Growth and characterization of human skin epithelial cell cultures

Abstract

In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epilthelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis.

Growth of cultured human epidermal cells into multiple epithelia suitable for grafting.

“…Although there were difficulties in expanding an epidermal cell population in this way and there was no effective means of controlling fibroblast proliferation, such cultures were shown to be transplantable onto animals by Medawar (2) and Karasek (3), and hopes were expressed very early for application to human transplantation (4, 5). Methods of expanding the size of an epidermal cell population by growth from tissue fragments have since been improved by Freeman et al (6,7); the proliferated epithelium remained transplantable (8).…”

“…Although there were difficulties in expanding an epidermal cell population in this way and there was no effective means of controlling fibroblast proliferation, such cultures were shown to be transplantable onto animals by Medawar (2) and Karasek (3), and hopes were expressed very early for application to human transplantation (4, 5). Methods of expanding the size of an epidermal cell population by growth from tissue fragments have since been improved by Freeman et al (6,7); the proliferated epithelium remained transplantable (8).When the superiority of disaggregated cell culture for obtaining increased proliferation of other cell types was recognized, attempts were made to grow epidermal cells in this way by Prunieras et al (9) (13), and Marcelo et al (14). Some multiplication took place in such cultures, but the cells generally were not serially cultivable, either because of overgrowth of fibroblasts or possibly because the epidermal cells when pure were deprived of fibroblast support.…”

Heteroploid conversion of human skin cells by methylcholanthrene.

“…Some colonies, presumably derived from bits of dermis, seemed to consist entirely of fibroblasts. For two other experiments, 0.36-mm split-thickness skin tissue and full-thickness foreskin tissue were minced as described and planted on the dermal bed of dead sterile pigskin (9)(10)(11). A 1-to 2-cm-diameter clearing of the pigskin substrate, corresponding to the diameter of virtually pure epithelial cell outgrowth, occurred around the skin explants within 17 preparations at the Children's Hospital of Akron were made by the method of Kajii et al (14).…”

Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum-free media

“…However, in previous eulture systems, broad applieation of eultured HK (10,17,251 to investigations of human diseases has been severely restrieted by requirements for high eellular population densities, specialized substrates, or reeder interaetions with fibroblastie eells. Growth-promoting substrates that have been used include collagen-eoated dishes (10,16,171 and speeially processed pigskin (6,24). Feeder interaetions that have been employed inelude use of irradiated 3T3 eells (26) and an initial period of eo-culture with human fibrob|asts (20).…”