Developments in xenobiotic-free culture of human keratinocytes for clinical use.

We have recently reported that irradiated human fibroblasts can be used as a feeder layer, to expand keratinocytes under serum-free conditions, on a chemically defined plasma polymer surface developed for the culture and transfer of keratinocytes for clinical use. While this is a significant advance in developing a serum-free keratinocyte culture approach, the need to irradiate fibroblasts to growth-arrest them and prevent them from overgrowing the keratinocytes introduces another small, but potential, risk for the patient. The aim of the present study was to develop conditions for the coculture of normal human keratinocytes with nonirradiated normal human fibroblasts under serum-free conditions. We examined the fibroblast/keratinocyte relationship on three separate surfaces: tissue culture plastic, non-tissue culture plastic, and a plasma polymer surface designed for clinical use. We report that it is possible to achieve rapid and successful expansion of human keratinocytes under serum-free conditions on all three surfaces providing one uses a keratinocyte-friendly media, a minimum seeding density of keratinocytes, and a ratio of fibroblasts to keratinocytes that does not exceed 1:1. These results provide us with a rapid laboratory expansion of proliferative human keratinocytes, under completely defined culture conditions, without any xenobiotic cells (mouse fibroblasts) or material (bovine serum), for the treatment of patients with extensive skin loss.