Functional Melanocytes Obtained From Rabbit Hair Follicle Outer Root Sheath

Abstract BackgroundMelanocytes have been cultivated from the outer root sheath of hair follicles for two decades. So far, these isolation and culturing procedures have been carried out from human and mouse follicles. In this study, we have translated the established procedure for obtaining melanocytes from stem cells and precursors of human hair follicle to a rabbit species in order to isolate and cultivate rabbit melanocytes from whisker follicle outer root sheath (ORS), hereby named rMORS, and compare them to rabbit epidermal rabbit melanocytes (rEMs).ResultsThe rMORS were isolated and cultured by extracting whisker follicles from enzymatically digested skin and submitting them to the air-liquid interface hypoxic conditions. The cells were allowed to migrate from the follicle ORS onto the nylon mesh of Transwell inserts, collected and subcultured in melanocyte culture medium for 11 passages. From early passages on, the rMORS cells displayed typical melanocyte characteristics comparable to those of rEM used as experimental control. Melanocyte features were assessed on morphological level by the means of microscopy, functional and gene expression level. Functionality was qualitatively assessed by the Von Kossa and Nile Blue staining of melanin and by biochemically quantifying melanin content and Tyrosinase activity. Gene expression of neuroectodermal and melanocytic lineage markers NES, PAX3, MITF, TYR, CKIT and PMEL was determined by the means of qRT-PCR. ConclusionsWe concluded that the method of isolating and culturing rMORS efficiently and reproducibly corresponded to the prior art method established in human follicle, yielding melanocytes comparable to the rabbit epidermal melanocytes in all assessed features. This method is not crucial in terms of regenerative therapy, it rather paves way for a non-invasive ex vivo sampling of hair and it may give way to deeper insights into rabbit follicle biology or to postulating useful species-specific in vivo experimental models.