Permanent skin replacement using engineered epidermis containing fewer than 5% syngeneic keratinocytes.

This study was conducted to investigate permanent skin replacement using heterologous (syngeneic-allogeneic) epidermal substitutes containing fewer than 5% syngeneic keratinocytes. Keratinocytes were isolated from the skin of new-born Balb/c (B) and C3H/HeN (C) mice and cocultured in different ratios: 20%B-80%C, 10%B-90%C, 5%B-95%C, and 2%B-98%C (and vice versa). After 4 to 5 days, graftable epidermal substitutes were obtained and transplanted onto adult Balb/c or C3H/HeN male mice. Recipients always received the heterografts containing the lower percentage of their own keratinocytes. On Days 15 and 30 postgrafting, all heterografts showed significant graft take (> 75%) and skin replacement compared with allografts. Regenerated tissues were well structured and well vascularized. These tissues contained only syngeneic keratinocytes. These results led us to question whether this was an active immune situation. Structural analyses revealed the presence of leukocyte infiltration that was dependent on the percentage of allogeneic keratinocytes present in the heterologous implant. However, even with the 2% syngeneic-98% allogeneic implant, infiltration was lower than with the allograft. Leukocyte phenotyping confirmed the presence of immune cells infiltrating the heterologous implants and revealed the involvement of CD8+ and CD4+ lymphocytes in this immune activation. The percentages of these two cell populations were lower than those obtained with the allografts, suggesting moderate cellular activation after each heterograft compared with the allografts. In conclusion, it is possible to generate functional skin even after 2% syngeneic-98% allogeneic heterografts; there was moderate cellular immune activation compared with the allografts.