JPS62265978A - Culture medium for epidermal cell - Google Patents

Abstract

PURPOSE:The titled culture medium suitable for culture of artificial skin, providing epidermal cell membrane useful as artificial skin efficiently because of a large amount of the epidermal cell bonded to the bottom of the culture medium, obtained by coating the bottom part of the culture medium with atherocollagen irradiated by ultraviolet light rays having a specific concentration. CONSTITUTION:The bottom part of a bottomed culture medium for epidermal cell is coated with 5-500mug based on 1cm<2> bottom area of the culture medium of atherocollagen irradiated with ultraviolet light rays. To be concrete, for example, a solution of atherocollagen is put in the medium culture and irradiated with ultraviolet light rays to carry out coating. An athercollagen obtained by treating collagen with an enzyme and removing an antigen determining group-containing peropeptide is preferable as the atherocollagen.

Description

[Detailed description of the invention] 10 Background of the invention (Technical field) The present invention relates to an improved epidermal cell culture device.

More specifically, the present invention relates to an epidermal cell culture vessel in which the bottom of the culture vessel is coated with infrared irradiated atelocollagen of a specific heart disease.

When the skin is damaged by a wound, burn, etc., it is most desirable to collect one's own skin from another site and treat it with f3ta. However, there are limits to where and how much skin can be collected, so research into cultured artificial skin has been actively conducted in recent years. The incubator of the present invention is advantageously used for culturing such artificial skin.

(Prior art) To culture epidermal cells for the purpose of artificial skin, a culture solution containing epidermal cells is placed in a culture container, cultured at constant temperature for a certain period of time,
This is done by ingesting epidermal cells that adhere to and proliferate on the bottom of the vessel. Therefore, cells that do not adhere to the bottom of the vessel are not used, and during the culture period, the culture medium is replaced with a new one as appropriate, but at this time, cells that do not adhere to the bottom of the vessel are discarded together with the old culture medium. For these reasons, in culturing epidermal cells, importance is placed on the amount of adhesion of cells to the bottom of the vessel, but in conventional culture vessels, the amount of adhesion is not always sufficient, and improvements have been desired.

41 OBJECTS OF THE INVENTION Therefore, an object of the present invention is to provide an epidermal cell culture device in which the adhesion of epidermal cells to the bottom of the culture device is increased. This object of the present invention is achieved by an epidermal culture vessel in which the bottom of eight epidermal cell culture vessels with a bottom is coated with ultraviolet irradiated atelocollagen at a concentration of 5 to 500 μ9/cm 2 .

■1 Detailed explanation of the invention That is how the cell culture vessel is coated with atelocollagen.

This is carried out by methods known per se. For example, put a solution containing a calculated amount of atelocollagen into a cell culture vessel,
By air-drying and then irradiating with ultraviolet rays, the bottom of the culture vessel can be easily coated with ultraviolet-irradiated atelocollagen. Preferably, an appropriate liquid medium is further added to restore the atelocollagen to a gel state and reconstitute the collagen fibers.

As the cell culture vessel, conventionally known ones can be used without particular limitation, but dish-shaped ones are preferred.

Atelocollagen that has been conventionally used as a substrate for cell culture can be used, and atelocollagen for tissue culture, which is obtained by enzymatically treating collagen to remove peropeptide having antigenic determinants, is preferably used. Atelocollagen is 5 to 500 per 1 cm of bottom area of culture vessel.
μ it is necessary to be coated with this concentration (density). This concentration is critical and is less than 5μ97cm2 or 50
At a concentration of 0μ97cm2 or higher, the amount of adhesion of epidermal cells to the vessel bottom cannot be improved. Although the amount of ultraviolet rays irradiated to atelocollagen is not critical, it is suitable, such as the amount equivalent to irradiation of 15 cdlL from a 6 watt germicidal lamp.

The epidermal cell culture device of the present invention is used in the same manner as conventionally known culture devices. For example, a predetermined amount of epidermal cells are suspended in a culture solution and cultured at a constant temperature. The cells are cultured for a predetermined period of time while changing the culture medium as appropriate, and the epidermal cells that adhere and proliferate on the ultraviolet-irradiated atelocollagen at the bottom of the vessel are collected.

Next, the present invention will be explained in more detail with reference to Examples and Test Examples.

Example (1) Preparation of ultraviolet irradiation atelocollagen-coated culture vessels 1 ml of atelocollagen aqueous solutions with concentrations of 0.5%, 0.05%, 0.005% and 0.0005% were placed in each culture vessel, air-dried at room temperature, and then cooled at low temperature. Under nitrogen gas, ultraviolet rays were irradiated from a 6-watt stage lamp at a depth of 15 cm. Next, a culture solution was added and collagen fibers were reconstituted at 37°C. Atelocollagen was extracted and purified from calf skin by the Pepun digestion method, and a tissue culture petri dish with a diameter of 35 mm was used as the incubator.

After 30 minutes of cell seeding, 1 m (2 ml) of serum-free culture solution was dispensed into each incubator and precultured at 37°C.

(2) Culture of epidermal cells Br, J, Dermatol, 1013.555-5
60 (1983) using the method of Kitano et al.
Epidermal cells were collected from 4-day-old newborn rats. The obtained cells were placed in the culture vessel at a rate of 1 cm per 1 cm of base area.
Seed at a density of
% air for 2 days. 10% FCS (
Fatal Calf Serum, Commonwe
alth Lot, 971-405)! +119
9 medium (manufactured by GI BCO, catalog No. 400-1
200) was used. After the culture was completed, the epidermal cells adhered to the ultraviolet irradiated atelocollagen at the bottom of the vessel were collected, and the amount of DNA contained therein was quantified and determined as the amount of adhesion.

The relationship between the concentration of ultraviolet irradiated atelocollagen and the amount of adhesion is shown graphically in FIG. As is clear from Figure 1, 5 to 500μ of ultraviolet irradiated atelocollagen was added to the bottom of the culture vessel.
In the epidermal cell culture vessels of the present invention coated with a concentration of 97cm'', the DN, Affi (indicated by ○) was approximately 7 cm per culture vessel.
DNAff1 (indicated by △) in a culture vessel coated with UV-irradiated atelocollagen at a concentration of 05μ97cm2 or less, or DNAff1 in a culture vessel not coated with UV-irradiated atelocollagen at all.
(indicated by a seal) was 6.6 μg or less.

(1) Effects of the Invention According to the present invention, there is provided an epidermal cell culture vessel in which the bottom of the epidermal cell culture vessel is coated with ultraviolet irradiated atelocollagen at a concentration of 5 to 500 μ9/Cm2.

Since the culture vessel of the present invention has excellent adhesion of cells to the bottom of the vessel, when epidermal cells are cultured using this culture vessel, an epidermal cell membrane suitable for artificial skin can be efficiently obtained.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the concentration of atelocollagen and the amount of adhesion of epidermal cells to the bottom of the vessel when epidermal cells are cultured in a culture vessel coated with ultraviolet irradiated atelocollagen.

Claims (1)

[Scope of Claims] 1) An epidermal cell culture device with a bottom, the bottom of which is coated with atelocollagen irradiated with ultraviolet rays at a concentration of 5 to 500 μg/cm^2. 2) The epidermal cell culture device according to claim 1, wherein the amount of ultraviolet irradiation is equivalent to that when placed 15 cm away from a 6 watt germicidal lamp.