Abstract
Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmeticsin vitrotesting and for the development of complex skin wound therapeutics.In vitrothree-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamentalin vitrotool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection.
Highlights
Human skin constructs (HSCs) are three-dimensional (3D) in vitro tissue-engineered human skin, which may find application in regenerative medicine and as in vitro models for fundamental research and industrial uses, for example for cytotoxicity analysis, and testing new therapeutic approaches
normal human dermal fibroblasts (NHDFs) and normal human epidermal keratinocytes (NHEKs) were maintained in CnT-Prime fibroblast medium (CnT-PR-F, CELLnTECH) and CnTPrime epithelial culture medium (CnT-PR, CELLnTECH) respectively under the physiological culture conditions (37 °C, 5% CO2), and sub-cultured using DetachKit-PromoCell HEPES BSS (2-[4-(2-hydroxyethyl)piperazine-1-yl] ethanesulfonic acid buffered saline solution); 0.04% Trypsin/0.03% EDTA; and TNS (Trypsin Neutralizing Solution) containing 0.05% trypsin inhibitor from soybean/0.1% bovine serum albumin
Different media were used to optimize the development of human skin equivalent (HSE): i) CnT-PR-F (CnT-Prime fibroblast medium, CELLnTECH) named as Medium A; ii) CnT-PR (CnTPrime epithelial culture medium, CELLnTECH) named as Medium B; iii) CnT-PR-3D (CnT-Prime-3D barrier medium, CELLnTECH) named as Medium C; iv) CnTPR-FTAL (CnT-Prime-full thickness airlift medium, CELLnTECH); v) KGM2 (Keratinocyte Growth Medium 2, i.e. Basal medium supplemented with Bovine Pituitary Extract 0.004 mL/mL, human EGF 0.015 ng/mL, human Insulin 5 lg/mL, Hydrocortisone 0.33 lg/mL, Epinephrine 0.39 lg/mL, Transferrin 10 lg/mL, CaCl2 0.06 mM; PromoCell); vi) High calcium KGM2 (KGM2 + 1.2 mM CaCl2, Sigma); vii) FGM2 is a low serum media (2% v/v) that contains: 0.02 mL/mL foetal calf serum (FCS), 1 ng/mL basic fibroblasts growth factor, 5 lg/mL Insulin (PromoCell)
Summary
Human skin constructs (HSCs) are three-dimensional (3D) in vitro tissue-engineered human skin, which may find application in regenerative medicine and as in vitro models for fundamental research and industrial uses, for example for cytotoxicity analysis, and testing new therapeutic approaches. In advanced 3D in vitro systems, keratinocytes are able to develop well-ordered epidermis and basement membrane features [1,2,3], to closely recapitulate the characteristics of native human skin (NHS). Fibroblasts have an important role in enhancing the keratinocytes resistance towards toxic compounds [4]. This suggests that a single compartment skin model may not be predictive for in vitro toxicological studies. As HSCs are physiologically more similar to the NHS, they offer advanced 3D testing systems as an alternative to animals for drugs and cosmetics evaluation, irritancy and toxicity testing, wound healing studies, cancer research, skin infection studies, and research on other skin diseases [5,6,7]. Mouse skin is effectively able to regenerate after wounding, while human skin damage may lead to hypertrophic scar (keloids) formation [9]
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