JPH0654899A - Production of two-layer type protein sheet - Google Patents

Abstract

PURPOSE:To industrially and inexpensively provide the two-layer type protein sheet having excellent cytoaffinity and bioaffinity by superposing a porous sheet of protein on one surface of a sheet-like gel of protein and treating the gel so as to dry the gel and form the thin film thereof. CONSTITUTION:The two-layer type protein sheet which has the affinity to animal cells and is adequate as a culture base material and biomaterial is formed by superposing the porous body of the protein, at least one surface 2 of which is formed of wall surfaces 3 having open cells, on one surface of the sheet-like gel of the protein in such a manner that the surface having the open cells remains, drying the gel without making the gel to a sol or soln. state to form the thin film 1 and joining and integrating the thin film to the superposed porous sheet. Gelatin, collagen, figroin, etc., are used in terms of ease of gelatinization as the protein to be used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a bilayer protein sheet, and particularly to a method for producing a bilayer protein sheet which has an affinity for animal cells and is suitable as a culture substrate or biomaterial. Regarding

[0002]

2. Description of the Related Art In culturing animal cells, from basic research such as developmental organism research and research on response of physiologically active substances and cells to industrial production of physiologically active substances such as interferon and TPA, development of hybrid artificial organs, etc. Active research is being conducted. Since most animal cells are adhesion-dependent, they have a high affinity for cells during culture,
Moreover, a base material that allows the cultured cells to maintain the proliferation and differentiation functions is required.

Hitherto, as a membrane-shaped substrate for culturing animal cells, a porous membrane filter such as a cellulose-based or polycarbonate-based material, or a porous membrane filter coated with collagen or a collagen film has been used. However, the one using a membrane filter is opaque so that microscopic observation of cells is not possible, and in the application to hybrid artificial organs, for example, skin cells are cultured on a substrate and transplanted to a living body as artificial skin. However, it has a drawback that it cannot be used due to lack of biocompatibility and biodegradability.

On the other hand, it is possible to produce a collagen membrane, which is a protein derived from a living body, which overcomes the above-mentioned drawbacks to some extent.
In the 266064 publication, the manufacturing method is proposed. However, the performance is still not satisfactory.

In the collagen type, in recent years, collagen and chondroitin-6 have been used as a hybrid type artificial skin substrate.
-A material having a thin film of sulfuric acid and a porous layer (Steve
nT. Boyce, Deborah J. Christ
ianson, and John F. Hansbro
Ugh, J. et al. Biomed. Mater. Res. , V
ol. 22, 939-957 (1988)) and collagen having a similar structure (Norimitsu Kuroyanagi et al., No. 1).
8th Symposium on Medical Polymers 31, JP 2
-71749) has been studied or proposed,
It is reported to have excellent performance.

The manufacturing method of these materials having a thin film and a porous layer is as follows. In the material of Boyce et al., A suspension of collagen and chondroitin-6-sulfate co-precipitate containing dimethylsulfoxide was sprayed onto a smooth, non-sticky surface and then dehydrated using the same material. After stacking the sponge bodies of, freeze-dry. This method can change the thickness of the thin film by changing the spray amount. However, since it is spraying, it is difficult to obtain uniform film thickness.
Especially when the film thickness is small or when making a large area,
It is difficult to obtain uniformity.

In the material of Kuroyanagi et al., Atelocollagen is treated with a homogenizer to form a cream, which is neutralized and gelled in an ammonia atmosphere, washed with water, and then at -80 ° C.
Quench and vacuum freeze dry. After that, it is crosslinked with an aqueous solution of glutaraldehyde, washed with water and freeze-dried. This method does not describe adjustment of thin film thickness,
Adjustments in this way can be difficult.

In the method described in JP-A-2-71749, a collagen film is first prepared, and a mixed solution of fibrotic collagen and denatured collagen is poured on the collagen film,
Freeze at 30 ° C. and lyophilize. Although this method can adjust the thickness of the thin film, it has a drawback that the thin film becomes difficult to handle because it is prepared separately.

As described above, a material having a collagen-based thin film and a porous layer has excellent characteristics as a cell culture substrate or a biomaterial, but there are various problems in its production method, and thus there are various problems. It has been difficult to manufacture high-performance materials of this kind at high cost and at low cost.

[0010]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and an object thereof is to have excellent cell affinity and biocompatibility, and in particular, a culture substrate for animal cells. Another object of the present invention is to provide a method for industrially and inexpensively producing a bilayer protein sheet having a thin film and a porous layer which are excellent as biomaterials.

