer. Is what it means.

The hyaluronic acid gel referred to in the present invention can be decomposed and solubilized by treating the hyaluronic acid gel under the conditions of the accelerated acid hydrolysis reaction of hyaluronic acid. When the solubilized hyaluronic acid retains a crosslinked structure,
Hyaluronic acid having a branch point can be distinguished from linear hyaluronic acid in terms of polymer solution theory.

The conditions for the accelerated acid hydrolysis reaction of hyaluronic acid referred to in the present invention include a pH of an aqueous solution of 1.5 and a temperature of 60 ° C.
Is appropriate. The main chain cleavage reaction due to hydrolysis of the glycosidic bond of hyaluronic acid, compared with neutral aqueous solution,
Significantly promoted in acidic or alkaline aqueous solutions. Further, the acid hydrolysis reaction is promoted at a higher reaction temperature.

In the present invention, the GPC-MALLS method is used,
The molecular weight and the degree of branching of the molecular weight fraction separated by GPC were continuously measured online. In the present invention, the degree of branching is measured by using the elution volume method of calculating the degree of branching by comparing the molecular weight of the solubilized hyaluronic acid of the fraction having the same elution volume with the molecular weight of the linear hyaluronic acid as a control. Was. The degree of branching is the number of branch points present per one polymer chain of solubilized hyaluronic acid, and is plotted against the molecular weight of solubilized hyaluronic acid.

The solubilized hyaluronic acid was diluted with a GPC solvent to adjust the concentration, filtered through a 0.2 μm membrane filter, and used for measurement. In the case where the hyaluronic acid gel referred to in the present invention has a cross-linked structure that is stably present even under the accelerated acid hydrolysis conditions of hyaluronic acid, a branched structure is confirmed in the solubilized hyaluronic acid in terms of a polymer solution. The degree of branching of the hyaluronic acid gel referred to in the present invention is 0.5
That is all.

As the acid used for adjusting the pH of the aqueous solution of hyaluronic acid, any acid can be used as long as the pH can be adjusted to 3.5 or less. In order to reduce the amount of acid used, it is desirable to use a strong acid, for example, hydrochloric acid, nitric acid, sulfuric acid and the like.

The pH of the aqueous solution of hyaluronic acid is such that the carboxyl groups of hyaluronic acid are protonated at a sufficient rate.
Adjust to H. The pH to be adjusted is appropriately determined according to various kinds of properties such as the type of the counter ion of the hyaluronic acid salt, the molecular weight of the hyaluronic acid, the concentration of the aqueous solution, the conditions for freezing and thawing, and the strength of the gel to be formed. The pH is adjusted to 0.5 or less, preferably to 2.5 or less.

For freezing and thawing, an operation of placing an acidic aqueous solution of hyaluronic acid in an arbitrary container, freezing it at a predetermined temperature, and thawing at a predetermined temperature after freezing is performed at least once. . The temperature and time for freezing and thawing are appropriately determined within the range of the temperature and time for freezing and thawing the acidic aqueous solution of hyaluronic acid depending on the size of the container and the amount of the aqueous solution. Is preferred. Freezing and thawing time can be shortened,
More preferably, a freezing temperature of −5 ° C. or less and a thawing temperature of 5 ° C. or more are selected. The time is not particularly limited as long as it is equal to or longer than the time at which freezing and thawing is completed at that temperature.

The number of repetitions of the operation of freezing and then thawing the adjusted acidic aqueous solution of hyaluronic acid depends on the molecular weight of the hyaluronic acid used, the concentration of the aqueous solution, the pH of the aqueous solution, the temperature and time of freezing and thawing, and the gel formed. It is appropriately determined according to various characteristics such as the strength of the rubber. Normally, it is preferable to repeat it one or more times. Further, each time the operation of freezing and thawing is repeated, the temperature and time of freezing and thawing may be changed.

The hyaluronic acid gel obtained by freezing and thawing the adjusted acidic solution of hyaluronic acid needs to exclude components such as acids used for adjusting the acidity in order to avoid acid hydrolysis of hyaluronic acid. is there. In order to remove components such as acids, washing or dialysis is usually performed with an aqueous solvent. The solvent to be used is not particularly limited as long as it does not impair the function of the hyaluronic acid gel, for example, water, saline,
Phosphate buffer and the like are used, and preferably, physiological saline, phosphate buffer and the like are used.

The washing / dialysis method is not particularly limited, but is usually a batch method, a filtration method, a method of filling a column or the like, and passing the solution through. A method using a filtration membrane or the like is suitably used. These conditions are:
Any conditions can be used as long as the components to be removed, including the amount of liquid and the number of times, can be reduced to the target concentration or less, and can be appropriately selected depending on the form and use of the hyaluronic acid gel.

