Organotypic (raft) epithelial tissue culture system for the differentiation-dependent replication of papillomavirus

Abstract

We have developed an in vitro organotypic (raft) culture system capable of reproducing the differentiation-dependent replication cycle of human papillomavirus. A dermal equivalent is made from a mixture of type I collagen and fibroblasts. Cells of a line derived from a cervical intraepithelial neoplasia type 1 (CIN-1) are placed on top of the dermal equivalent and while submerged allowed to grow to confluence. The dermal equivalent with epithelial cells on top is then lifted onto a wire grid where it remains at an air-liquid interface. From this point on the epithelial cells never come in contact with the culture media. Feeding of the epithelial cells is done by diffusion through the dermal equivalent similar to the in vivo situation. The epithelial cells under these conditions will stratify and differentiate over approximately a two week period. When an inducer of protein kinase C is added to the media our CIN-1 derived cell lines, which maintains episomal copies of a high risk human papillomavirus will biosynthesize virion. We describe in detail how to perform this procedure and provide information on how to use this system for the study of the interaction of human papillomavirus with its host tissue, squamous epithelium.