[0011]

Means for Solving the Problems The above-mentioned object is to provide a protein porous sheet formed by a wall surface having open pores on at least one side of a sheet-like gelled product of a protein, and a surface having open pores. Of the two-layer protein sheet comprising a thin film and a porous layer, characterized in that the gelled product is dried to form a thin film, and the thin film and the porous sheet are bonded and integrated together. This is achieved by the manufacturing method.

The present invention will be described in detail below. The sheet-like gelled product of the protein used in the present invention refers to a product obtained by casting a protein solution on a suitable base material, for example, a plate-shaped mold, and gelling it into a sheet. Any protein may be used as long as it has excellent cell affinity and biocompatibility, and gelatin, collagen, and fibroin are preferable because gelatinization is easy, and among these, gelatin and collagen are particularly preferable.

Gelatin may be industrially obtained mainly from beef bone, cowhide or pig skin by an alkali method or an acid method, and these gelatins may be further purified, for example, gelatin of the Japanese Pharmacopoeia or purified gelatin. Those that meet the standards are preferable. Further, gelatin obtained by thermally denaturing commercially available collagen can also be used.

Various types of collagen are commercially available, and these can be used in the production method of the present invention if a gel is formed by an appropriate method. Above all,
From the viewpoint of biocompatibility, it is preferable to use so-called atelocollagen obtained by treating collagen derived from cowhide or pigskin with acid or alkali and then treating with protease or pepsin to digest and remove the telopeptide at the molecular end.

When these proteins are cast in a mold,
Prepare a solution or dispersion by a method suitable for each. The concentration of protein is preferably 10% by weight in the case of gelatin.
In the case of collagen, it is preferably 5% by weight or less, more preferably 1% or less.

In the case of gelatin, for example, gelatin may be dissolved in water as it is to form an aqueous solution or in a buffer solution. In the case of collagen, atelocollagen is generally used as an acid solution, but it may be mixed with a balanced salt solution such as a phosphate buffer solution immediately before casting. However, when the solution is mixed with a balanced salt solution and the pH is adjusted to around neutrality, gelation occurs when the temperature rises, so it is necessary to keep the solution at a low temperature (about 4 ° C.).

A cross-linking agent may be added to the above-mentioned protein solution for the purpose of cross-linking for imparting water resistance and increasing stability in vivo. As the crosslinking agent, a water-soluble crosslinking agent, for example,
Water-soluble epoxy compounds and aldehydes are suitable.
The addition amount can be appropriately selected, but is usually preferably 1 to 20% by weight based on the protein.

The mold used for casting the protein solution is a suitable plate-like body having a hollow so that the protein can be held, or a frame framed with an adhesive tape or the like.
It is necessary that the material of the plate-shaped body is such that proteins do not adhere to it after drying.

As an example of such a plate-like body, the liquid contact surface is
Examples thereof include plate-like members made of fluororesin such as poly-4-fluoroethylene, polyethylene, polypropylene, acrylic resin, polyvinyl chloride and polystyrene. However, the material is not limited to these, and other materials can be used as long as they satisfy the above conditions.

In order to obtain a bilayer protein sheet having a uniform thin film, it is necessary that the gelled product in sheet form has a uniform thickness. For this, the formwork must be kept horizontal. To keep the formwork horizontal,
For example, there is a method of placing a formwork on a surface plate with feet whose height can be adjusted and keeping it horizontal, but other methods can also be used.

As described above, the amount of the protein solution that is cast on the horizontal mold is appropriately adjusted so that the thickness of the thin film formed after drying falls within the preferable range described later.

As the gelation method, a method suitable for each protein is used. In the case of gelatin, it can be gelled by cooling, for example. The cooling temperature may be below the gelling temperature of the gelatin used and above the freezing temperature of the gelatin solution.

In the case of collagen, for example, a gel can be formed by casting an acidic solution and then contacting it with ammonia gas in the range of 25 to 37 ° C. Further, the low-temperature collagen solution which is mixed with a balanced salt solution or the like before casting to have pH near neutrality can be gelated by holding it at 25 ° C to 37 ° C after casting.

Next, the protein that constitutes the porous sheet of protein formed by the wall surface having open pores on at least one surface may be any protein as long as it has excellent cell affinity and biocompatibility, and particularly gelatin, Collagen is preferred.
As the gelatin or collagen, the same ones as those used for preparing the sheet-like gelled product can be preferably used.

