JPS61207340A - Treatment of collagen substrate - Google Patents

Abstract

PURPOSE:A collagen substrate for cell culture or hybrid organ is modified to attain high cell proliferability and is used in the field of biotechnology or hybrid organ. CONSTITUTION:A collagen substrate such as acid-soluble collagen, atelocollagen obtained by removing telopeptide by pepsin treatment, etc. is applied to the surface of a culture medium in an amount of 0.2-50mug per 1cm<2>, or a collagen substrate for hybrid organ is modified. The modification treatment is carried out by irradiating the collagen substrate with ultraviolet radiation or gamma ray, or exposing the substrate to ethylene oxide gas, hexamethylene diisocyanate or an aldehyde. The cell-proliferation activity of collagen can be improved by this process.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for treating a collagen matrix used in the biotechnology field or the hybrid organ field, and particularly relates to a method for treating a collagen matrix that has a high cell proliferation property in the collagen matrix. Regarding the processing method for endowment.

(Prior art and problems to be solved) In recent years, in the field of biotechnology, which is a cutting-edge technology, it has become possible to take animal cells out of the body, culture them in large quantities in vitro, and produce useful substances produced by the cells. Furthermore, in the field of artificial organs, artificial organs are not based on mere physical functions such as membrane permeation or physical adsorption.
Research into so-called hybrid organ cancers, which incorporate cells to effectively utilize their physiological activity, is intensifying, and is gradually approaching the stage of practical application. However, in the field of sea urchin biotic plants and hybrid organs, inν1tro r#t! There is a strong demand for the development of a technology that allows cells to easily grow. On the other hand, various studies on cell culture have been conducted, and culture is usually carried out in glass or plastic dishes, but depending on the nature of the container, cells may not be suitable for adherent growth.
In such cases, the inner surface of the culture dish is coated with a substance that is convenient for cell culture, and various studies have been conducted to increase the adhesion and growth of various cells.

By the way, collagen is also known to play a role as a scaffolding material for cells as an intercellular matrix in the living body, and is also closely involved in the formation of differentiated organs of cells. Therefore, a collagen matrix under the same conditions as in the living body is the most ideal substrate for maintaining cells in a state similar to that in the living body, but cells under the same conditions as in the living body usually do not proliferate. They are not active and are disadvantageous when producing useful substances by mass culturing cells or when growing cells in culture dishes. Therefore, when collagen is used as a substrate, it is necessary to perform some kind of 4G decoration treatment on the collagen in order to inhibit the proliferation ability of cells. Thus, the present invention relates to a method of treating a collagen matrix to provide collagen favorable for cell proliferation.

(Means for Solving the Problems) The present invention is a method for processing a collagen matrix, which is characterized by subjecting a collagen matrix for cell culture or hybrid organ to a modification treatment to increase cell proliferation. Examples of modification treatments include irradiating the collagen matrix with ultraviolet rays or thermal rays, and treating the collagen matrix with ethylene oxide gas, hexamethylene diisocyanate, or aldehydes. Proliferative ability is sufficiently impaired. Types of collagen used as a collagen matrix include acid-soluble collagen, atelocollagen from which telopeptides have been removed by pepsin treatment, or insoluble collagen #a.
Any fiber dispersion liquid or the like may be used.

The collagen matrix to be processed can be a 7M 7A collagen membrane in which collagen molecules are randomly distributed in a dry state, a collagen fibrous membrane, or a collagen matrix in any state, such as a wet state, to allow cell proliferation. ability increases. Furthermore, it was particularly effective against cells such as fibroblasts, epidermal cells, and hepatocytes. When coating a culture dish with collagen as a substrate, 0.2 μg to 4.50 μg per 11qlcu+2 of the culture dish surface.
of collagen, preferably 0.2 to 50μ
g/am2. In addition, it can also be applied to the collagen micro 7 air matrix used in Susbenzidine culture and the collagen matrix of hybrid organs. The collagen micros 7 air matrix is a JJ-shaped matrix with a diameter of 100 to 1000μ, and cells are adhered to the surface and grown in Susbenz old culture.
Collagen matrices of various shapes that are optimal for the purpose are required, such as er). For example, in the case of artificial skin, it is desirable to make the part corresponding to the dermis a sponge appendage, and in the case of a hybrid artificial liver or artificial pancreas, cells are grown on the outer surface of the collagen capillary of the semipermeable membrane, and blood is drawn on the inner surface. Flowing is done. A collagen matrix having any of these shapes can be subjected to modification treatment to increase cell proliferation.

