JPS62262986A - Culture device for epidermal cell - Google Patents

Abstract

PURPOSE:To obtain epidermal cell suitable as artificial skin, in high efficiency, by coating a bottom part of a bottom-closed vessel for culture of epidermal cell with an atherocollagen, seeding epidermal cells on the coating layer and carrying out the cell culture. CONSTITUTION:The bottom of a bottom-closed vessel (e.g. dish) for the culture of epidermal cell is coated with an atherocollagen for tissue culture at a concentration of 0.15-500mug/cm<2>. The atherocollagen is free from peropeptide having antigen determinant group and obtained by the enzymatic treatment of collagen. Epidermal cells (e.g. collected from a rat neonate) are scattered on the coating layer in the culture vessel and cultured at a constant temperature for a prescribed period. After the culture, the proliferated epidermal cells adhering to the atherocollagen at the bottom of the vessel are collected.

Description

[Detailed description of the invention] 19 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 atelocollagen at a specific concentration.

When the skin is damaged by a wound, burn, etc., it is most desirable to collect one's own skin from another site and transplant it for treatment. But l h-IJ, -rh+-#
*-ta+vtt<72 mlh mT-T-IIE
Because of this, 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, when culturing epidermal cells, the amount of adhesion of cells to the bottom of the vessel is important, but in conventional culture vessels, the amount of adhesion is not necessarily sufficient, and improvements have been desired.

(1) Purpose of the Invention Therefore, an object of the present invention is to provide an epidermal cell culture vessel in which a large amount of epidermal cells adhere to the bottom of the culture vessel. The purpose of the present invention is to place zero atelocollagen at the bottom of a cell culture vessel.
．． This is achieved by coating epidermal cell culture vessels with a concentration of 15-500 μ9/cm2.

(5) Detailed Description of the Invention The cell culture vessel is coated with atelocollagen by a method known per se. For example, the bottom of the culture vessel can be easily coated with atelocollagen by placing a solution containing a calculated amount of atelocollagen in a cell culture vessel and air-drying the solution. 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 peripeptides having antigenic determinants, is preferably used. Atelocollagen is 0.15 to 1 cn + 1 bottom area of culture vessel.
500 μ9, particularly preferably a concentration of 0.5 to 50 μ9 (
density). Is this concentration critical and 0.15μ? /cm' or less or 500μ9
/cI11! The amount of adhesion of epidermal cells to the bottom of the vessel cannot be improved with the above one-time treatment.

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 to and proliferate on the 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 (+) Preparation of atelocollagen coated culture vessel 0.5.0.
Atelocollagen aqueous solutions 1- with concentrations of 0.05, o, oos, and 0.0005% were placed in each incubator, air-dried at room temperature, and then culture solution was added to reconstitute collagen fibers at 37°C. Atelocollagen is extracted and purified from calf skin using the pepsin digestion method, and is placed in an incubator with a diameter of 3.
A 5nv tissue culture petri dish was used.

Thirty minutes before cell seeding, serum-free culture solution 1III2 was dispensed into each culture vessel, and preculture was performed at 37°C.

(2) Culture of epidermal cells Br, J, Dermatol, 108.555-56
F344 according to the method of Tatsuno et al.
Epidermal cells were collected from newborn rats on the fourth day after birth.

The obtained cells were placed in the culture vessel at a rate of lX per 1 am' of base area.
Seed at a density of 10' seeds at 37°C, 5% CO, -95%
Cultured in air for 2 days. 10% FC3 (F
atal CalrSerum, Commonweal
th Lot, 971-405) containing M199 medium (
(manufactured by GIBCO, catalog No. 400-1200) was used. After culturing, the epidermal cells adhered to the atelocollagen at the bottom of the vessel were collected, and the amount of DNA contained therein was quantified.
It was defined as the amount of adhesion. The relationship between atelocollagen concentration and adhesion amount is shown graphically in FIG. As is clear from Figure 1,
Culture vessel/7) E @ r-mer; ry 14
- /y' ・i mei n + E-,
Amount of DNA in the epidermal cell culture vessel of the present invention coated with a concentration of -1; An rr O/sm (indicated by a circle)
is about 7 μg or more per culture vessel, whereas the amount of DNA in a culture vessel coated with atelocollagen at a concentration of 0.05 μg/cm' (indicated by Δ) or the amount of DNA in a culture vessel not coated with atelocollagen at all (indicated by the old mark) ) was 6.6 μg or less.

(2) Effects of the Invention According to the present invention, there is provided an epidermal cell culture device with a bottom, the bottom of which is coated with atelocollagen at a concentration of 0.15 to 500 .mu.9/cm''.

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 the 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 atelocollagen.

Claims (1)

[Scope of Claims] 1) An epidermal cell culture device with a bottom, the bottom of which is coated with atelocollagen at a concentration of 0.15 to 500 μg/cm^2. 2) Atelocollagen concentration is 0.5-50μg/cm^
2. The epidermal cell culture device according to claim 1, which is