Volume 4, 2021
|Number of page(s)||21|
|Section||Life Sciences - Medicine|
|Published online||15 February 2021|
Fundamental in vitro 3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments
Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, 10129 Turin, Italy
2 Universitätsklinikum Knappschaftskrankenhaus Bochum, Department of Surgery, Hospital of the RUHR-University, 44892 Bochum, Germany
3 Department of Experimental Surgery, Centre for Clinical Research, RUHR-University, 44801 Bochum, Germany
4 Institute of Pathology, RUHR University, 44789 Bochum, Germany
5 Interuniversity Centre for the Promotion of the 3Rs Principles in Teaching and Research, Centro 3R, 56122 Pisa, Italy
Accepted: 3 February 2021
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.
Key words: 3D / Actin / Animal model / Biocompatibility / Biomaterials / Collagen / Culture / Dermal / Dermatoblasts / Development / ECM / Electron microscopy / Extracellular matrix / Effectiveness / Engineering / Epidermal / Equivalent / Fibroblasts / Flg / Human skin equivalent / HSE / Inv / iPSC / Keratinocytes / KGF / Lam / Layer / Lor / Maturation / Medium / Microenvironment / Organoid / NHDF / NHEK / Pharmacotoxicity / Proliferation / Regenerative medicine / SE / Signaling / TEM / Tissue engineering / Wound healing
© A. Idrees et al., Published by EDP Sciences, 2021
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.
Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.
Initial download of the metrics may take a while.