RETROVIRAL GENE-TRANSFER INTO PORCINE KERATINOCYTES FOLLOWING IMPROVED METHODS OF CULTIVATION

We embarked on a program examining the application of cultured epithel ial sheets to skin wounds in pigs using retroviral gene transfer as a means to follow, the grafted cells. In the past similar studies have b een hampered by an inability to grow porcine keratinocytes without see ding at an extremely high density. In this study we found that excelle nt results could be achieved with Opti-MEM(R)1 (Gibco BRL Life Technol ogies) containing 1 per cent foetal calf serum, 0.5 mM Ca2+ and no oth er growth factors or stimulants. Keratinocytes were plated on gamma-ir radiated 3T3 feeders on surfaces which had previously been coated with rat tail collagen I. Keratinocyte cultures were established at a seed ing density of 5 x 10(4) cm(-2). The yield of cells from I cm(2) of sk in was sufficient to set up a 75 cm(2) flask. Cultures reached 80-90 p er cent confluence in 7-10 days, after which they were passaged 1:3 mu ltiple times, faking 3-4 days to reach the same confluency. Allowing c ultures to remain confluent for 1 week was sufficient to allow Dispase (R) removal of an intact sheet. Using these techniques porcine keratin ocytes 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 + enzAm12. (C) 1997 Elsevier S cience Ltd for ISBI. All rights reserved.