A 2D and 3D melanogenesis model with human primary cells induced by tyrosine

Abstract

Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH₄Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH₄Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation.

Keywords: co-culture; keratinocyte; melanin; melanocyte; pigmentation.

Figure 1.

The melanogenesis model using melanocytes plus keratinocytes. A. Melanin content (μg/ml) in melanocytes treated or not with tyrosine (0.25 mM) + NH₄Cl (5 mM) in the presence and absence of KGF (20 ng/ml) in 254CF medium for 5 d (n = 6). B. Melanocytes count after 5 d in 254CF, KGM Gold, or 1:1 254CF:KGM Gold medium (n = 3). C. Melanocytes + keratinocytes count and microscopy (40x) in co-culture stimulated with tyrosine (0.25 mM) + NH₄Cl (5 mM) for 5 d (n = 6). D. Melanin content in melanocytes plus keratinocytes cocultures (2.5 × 10⁵ cells each) in presence of tyrosine (0.1 and 0.25 mM) and NH₄Cl (2 and 5 mM) after 5 d and pellets aspect for each condition (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control. ^#P < 0.05 compared with the condition of lowest concentration.

Figure 2.

Gene expression of melanogenesis markers. Gene expression of MITF (A), TYR (B), TYRP1 (C), and Melan-A (D) in melanocytes plus keratinocytes co-cultures (2.5 × 10⁵ cells each) stimulated with tyrosine (0.25 mM) and NH₄Cl (5 mM) for 8 h, 24 h, and 5 d (n = 6). GAPDH was used as the reaction calibrator. Comparative method (2^-ΔΔCT) was used to analyze the levels of mRNA expression. The white bars refer to the control, and the black bars refer to conditions stimulated with tyrosine plus NH₄Cl. ***P < 0.001.

Figure 3.

Protein expression of TYR and melanin content over time in melanocytes plus keratinocytes co-culture (2.5 × 10⁵ cells each) stimulated with tyrosine (0.25 mM) and NH₄Cl (5 mM) for 8, 24, 48 and 72 h. A. Relative melanin content from 8 h to 5 d (n = 6). B. Relative intensity of TYR bands and representative western blot. The endogenous antibody used was anti-vinculin (n = 4). The white bars refer to the control, and the black bars refer to the conditions stimulated with tyrosine plus NH₄Cl. ***P < 0.001.

Figure 4.

3D skin model treated with tyrosine (0.25 mM) + NH₄Cl (5 mM). Pigmentation was analyzed macroscopically and on histological sections stained with Fontana-Masson. The arrows indicate the melanin granules in the basal layer of the epidermis.