Quantification of Melanosome Transfer Using Immunofluorescence Microscopy and Automated Image Analysis.

The study of skin pigmentation requires determining the rate of melanin production in melanocytes and quantifying the rate of melanosome transfer to keratinocytes. Here, we describe a method to quantify melanosome transfer using immunofluorescence microscopy coupled with automated image analysis of in vitro human melanocytes and keratinocytes in co-culture. In this method, the number of melanin capped keratinocyte nuclei is quantified.

Inhibition of melanin synthesis and melanosome transfer by chitosan biomaterials.

Decreasing skin pigmentation is desirable for various medical or cosmetic conditions. Although numerous pharmaceutical agents are currently available, their depigmentation effects are still not satisfactory. In this study, we investigated the effects of chitosan, a natural marine product, on melanin synthesis and melanosome transfer. Treating B16F10 melanoma cells caused the inhibitory effect of chitosan on melanogenesis to be more prominent under α-melanocyte-stimulating hormone (α-MSH) stimulation. Chitosan samples of different molecular weights inhibited melanogenesis to a comparable extent, whereas increasing the deacetylation of chitosan enhanced its depigmentation effects. Chitosan was found to effectively reduce basal or α-MSH-stimulated melanogenesis by suppressing the expression of melanogenic-related proteins (microphthalmia transcription factor, tyrosinase, and tyrosinase-related protein-1 and protein-2) as well as inhibiting tyrosinase activity. Moreover, the inhibitory effect of chitosan on melanogenesis in human melanocytes was confirmed. A transwell coculture system using permeable inserts was designed to allow the contact of human melanocytes and human HaCaT keratinocytes through the tiny holes on the membrane. When chitosan was added to this melanocyte-keratinocyte coculture system, we observed decreased melanosome release from melanocytes. Reduced melanosome uptake by keratinocytes was also observed, and was probably mediated by inhibiting protease-activated receptor 2 expression. Many skin-whitening agents can modulate the process of melanogenesis, but few have been shown to inhibit the melanosome transfer and uptake process. We demonstrated that chitosan exhibits a robust effect on depigmentation by inhibiting melanogenesis as well as melanosome transfer and uptake. Therefore, chitosan represents a potential therapeutic agent for hyperpigmentation disorders.