The porous sheet suitable for use in the present invention can be manufactured, for example, as follows. That is, first, a protein solution containing a crosslinking agent is prepared. The same protein solution and cross-linking agent as those used for preparing the sheet-like gelled product can be preferably used. Next, the mixed solution of the protein and the cross-linking agent is cast on, for example, a plate-shaped mold. The mold used at this time is also preferably the one described above.
Then, the mold in which the protein cross-linking agent mixed solution is cast is placed on a table kept below the freezing temperature of the protein solution and cooled.

Examples of such a stand include a plate in which a refrigerant is circulated inside and a cooling plate using dry ice or another cryogen. The pedestal is preferably kept horizontal so that the porous sheet has a uniform thickness. If it is frozen in an environment such as a freezer that is cooled from the surroundings, the amount of open pores formed on the air surface is reduced.

The cooling temperature needs to be a temperature at which the protein solution freezes. This cooling temperature and cooling rate are important factors that control the pore size. The smaller the cooling temperature and the faster the cooling rate, the smaller the pores. Conversely, the pore size can be adjusted by appropriately selecting the cooling temperature and the cooling rate.

In the production of this porous sheet, the protein solution cast on the mold may be gelled prior to cooling and freezing. As the gelling method, the method used for preparing the above-mentioned sheet-like gelled product can be used.

Next, the frozen product is dried by vacuum freeze drying. Although general methods and conditions can be applied to the vacuum freeze-drying, in the method of the present invention, it is necessary to keep the temperature as low as possible in order to prevent loss of the crosslinking agent during drying. 3 during the drying period
It is preferably 0 ° C. or lower.

After freeze-drying, heat treatment is preferably performed to accelerate the crosslinking reaction. If the heat treatment temperature is higher, the reaction is accelerated, but if the heat treatment temperature is too high, the sheet is colored when heat treated in air. 150 for air treatment
C. or less is preferable. The heat treatment time is determined by the relationship with the treatment temperature. The higher the temperature, the shorter the crosslinking progresses and the insoluble in water, but the lower the temperature, the longer the time.

The porous body can be produced by the same method even if a protein solution containing no crosslinking agent is used. However, in this case, it is preferable that the resin is finally crosslinked by a suitable method to impart water resistance. The porous sheet produced in this manner has open pores on the air side, and the pore walls stand vertically. On the other hand, the mold side is a non-porous or porous thin film having a thickness of 2 μm or less.

The production method of the present invention is carried out, for example, as follows, using the above-mentioned sheet-like gelled product of protein and a porous protein sheet having at least one surface formed by a wall surface having open pores. . That is, first, a porous sheet of protein having at least one surface formed by a wall surface having open pores is laid on one surface of the sheet-like gelled product of protein so that the surface having open pores remains. Next, the gelled product is dried without forming a sol or solution to form a thin film and is joined and integrated with the superposed porous sheet.

Here, it is important to dry the gelled sheet without making it into a sol or solution. When the sol or solution is used, this sol or solution is
It is absorbed in the porous sheet, and a bilayer protein sheet having a desired thin film and porous layer cannot be stably obtained.

In order to dry the sheet-like gelled product without making it into a sol or solution form, in the case of gelatin, the gelled product is dried at a temperature below the melting point. The melting point changes depending on the type and concentration of gelatin. In the case of the present invention, it is preferable to dry at a temperature not to be frozen of 25 ° C or lower, more preferably 20 ° C or lower.
In the case of collagen, it may be dried at a temperature of 37 ° C. or lower that does not freeze in order to prevent deterioration.

As a result, the sheet-like gelled product of the protein is bonded as a thin film to the superposed protein porous bodies to form a bilayer protein sheet. In the method of the present invention, when the gelled sheet of protein contains a crosslinking agent, the crosslinking reaction may be promoted by heat treatment after drying. The conditions for the heat treatment may be the same as those for preparing the porous body.

In the method of the present invention, when the sheet-like gelled product does not contain a cross-linking agent, it may be cross-linked by a suitable method after forming the bilayer protein sheet. The bilayer protein sheet produced as described above may be washed with water, alcohol, a water / alcohol mixture, or other suitable solvent, if necessary.

