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 research. A modified fast and simple method for isolation, growth and passage 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. The epithelium could be seperated easily from the mucosa after incubation the biopsies in dispase over night. Subsequently, keratinocytes were isolated enzymatically by dissociation of epidermal sheets in trypsin, resulting a suspension of highly proliferating keratinocytes without contaminating fibroblasts (2 x 10(6) keratinocytes/biopsy). The cells 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. Results: An average plating efficiency of 60% in primary cultures and 85% in subcultures was obtained. Passaging was possible every 10-13 days when keratinocytes reached sufficient confluency. The cells could be subcultured up to eight times (lifespan of 120 days), and exhibited the typical epithelial morphology during cultivation. Conclusion: Because of the modified pretreatment of the keratinocytes before plating, this culturing protocol for keratinocytes derived from the upper aerodigestive tract enables easy and fast cultivation of keratinocytes, further simplifying currently available methods.