JPH05336996A - Method for testing cytotoxicity with cultured cell - Google Patents

Abstract

PURPOSE:To evaluate the toxicity of a specimen against skin tissues by separately culturing fibroblasts and epidermal cells in a matrix produced by filling a collagen gel in a collagen sponge, fixing the matrix on the interface between a medium and air and subsequently testing a specimen on the fixed matrix. CONSTITUTION:The evaluation of the cytotoxicity is performed by treating collagen with an enzyme to produce atherocollagen reduced in antigenicity, preparing the aqueous solution of the atherocollagen, adjusting the pH of the aqueous solution so as to increase the viscosity of the aqueous solution, homogenizing the aqueous solution into a creamy solution, pouring the creamy solution in a container whose bottom surface has preliminarily been coated with an aqueous solution of atherocollagen and then dried with air to form a thin coating film, gelating the poured creamy solution in the foam-containing state, vacuum-drying the gelation product to produce a collagen sponge, filling a collagen gel in the produced collagen sponge to produce a matrix, separately culturing fibroblasts and epidermal cells in the matrix, fixing the culture matrix to the interface between a medium and air, putting a specimen on the culture matrix and, after a constant time, counting the number of the alive cells.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cytotoxicity test method for testing the toxicity of a specimen to cells using cultured cells, and more particularly to a cytotoxicity test method for testing the toxicity of a specimen to skin tissue.

[0002]

2. Description of the Related Art Animal tests have been conducted as toxicity tests on cells of specimens, but the toxicity tests using animals are not only time-consuming and expensive, but recently, other test methods have been gradually adopted from the viewpoint of animal welfare. Has been adopted. One of them is a cytotoxicity test method. As the cytotoxicity test method, methods using cultured cells such as ⁵¹ Cr elution test, agar overlay method, Millipore filter-overlay method and the like are known. ^{The 51} Cr elution test method is based on ⁵¹ Cr in advance.
When labeled cells that have taken up a compound are exposed to a dental material that is a sample, ⁵¹ Cr is released from the cells due to damage, and the dental material is measured by measuring the radioactivity of ⁵¹ Cr released in the medium. It is a method for quantitatively detecting and evaluating the cytotoxicity of. In the agar overlay method, agar layer containing the culture solution is placed on the cells cultured in a single layer on a Petri dish, and a solid or liquid sample is placed on the solidified agar layer to perform culture, and then neutralized. It is a method of evaluating toxicity by using the degree of red staining of cells alive and the cells disappearing or dissolving when they are damaged. Millipore filter
In the overlay method, the cultured cells are preliminarily cultivated in a monolayer on the Millipore filter, and the Millipore filter is placed on the agar medium so that the cell layer is on the lower side, and the specimen is placed on the filter. After this time, the succinate dehydrogenase activity of the cell layer is colored cytochemically, and the inhibition zone of color development by the sample is measured to evaluate toxicity. These methods are expected to become increasingly important as alternatives to animal experiments.

By the way, the above-mentioned cytotoxicity test is carried out using a drug applied to the skin as a sample to test the toxicity of the drug to the skin. The conventional method is a method of inspecting the growth behavior of cells by adding a certain amount of a drug to a culture dish on which cells have been seeded.
However, the drug applied to the skin is usually applied directly in the form of a drug-containing ointment or a covering material, and therefore the effect on cells in an experimental system using cultured skin in which cells are three-dimensionally assembled as a cytotoxicity test. The method of Bell et al. Is currently known (US Pat. No. 4,835,35).
102). In this method, epidermal cells and fibroblasts, which are dermal cells, are cultured in a collagen matrix to make a model close to skin, and the specimen is placed on this model and observed and evaluated by a tissue section after a certain period of time to evaluate the specimen. This is a method of examining toxicity.

By the way, the most important test method for evaluating the toxicity of a drug is to count the number of viable cells, but the method of Bell et al., Mentioned above, uses epidermal cells and fibroblasts in a collagen matrix. Although it is possible to observe the condition of the tissue section for culturing the cells together with the cells for toxicity test of the specimen, it was impossible to count the number of viable cells, which is important for evaluation of toxicity.

