Organotypic epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents

Organotypic epithelial raft cultures accurately reproduce the process of epithelial differentiation in vitro and can be prepared from normal keratinocytes, explanted epithelial tissue, or established cell lines. Normal primary human keratinocytes (PHKs) stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while transformed cell lines exhibit dysplastic morphologies similar to the (pre)neoplastic lesions seen in vivo. This three-dimensional (3D) culture system provides an essential tool for either alone or co-cultured with PHKs, tumor biology and selectivity of antitumor agents can be analyzed. For the preparation of dermal equivalents, a collagen matrix solution is made with rat-tail collagen mixed on ice with 10-fold concentrated Ham's F-12 medium, 10-fold concentrated reconstitution buffer, and 3T3 J2 murine fibroblasts. One milliliter of the collagen matrix solution is poured into 24-well plates. After equilibration of the gel with 1 ml of growth medium overnight at 37°C, 2.5 × 105 PHKs isolated from neonatal foreskins are seeded on the tops of the gels and maintained submerged for 24 to 48h. The collagen rafts are raised and placed onto stainless steel grids at the interface between the air and the liquid culture medium. The raft cultures can be infected with epitheliotropic viruses at different stages of epithelial cell differentiation. The epithelial cells are then allowed to stratify for 10 to 12 days. The cultures can be harvested at 10-12 days post-lifting and analyzed to check epithelial differentiation by histological examination and viral replication. Cultures can also be prepared using primary epithelial cells derived from tonsillectomies. Tumor cells can be used to prepare the raft cultures, for instance, cell lines transformed with human papillomavirus (HPV) transformed cells (such as HeLa and SiHa) or Merkel cell carcinomas (MCCs), melanoma cell lines, etc. The tumor cells are seeded alone on top of the gels or in combination with PHKs, resulting, respectively, in the production of 3D cultures with dysplastic morphology (tumor cells monoculture) or 3D cultures with patches of dysplastic morphology in the differentiated epithelial cells.