[Efficient production of transfected human keratinocytes under serum-free and feeder layer-free conditions]

Keratinocyte transplantation after burn injury and in chronic wound treatment is a potentially useful method in clinical practice. As transfer of keratinocytes is easily monitored and gene expression is controllable by topical administration of inductors, keratinocyte cultures are an especially interesting medium for gene therapeutic approaches far above of wound healing applications. A major obstacle is the standardization of keratinocyte preparation and maintenance of pure proliferative cultures for clinical application. The best outcomes in previous protocols were obtained using fibroblasts as a feeder layer, a requirement for long-term expanded cultures. Cell expansion and a high purity of keratinocytes are prerequisites for clinical transfer studies. Here, we describe a human keratinocytes preparation method that allows cell proliferation and expansion in culture without a feeder layer.

Human keratinocytes were prepared from skin biopsies and cultured on untreated plastic culture dishes using Waymouth medium the first days followed by a change to a commercially available serum-free keratinocyte medium. The cells were characterized morphologically followed by transfection. For positive selection, transfected cells were selected by the cotransfection system pMACS Kk and magnetic cell sorting.

Transfection rates were determined by expression of GFP vector which were 35%. The usage of magnetic cell sorting resulted in positive selection of transfected cells. Positive cells were able to adhere and proliferate after the sorting procedure. High viability and expansion of plastic adherent keratinocytes was achieved allowing up to 5 passages without signs of senescence and the doubling times were 3-5 days. The cells displayed typical keratinocyte morphology and immunostaining confirmed high keratinocyte purity. The number of contaminating fibroblasts was low.

Here, we describe an efficient and inexpensive method for a standardized human keratinocyte isolation without the need of a fibroblast feeder layer. This protocol may facilitate the clinical application of cell based therapies in burn injuries or chronic wounds using keratinocytes.