The washed and dialyzed hyaluronic acid gel may be immersed in a solvent, wet with a solvent, air-dried, dried under reduced pressure or freeze-dried depending on the purpose of use. To serve as a substrate for cell culture.

Processing such as molding of hyaluronic acid gel is as follows:
At the time of preparation, the desired form of sheet, film, crushed, sponge, fiber, or tube-shaped hyaluronic acid gel can be prepared by selecting the container and method for freezing the adjusted acidic solution of hyaluronic acid. It is possible. For example, a film-like or sheet-like form can be obtained by casting and freezing on a plate, or a crushed form can be obtained by freeze-thawing with vigorous mixing and stirring with an organic solvent immiscible with water.

By changing the conditions such as the molecular weight and concentration of hyaluronic acid, various poorly water-soluble hyaluronic acid gels having different bioabsorbability can be obtained.

The substrate for cell culture containing the poorly water-soluble hyaluronic acid gel of the present invention can be used by itself or in combination with other physiologically active substances, biocompatible substances, antibiotics and the like.

As the physiologically active substance, factors that promote the growth of cells to be cultured are important. Examples of such factors include insulin, hepatocyte growth factor, torus forming growth factor, and the like.
Examples include nerve growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, fibroblast growth factor, colony stimulating factor, erythropoietin, transferrin, interleukin, interferon and the like.

As the biocompatible substance, a substance that promotes adhesion of cells to be cultured is important, and examples thereof include collagen, fibronectin, vitronectin, laminin, albumin, chondroitin sulfate, keratan sulfate, heparan sulfate, and hyaluronic acid. No.

As antibiotics, streptomycin,
Penicillin, tobramycin, amikacin, gentamicin, neomycin, amphotericin B and the like.

The substrate for cell culture containing the poorly water-soluble hyaluronic acid gel of the present invention allows various cells to be cultured inside and / or on its surface, and can be used as a material for constructing artificial organs such as artificial skin. It can also be used for producing useful substances by cell culture.

Autologous skin cells can be cultured on the cell culture substrate of the present invention and used as an autograft in the treatment of skin wounds caused by burns, abrasions, ulcers and the like. Similarly, autologous mucosal cells can be cultured and used for treatment of damage to mucous membranes in the oral cavity and the like.

[0032]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

Preparation Example 1: Preparation of a sheet-shaped poorly water-soluble hyaluronic acid gel Hyaluronic acid having a molecular weight of 2 × 10 ⁶ daltons was dissolved in distilled water to prepare a 1% solution. After adjusting the pH to 1.5 by adding 1N nitric acid to the aqueous solution, the solution was placed in a flat-bottomed container and frozen in a freezer set at -20 ° C. After 5 days, it was taken out and thawed at 25 ° C to obtain a poorly water-soluble hyaluronic acid gel. Next, the gel was immersed and neutralized in 100 ml of 25 mM phosphate buffered saline having a pH of 7 for 24 hours, then sufficiently filtered and washed with distilled water, and dried to obtain a sheet-like poorly water-soluble hyaluronic acid gel.

Example 1: Solubility test of poorly water-soluble hyaluronic acid gel A phosphate buffer component was added to physiological saline at a concentration of 50 mM, and p
H7 phosphate buffered saline was prepared. The poorly water-soluble hyaluronic acid gel prepared in Preparation Example 1 was immersed in 50 ml of phosphate buffered saline and gently stirred. The percentage of hyaluronic acid eluted in phosphate buffered saline at 37 ° C.
It was determined from the concentration of hyaluronic acid in phosphate buffered saline.

Measurement of Hyaluronic Acid Concentration Hyaluronic acid concentration in phosphate buffered saline was measured by GPC
Was determined from the peak area of the differential refractive index detector.

As a result, the dissolution rate of the hyaluronic acid gel obtained in Preparation Example 1 was 2% after 12 hours,
It was 4% after 24 hours, and 86% of the hyaluronic acid gel remained after 24 hours. Therefore, it is suggested that the hyaluronic acid gel obtained in Preparation Example 1 is poorly soluble in a neutral aqueous solution.

Example 2: Measurement of degree of branching of poorly water-soluble hyaluronic acid gel The poorly water-soluble hyaluronic acid gel obtained in Preparation Example 1 was subjected to pH
It was immersed in 15 ml of 1.5 aqueous hydrochloric acid solution and hydrolyzed at 60 ° C. for 6 hours. The gel was solubilized by hydrolysis, and diluted twice with GPC solvent to a concentration of 0.05.
% By weight, filtered through a 0.2 μm membrane filter, injected 0.1 ml, and measured for GPC-MALLS. As a result, the degree of branching was 0.5 or more.

