In vitro Isolation and Cell Culture of Vestibular Inner Ear Melanocytes

Abstract

Introduction: Melanocytes of the membranous labyrinth of the inner ear have been described morphologically in various contexts. Nature and functions of these cells are as yet not completely clear, even though several hypotheses exist regarding the same. The limited knowledge is due in part to a lack of methods regarding in vitro cell culture. The aim of this study was to describe conditions for the successful cell culture of vestibular inner ear melanocytes (VIEM), to compare their growth properties with those of epidermal melanocytes, and to characterize them immunohistochemically. Materials and Methods: Membranous labyrinth cells from freshly slaughtered sheep were isolated, and melanocytes and fibroblasts subsequently cultured. In addition, melanocytes from the skin of the same sheep were cultured. Antibodies specific to tyrosinase, tyrosinase-related protein 1 (TRP-1/Mel-5), and melanoma-specific antigen A (Melan A) were used to analyze the cultured cells. Results: The proliferation of VIEM was retarded in comparison to epidermal melanocytes. After 14 days, VIEM began to proliferate for the first time, whereas epidermal melanocytes proliferated already after 7 days. In contrast to epidermal melanocytes, the culturing process of VIEM seemed to be dependent on the presence of fibroblasts, and VIEM often accumulated in the vicinity of fibroblasts forming three-dimensional clusters. Moreover, VIEM showed a higher ratio of highly pigmented cells with a round cell shape and small dendrites in comparison to epidermal melanocytes. Immunohistochemical techniques proved the VIEM to be positive for Melan A, TRP-1 and, in the majority of cases, also for tyrosinase. Conclusion: We successfully cultured melanocytes of the inner ear vestibular labyrinth for the first time and demonstrated melanocytic characteristics of these cells. This accomplishment will provide the opportunity to investigate VIEM in more detail in future experiments.

Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures

“…Variable pigmentation intensities were also previously reported in cultured primary human melanocytes (Hanke et al 2005). The level of pigmentation was not as intense as in the excised tissue, and this can be attributed to the fact that melanoma cell cultures may rapidly alter mRNA and protein expression due to a lack of cell-cell interactions as previously indicated (Hofbauer et al 2001).…”

What are melanocytesreallydoing all day long…?

“…Melanocytes, however, are not restricted to the skin and can be found in the inner ear and the leptomeninges (5,6). The persistence of melanocytes in these non‐sun‐exposed organs throughout evolution suggests that melanocytes might have a role other than protection from sun radiation.…”

Upregulation of the Nr2f1-A830082K12Rik gene pair in murine neural crest cells results in a complex phenotype reminiscent of Waardenburg syndrome type 4

“…The mechanotransduction function of these sensory cells is crucially dependent on the unusual K + -enriched ionic composition of endolymph, on which cochlear melanocytes (also called intermediate cells) of the stria vascularis are known to play a key role (Tachibana, 1999; Zdebik et al, 2009). Although the similar distribution of melanocytes around the utricle and ampullae has been described a long time ago in both human and rodents (Escobar et al, 1995; LaFerriere et al, 1974; Masuda et al, 1995), the function of such vestibular melanocytes remains currently unclear (Barozzi et al, 2015; Masuda et al, 2001; Sanchez Hanke et al, 2005). …”