Pigmentation defects in tissue-engineered skin substitutes are induced by prolonged epithelial cell culture and not cryopreservation

Summary: Abstract Body: Severe burn patients can be treated with tissue-engineered skin substitutes (TESs). However, pigmentation is not fully restored after TES grafting, resulting in hypopigmentation with pigmented spots in treated patients. We hypothesized that these defects are caused by the culture conditions. The aim of this project was to evaluate the effect of culture time and cryopreservation of epithelial cells used in TESs production. The subsequent pigmentation of TESs was assessed after grafting onto athymic mice. Briefly, epithelial cells were freshly isolated, minimally amplified over a period of 4 days or for a longer time (12 days) and cryopreserved or not. Epithelial cells were seeded onto a dermal component and the resulting TESs were matured at the air-liquid interface for 12 days before grafting onto athymic mice for 45 days. Our results showed that pigmentation in TES with minimally amplified epithelial cells, cryopreserved or not, appeared 7 days after grafting. Pigmentation then became homogenous 28 days after grafting. In contrast, TES produced with epithelial cells cultured for 12 days, cryopreserved or not, did not show homogenous pigmentation even 45 days after grafting. Histological and immunofluorescence analyzes of the pigmented TESs showed basal epithelial cells presenting nuclear caps of melanin, suggesting that the melanocytes were functional. These results showed that cryopreservation of epithelial cells does not affect TES pigmentation while prolonged culture time causes pigmentation defects. Ultimately, changes in epithelial cell culture conditions, such as reducing the culture time of epithelial cells or modifying the culture medium, could lead to restoration of a homogeneous pigmentation after TES grafting onto severe burn patients. Karel Ferland^{1, 2, 3}, Henri De Koninck^{1, 2, 3}, Brice Magne^{1, 2, 3}, Amélie Morissette^{1, 2, 3}, Danielle Larouche^{1, 2, 3}, Lucie Germain^{1, 2, 3} 1. Faculty of Medicine, Universite Laval, Québec City, QC, Canada. 2. Centre de recherche en organogénèse expérimentale de l'Université Laval (LOEX), Québec, QC, Canada. 3. Centre de recherche du CHU de Quebec-Université Laval, Québec, QC, Canada. Pigmentation, Melanoma, and Melanoma Immune Surveillance