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

Abstract

Owing to several recent developments, the cultivability of epidermal keratinocytes, particularly those of the human, has been greatly improved. Under the conditions used, single cultured cells generate stratified colonies that ultimately fuse and form an epithelium that is a reasonable approximation of the epidermis. It will be shown here that large amounts of cultured epithelium can be generated from a small piece of epidermis in a short time. We wish to bring to the attention of surgeons and cell biologists the possibility of using culture-gown epithelium derived from the same individual to restore defects in the epidermis.General description of behavior of cultured human epidermal cells There have been many studies of the epidermis in culture (for review of early work, see ref. 1). Most studies were carried out on explanted fragments, a form of cultivation in which multiplication is usually limited. 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. In such systems the inoculation density was high, and separated single cells were probably not able to give rise to colonies efficiently.In studies of a keratinocyte line of teratomal origin by Rheinwald and Green (15), it was discovered that the cells could be cultivated serially from small inocula by supporting them with cocultivated lethally irradiated 3T3 cells or other fibroblasts. It was then found (16) that human epidermal cells could be serially cultivated under the same conditions. The following points were established.(i) By use of supporting 3T3 cells, epidermal cells of newborn infants can be serially transferred through numerous subcultures.(ii) Each colony initiated by a single cell forms a stratified squamous epithelium. The basal cells (those attached to the surface of the culture vessel) resemble basal cells of the epidermis in that they account for all cell multiplication, whereas the cells that leave the basal layer become terminally differentiated.(iii) Living fibroblasts, which were present in the original tissue and whose multiplication in culture was formerly uncontrollable, can be controlled by ...

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