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.

CMT-308, a Nonantimicrobial Chemically-Modified Tetracycline, Exhibits Anti-Melanogenic Activity by Suppression of Melanosome Export

“…Pigmentation in cocultures in the absence of an external stimulus is prolonged and can take >10 d [84]; external factors such as UVB irradiation or α-MSH are necessary to stimulate melanogenesis and melanosome transfer in HEMn-DP cocultures [85], although HEMn-DP cells are unresponsive to α-MSH-stimulation in monocultures [86]. Moreover, a recent study demonstrated that a mild stimulus involving a combination of l-tyrosine and NH 4 Cl stimulated pigmentation in HEMn-DP cocultures within a short duration of 5 d [87]. Further studies employing the use of such coculture methods with external stimulators to validate whether CMT-308 could in fact suppress the transfer of melanosome in recipient keratinocytes are warranted.…”

Kynurenine inhibits melanogenesis in human melanocyte‐keratinocyte co‐cultures and in a reconstructed 3D skin model

“…For co‐cultures, keratinocytes (2.5 × 10 5 ) were seeded in medium KBM Gold supplemented with KBM Gold Single Quots, plated on 100 mm Petri plates (Corning ® ) for 24 h; then, melanocytes (2.5 × 10 5 ) were added in 10 ml of the same medium. Pigmentation was induced with tyrosine (0.25 mM) and ammonium chloride (5 mM) after 24 h 21 . Unless otherwise stated, co‐cultures were incubated for 5 days at 37°C in a humidified incubator with a 5% CO 2 atmosphere without further handling.…”

“…After 2 h, keratinocytes (2.5 × 10 5 ) and melanocytes (1.7 × 10 5 ) were seeded on the top of each construct in RAFT: KGM‐Gold Bullet Kit Medium (1:1). After 24 h, the construct was transferred and maintained at an air‐liquid interface for 10 days in a 5% CO 2 incubator with low humidity, and the medium supplemented with tyrosine (0.25 mM) + NH 4 Cl (5 mM) was replaced every 2 days 21 . After stratification, skins were fixed in 10% buffered formalin at 4°C for 12 h, followed by dehydration in solutions containing increasing concentrations of alcohol and xylene for paraffin inclusion.…”