Organotypic (raft) culture of biopsy-derived upper respiratory epithelium

OBJECTIVE: The objective of this study was to derive a reliable technique for culturing biopsy-derived upper respiratory epithelium in a system that supports epithelial differentiation and simulates the normal epithelial life cycle. STUDY DESIGN: The authors conducted a prospective study of modification and development of an in vitro tissue culture method. METHODS: Thirty biopsy specimens from 16 individuals with recurrent respiratory papillomatosis and chronic tonsillitis, pretreated to prevent bacterial and fungal overgrowth, were digested with trypsin to create a supernatant of individual cells. The cells were plated and incubated. At 14 to 16 days, the resulting colonies were placed on a wire cloth raft and fed through diffusion from the underlying culture medium in an air-liquid interface. RESULTS: Eight specimens were successfully cultured for an average of over 32 days. The longest duration of sustained growth was 60 days. Low-risk human papillomavirus specimen-based cultures reproduced infection in cultured squamous epithelium with corresponding histopathologic features indicating a high level of stratification and differentiation. CONCLUSIONS: Unlike commercially available cell lines, biopsy-derived material is predisposed to contamination, and successful in vitro culture and experimentation creates many unique challenges. An organotypic culture system, capable of reproducing the differentiation-dependent replication cycle of human papillomavirus, may be used for culturing biopsy-derived specimens for a variety of studies.