ABSTRACT
HUMAN KERATINOCYTE GRAFTS: BACKGROUND AND DEVELOPMENT In 1975, Rheinwald and Green reported a cell culture method that made rapid in vitro propagation of human keratinocytes possible for the first time. Human keratinocytes had previously proven difficult merely to maintain in tissue culture, and they had never before been serially subcultivated (Andreassi, 1992). Thus, the method represented a significant breakthrough in cell cultivation technology. The culture technique was based on the use of sublethally irradiated 3T3 fibroblast feeder layers and of culture medium supplemented with ingredients such as fetal calf serum, glucocorticoids, cholera toxin (Green, 1978), and epidermal growth factor (Rheinwald and Green, 1977). The system so efficiently supported keratinocyte growth that large quantities of cultured epithelium could be produced within a relatively short period from a small sample of normal skin. In fact, the surface area of a donor skin biopsy could be expanded 5,000-to 10,000fold within two to three weeks by this technique, making it possible to produce sheets of keratinocytes with an aggregate surface area large enough to cover the entire human body from a skin biopsy the size of a postage stamp (Green et al., 1979). In 1979, Green, Kehinde and Thomas reported that a confluent culture of keratinocytes grown by this method could be released intact from the culture vessel by the neutral proteinase Dispase. This enzyme had the ability to selectively disrupt the strong bond between the basal keratinocytes and the culture vessel surface without disrupting intercellular junctions within the cell sheet. The result was a potentially graftable epithelial unit. However, it remained to be demonstrated that these delicate keratinocyte sheets were capable of surviving transplantation to the relatively hostile environment of a living wound. In order to test the performance of human keratinocyte sheets as grafts, short-term pilot studies were performed in athymic mice (Banks-Schlegel and Green, 1980). In these experiments, the cell sheets not only survived transplantation, they appeared to attach spontaneously the surface of a fresh cutaneous wound and to rapidly undergo both stratification and terminal differentiation.
