Department of Molecular Biology, Institute of Genetics and Animal Biotechnology, Polish Academy of Science, Postępu 36A, 05-552 Magdalenka, Poland.
Department of Regenerative Medicine and Cell Biology, Military Institute of Hygiene and Epidemiology Kozielska 4, 01-163 Warsaw, Poland.
Int J Mol Sci. 2020 Jun 30;21(13):4644. doi: 10.3390/ijms21134644.
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.
对于从事皮肤科研究的研究人员来说，一个重要的问题是制备人皮肤等效物（HSE）。在这里，我们描述了一种从市售细胞系：角质形成细胞、成纤维细胞和黑素细胞制备皮肤模型的简单可靠的方案。重要的是，在我们的 3D 模型中，角质形成细胞是多样化的，这使得该模型更接近天然皮肤。为了生产 HSE，我们使用了市售的原发性 PCS-200-010、PCS-201-010、PCS-200-013 和永生化的 CRL-4048 和 CRL-4001 细胞系。我们使用了必需的用于胶原蛋白交联的京尼平，并研究了其对角质形成细胞和成纤维细胞的细胞毒性。在构建物培养的第 12 天，添加 20 μM 京尼平可将胶原的收缩率从 59%降低至 24%。浓度较高（80-200μM）的京尼平可使收缩率平均降低 14%。浓度为 10μM 及以下的京尼平对角质形成细胞无细胞毒性，150μM 及以下的对成纤维细胞无细胞毒性。苏木精和伊红染色显示，HSE 的形态与天然人皮肤相同。构建物的免疫组织化学染色显示，皮肤层中有波形蛋白阳性的成纤维细胞，而黑素细胞位于表皮和基底层。我们观察到，构建物的分化时间越长，GM-CSF、IL-10、IL-15、IL-1α、IL-6、IL-7、IL-8 和 MCP-1 的分泌量越高。我们还观察到，分化时间越长，所有分析物的分泌越稳定，这反映在变异系数上。我们在这里描述了一种简单、可靠且具有成本效益的全厚人皮肤等效物的生产方法，可用于研究和工业。随着全球减少动物在研究和测试中的使用的趋势，我们的 HSE 可以成为一种有用的测试工具和替代研究模型。
