CULTIVATION OF HUMAN KERATINOCYTES OF THE UPPER AERODIGESTIVE TRACT

Background: Cultivation of benign epithelial cells under standardized conditions is of major interest in many fields of clinical and basic r esearch. A modified fast and simple method for isolation, growth and p assage of epidermal cells has been developed with consideration given to the complex environment of the upper aerodigestive tract. Methods: Normal human mucosa of the upper aerodigestive tract was taken from 15 patients (4-73 years) during diagnostic and therapeutic operations. T he epithelium could be seperated easily from the mucosa after incubati on the biopsies in dispase over night. Subsequently, keratinocytes wer e isolated enzymatically by dissociation of epidermal sheets in trypsi n, resulting a suspension of highly proliferating keratinocytes withou t contaminating fibroblasts (2 x 10(6) keratinocytes/biopsy). The cell s were washed several times in fresh fetal calf serum before they were plated in untreated culture flasks. The keratinocytes were cultivated in serumfree medium supplemented with epidermal growth factor, bovine pituitary extract, amphotericin B, and penicillin/streptomycin. Resul ts: An average plating efficiency of 60% in primary cultures and 85% i n subcultures was obtained. Passaging was possible every 10-13 days wh en keratinocytes reached sufficient confluency. The cells could be sub cultured up to eight times (lifespan of 120 days), and exhibited the t ypical epithelial morphology during cultivation. Conclusion: Because o f the modified pretreatment of the keratinocytes before plating, this culturing protocol for keratinocytes derived from the upper aerodigest ive tract enables easy and fast cultivation of keratinocytes, further simplifying currently available methods.