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.
Highlights
One of the biggest challenges for the researchers working in the field of dermatology is the construction of a model of human skin
We present a method for the generation of functional full-thickness human skin equivalents which is simple, cost-effective, and reproducible
The authors observed that 0.4 mmol/L genipin is non-toxic for the fibroblasts and greatly reduces the contraction of 3D skin equivalents from 87% to 28% (n = 9, p < 0.05) over a 21-day follow-up period
Summary
One of the biggest challenges for the researchers working in the field of dermatology is the construction of a model of human skin. Most skin models are prepared from human skin biopsies, which may be quite problematic because of the individual differences between the donors. The first noticeable difference between the individuals is skin pigmentation [1] and its elasticity, not to mention structural differences between old and young skin [2]. The main problem of recreating the human skin is the inclusion of anciliary components such as dermal glands, and hair follicles, simple full-thickness skin models having both dermal and epidermal components are available [3]. The first studies on the development of the epidermis met with deep skepticism among clinical practitioners [4]. Some of the studies described only ex vivo reconstructions of the epidermis with keratinocytes and melanocytes [5,6,7]. Because the two-dimensional monocultures don’t give satisfying results [8], the three-dimensional (3D) skin models are becoming more desirable for future therapeutic applications, for example in skin repair or tumor models, and for the testing of medications and cosmetics
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