JP2511834B2 - Bilayer gelatin sheet and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a bilayer gelatin sheet and a method for producing the same, and particularly to a bilayer having affinity for animal cells and suitable as a culture substrate or biomaterial. -Related gelatin sheet and its manufacturing method.

(Prior art) From the basic research such as developmental organism research, physiologically active substance and cell response research to animal cell culture, interferon, TPA
In recent years, research has been conducted on the development of industrial production of physiologically active substances and applied research such as the development of hybrid artificial organs.

Since most animal cells are adhesion-dependent, a substrate that has a high affinity for the cells and is capable of maintaining the proliferation and differentiation functions of the cultured cells is required in the culture.

Heretofore, as a membrane-shaped substrate for culturing animal cells, a cellulose-based or polycarbonate-based porous membrane filter, a collagen membrane coated on these porous membrane filters, or a collagen film has been used.

However, the one using a membrane filter is opaque, so it is not possible to observe the cells under a microscope, and in the application to a hybrid artificial organ, for example, culturing skin cells on a substrate and transplanting it into a living body as artificial skin. In that case, it has a drawback that it cannot be used in terms of biocompatibility and biodegradability.

On the other hand, it is possible to manufacture a collagen film which can solve the above-mentioned drawbacks to some extent, and for example, a manufacturing method has been proposed in JP-A-62-266064. However, the performance is still not satisfactory.
In addition, in the collagen system, a material having a thin film of collagen and chondroitin-6-sulfate and a porous layer has recently been used as a hybrid artificial skin substrate (Steven T. Boyce, Debor.
ah J. Christianson, and John F. Hansbrough, J.Biomed.M
ater.Res., Vol.22, 939-957 (1988)) and collagen with a similar structure (Kuroyanagi Nomitsu et al., 18th Symposium on Medical Polymers, P.31, JP-A-2-71749). Researched or proposed. All of these have relatively good performance, but the manufacturing process is complicated, and some have a highly toxic chemical such as glutaraldehyde as a cross-linking agent. Moreover, collagen is difficult to handle because it is easily denatured, and is very expensive.

(Problems to be Solved by the Invention) The present inventors have made diligent research by focusing on gelatin, which has characteristics superior to collagen in terms of morphology of adherent cells, proliferative property, and degree of suppression of collagenase expression, and which is inexpensive. As a result, they have found that a structure suitable as a cell culture substrate can be formed without impairing the original cell affinity of gelatin, and completed the present invention.

An object of the present invention is to provide at low cost a substrate having excellent cell affinity and biocompatibility, particularly a substrate for culturing animal cells or a biomaterial.

(Means for Solving the Problems) The above-mentioned object is a bilayer sheet made of cross-linked gelatin, one surface of which is formed of a dense thin film,
A bilayer gelatin sheet characterized in that the other surface is formed by a wall surface having open holes, and one surface of a sheet-like cooled gelled product of a gelatin aqueous solution containing a cross-linking agent is below the freezing temperature and the other surface. Is maintained at a freezing temperature or higher, and the sheet-like cooled gelled product is frozen while a temperature gradient is provided so as to gradually increase from one surface to the other surface, and subsequently freeze-dried. This is achieved by the method for producing a layered gelatin sheet.

The bilayer gelatin sheet of the present invention will be described with reference to the drawings. FIG. 1 is an explanatory view showing a cross-sectional structure of a bilayer gelatin sheet of the present invention, and FIG. 2 is a plan view of the bilayer gelatin sheet of the present invention as seen from a surface having open holes. In the figure, (1) is a dense thin film, (2) is an open hole surface,
(3) represents a wall surface, and (4) represents an open hole.

As shown in FIGS. 1 and 2, the bilayer gelatin sheet of the present invention comprises a dense thin film (1) on one surface and an open hole surface (2) having open holes (4) on the other surface. Has been done. The dense thin film (1) and wall surface (3) are made of cross-linked gelatin as described later. 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 separate cells are cultured on both sides of the gelatin sheet of the present invention. Considering such a case, it is preferable that the dense thin film is non-porous or has pores of 1 μm or less.

The dense thin film is preferably thick from the viewpoint of handleability of the sheet, but is preferably thin from the viewpoint of material permeability. Usually, it is preferably 200 μm or less, more preferably 0.1 to 50 μm.