The bilayer protein sheet obtained by the production method of the present invention has a structure as shown in FIGS. FIG. 1 is an explanatory view showing a cross-sectional structure of the bilayer protein sheet of the present invention, and FIG. 2 is a plan view of the bilayer protein sheet obtained by the production method of the present invention as seen from the side having open holes. In the figure, (1) is a dense thin film, (2) is an open hole surface, (3) is a wall surface, and (4) is an open hole.

As shown in FIGS. 1 and 2, the bilayer protein sheet according to the production method of the present invention has an open pore surface having a dense thin film (1) on one surface and open pores (4) on the other surface. (2)
(Porous layer). The dense thin film (1) and wall surface (3) are made of protein. The dense thin film (1) is a membranous body that does not substantially contain pores or the number or size of pores is overwhelmingly smaller than that of the porous portion, and it is separated on both sides of the protein sheet by the production method of the present invention. Considering the case of culturing cells, the dense thin film is preferably non-porous or has pores of 1 μm or less.

The thickness of the dense thin film is preferably thick from the viewpoint of handleability of the sheet, but is preferably thin from the viewpoint of permeability of the substance. Usually, it is preferably 200 μm or less, more preferably 50 μm or less.

The wall surface (3) which is integrated with the dense thin film (1) and constitutes the other surface on one surface of the bilayer protein sheet according to the production method of the present invention is formed with open holes (4). The open hole is an end surface of the wall surface (3) forming the bilayer protein sheet. The open pores are preferably large for cell culture, and preferably 50 μm or more on average.

The size of the open hole was determined by taking a photograph of the surface having the open hole with a scanning electron microscope (SEM) and using the obtained photograph as follows. That is, the number of aperture openings on a long line having a constant length in the longitudinal direction and the lateral direction of the photograph was determined, and the length of the straight line was divided by this value to obtain the average pore diameter. In the present invention, 5 in either direction
It is preferably 0 μm or more.

The bilayer protein sheet according to the production method of the present invention may contain closed pores in addition to the open pores described above. The number of closed pores is not particularly limited, but it is preferably as small as possible in consideration of cell invasion and medium permeation. Further, the pore shape is preferably such that the wall surface (3) stands in the vertical direction, and ideally one pore is continuous up to the thin film (1), that is, it is preferable that the closed pore is not included. . Further, the types of proteins used for the sheet-like gel product to be superposed and the porous sheet having at least one surface formed by the wall surface having open pores may be the same or different.

[0043]

INDUSTRIAL APPLICABILITY As described above, according to the method for producing a bilayer protein sheet of the present invention, a gel sheet of a protein, which is prepared in advance to a uniform thickness, is dried without forming a sol or solution. Then, since a thin film is formed, a bilayer protein sheet having a thin film having a uniform thickness can be obtained. In addition, since the sheet-like gelled product is prepared by casting the protein solution on an appropriate base material and then gelling it, it is easy to increase the area.

Further, the thickness of the thin film can be easily changed by changing the thickness of the gelled product in sheet form or the concentration of the protein constituting it. According to the production method of the present invention, a bilayer protein sheet can be produced in which most of the porous layer is open pores and the wall surface forming the pores stands in the direction perpendicular to the thin film.

This bilayer protein sheet is excellent in cell invasion and medium penetration in cell culture. In addition, cell culture is possible on both the thin film and the porous layer. Further, it can be used for the purpose of studying the interaction between cells by culturing two types of cells separately on both sides.

According to the manufacturing method of the present invention, since the thin film and the porous layer can be formed in an integrated form, there is an advantage that the thin film can be handled even if it is made as thin as possible. Furthermore, according to the production method of the present invention, different proteins can be used in the thin film and the porous layer, and a film having different properties between the thin film and the porous layer can be produced.

Next, the present invention will be specifically described with reference to examples.

[Examples 1 and 2]

[Preparation of Porous Sheet] Commercially available gelatin [viscosity 28 mp, jelly strength 96 g (6.66%),
5% of [Nippi Co., Ltd.] was added to the aqueous solution as a cross-linking agent of glycerol polyglycidyl ether (Nagase Kasei Co., Ltd.).
10% by weight of gelatin was added and dissolved. This mixed solution was cast on a polymethylmethacrylate plate having a thickness of 2 mm and framed with an adhesive tape in an amount of ² g / 64 cm ² , and then cooled and gelled for 20 minutes on a horizontal table kept at 5 to 10 ° C. After that, it was placed on a plate kept at -70 ° C with dry ice, cooled from the lower surface and frozen, and then 2
Lyophilization was performed at 5 ° C or lower. After completion of drying, heat treatment was performed at 110 ° C. for 3 hours.

