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.

Keywords: 3D model; commercially available cell lines; cytokines; differentiated keratinocytes; skin equivalent.

Figure 1

Effect of genipin on keratinocytes and fibroblasts. The cytotoxic effect was measured by MTT test according to ISO 10993-5 protocol.

Figure 2

Human skin equivalent generated using PCS-200-010 and PCS-201-010 cell lines and differentiated for 14 days. Sections of the formalin-fixed, and paraffin-embedded, 3-dimensional (3D) skin models. (A) Hematoxylin and Eosin staining. HSEs consist of well-differentiated epidermis on top of a fibroblast-populated dermis. (B) Fluorescent immunostaining. Green—Vimentin (Biolegend 677807), Red—Cytokeratin 1 (LSBio LS-C180221), White—Cytokeratin 14 (Abcam ab77684), Blue—Cell nucleus (DAPI). Cytokeratin 14 is strongly expressed in keratinocytes forming a basal layer with lower expression in the keratinocytes of the more apical layers, stratum spinosum, and stratum granulosum. Cytokeratin 1 expression is detected in the fully differentiated epidermis.

Figure 3

Human skin equivalent generated using PCS-200-010, PCS-200-013, and PCS-201-010 cell lines and differentiated for 14 days. Formalin-fixed, paraffin-embedded 3-dimensional (3D) skin models. Fluorescent immunostaining. Green—Cytokeratin 14 (Abcam ab77684). Melanocytes are seen as the dark color of the entire surface of the epidermis (green) layer. (A) brightfield, (B) Cytokeratin 14 (green), (C) merge.

Figure 4

Comparison of GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-10, IL-12p70, IL-15, IL-1α, IL-6, IL-7, IL-8, (interleukins), MCP-1 (monocyte chemoattractant protein-1), TNFα (tumor necrosis factor α), and VEGF (vascular endothelial growth factor) levels in 24 h culture supernatant between 12 and 14 days differentiated HSEs. Results presented as Mean ± SEM [pg/mL/cm²]. The variability between the samples is measured by the coefficient of variation.