In vitro production and transplantation of immunologically active skin equivalents

In this study, we produced in vitro epidermal equivalents (EE) and skin equivalents (SE) with and without spleen lymphocytes. These skin substitutes were used for in vitro and in vivo (after isograft) histologic studies of cell and extracellular matrix organization and for protein synthesis. Then, using spleen lymphocytes in syngeneic and allogeneic SE, we assessed the immunogenicity of these skin substitutes after transplantation. In vitro histologic analyses showed that the epidermal organization of EE was comparable to that of SE. Fibroblasts and spleen lymphocytes were present in the extracellular matrix, as is the case in normal skin. Comparative immunohistologic studies after EE and SE isografting showed that the newly generated cutaneous tissues were well structured and vascularized. There were indications of physiologically active skin. The dermal component in these regenerated skins was, however, more organized after SE than after EE isografting, which indicates the importance of the dermis. Lastly, allografting of SE with and without spleen lymphocytes showed interesting results. Indeed, 10 days after allografting, all SE allowed skin regeneration comparable to isografts. Moreover, leukocyte infiltration in allografts was observed as early as 10 days and increased during the postgrafting period. Also, the presence of allogeneic spleen lymphocytes alone in syngeneic SE initiated recipient immune activation and induced leukocyte infiltration and graft rejection. The density of infiltrating leukocytes was higher in the complete allograft (allogeneic keratinocytes, fibroblasts, and spleen lymphocytes) compared with the partial allograft (only spleen lymphocytes were allogeneic), with the allograft (allogeneic keratinocytes and fibroblasts), and with the partial isograft (presence of syngeneic lymphocytes with allogeneic keratinocytes and fibroblasts). Mac-1+ and CD8+ cells were present in these leukocyte infiltrations, which indicates recipient immune system activation and allograft rejection. CD4-positive cells were not, however, seen in these leukocyte infiltrations. These results suggest that the incorporation of spleen lymphocytes in SE enhanced their immunogenicity as judged by leukocyte infiltration and the presence of CD8+ cells in the implants.