Development and validation of a simple method for the extraction of human skin melanocytes

Abstract

Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products. Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as melanocytes, are described in literature. They are usually based on two enzymatic steps, Trypsin in combination with Dispase, in order to separate dermis from epidermis and subsequently to provide a suspension of epidermal cells. The objective of this work was to develop and validate an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. TrypLE™ product was tested on very limited size of human skin, equivalent of multiple 3-mm punch biopsies, and was compared to Trypsin/Dispase enzymes. Functionality of extracted cells was evaluated by analysis of viability, morphology and melanin production. In comparison with Trypsin/Dispase incubation method, the main advantages of TrypLE™ incubation method were the easier of separation between dermis and epidermis and the higher population of melanocytes after extraction. Both protocols preserved morphological and biological characteristics of melanocytes. The minimum size of skin sample that allowed the extraction of functional cells was 6 × 3-mm punch biopsies (e.g., 42 mm²) whatever the method used. In conclusion, this new procedure based on TrypLE™ incubation would be suitable for establishment of optimal primary melanocytes cultures for clinical applications and research.

Keywords: Cell culture; Extraction; Melanocytes; Pigmentation; Skin.

Fig. 1

Schematic diagram for the isolation of melanocytes from small amount of human skin, via the TrypLE™ method. The initial seeding density for initiating the primary culture is 2–4 × 10⁴ cells per 25 cm² tissue culture flask. Generally, one such flask is set up from an initial yield of 4–10 × 10⁵ cells isolated from about 42–70 mm² piece of skin

Fig. 2

Epidermal-dermal separation using TrypLE™ enzyme 1X (a) and Dispase II (b). After TrypLE™ incubation, epidermis (arrow) was completely separated from dermis, whereas after Dispase treatment, it was not perfectly detached

Fig. 3

Skin epithelial cells isolated from multiple 3-mm punch biopsies. Comparison of number of cells using TrypLE™ and Dispase/Trypsin methods. Cell counts were performed at the extraction day and the first passage (P1). Histograms are mean ± SE of five independent experiments with skin from different donors. Whatever the method used, there was no significant difference in cell number from 6 biopsies to 10 biopsies (P > 0.05)

Fig. 4

Morphology of cells extracted from 10 punch biopsies of skin, by TrypLE™ (a, c) and Dispase/Trypsin (b, c), at 2 days (a, b) and 10 days (c, d) after cell isolation. Extracted cells attached to the culture flask within 2 days. After 10 days of incubation, melanocyte-like morphology (dendritic cells) was dominant with TrypLE™ method whereas melanocyte and keratinocyte-like morphologies (dendritic and polygonal cells) were observed with Dispase/Trypsin method. Bar 100 µm

Fig. 5

MITF expression profiles of cells extracted from 10 punch biopsies of skin, by TrypLE™ (a, c) and Dispase/Trypsin (b, c), at 2 days (a, b) and 10 days (c, d) after cell isolation. The percentage of cells expressing MITF (a marker of melanocytes) is indicated in cytometry histograms. With TrypLE™ method, 94–98% of cells were positive whereas with Dispase/Trypsin method, 53–57% of cells were positive

Fig. 6

Number of viable melanocytes extracted by TrypLE and Dispase/Trypsin methods. Melanocytes at passage 2 were deposited in 12-well plates at a density of 6 × 10⁴ cells/well. Cell numbers were measured after 1, 2, 3 and 4 days of culture. Data are expressed as the mean ± SE of 5 independent experiments carried out in duplicate. No difference was found (P > 0.05)

Fig. 7

Intracellular melanin content normalized to the amount of protein. Melanocytes at passage 2 were deposited on 25 cm² flask at a density of 4 × 10⁵ cells/flask. Melanin and protein contents were measured after 5 days of culture. Data are expressed as the mean ± SE of 5 independent experiments carried out in duplicate. No difference was found between TrypLE and Dispase/Trypsin methods (P > 0.05)

Fig. 8

Immunostaining of cells extracted from 10 punch biopsies of skin, by TrypLE™ (a) and Dispase/Trypsin (b), and cultured at passage 2. MITF (in red) was used as a characteristic melanocyte marker while F actin (green) as a cell shape marker. Cells extracted by both methods positively expressed MITF and F actin. Bar 50 µm. (Color figure online)