Optimizing in vitro culture conditions leads to a significantly shorter production time of human dermo-epidermal skin substitutes
- 911 Downloads
Autologous dermo-epidermal skin substitutes (DESS) generated in vitro represent a promising therapeutic means to treat full-thickness skin defects in clinical practice. A serious drawback with regard to acute patients is the relatively long production time of 3–4 weeks. With this experimental study we aimed to decrease the production time of DESS without compromising their quality.
Two in vitro steps of DESS construction were varied: the pre-cultivation time of fibroblasts in hydrogels (1, 3, and 6 days), and the culture time of keratinocytes (3, 6, and 12 days) before transplantation of DESS on nude rats. Additionally, the impact of the air–liquid interface culture during 3 days before transplantation was investigated. 3 weeks after transplantation, the macroscopic appearance was evaluated and histological sections were produced to analyze structure and thickness of epidermis and dermis, the stratification of the epidermis, and the presence of a basal lamina.
Optimal DESS formation was obtained with a fibroblast pre-cultivation time of 6 days. The minimal culture time of keratinocytes on hydrogels was also 6 days. The air–liquid interface culture did not improve graft quality.
By optimizing our in vitro culture conditions, it was possible to very substantially reduce the production time for DESS from 21 to 12 days. However, pre-cultivation of fibroblasts in the dermal equivalent and proliferation of keratinocytes before transplantation remain crucial for an equilibrated maturation of the epidermis and cannot be completely skipped.
KeywordsTissue engineering Dermo-epidermal skin substitutes Skin reconstruction Air-liquid interface Collagen hydrogels
- 11.Pontiggia L, Biedermann T, Meuli M, Widmer D, Bottcher-Haberzeth S, Schiestl C, Schneider J, Braziulis E, Montano I, Meuli-Simmen C, Reichmann E (2009) Markers to evaluate the quality and self-renewing potential of engineered human skin substitutes in vitro and after transplantation. J Invest Dermatol 129:480–490PubMedCrossRefGoogle Scholar
- 12.Braziulis E, Diezi M, Biedermann T, Pontiggia L, Schmucki M, Hartmann-Fritsch F, Luginbuhl J, Schiestl C, Meuli M, Reichmann E (2012) Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes. Tissue Eng Part C Methods 18(6):464–475PubMedCrossRefGoogle Scholar
- 20.Wood FM (2003) Clinical potential of autologous epithelial suspension. Wounds 15:16–22Google Scholar
- 25.Stark HJ, Willhauck MJ, Mirancea N, Boehnke K, Nord I, Breitkreutz D, Pavesio A, Boukamp P, Fusenig NE (2004) Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture. Eur J Cell Biol 83:631–645PubMedCrossRefGoogle Scholar