1435 Enzymatic digestion of scalp hair follicle bulge allows to have more bulge-derived stem keratinocytes than explant isolation

The hair follicle bulge is a niche for stem keratinocytes which can differentiate into epidermis and hair lineage. Even if reconstructed skin substitutes produced by the self-assembly approach have several advantages, skin appendages are lacking. To study the potential of bulge-derived keratinocytes to differentiate into hair follicles within skin substitutes, keratinocytes from the bulge must be extracted. As the commonly used explant culture is slow and offers a low yield, an enzymatic approach is proposed. We hypothesize that trypsin digestion of microdissected bulges would provide a higher yield of stem keratinocytes than the explant technique. The objective of this study was to optimize the isolation procedure of keratinocytes from the bulge to obtain cultures enriched in bulge-derived stem keratinocytes. Biopsies from adult scalps were incubated with thermolysin to separate the dermis from the epidermis. Bulge were separated by microdissection and cultured either by explant or after trypsin digestion. In parallel, simultaneous extraction of epidermal and hair keratinocytes (total extraction) was conducted. These populations were compared. After the first passage, bulge-derived keratinocytes extracted by digestion were in higher quantity and showed a shorter doubling time than those of the explant method. Furthermore, a higher number of colonies was obtained, suggesting that the digestion method enriches the culture with stem keratinocytes. In conclusion, the dissociation of bulge using trypsin to extract bulge-derived keratinocytes is a faster and easier method compared to the explant technique. Reconstructed skin substitutes will be produced with these cells to evaluate their potential for follicular differentiation.