Organotypical engineering of differentiated composite-skin equivalents of human keratinocytes in a collagen-GAG matrix (INTEGRA Artificial Skin) in aperfusion culture system

Background: The production of autologous composite skin equivalents for the treatment of full-thickness skin defects in bums is time consuming and cos tly because of laboratory procedures which have to be performed manually. I n the present study keratinocytes were seeded into INTEGRA Artificial Skin and placed in a perfusion culture system in order to evaluate the possibili ty of producing composite grafts in an automated system with the aim of est ablishing a cost-effective method of industrial production. Methods: Compos ite grafts of INTEGRA and human keratinocytes were raised in perfusion cult ure and grafted onto athymic mice to evaluate their potential to reconstitu te a full-thickness skin substitute in vivo compared to grafts from standar d stagnant cultures. Results: Cultured composites from perfusion cultures s howed no significant histological differences compared to those from stagna nt cultures; however, a tendency of improved cell growth and a more surface -oriented localization was observed. Cell proliferation and surface-bound d ifferentiation were not impaired by the use of carbonate-independent buffer ing (HEPES), which is necessary for perfusion culture. The composite grafts from perfusion culture exhibited identical wound adherence and complete he aling and histologically represented a multi-layered, keratinizing human ep idermis. Conclusion: Engineering of differentiated composite skin equivalen ts is possible in a perfusion culture system, which offers technical and pr ocedural and possibly even biological advantages compared to standard stagn ant culture methods. The development of automated perfusion culture systems for the production of composite grafts in sizes required clinically (scale -tip) will be the next step in the cost-effective engineering of large-scal e composite skin equivalents.