The wall surface (3) which is integrated with the dense thin film (1) and constitutes the other surface of the bilayer gelatin sheet of the present invention forms an open hole (4). An open hole is a wall surface (3) forming a double-layered gelatin sheet having an open end surface. It is preferable that the open pores are large for cell culture, and average 50 pores.
It is preferably at least μm. Here, 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 from the obtained photograph as follows.
That is, the number of aperture openings on a straight line having a constant length in the vertical and horizontal directions 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, it is preferably 50 μm or more in both directions.

The bilayer gelatin sheet 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. 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.

The gelatin constituting the bilayer sheet in the present invention may be industrially obtained mainly from cow bone, cowhide or pig skin by an alkali method or an acid method, but these gelatins are further purified, for example, according to the Japanese Pharmacopoeia. Those that meet the specifications of gelatin or purified gelatin are preferable. Further, gelatin obtained by thermally denaturing commercially available collagen can also be used.

Since the bilayer gelatin sheet of the present invention is water-insoluble, gelatin needs to be crosslinked with a crosslinking agent.
Examples of the crosslinking agent used for this crosslinking include glycerol polyglycidyl ether, sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene. Water-soluble epoxy compounds such as glycol diglycidyl ether, aldehydes such as glutaraldehyde, formaldehyde and gluoxal, diisocyanates such as P, P'-diphenylmethane diisocyanate and hexamethylene diisocyanate,
Carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride are included.
Of these, water-soluble cross-linking agents, water-soluble epoxy compounds, aldehydes, and carbodiimides are preferable, and above all, water-soluble epoxy compounds are preferable because they are easy to handle and have low toxicity, and particularly cross-linked with glycerol polyglycidyl ether. The most preferable one is.

 Next, the manufacturing method of the present invention will be described.

In the method of the present invention, first, a sheet-like cooled gel product of an aqueous gelatin solution containing a crosslinking agent is produced. In this case, the mixed aqueous solution of gelatin and the crosslinking agent may be produced by any method. For example, there may be mentioned a method in which gelatin is first made into an aqueous solution, and a predetermined amount of a crosslinking agent or an aqueous solution thereof is added, mixed and dissolved. The concentration of gelatin varies depending on the type of gelatin to be applied, the purpose of the sheet, etc., and cannot be specified unconditionally, but usually 10% or less is preferable.

The addition amount of the cross-linking agent also differs depending on the cross-linking agent used and cannot be generally stated, but in the case of the aqueous epoxy compound, it is preferably 1 to 20% by weight, more preferably 3 to 10% by weight, based on gelatin.

In the present invention, the gel aqueous solution prepared by mixing the gelatin and the cross-linking agent prepared as described above is cooled and gelated into a sheet by a method such as casting in a mold. In this case, in the case of using the form method, a plate-like one having one side open is preferable. An example of such a mold is an appropriate plate-shaped body which is framed with an adhesive tape or the like so as to have a predetermined thickness. It is necessary that the plate-like material used is one that does not adhere to the gelatin and the cross-linking agent mixture after drying.
Examples of such a plate-like body include a plate-like body whose liquid contact surface is a fluororesin such as poly-4-fluoroethylene, polyethylene, polypropylene, an acrylic resin, polyvinyl chloride, or polystyrene. However, the material is not limited to these, and other materials can be used as long as they satisfy the above conditions.

It is preferable that the sheet-like cooled gelated product has a uniform thickness.

Although various methods can be adopted as the cooling method,
In order to obtain a sheet having a uniform thickness, it is necessary to gel a mixed aqueous solution containing gelatin and a cross-linking agent while keeping it horizontal. The gelling temperature is preferably in the range of 0 to 20 ° C. Although the gelling time can be set as appropriate, it is usually preferably 2 hours or less because the formation of open pores decreases if the gelling state is maintained for a long time.

In the method of the present invention, as described above, one surface of the sheet-like cooled gelated product is kept at a freezing temperature or lower and the other surface is kept at a freezing temperature or higher, and the temperature is gradually increased from one surface to the other surface. The sheet-like cooled gel product is frozen while a gradient is provided. As an example of a method of freezing in this way, for example, a mixed aqueous solution of gelatin and a cross-linking agent is cast into a mold to form a sheet-like cooled gelled product, and then the mold is placed on a table below the freezing temperature. Cool while keeping the surroundings above the freezing temperature. Examples of the table having a cooling capacity include a plate in which a refrigerant is circulated and a cooling plate using dry ice or another cryogen.

