JPH0655218B2 - Artificial basement membrane and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an artificial basement membrane and a method for producing the same. The basement membrane is a cell stroma mainly composed of collagen and glycoprotein existing on the basal surface of epithelial cells, endothelial cells and mesothelial cells. At present, it has been revealed that the basement membrane plays an important biological role such as metastasis of cancer and regulation of growth control of epithelial cells in epithelial-mesenchymal yarn. However, it is generally extremely difficult to artificially make this basement membrane in vitro, and laminin type IV collagen, HSPGS, etc.
In the method of reconstituting (heparan sulfate, proteoglycan, etc.) as the main component, the artificial basement membrane produced was remarkably different in structure from that in the living tissue.

In view of the above, the present inventors have established an artificial basement membrane structurally extremely equivalent to the natural basement membrane by layer-culturing the basement membrane elements on the cohesive weave in which fibroblasts are embedded in the collagen gel. Succeeded in manufacturing. When skin-derived epithelial cells were cultured on the reconstituted basement membrane prepared by the above method,
It showed remarkably excellent proliferative ability, and after 3 weeks, formation of a uniform multi-layer structure of epithelial cells could be observed by light and electron microscopy. Furthermore, when the above-mentioned cultured skin was transplanted into a rat, wound healing was markedly promoted and no keloid scar was observed at all.

The constitution of the present invention will be described in detail below.

Basement Membrane Constituent In the present invention, the basement membrane constituent is a MEM medium 10% FCS containing appropriate amounts of type IV collagen (manufactured by BRL), laminin (manufactured by BRL), and fabronectin (manufactured by BRL).
It was a solution.

In addition, as a fibroblast-containing gel that serves as a substrate, an appropriate amount of ty
MEM containing peI collagen (or atelocollagen)
A medium 10% FCS solution was used. It was also confirmed that the artificial basement membrane can be produced better by adding a small amount of heparan sulfate, chondroitin sulfate, nidogen, entactin, etc. to the above two solutions.

Manufacture of reconstituted basement membrane It is necessary to select the animal species of the reconstituted basement membrane according to the intended use, but considering cell-mediated immunity, it is desirable to contain as few cells as possible in the basement membrane. .

From the above viewpoint, the basement membrane was produced by the following method.

FIG. 1 schematically shows one of the methods for producing the reconstituted basement membrane of the present invention, in which type I collagen gel 2 containing no cells is layered on type I collagen gel 1 incorporating fibroblasts, and further, The basement membrane constituent 3 is layered thereon. The culture conditions at this time are 5% CO ₂ , 95% air, and 37 ° C.

At this time, if a Millipore filter (manufactured by Millipore) is sandwiched between the first layer and the second layer at equal intervals, it is possible to further prevent the cells from being mixed into the basement membrane.

In addition, as schematically shown in FIG. 2, basement membrane component 3 was overlaid on typlI collagen gel 1 incorporating fibroblasts, and cultured for 5 days in 5% CO ₂ and 95% atmosphere, and then 5% CO 2. ₂ ,
A method of culturing for 3 days under different conditions of 50% O ₂ and 45% N ₂ is also effective. This utilizes the property that fibroblasts are growth-inhibited under high oxygen concentration. According to this method, compared to the above method, there are many opportunities for the fibroblasts and the reconstituted components to come into direct contact with each other, so that the structure of the artificial basement membrane can be made robust, and further, the fibroblasts are mixed in the reconstituted basement membrane. Can be prevented.

Hereinafter, the present invention will be described in detail with reference to experimental examples.

Experimental Example 1 6 × 10 ⁶ fibroblasts were planted per one sheet on 6 ml of the reconstituted basement membrane prepared by the above method, and the contracted state of the gel was observed at 37 ° C. in 5% CO ₂ , 95% atmosphere. . It is known that the degree of gel contraction is related to the activity of cells cultured on the basement membrane. The results are shown in FIG. 3, in which the horizontal axis represents the number of days after transplantation and the vertical axis represents the gel diameter (cm).

Experimental Example II When guinea pig skin epithelial cells were cultured on the reconstituted basement membrane produced by the above-mentioned method, they proliferated significantly, and a uniform multi-layer structure of epithelial cells was formed after 3 weeks. It was observed under a microscope. Furthermore, when the above cultured epithelial cells were transplanted to the back of the guinea pig, normal keratin formation was observed, and when the observation was continued for 4 weeks after transplantation, there was no rejection of the transplant, and on keloids such as the skin's plaque. No scar formation was observed.

As is apparent from the above, the present invention provides an artificial basement membrane structurally extremely equivalent to a natural basement membrane and a method for producing the same, and it can be said that the present invention has a remarkable effect in culturing epithelial cells.

[Brief description of drawings]

1 and 2 are explanatory views showing the method for producing an artificial basement membrane of the present invention. FIG. 3 is an explanatory diagram showing the results of Experimental Example I.

Claims (1)

[Claims] 1. A fibroblast cultured in a collagen gel is used as a substrate, and constituent components including type IV collagen, glycoprotein, and mucopolysaccharide are layered thereon, and gelled under physiological conditions. A method for producing an artificial basement membrane.