Development of a short-term human full-thickness skin organ culture model in vitro under serum-free conditions

Abstract

Skin exerts a variety of important functions to maintain its integrity and viability. It can be used as an experimental ex vivo model to study wound healing, oxidative stress, skin aging, carcinogenesis as well as topical or “systemic” therapeutic intervention in vitro. This report aims to validate a serum-free human full-thickness skin organ culture model with regard to dependency of skin viability on culture duration and location of skin cross-section (1–5) from the outer (section 1) to the inner side (section 5) of a skin specimen (5 × 10 mm). Cultured skin was analyzed in time-dependent manner for structural damage (H&E staining) and ‘balance’ between proliferation (Ki67) and apoptosis [cleavage of caspase-3, lactate dehydrogenase (LDH), TUNEL]. First structural disturbances were observed at 48 h (section 3; middle part), increasing with prolonged culture time. Cleavage of caspase-3 and appearance of apoptotic [TUNEL(+)] cells showed significant increase at 72 h in sections 4 and 5, respectively. This correlated to increasing LDH release. Parallel analysis of proliferating [Ki67(+)] cells revealed simultaneous down-regulation within the first 48 h reaching complete absence of Ki67(+) cells at 72 h. These data define an accurate, standardized and robust serum-free short-term ex vivo human full-thickness skin model which is suitable for experimental studies of up to 48 or 72 h in vitro. This model therefore might be used for research related to, e.g., short-term experimentally induced inflammation, UV-induced structural and functional damage, wound healing and substance penetration.