General conditions for modification processing that can be employed in the present invention will be explained. For ultraviolet irradiation, when the irradiation distance for IOW ultraviolet sterilization is about 10 cm, it may take about 1 minute to 60 minutes, preferably 10 to 20 minutes, and for gamma ray irradiation, 0.01 to 5 M rad, preferably 0 , 1~I
In the ethylene oxide treatment, a culture vessel coated with collagen is placed in a polyethylene bag, and ethylene oxide gas is blown into the bag at room temperature under reduced pressure to remove air for 0.5 to 20 hours, preferably 1 to 20 hours. Exposure to ethylene oxide for 4 hours enhances cell proliferation ability, and when treated with hexamethylene diinocyanate (HMDIC), HMD IC concentration 7ffi 0.01-1% is good*L<l*0 .05%~0
, 1% solution is poured onto the collagen footing scraps and left in contact at room temperature for 10 minutes to 5 hours, preferably 30 minutes to 2 hours, the cell proliferation effect can be sufficiently enhanced.

Furthermore, examples of aldehydes that can be used in the aldehyde treatment include formaldehyde, glyoxal, acetaldehyde, propionaldehyde, and glutaraldehyde, with glutaraldehyde being the most preferred. The processing conditions are 0.01-1% glutaraldehyde at room temperature.
, preferably a 0.01-0.1% solution, and the pH of the solution is 5-10. Preferably it was 5-7. Processing time is 10-20 minutes.

These modification treatments can be used in combination; for example, the effect can be further enhanced by further treatment with HMD IC after UV irradiation.

The present invention will be explained below with reference to Examples.

Example 1 The dermal layer of fresh calf skin was aseptically removed, cut into pieces, and treated with pepsin (pH 3.0, pepsin addition amount: O-5% of collagen weight) at 20-25"C for 3 hours under aseptic conditions. The insoluble collagen was all solubilized and became a viscous solution. This solution was adjusted to pH 7.5 with Na0Hffi solution to precipitate the collagen. After collecting the precipitate with a centrifuge, it was washed with water. Dissolve again in HCl at pH 3. Pass through 1μ, 0.65μ, and 0.45μ Millipore filters.
Finally, 0.45μ sterile filtration was performed to obtain a 0.45μ sterile filter.
A 0.2% sterile, pyrogen-free atelofluden solution was obtained. Cell culture dish (7 Alfon cell culture dish,
1+nl of the above atelocollagen solution was poured into a dish (35+nm in diameter) to uniformly wet the bottom surface of the dish, and then air-dried under sterile conditions. Next, after UV irradiation (10W), 10
Irradiate for 10 minutes at a distance of cm.

When human skin fibroblasts, murine hepatocytes, and murine epidermal cells were cultured using the collagen-coated culture dishes obtained in this way, it was found that compared to dishes without collagen coating and dishes coated with collagen but not irradiated with ultraviolet rays, The proliferation rate of these cells was high.

Example 2 A tissue culture dish is coated with 7telocollagen according to the method of Example 1 and air-dried.

After irradiation with 0.5 Mrad gamma rays, three types of cells were cultured in the same manner as in Example 1. As a result, cell proliferation was significantly superior to those without collagen coating and those with collagen coating only. The rate was shown.

Example 3 A ML tissue culture dish was coated with atelocollagen according to the method of Example 1, and a 0.05% HMDIC methanol solution was added to the dish and treated at room temperature for 1 hour. After treatment, after washing with methanol and thoroughly washing with phosphate buffer 7-saline water, a culture test was conducted using three types of cells in the same manner as in Example 1.
It showed superior proliferation compared to those coated with collagen alone.

Example 4 A dish is coated with atelocollagen according to the method of Example 1 and air-dried. Add 0.01% glutaraldehyde solution (phosphate buffer, pH 7.0) to the dish and treat for 10 minutes at room temperature. After washing repeatedly with phosphate buffered saline, three types of cells were added in the same manner as in Example 1. When we conducted a culture test using the same material, superior cell proliferation was observed compared to those not coated with collagen, and those coated with collagen either immediately or later.

