Isolation and Culture of Amelanotic Melanocytes from Human Hair Follicles

Abstract

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.

Stem Cell Factor Combined with Matrix Proteins Regulates the Attachment and Migration of Melanocyte Precursors of Human Hair Follicles <i>in Vitro</i>

“…MPs were cultured as described previously but with improvements to the culture medium. 4,6) Normal human scalp skin (approximately 15-20 cm 2 ) was obtained from two cadaveric donors (men aged 49 and 32 years) within 1 h of death and was immediately immersed in MCDB-153 medium (Sigma) supplemented with 1000 U/mL penicillin and 1000 µg/ mL streptomycin. Before the scalp samples were collected, the two scalp donors were asked to sign informed consent forms.…”

“…[3][4][5][6] When cells migrate, they synchronously attach and detach to various matrix proteins. During the repigmentation of vitiliginous skin, it is thought that MPs become activated, migrate along the basement membrane under the ORS into depigmented skin and then differentiate into mature melanocytes.…”

“…4) Here, we will investigate the effects of SCF combined with matrix proteins FN, LN and CIV on the attachment and chemotaxis of MPs. In addition, the changes in the MPs cytoskeleton will also be analyzed to clarify the mechanism of how these proteins affect MPs.…”

“…2) Although this phenomenon has been clinically well supported and MPs have been cultured successfully in vitro, little previous work has been performed to evaluate the migration capability of MPs. [3][4][5][6] When cells migrate, they synchronously attach and detach to various matrix proteins. During the repigmentation of vitiliginous skin, it is thought that MPs become activated, migrate along the basement membrane under the ORS into depigmented skin and then differentiate into mature melanocytes.…”

A human serum‐enriched medium formulation supports high viability and marker expression in primary melanocyte cultures from the outer root sheath and epidermis

“…[1][2][3][4][5][6][7][8][9][10][11] However, the emerging demand for cell therapies for skin regeneration and repigmentation requires conformity with medicinal product-grade melanocytes.…”