Xenogeneic Mouse Fibroblasts Persist in Human Cultured Epidermal Grafts: A Possible Mechanism of Graft Loss

Recent reports suggest that long-term graft take of cultured epidermal autografts (CEAs) is less than 50% when late graft loss is considered. The characteristics of late CEA loss suggest that it may occur as a result of an immunologic reaction to persistent xenogeneic cells and/or proteins used to grow CEA. In this study we examined whether immunologically reactive, mouse 3T3 fibroblasts used as feeder layers can persist in primary, secondary, and tertiary human CEA. We cocultured keratinocytes from 11 separate burn patients with growth-arrested 3T3 fibroblasts. After removing visible 3T3 fibroblasts from CEA with trypsinization, we allowed CEA to reach confluence. We then harvested CEA either as primary, secondary, or tertiary cultures. We detected mouse fibroblasts using fluorescence activated cell sorting (FACS) with a monoclonal antibody specific for mouse major histocompatibility (MHC) antigens. We detected mouse MHC class II antigens by performing Western immunoblotting with another mouse MHC-specific monoclonal antibody. By FACS we identified mouse fibroblasts in 100, 75, and 62.5% of primary, secondary, and tertiary passage CEAs, respectively. Similarly by immunoblotting we found mouse MHC class II antigen in 100, 80, and 66.7% of primary, secondary, and tertiary CEAs. These results demonstrate that xenogeneic fibroblast feeder layers capable of generating immunogenic transplantation antigens persist in CEAs. The persistence of these cells and their antigen expression may contribute to CEA loss.