Cardiovascular Research Center, The Icahn School of Medicine at Mount Sinai;
Cardiovascular Research Center, The Icahn School of Medicine at Mount Sinai; Diabetes, Obesity and Metabolism Institute, Department of Medicine, The Icahn School of Medicine at Mount Sinai; Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai.
J Vis Exp. 2021 May 10(171). doi: 10.3791/62419.
The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.
主动脉瓣细胞的钙化是主动脉瓣狭窄的标志，并与瓣叶纤维化有关。 瓣膜间质细胞（VIC）通过激活其向成骨样细胞的去分化程序，在主动脉瓣狭窄的钙化过程中发挥重要作用。 小鼠 VIC 是阐明驱动主动脉瓣细胞矿化的信号通路的良好体外工具。 本文作者成功使用了所描述的方法来获得新鲜分离的细胞。 采用 1mg/mL 和 4.5mg/mL 的两步胶原酶法进行处理。 第一步对于去除内皮细胞层并避免任何污染至关重要。 第二步胶原酶孵育有助于 VIC 从组织迁移到平板上。 此外，还讨论了用于分离的小鼠瓣膜细胞表型特征鉴定的免疫荧光染色程序。 此外，通过使用钙试剂测量程序和茜素红染色，在体外进行钙化测定。 体外抑制细胞矿化的新药理靶点的测试，使用小鼠瓣膜细胞原代培养是必不可少的。