[0005]

DISCLOSURE OF THE INVENTION As a result of various studies to improve this drawback and count the number of viable cells, the present inventors have found that collagen is used as a matrix and epidermal cells and fibroblasts are separated from each other. It is possible to evaluate cytotoxicity by culturing cells in a matrix, fixing the culture matrix to the interface between the medium and air, placing a sample, and culturing for a certain period of time, and then observing the number of viable cells and tissue sections in each matrix. The present invention has been completed and the present invention has been completed, and an object of the present invention is to provide a cytotoxicity test method for testing the toxicity of a specimen to skin tissue.

[0006]

The gist of the present invention is to
After fibroblasts and epidermal cells were separately cultured in a matrix filled with collagen gel in Gensponge, the culture matrix was fixed at the interface between the medium and air, and the specimen was placed on the culture matrix to This is a cytotoxicity test method for evaluating cytotoxicity. As a method for evaluating cytotoxicity, cell evaluation is performed by the number of living cells in a culture matrix and a tissue section.

That is, according to the present invention, fibroblasts and epidermal cells are separately cultured in a matrix to form a composite cultured dermal cell and a composite cultured epidermal cell. The number of cells is counted, and at the same time, the tissue section is observed.

The present invention will be described in detail below. The cultured skin matrix of the present invention can be produced, for example, by the method described below.

First, collagen is enzymatically treated by a well-known method to prepare atelocollagen having less antigenicity, then an aqueous solution of atelocollagen is prepared, and then pH is adjusted so as to increase its viscosity. Using a homogenizer, make a cream solution. Next, in advance on the bottom
Gen solution was applied and air-dried, and the above-mentioned cream-like solution was poured into a thin film-coated container in a thin film form, and allowed to stand in an alkaline atmosphere to cause gelation in the state of containing bubbles,
A quick-freeze, vacuum-dried filter with a thickness of 1 to 2 mm.
A sheet having Gensponge as a skeleton is prepared.

Both surfaces of the sheet are irradiated with ultraviolet rays to partially introduce intermolecular crosslinks, and then sterilized with ethylene oxide gas. Next, a matrix used in the present invention is obtained by impregnating a neutral solution containing a tissue culture type atelocollagen and allowing it to stand in an incubator for gelation (fiber formation by aggregation of collagen molecules). To make.

Then, dermal fibroblasts and epidermal cells are seeded on the above matrix and cultured in a medium containing serum. At this time, the fibroblasts are in a state of entering the matrix gel.

In the culture matrix of fibroblasts, in order to avoid the risk that the fibroblasts are directly exposed to air and damaged, the culture matrix is turned upside down so that the back surface of the matrix is the interface between the medium and air. A sample for evaluating cytotoxicity is placed on this. On the other hand, in the epidermal cell culture matrix, keratinization is promoted by the contact of the stratified epidermal cells with air, so that the epidermal cell layer is brought into contact with air and a sample for evaluating cytotoxicity is placed thereon. This matrix is placed on a stainless steel net several millimeters above the bottom of the dish. After this, a drug for evaluating toxicity was added to gase and the like and placed on the upper surface of the culture matrix. Then, after culturing for a certain period of time, a part of the matrix is dissolved using collagenase, only viable cells are stained, and the number of cells is measured. In addition, a part of the tissue is fixed with formalin or the like to prepare a tissue section, which is then evaluated histologically. Next, the present invention will be described more specifically with reference to Examples.

[0013]

【Example】

Example 1 Preparation of cultured skin matrix: Atelocollagen prepared by enzymatic treatment of cowhide-derived collagen (manufactured by Koken Co., Ltd.)
1% aqueous solution of was adjusted to pH 4 and homogenized with a homogenizer 15
The mixture was stirred at 000 rpm for 3 minutes to form a cream. 0.5m of 0.5% atelocollagen aqueous solution (pH 4) in advance
Polystyrene container (6c
15 ml of the above-mentioned cream-like solution was poured into (m × 9.5 cm) and allowed to stand for 1 hour in an ammonia atmosphere to cause gelation. Then, after thoroughly washing with water, it was rapidly frozen at -70 ° C and vacuum dried to prepare a sheet-like sponge.

