HSV-infected human epidermal keratinocyte lines. In an initial study, this technique hasenabled us to compare various aspects of the processing of UV-damaged HSV in primary fibroblasts and epidermal keratinocytes cultured from the skin of the same donor. MATERIALS AND METHODS

Human fibroblast strains, 48BR, GMO73OA, and IBR/3 (from the MRC Cell Mutation Unit, Brighton, England), were obtained from skin biopsy of normal adult individuals aged 46 (female), 45 (female), and 33 (male), respectively. EK13 and EK4 fibroblasts were derived from foreskin explants in this laboratory(see below). Fibroblast cultures were grown in Earle's modified MEM (Gibco) supplemented with penicillin, streptomycin, sodium bicarbonate, glutamine, and 15% fetal calf serum. Cells were passaged at weekly intervals by trypsinization. The transformed epidermal keratinocyte line (SV61 BaIn/HFK) was developed by Dr. P. H. Gallimore (Cancer Research Campaign Labo ratories, Birmingham, England) by transfection of human fetal kerati nocytes with the DNA of SV61, a mutant of SV4O which lacks the origin of DNA replication (12). They were grown in the same medium as the primary fibroblasts, except that only 10% fetal calf serum was added, and the medium was supplemented with 4 @g of hydrocortisone per ml. This concentration of hydrocortisone (10 times that used for the primary keratinocytes, see below) was found to be optimal for this transformed line. To maintain healthy rapid growth without feeder, cells were seeded at high density (5 x 10―/cm2plastic) and split twice perweekata 1:2 ratio. Swiss 3T3 fibroblasts (Flow) were cultured in Dulbecco's MEM (Flow) with 10% fetal calf serum, and African green monkey kidney cells (CV-ls) were cultivated in Earle's MEM supplemented with 1% glucose and 5% fetal calfserum. Both cell types were passaged at weekly intervals.