A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture

Abstract

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 μM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80–200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 μM and below was not cytotoxic to the keratinocytes, and 150 μM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

Effect of Different Wavelengths of Laser Irradiation on the Skin Cells

“…These results were confirmed by Penna et al [ 32 ]. Taken together, these confirm the hypothesis that in some cases reconstructed skin tissues may be a good substitute for conducting effective skin tests, especially when the morphology of natural skin is preserved, [ 33 ]. It has been shown that exposure to UV light might regulate epidermal keratinocytes autophagy through selective down-regulation of key autophagy-related genes including ULK1, ATG3, ATG5, and ATG7 [ 34 ].…”

A review of diabetic wound models—Novel insights into diabetic foot ulcer

“…There is an emerging consensus that 3D skin models are now favored over both animal models and 2D cellular models due to their architecture that physiologically resemble the native human skin (Klicks et al, 2017;Szymański et al, 2020). The biggest advantage of 3D skin models over 2D models is their 3D assembly that reconstructs the physiological features found in vivo (Klicks et al, 2017).…”

A Journey on the Skin Microbiome: Pitfalls and Opportunities

“…The development of advanced 3D-culture methods, with well-differentiated cell types (e.g., keratinocytes, fibroblasts, and immune cells) representing an artificial replicate of multi-layered human skin, has been particularly relevant for the topics treated in this work, as it allowed for the study of interactions among commensal, opportunistic, and pathogenic bacteria, and the investigation of critical processes such as wound healing and biofilm formation [ 166 , 167 ].…”