A modified culture system for epidermal cells for grafting purposes: an in vitro and in vivo study

A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air–liquid interface. In addition, after 2 weeks of culture, hemidesmosome‐like structures were formed along the basal area of the plasma membrane of the basal cells at the cell–filter interface. When grafted onto full‐thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase‐treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell–“neodermis” interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air–liquid interface and detached from the substrate by dispase‐treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase‐treatment appeared in our hands the only method for successful and complete epithelial regeneration in full‐thickness wounds.