15.    Towards the development of a simplified long-term organ-culture method for human scalp skin under serum-free conditions. 
Zhongfa Lu(1), Ralf Paus(2), Klinik Dr. Koslowski(3),1.Department of Dermatology, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.2. Dept. of Dermatology, University Hospital Schleswig-Holstein, University of Luebeck, Luebeck Germany. 3. Munich, Germany

Organ-culture of human scalp skin is usually performed with serum-containing medium, which limits its analytical usefulness. However, without serum, long-term survival of the skin and its appendages is thought to be problematic. Here, we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum-free William’s E medium for up to several weeks. Active hair shaft growth was visible until day 16 and was significantly faster than in MEM +10% FBS. LDH release into the medium (as an indicator of cell death) revealed a first peak on day 2, stabilized LDH values between day 3 and day 12, and a second LDH peak around day 15 (indicating progressive organ decay). By quantitative immunochemistry, proliferating (Ki-67+) cells could still be observed in the epithelium of hair follicles even on day 12, and day 17 of serum-free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Before day 12, William’s E better protected against cell death than MEM+10% FBS. Giemsa stains revealed mature skin mast cells were observed even after 13 days in culture. The percentage of surviving hair follicles with catagen- or telogen-like morphology gradually increased over time, while after 17days of culture most hair follicles were found to be in telogen. While epidermis and hair follicle epithelium showed increasing atrophy over time, some long term-surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum-free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than two weeks under well-defined experimental conditions. This pragmatic assay should become a valuable tool for both skin and hair research.