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Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro three-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 fundamental in vitro tool 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.

Micropatterning techniques and substrate engineeringare becominguseful tools to investigate several aspects of cell–cell interactionbiology. In this work, we rationally study how different micropatterninggeometries can affect myoblast behavior in the early stage of in vitromyogenesis. Soft hydrogels with physiological elasticmodulus (E= 15 kPa) were micropatterned in parallellanes (100, 300, and 500 μm width) resulting in different localand global myoblast densities. Proliferation and differentiation intomultinucleated myotubes were evaluated for murine and human myoblasts.Wider lanes showed a decrease in murine myoblast proliferation: (69± 8)% in 100 μm wide lanes compared to (39 ± 7)% in500 μm lanes. Conversely, fusion index increased in wider lanes:from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from(15 ± 3)% to (36 ± 2)% for human primary myoblasts, usinga patterning width of 100 and 500 μm, respectively. These resultsare consistent with both computational modeling data and conditionedmedium experiments, which demonstrated that wider lanes favor theaccumulation of endogenous secreted factors. Interestingly, humanprimary myoblast proliferation is not affected by patterning width,which may be because the high serum content of their culture mediumoverrides the effect of secreted factors. These data highlight therole of micropatterning in shaping the cellular niche through secretedfactor accumulation, and are of paramount importance in rationallyunderstanding myogenesis in vitrofor the correctdesign of in vitroskeletal muscle models. [ABSTRACT FROM AUTHOR]