When placed in an environment such as a refrigerator that is cooled from the surroundings and frozen, both surfaces of the sheet-like cooled gelated product are cooled to substantially the same temperature, so that the amount of open pores formed is reduced. The cooling temperature needs to be a temperature at which one surface of the sheet-like cooled gelled product is frozen. 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.

Next, the frozen gel is freeze-dried under vacuum. 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. The temperature is preferably 30 ° C. or lower during the drying period.

In the method of the present invention, the crosslinking reaction can be promoted by heat treatment after freeze-drying. The reaction is promoted when the heat treatment temperature is higher, but if the heat treatment temperature is too high, the sheet is colored when heat treated in air. 150 ° C for air treatment
The following are preferred.

The heat treatment time is determined by the relationship with the treatment temperature, and the higher the temperature, the shorter the crosslinking progresses and the insoluble in water, but the lower temperature requires a longer time. When glycerol polyglycidyl ether is used as the cross-linking agent, a treatment at a treatment temperature of 80 to 150 ° C for 10 hours or less is sufficient.

The gelatin porous sheet produced as described above can be washed with water, a water / alcohol mixture or other suitable solvent to remove uncrosslinked gelatin and the remaining crosslinking agent, if necessary.

(Effect of the Invention) The bilayer gelatin sheet of the present invention is excellent in cell affinity and biocompatibility because it is based on gelatin. Further, since one side of the sheet forms a dense thin film, it is easy to handle even if the thin film is very thin, and since the other side has open pores, the penetration of medium is not hindered and transparency is high. Therefore, after the cell culture, the cells can be easily observed under a microscope by staining the sheet to which the cells are attached.

Needless to say, cell culture is possible on both the thin film and the open pore surface, and cell culture is also possible inside the porous body. It is also possible to study the interaction between the cells by culturing the two types of cells separately on both sides.

The bilayer gelatin sheet of the present invention is extremely useful because it can be used not only as a cell culture material but also as a base material for artificial skin and artificial blood vessels, and can be applied as a wound dressing material.

The production method of the present invention is a very excellent method for producing a bilayer gelatin sheet having a thin film on one side and open holes on the other side in one process without dividing into two steps of a thin film and a porous body. . Further, since gelatin and the cross-linking agent are premixed in a fixed ratio, it is not necessary to use an extra cross-linking agent. Therefore, the unreacted cross-linking agent does not remain, or even if it is present, the amount is extremely small, so that it can be easily purified and removed. Therefore, it is an extremely excellent manufacturing method without the problem of toxicity due to the residual crosslinking agent.

 Next, the present invention will be described in more detail with reference to examples.

Example 1 Commercially available gelatin [viscosity 28 mp, jelly strength 96 (6.66
%), Manufactured by Nippi Co., Ltd.], and 10% of glycerol polyglycidyl ether (manufactured by Nagase Kasei Co., Ltd.) as a crosslinking agent was added and dissolved.

This mixed solution was cast on a stainless steel plate with a thickness of 2 mm and 5 mm coated with poly-4-fluoroethylene 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. It was After this, put it on a plate kept at -70 ° C with dry ice, cool it from the bottom and freeze it at 25 ° C.
Lyophilized below. 11 sheets formed after drying
It was treated at 0 ° C. for 3 hours. Treated sheet in water at 50 ℃ 5
After soaking and washing for a period of time, it was freeze-dried again. This sheet
As a result of SEM observation, it was a porous sheet as a whole, and it was a structure in which one surface had a substantially non-porous thin film and the other surface had many open pores. The thickness of each thin film was around 1 μm. The average hole diameter of the open holes measured from the SEM photograph is 72μm × 69μm, 5mm thickness when the thickness of the stainless plate is 2mm.
In the case of, it was 89 μm × 84 μm. The pore walls were mainly oriented perpendicular to the sheet surface.

Example 2 A polymethylmethacrylate plate having a thickness of 2 mm (P
MMA), polyvinyl chloride plate (PVC), polyethylene plate (P
A gelatin sheet was prepared in the same manner as in Example 1 except that E) was used.