[Production of Bilayer Sheet] A mixture of gelatin and a cross-linking agent used for producing the porous sheet was applied to the same polymethylmethacrylate plate as used for producing the porous sheet.
cast in an amount of g / 64 cm ² or 2 g / 64 cm ² , 5
It was cooled and gelled for 25 minutes on a horizontal table kept at -10 ° C.

Next, the porous sheet prepared by the above method was placed on the gelled product so that the surface in contact with the polymethylmethacrylate plate was in contact with the gelled product, and left for 10 minutes. Then, after air-drying at 110 ° C. × 65% RH overnight, heat treatment was performed at 110 ° C. for 3 hours. Further, after immersing and washing in water at 50 ° C. for 5 hours, it was freeze-dried again.

SEM observation of this sheet revealed that it was a porous sheet as a whole and had a non-porous thin film on one side.
The other side was a bilayer structure with a large number of open pores. SE
The average pore diameter of the open pores measured from the M photograph is 96 μm × 91.
was μm. On the other hand, the thickness of the thin film was 5.8 μm for the film formed at 1 g / 64 cm ² and 8.8 μm for the film formed at ² g / 64 cm ² .

(Cell culture experiment) This sheet has a diameter of 30 m.
It was cut out into a circle of m and was adhered to the lower surface of a cylindrical cup with a foot of 5 mm in height to give a culture cup. Culture cup
Two kinds of porous sheets were prepared, one with the thin film surface facing up and one with the open pore surface facing up.

Human fibroblasts were cultured using this cup. Put the cup in a dish with a diameter of 35 mm,
A suspension of 4 × 10 ⁵ human fibroblasts in 1 ml of culture medium was poured into a cup. Further, 2 ml of the culture solution was added, and the cells were cultured at 37 ° C for 1, 4 and 8 days. After a predetermined period,
The cells were fixed with methanol, stained with Giemsa, air-dried, and then the cell morphology was observed with an optical microscope. The culture medium used was Dulbecco's Modified Eagles Medium.

Good results were obtained in all the films. That is, a large number of cells adhered and spread on both the thin film surface and the open pore surface and maintained a good condition for up to 8 days. The cells cultivated on the open pore surface were three-dimensionally cultivated with cells attached to the wall surface.

[0055]

Examples 3 to 5 2.5% aqueous solution of commercially available gelatin [viscosity 52 mp, jelly strength 220 g (6.66%), manufactured by Nippi Corporation] was added as a cross-linking agent to glycerol polyglycidyl ether (Nagase Kasei Co., Ltd.). 10% of gelatin was added and dissolved.This mixed solution was applied to a 2 mm-thick polymethylmethacrylate plate framed with an adhesive tape to 2 g / 64 cm ² , 1 g / 64 cm ² or 0.5 g / 6.
It was cast in an amount of 4 cm ² and cooled on a horizontal table kept at 5 to 10 ° C. for 25 minutes.

The porous sheet prepared in Example 1 was placed on the gelled product so that the surface in contact with the polymethylmethacrylate plate was in contact with the gelled product and left for 10 minutes. Thereafter, a bilayer sheet was manufactured in the same manner as in Example 1.

SEM observation of this sheet revealed that it was a porous sheet as a whole and had a non-porous thin film on one side.
The other side was a bilayer structure with a large number of open pores. SE
The average pore size of the open pores measured from the M photograph is 98 μm × 93
was μm. On the other hand, the thin film having a thickness of 2 g / 64 cm ² is 5.8 μm, the film having a thickness of 1 g / 64 cm ² is 4.0 μm, and the film having a thickness of 0.5 g / 64 cm ² is 2.4 μm. Met. A cell culture experiment was carried out in the same manner as in Example 1 using these sheets. The same favorable results as in Example 1 were obtained for all the films.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a cross-sectional structure of a bilayer protein sheet of the present invention.

FIG. 2 is an explanatory view of a plane of the bilayer protein sheet of the present invention viewed from a surface having open holes.

[Explanation of symbols]

 1 Dense thin film 2 Open hole surface 3 Wall surface 4 Open hole

Claims (1)

[Claims] 1. A gel sheet of protein is laminated on one side with a porous sheet of protein having a wall surface having open pores on at least one side such that the surface having open pores remains. A method for producing a bilayer protein sheet comprising a thin film and a porous layer, wherein the compound is dried to form a thin film, and the thin film and the porous layer are joined and integrated with each other.