Retroviral gene transfer into porcine keratinocytes following improved methods of cultivation

We embarked on a program examining the application of cultured epithelial sheets to skin wounds in pigs using retroviral gene transfer as a means to follow the grafted cells. In the past similar studies have been hampered by an inability to grow porcine keratinocytes without seeding at an extremely high density. In this study we found that excellent results could be achieved with Opti-MEM-1 (Gibco BRL Life Technologies) containing 1 per cent foetal calf serum, 0.5 mM Ca2+ and no other growth factors or stimulants. Keratinocytes were plated on gamma-irradiated 3T3 feeders on surfaces which had previously been coated with rat tail collagen I. Keratinocyte cultures were established at a seeding density of 5 x 10(4) cm-2. The yield of cells from 1 cm2 of skin was sufficient to set up a 75 cm2 flask. Cultures reached 80-90 per cent confluence in 7-10 days, after which they were passaged 1:3 multiple times, taking 3-4 days to reach the same confluency. Allowing cultures to remain confluent for 1 week was sufficient to allow Dispase removal of an intact sheet. Using these techniques porcine keratinocytes were transduced at an average frequency of 25.3 per cent (+/- 14.0 SEM) with the retroviral vector MFG lacZ nls by growth on the gamma-irradiated retroviral producer line GP + envAm12.