Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

Abstract

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full-thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS-P) before transplantation. ESS-P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100-fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS-P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal-epidermal junction of ESS-P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS-P efficiently to restore cutaneous pigmentation and UV photoprotection after full-thickness skin loss conditions.

Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds

“…Primary fibroblasts and keratinocytes were isolated and cultured as previously described [37–39]. Full-thickness skin was cut to narrow strips (2–3 mm wide), which were incubated overnight at 4° C in Dispase (Roche Life Sciences, Indianapolis, IN) to separate dermal and epidermal layers.…”

“…Keratinocytes were inoculated onto the dermal substrates 24 hours later at 1.0 X 10 6 /cm 2 and ESS were transferred to cotton pads supported by perforated stainless steel platforms for incubation at the air-liquid interface (37°C, 5% CO 2 ). Culture medium for ESS consisted of DMEM/F12 (Sigma-Aldrich) supplemented with 1 mM strontium chloride, 0.3% FBS, 1X ITS Supplement (Sigma Aldrich), 10 μg/ml linoleic acid, 0.1 mM AA2P, 20 pM triiodothyronine, 0.5 μg/ml hydrocortisone, 5 ng/ml keratinocyte growth factor (Peprotech, Rocky Hill NJ), 1 ng/ml basic fibroblast growth factor (Peprotech), and 1X PSF [37]. ESS were cultured in vitro for 10 days prior to transplantation to mice.…”

Analysis of chromatin accessibility in human epidermis identifies putative barrier dysfunction-sensing enhancers

“…24 hours later primary human epidermal keratinocytes were added at 1x10 ^6 / cm 2 . The collagen and cells were cultured at the air-liquid interface for 14 days in University of Cincinnati Dermatology Medium-1 (UCDM-1) with daily media changes [ 86 ].…”

Development and use of biomaterials as wound healing therapies

“…Also missing from scar tissue are melanocytes, the cells that produce pigments that give the skin its color. No skin substitutes to date contain these cells, but research in mice using skin substitutes containing melanocytes suggest that skin tone can be regained [54]. Incorporation of adipose-derived stem cells into a recombinant collagen scaffold demonstrated superior wound healing when compared to the recombinant protein scaffold alone [55].…”