Both sides of the sheet-like sponge were irradiated with ultraviolet rays at a distance of 15 cm for 1 hour using a 15 W ultraviolet lamp to partially introduce intermolecular crosslinks,
Through holes having a diameter of 0.5 mm were provided at intervals of 4 mm. Then, this sheet was put into a polystyrene container, sterilized with ethylene oxide gas, and Hank's solution (buffer solution).
After washing with water to remove air bubbles, 2 ml of 0.2% type I telocoragen neutral Hank's solution for tissue culture [CELLGEN (trade name of Koken Co.)] was added, and the temperature was 37 ° C.
The mixture was allowed to stand in an incubator for 2 hours for gelation to obtain the cultured skin matrix of the present invention in which the inside of the through-hole and the surface of the sheet were coated with a gel-like atelocollagen.

Fibroblasts are obtained by treating the dermis obtained by exfoliating the skin strips that have become excessive during skin grafting into the epidermis and dermis by treatment with disperse with a mixed solution of collagenase and hyaluronidase. Was collected and contained 10% fetal bovine serum in Dulbecco's modified Eagle.
Subculture was performed using e's Medium as a medium.

Collagen matrix (10 cm x 1
8 cm) subcultured fibroblasts to 5 × 10 ⁵ c
The composite dermis was prepared by seeding at a density of ells / cm ² and culturing for 1 week. A stainless mesh with a height of 3 mm is placed on a culture dish with a diameter of 90 mm, and the cut composite culture dermis (4 cm x 4 cm) is turned upside down on the mesh so that the composite culture dermis is located at the interface between the medium and air. 25 ml of medium was added.

In this state, an antibacterial agent-containing ointment coating gauze (4 cm × 4 cm) or an antibacterial agent-containing coating material (4 cm × 4 cm) was placed on the complex cultured dermis, and the incubator at 37 ° C. It was left to stand for 4 days. After that, the composite cultured dermis was cut into a size of 3 cm × 3 cm, placed in 5 ml of 0.5% collagenase solution and allowed to stand at 37 ° C. for 30 minutes, and then stirred to prepare a cell suspension and trypan bleed.
After staining with, the number of viable cells was measured using a hemocytometer. Further, a part of the composite cultured dermis was fixed with 10% formalin to prepare a tissue sample, which was stained with hematoxylin / eosin for histological examination.

Excessive skin debris during skin grafting was separated into epidermis and dermis by disperse treatment, and the resulting epidermis was trypsinized to collect epidermal cells, and 3T3 was prepared according to Green's method. Epidermal cells were cultured in the presence of feeder layer. 5 × 10 ⁵ cells of epidermal cells were placed on a collagen matrix (10 cm × 18 cm).
A composite culture epidermis was prepared by seeding at a density of 11s / cm ² and culturing for 1 week. Similarly to the above, the composite culture epidermis is placed on the interface between the medium and the air, and an antibacterial agent-containing ointment coating gauze or an antibacterial agent-containing covering material is placed thereon, and the temperature is 37 ° C
It was allowed to stand in the incubator for 4 days. Then, a cell suspension was prepared and the number of viable cells was measured in the same manner as described above. In addition, hematoxylin / eosin staining was carried out for histological examination.

As a sample, a coating material containing silver sulfadiazine, which was recently put on the market, was used, the coating material was placed on the above-mentioned complex cultured dermis, and the number of viable cells was counted after 3 days of culture, and it was about 40%. The number of viable cells was 0% when the sample coated with the cream containing silver sulfadiazine was used as a sample.

In the composite culture epidermis, epidermal cells are layered in 3 to 4 layers on the surface of the collagen matrix, and the specimen is directly placed on the epidermal cells, so that the drug released from the specimen directly affects the epidermal cells. Exert. When the above-mentioned coating material containing silver sulfadiazine is used as a specimen, the number of viable cells is about 50% after 3 days of culturing, and when a sample coated with the same amount of cream containing silver sulfadiazine is used as a specimen, viable cells are used. Number is 0
%Met.

[0021]

As described above, according to the present invention, the combined culture epidermis and the combined culture dermis similar to the skin tissue are used,
Since the number of surviving cells is measured by placing a sample on this, it is possible to know the toxicity of the sample to the cells with much higher accuracy than in conventional methods.

Claims (2)

[Claims] 1. A culture medium in which fibroblasts and epidermal cells are separately cultured in a matrix in which collagen gel is filled in collagen sponge, and the culture matrix is fixed on the interface between a medium and air. A cytotoxicity test method in which a sample is placed on and the cytotoxicity is evaluated. 2. The method of the cytotoxicity test according to claim 1, wherein the cell evaluation is performed by the number of viable cells in the culture matrix and the tissue section.