As a result of SEM observation, all of these were porous sheets as a whole and had a structure in which one surface was a substantially non-porous thin film and the other surface had a large number of open pores. The thickness of each thin film was about 1 μm. The pore walls were oriented perpendicular to the sheet surface. The average pore diameter measured from the SEM photograph is PMMA 53μm × 51μm, PVC 67μm × 65μm, PE 74μm
It was × 66 μm.

Example 3 A gelatin sheet was prepared in the same manner as in Example 1, using polytetrafluoroethylene plates having a thickness of 5 mm and 10 mm as a mold. It was confirmed by SEM observation that the structure was similar to those in Examples 1 and 2. The average hole diameter of open holes is 105 when the thickness is 5 mm.
In the case of × 103 μm and 10 mm, it was 173 × 153 μm. The pore walls were mainly oriented perpendicular to the sheet surface.

Example 4 A gelatin sheet was prepared in the same manner as in Example 1 except that PMMA plates having thicknesses of 2 mm and 5 mm were used and the freezing temperature was -70 ° C or -39 ° C. Each had a thin film on one surface and open pores on the other surface, and the average pore diameter was as shown in the following table.

Comparative Example 1 A gelatin sheet was prepared in the same manner as in Example 1 except that freezing was performed in a freezer at -20 ° C. As a result of SEM observation, a continuous film, which was a porous body as a whole but had few open pores on both surfaces, was formed.

Example 5 A gelatin sheet was prepared in the same manner as in Example 1.
However, the gelation time was changed to 20 minutes, 2 hours, 5 hours, and 24 hours. When it was more than 2 hours, it was put in a polyethylene bag, sealed and stored to prevent it from drying. According to SEM observation, all were porous bodies, but both surfaces became thin with extension of gelation time, and after 5 hours or more, both surfaces became completely continuous films.

Example 6 A bilayer gelatin sheet was prepared in the same manner as in Example 1 using a PMMA plate having a thickness of 2 mm. The average pore size of this sheet was 112 μm × 101 μm. This sheet has a diameter of 30 mm
It was cut out into a circular shape and adhered to the lower surface of a cylindrical cup with a foot of 5 mm in height to give a culture cup. Two types of culture cups were prepared, one with the thin film surface of the porous sheet facing up and one with the open pore surface facing up. Human fibroblasts were cultured using this cup. The cup was placed in a dish having a diameter of 35 mm, and 4 × 10 ⁵ human fibroblasts suspended in 1 ml of a culture solution were poured into the cup. After further adding 2 ml of the culture solution, the cells were cultured at 37 ° C for 1, 4 and 8 days. After a predetermined period of time, the cells were fixed with methanol, stained with Giemsa, air-dried, and then observed for cell morphology with an optical microscope. The culture medium used was Dulbecco's Modified Eagles Medium.

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.

Example 7 Human epidermal cells (Epipack, manufactured by Clonetex) were cultured and observed using the culture cup prepared in the same manner as in Example 6. 1.5 × 10 ⁵ cells were suspended in 1 ml of culture medium (Dulbecco's modified Eagle's medium), poured into a cup, 3 ml of culture medium was added, and the cells were cultured at 37 ° C. for 6 days. After fixation with methanol and staining with Giemsa, observation with an optical microscope was performed. It was found that a large number of cells adhered and spread on both the thin film surface and the open pore surface, and showed colony formation. In addition, the continuous membrane surface was cultured as a single layer, and the wall surface including the open pores was three-dimensionally cultured.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a cross-sectional structure of a bilayer gelatin sheet of the present invention, and FIG. 2 is a plan view of the bilayer gelatin sheet of the present invention as seen from a surface having open holes. 1 ... Dense thin film, 2 ... Open hole surface, 3 ... Wall surface, 4 ... Open hole.

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Claims (2)

(57) [Claims] 1. A two-layer sheet made of cross-linked gelatin, wherein one surface is formed of a dense thin film and the other surface is formed of a wall surface having open holes. Layered gelatin sheet. 2. A sheet-like cooled gelled product of a gelatin aqueous solution containing a cross-linking agent, wherein one surface is kept at a freezing temperature or lower and the other surface is kept at a freezing temperature or higher, and gradually increases from one surface to the other surface. A method for producing a bilayer gelatin sheet, which comprises freezing the sheet-like cooled gelated product while providing a temperature gradient as described above, and subsequently freeze-drying.