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

Formulating clinically relevant melanocyte cultivation media that maintain the balance between proliferation and maturation to functional melanocytes is a major experimental and regulatory challenge. Within the translation of human melanocytes from the outer root sheath of human hair follicle (HUMORS), we developed a melanocyte medium free of chemical mitogens, chemical melanogenesis enhancers and bovine products, enabling proliferation as well as melanotic differentiation. The formulation involved the replacement of bovine pituitary extract (BPE) and bovine serum (FBS) with human serum (HS) combined with ascorbic acid, CaCl2 , epinephrine, L-glutamine, insulin and fibroblast growth factor. The cultivation efficiency was characterized through proliferation and exertion of melanotic phenotype, gene and protein expression of melanotic markers and melanin content. Having established an application-directed BPE-free formulation, we then re-formulated a research-grade medium with BPE for purposes of even more effective in vitro cultivation, adjusted to specific requirements of HUMORS and normal human epidermal melanocytes (NHEM).

The role of ADGRE5/CD97 in human retinal pigment epithelial cell growth and survival.

Cell surface molecules of retinal pigment epithelial (RPE) cells participate in the pathogenesis of retinal diseases. In an attempt to identify cell surface proteins that play a role in RPE cell-cell interactions, we have considered studying the expression, regulation, and signaling of ADGRE5/CD97, an adhesion G protein-coupled receptor family member, based on its known adhesive function in other cell types such as leukocytes. We showed that RPE cells express three isoforms of CD97 and identified inflammation-related cytokines as important mediators regulating CD97 expression. Whereas TNF-α and IFN-γ upregulated CD97, TGF-ß decreased CD97 expression. Due to interaction with CD55, ARPE-19 cells firmly adhered to monocytes and T lymphocytes when overexpressing CD97, suggesting a role for CD97 in controlling leukocyte infiltration across the RPE-based blood-retinal barrier. CD97-mediated signaling acted synergistically with PDGF-BB and IFN-γ to regulate cell growth and survival, ensuring a cellular balance under inflammatory conditions. These findings suggest that CD97 on RPE cells serves to control leukocyte activation and trafficking in uveoretinal inflammation while at the same time regulating second messenger-mediated gene expression, cell growth, and survival.

Polycaprolactone fiber meshes provide a 3D environment suitable for cultivation and differentiation of melanocytes from the outer root sheath of hair follicle.

Melanocytes differentiated from the stem cells of human hair follicle outer root sheath (ORS) have the potential for developing non-invasive treatments for skin disorders out of a minimal sample: of hair root. With a robust procedure for melanocyte cultivation from the ORS of human hair follicle at hand, this study focused on the identification of a suitable biocompatible, biodegradable carrier as the next step toward their clinical implementation. Polycaprolactone (PCL) is a known biocompatible material used for a number of medical devices. In this study, we have populated electrospun PCL fiber meshes with normal human epidermal melanocytes (NHEM) as well as with hair-follicle-derived human melanocytes from the outer root sheath (HUMORS) and tested their functionality in vitro. PCL fiber meshes evidently provided a niche for melanocytes and supported their melanotic properties. The cells were tested for gene expression of PAX3, PMEL, TYR and MITF, as well as for proliferation, expression of melanocyte marker proteins tyrosinase and glycoprotein 100 (gp100), L-DOPA-tautomerase enzymatic activity and melanin content. Reduced mitochondrial activity and PAX-3 gene expression indicated that the three-dimensional PCL scaffold supported differentiation rather than proliferation of melanocytes. The monitored melanotic features of both the NHEM and HUMORS cultivated on PCL scaffolds significantly exceeded those of two-dimensional adherent cultures.

The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts.

Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices were homogenized and extracted by high detergent concentrations westernblot analyses revealed cytosolic and cytosceleton proteins like GAPDH and smooth muscle actin which were depleted to 4.1 ± 1.9% and 13.8 ± 0.55%, resp. Also putative immunogenic MHC I complexes and the alpha-Gal epitop were reduced to only 14.8 ± 1.2% and 15.1 ± 2.05%. Mass spectrometry of matrix extracts identified 306 proteins belonging to cytosol, organelles, nucleus and cell membrane. Moreover, aqueous matrix extracts evoked a pronounced antibody formation when administered in mice and thus display high immunogenic potential. Our data indicate that an established decellularization protocol which results in acellular matrices evaluated by low DNA content reduces but not eliminates cellular components which may contribute to its immunogenic potential in vivo.