Study of the human keratinocyte isolation methods and in vitro culture techniques in a single laboratory

The survey was conducted to analyze different human keratinocyte isolation methods and in vitro culture techniques used in current practice. The aim was to find which would work best in our laboratory to obtain epidermal sheets suitable for grafting. The techniques for epidermal removal and keratinocyte isolation involve dispase II or trypsin solutions at different concentrations and times. The number of cells and cellular viability were measured from different origins and size samples. The main approaches developed for in vitro culture of keratinocytes include the use of serum-containing media and of a feeder-layer of lethally irradiated mouse fibroblasts, serum-free medium in the absence of a feeder-layer and a commercially prepared serum-free medium. Other techniques include the use of different substrates such as laminin, fibronectin, collagen IV or fibrin glue to obtain more complete differentiation of epithelial sheets. The growth rate and cellular attachment have been studied using these media and support systems. In summary, for the isolation of keratinocytes, the best results were obtained with 0.17% trypsin solution overnight. The best attachment and growth rates of cell cultures have been obtained when using serum-containing medium and a feeder-layer of lethally irradiated mouse fibroblasts. Received: 15 January 1997 / Accepted: 30 April 1998