A.I. Virtanen Institute for molecular sciences, University of Eastern Finland, Yliopistonranta 1E, 70211, Kuopio, Finland.
BMC Mol Cell Biol. 2019 Aug 13;20(1):32. doi: 10.1186/s12860-019-0215-2.
Endothelial cells (ECs) play a key role in tissue homeostasis, in several pathological conditions, and specifically in the control of vascular functions. ECs are frequently used as in vitro model systems for cardiovascular studies and vascular biology. The porcine model is commonly used in human clinical cardiovascular studies. Currently, however, there is no robust protocol for the isolation of porcine heart ECs. We have developed a fast isolation protocol, which is cost effective, takes only 1-2 h, and produces EC purity of over 97%. This protocol is optimized for porcine hearts but can be adapted for use with other large animals.
Heart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig.
The swiftness of the protocol limits changes in the phenotype and RNA expression profile of the cells. Cells were identified as ECs with CD31 (PECAM-1) antibody immunostaining. Functionality of ECs were ensured with in vitro angiogenesis assay. The purity of the ECs was verified by using fluorescence assisted cell sorting (FACS) with the CD31 antibody.
We developed a new, fast, and cost-effective isolation method for pig heart ECs. Successful isolation of pure ECs is a prerequisite for several cardiovascular and vascular biology studies.
内皮细胞（ECs）在组织稳态、多种病理条件下发挥着关键作用，特别是在血管功能的控制方面。ECs 经常被用作心血管研究和血管生物学的体外模型系统。猪模型常用于人类临床心血管研究。然而，目前还没有用于分离猪心脏 ECs 的稳健方案。我们开发了一种快速分离方案，该方案具有成本效益，只需 1-2 小时，并且产生的 EC 纯度超过 97%。该方案针对猪心脏进行了优化，但也可以适应其他大型动物的使用。
心脏用 PBS 冲洗，然后用胶原酶孵育分离内皮细胞，孵育后可立即收集细胞，并在从猪中分离心脏后 1 小时内进行铺板。
该方案的快速性限制了细胞表型和 RNA 表达谱的变化。细胞通过 CD31（PECAM-1）抗体免疫染色鉴定为 ECs。通过体外血管生成试验确保了 ECs 的功能。使用 CD31 抗体的荧光辅助细胞分选（FACS）验证了 ECs 的纯度。
我们开发了一种新的、快速且具有成本效益的猪心脏 ECs 分离方法。成功分离出纯度高的 ECs 是进行多种心血管和血管生物学研究的前提。