Example 3 Bioabsorbability Test The poorly water-soluble hyaluronic acid gel of Preparation Example 1 or 1 ml 1% intraperitoneally intraperitoneally of a mouse (ICR @ 8 weeks old)
The hyaluronic acid solution was charged. After 2, 4 and 8 days after the operation, the abdomen was opened, the remaining gel was collected, and 2 ml of physiological saline was intraperitoneally injected.
And the mixture was stirred well to collect the liquid. The gel and the solution were alkali-treated (0.1 N NaOH, 2 h), neutralized, and the concentration of hyaluronic acid was measured by gel filtration (ShodexOHpakKB806) to calculate the residual ratio. Table 1 shows the results.

[0039]

[Table 1]

According to Table 1, the hyaluronic acid gel obtained in Preparation Example 1 had 41% remaining after 4 days, whereas the hyaluronic acid solution was 0%. It suggests a long residence time in the body. That is, compared with the hyaluronic acid solution, the absorption rate in the living body is slow, suggesting that it has a bioabsorbable property useful as a cell culture substrate.

Example 4: Cell culture test A sheet-like poorly water-soluble hyaluronic acid gel (2 × 2 cm) prepared in Preparation Example 1 was placed in a 35 mm cell culture dish.
A sheet (2 × 2 cm) obtained by freeze-drying the same amount of hyaluronic acid as in Preparation Example 1 was placed, and 2 × 10 ⁴ normal human neonatal foreskin-derived fibroblasts were placed at 37 ° C. in 10% FCS-DMEM medium. The cells were cultured in a CO ₂ incubator and compared with the case of a petri dish alone. After 3 and 5 days, trypsin /
The cells were collected by EDTA, and the cells were counted. Table 2 shows the results.

[0042]

[Table 2]

As a result, when observed with an inverted phase-contrast microscope, cells adhered to the petri dish alone, and in the case of poorly water-soluble hyaluronic acid gel, cells were observed on and inside the sheet. However, when the hyaluronic acid was freeze-dried, the sheet was dissolved, the original shape was not retained, and the cells were on the petri dish. These cell numbers are shown in Table 2,
There is no significant difference in the proliferative property, and the one obtained by culturing skin cells on a sheet-like poorly water-soluble hyaluronic acid gel is suitable as artificial skin.

[0044]

According to the present invention, it is possible to provide a cell culture substrate containing a poorly water-soluble hyaluronic acid gel formed of hyaluronic acid alone. Since such a cell culture substrate of the present invention does not use a crosslinking agent or the like, it is excellent in safety and biocompatibility, and by culturing various cells inside and / or on the culture substrate, artificial skin In addition to being usable as a material for constructing an artificial organ such as the above, the present invention has an effect that it can be used for producing a useful substance by cell culture.

Claims (6)

[Claims] 1. A cell culture substrate comprising a gel formed of hyaluronic acid alone, which is hardly soluble in a neutral aqueous solution. 2. A cell culture substrate comprising a gel formed of hyaluronic acid alone, which satisfies the following requirements (a) and (b). (A) 1 with a neutral 37 ° C. aqueous solution
(B) hyaluronic acid solubilized by treating the hyaluronic acid gel under accelerated acid hydrolysis conditions for hyaluronic acid having a dissolution rate of 50% or less in 2 hours has a branched structure; In the hyaluronic acid thus obtained, the degree of branching is 0.5
The above molecular weight fraction is partially contained. 3. A gel formed with hyaluronic acid alone,
3. The cell culture substrate according to claim 2, wherein the substrate is one selected from the group consisting of a sheet, a film, a crushed, a sponge, a lump, a fiber, and a tube. 4. A hyaluronic acid which has a solubility of 50% or less in 12 hours in a neutral aqueous solution at 37 ° C. and is solubilized by treating a hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid. A cell culture substrate comprising a hyaluronic acid gel partially containing a molecular weight fraction having a degree of branching of 0.5 or more and a non-gelled hyaluronic acid. 5. A cell culture containing a hyaluronic acid gel formed of hyaluronic acid alone in a sheet, film, crushed, sponge, massive, fibrous, or tubular form, and a non-gelled hyaluronic acid. Substrate. 6. The cell culture substrate according to claim 1, wherein the cell culture substrate is for artificial skin.