Example 5 The ultraviolet irradiated atelocollagen coated dish obtained in Example 1 was further treated with ethylene oxide gas. Place the dish in a polyethylene bag, reduce the pressure to remove the air, then blow in ethylene oxide gas (Guiside LS Daido Oxygen Co., Ltd.), seal the polyethylene bag, and leave it at room temperature for 3 hours. After thoroughly washing the oxide gas-treated dish with phosphate buffer 7-saline water, a culture test was performed using three types of cells in the same manner as in Example 1. As with dish 7, excellent cell proliferation was observed.

Example 6 The atelocollagen solution obtained in Example 1 is concentrated to a collagen concentration of 1%. Add NaOH solution to this I)
Neutralize to H7.0, immediately place in a flat container, cool and freeze with dry ice. A sponge is obtained by drying this frozen atelocollagen using a vacuum freeze dryer. Slice this sponge into thickness II, and heat it with an ultraviolet germicidal light (10W
), irradiate both sides with UV rays from a distance of 10c+o for 30 minutes on each side.

On the other hand, dilute the above 1% atelocollagen solution to 0.1%
When a collagen-concentrated solution is placed in a flat plate container of depth II and air-dried, an atelocollagen thin film with a thickness of approximately 1 μm is obtained. This thin film is wetted and applied to one side of a sponge having a thickness of II and air-dried. 10c on the thin film side after air drying
By irradiating ultraviolet rays for 10 minutes with an IOW ultraviolet germicidal lamp from a distance of m, an atelocollagen sponge (irradiated with ultraviolet rays) having a thin atelocollagen film on one side and a thickness of 1 mm was obtained. After disinfecting with ethylene oxide gas treatment, washing thoroughly with phosphate buffer, and culturing dermal fibroblasts on a sponge with the thin film side facing down, the fibroblasts enter the sponge mesh and proliferate. When epidermal cells are placed on a thin film with the thin film side up and cultured, the epidermal cells proliferate over the entire surface, creating a hybrid Auger 2 artificial skin with the sponge part as the dermis and the 1W membrane part as the epidermis. If used, hybrid artificial skin that will survive without rejection will be created.

Example 7 The atelocollagen solution obtained in Example 1 was concentrated to obtain a solution with a concentration of 5% after defoaming the viscous collagen concentration.
It is extruded through a double cylindrical nozzle into a coagulating liquid in saturated saline and solidified in a capillary shape. This is followed by an ultraviolet germicidal lamp (I
After irradiating with ultraviolet light for 20 minutes at a distance of 100I11 from OW), neutralize and wash with phosphoric acid buff 7- of pH 7.0, then equilibrate by immersing in 50% ethanol (ethanol:water = 1:1) and freeze. When dried, porous atelocollagen capillaries form. Proteins with a molecular weight of 50,000 or more do not pass through this membrane. This capillary is treated with ethylene oxide.
After λ disinfection and washing with phosphate buffered saline, hepatocytes were allowed to adhere to the outer surface of the capillary tube and cultured.
Cells showed excellent proliferative properties and grew on all external surfaces. This is a permeable hair with an excellent outer surface for cell proliferation.
lW is applicable to hybrid artificial liver and piperid artificial pancreas.

A hybrid organ device made of a material with a collagen concentration can be coated with collagen in the manner described in Examples 1 to 5 to improve cell proliferation.

Claims (1)

[Scope of Claims] 1. A method for processing a collagen matrix, which comprises subjecting a collagen matrix for cell culture or hybrid organ to a modification treatment to increase cell proliferation. 2. The method for treating a collagen matrix according to claim 1, wherein the modification treatment is ultraviolet irradiation or gamma ray irradiation. 3. The method for treating a collagen matrix according to claim 1, wherein the modification treatment is treatment with ethylene oxide gas. 4. The method for treating a collagen matrix according to claim 1, wherein the modification treatment is treatment with hexamethylene diisocyanate. 5. The method for treating a collagen matrix according to claim 1, wherein the modification treatment is treatment with aldehydes. 6. The method for treating a collagen matrix according to claim 5, wherein the aldehyde is glutaraldehyde.