Using Organotypic (Raft) Epithelial Tissue Cultures for the Biosynthesis and Isolation of Infectious Human Papillomaviruses

Abstract

Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitrowherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

A keratinocyte life cycle model identifies novel host genome regulation by human papillomavirus 16 relevant to HPV positive head and neck cancer

“…Organotypic raft culture, Immunofluorescence staining and FISH NOKs, NOKs+HPV16 and HTK+HPV16 cells were differentiated via organotypic raft culture as described previously [ 56 , 57 ]. Briefly, cells were seeded onto type 1 collagen matrices containing J2 3T3 fibroblast feeder cells.…”

MEK/ERK signaling is a critical regulator of high-risk human papillomavirus oncogene expression revealing therapeutic targets for HPV-induced tumors

“…W12-E and CIN-612 9E cells were investigated at <30 passages after cloning. All keratinocyte cell lines were co-cultured with mitomycin C-treated J2-3T3 fibroblast feeder cells in E medium containing 10% fetal bovine serum (FBS; Atlanta Biologicals) and 10 ng/ml murine epidermal growth factor (EGF; Corning) as described previously [ 93 , 102 ]. J2-3T3 fibroblasts were propagated in high-glucose DMEM (Irvine Sci.)…”

HPV16-Immortalized Cells from Human Transformation Zone and Endocervix are More Dysplastic than Ectocervical Cells in Organotypic Culture

“…All HPV16-immortalized cervical cells were tested at approximately 80 to 95 population doublings or about 25 to 30 passages. Rafts were submerged in raft medium 48 (1:3 mixture of F12/DMEM medium with 5% FBS plus Supplements) for 48 hours before being raised onto stainless steel grids in a 100 mm cell culture dish to form an air-liquid interface for epithelial cells. The medium was changed every 2 days and cultures were fixed after 10 days.…”