Univ. Bordeaux, Inserm, BMGIC, UMR1035, Bordeaux, France.
Department of Rheumatology, National Reference Center for Systemic Autoimmune Rare Diseases, Hopital Pellegrin, Bordeaux, France.
Front Immunol. 2020 Oct 7;11:566607. doi: 10.3389/fimmu.2020.566607. eCollection 2020.
Systemic Sclerosis (SSc) is a complex auto-immune connective tissue disease combining inflammatory, vasculopathic and fibrotic manifestations. Skin effectively recapitulates the main pathogenic processes and therefore is a good organ to decipher the disease pathophysiology, which remains unclear. However, culturing primary skin cells is SSc can be a major issue due to small sample size combined to skin fibrosis. Here, we present a protocol allowing to isolate and culture the four main types of skin cells: dermal cells (microvascular dermal endothelial cells-HDMECs-and fibroblasts) and epidermal cells (keratinocytes and melanocytes), from a single 4 mm-punch biopsy, at a low cost. The present protocol has been optimized to fit SSc skin cells particularities. Such technique allows to culture primary cells, crucial to study the disease pathophysiology, as well as to isolate cells in order to perform immediate molecular biology experiments such as single-cell transcriptomic. Cells grown from biopsies are also suitable for various types of experiments such as immunocytochemistry, Western blot, RT-qPCR or functional assays (angiogenesis, migration, etc.). Ultimately, they can be used for experimental 3D cell culture models such as reconstructed skin.
系统性硬化症（SSc）是一种复杂的自身免疫性结缔组织疾病，结合炎症、血管病变和纤维化表现。皮肤有效地概括了主要的发病过程，因此是破译疾病病理生理学的一个很好的器官，而疾病病理生理学仍然不清楚。然而，由于皮肤纤维化和样本量小，培养 SSc 的原代皮肤细胞可能是一个主要问题。在这里，我们提出了一种从单个 4 毫米皮肤活检中分离和培养四种主要皮肤细胞的方案：真皮细胞（微血管真皮内皮细胞-HDMECs-和成纤维细胞）和表皮细胞（角质形成细胞和黑素细胞），成本低。本方案已针对 SSc 皮肤细胞的特点进行了优化。这种技术可以培养原代细胞，这对于研究疾病病理生理学至关重要，还可以分离细胞以进行即时的分子生物学实验，如单细胞转录组学。从活检中生长的细胞也适合各种类型的实验，如免疫细胞化学、Western blot、RT-qPCR 或功能测定（血管生成、迁移等）。最终，它们可用于实验性 3D 细胞培养模型，如重建皮肤。
