Isolation of rat aortic endothelial cells by primary explant techniques and their phenotypic modulation by defined substrata.
Isolation of rat aortic endothelial cells by primary explant techniques and their phenotypic modulation by defined substrata.
复制标题
DOI：
--
复制
发表时间：
1987-07
期刊：
影响因子：
--
通讯作者：
P. McGuire;R. Orkin
中科院分区：
文献类型：
--
作者：
P. McGuire;R. Orkin
本文描述了一种从小动物（如大鼠）的大血管中分离和培养内皮的有效和可靠的方法，该方法利用内皮细胞与细胞外基质的相互作用来促进组织外植体细胞的生长。该方法也允许用非酶方法从其他血管和组织中分离内皮细胞。将主动脉组织环和打开的部分放置在各种基质上，包括：未经处理的组织培养塑料；纤维连接蛋白、层粘连蛋白、I型胶原蛋白和明胶薄膜；I型胶原蛋白凝胶（Vitrogen, collagen Corporation, Palo Alto, California）， EHS肉瘤基底膜成分凝胶（Matrigel, Collaborative Research Inc., Lexington, Massachusetts），琼脂糖凝胶。使用的培养基为OPTI-MEM或RPMI 1640 (Gibco实验室，Grand Island, New York)，添加3%或20%胎牛血清和50微克/毫升内皮细胞生长补充剂。只有在Vitrogen和Matrigel上的外植体产生了显著的和一致的细胞生长，这发生在外植体开始后不久。在其他基质上的外植体几乎没有生长，即使在培养10天后也是如此。在玻璃体凝胶上，细胞从外植体中生长为单星状和双极细胞，而在基质凝胶上，细胞从外植体边缘生长为链状和片状细胞。细胞从两种类型的凝胶传代到塑料或玻璃表面。从两种凝胶基质中分离出的传代细胞表现出特定的内皮细胞特征，包括融合时的“鹅卵石”形态，血管性血液病因子阳性染色，以及di - i- ac低密度脂蛋白的摄取。由于大鼠和其他小型动物的主动脉内皮细胞对酶处理的分离具有抗性，因此该技术提供了一种获得大量这种细胞类型的简单方法。此外，该方法允许在体外研究内皮细胞的功能，以及细胞外基质在这些过程中可能发挥的作用。
An efficient and reliable procedure for the isolation and culture of endothelium from large vessels of small animals (e.g., rat) is described, which takes advantage of endothelial cell-extracellular matrix interactions to promote the outgrowth of cells from tissue explants. The procedure may also permit the isolation by nonenzymatic means, of endothelial cells from other vessels and tissues. Rings and opened segments of aortic tissue were placed on a variety of substrates including: untreated tissue culture plastic; films of fibronectin, laminin, type I collagen, and gelatin; gels of type I collagen (Vitrogen, Collagen Corporation, Palo Alto, California), of basement membrane components derived from the EHS sarcoma (Matrigel, Collaborative Research Inc., Lexington, Massachusetts), and of agar and agarose. The medium used was OPTI-MEM or RPMI 1640 (Gibco Laboratories, Grand Island, New York) with 3% or 20% fetal calf serum, and 50 micrograms/ml endothelial cell growth supplement. Only explants on Vitrogen and on Matrigel produced a significant and consistent outgrowth of cells and this occurred shortly after the initiation of explants. Virtually no outgrowth occurred from explants on the other substrata, even after 10 days in culture. On Vitrogen gels, the cells emerged from the explants as single stellate and bipolar cells, whereas those on Matrigel grew as chains and sheets from the edges of the explant. Cells were passaged from both types of gels onto plastic or glass surfaces. The passaged cells isolated from both gel matrices exhibited specific endothelial cell characteristics including a "cobbled" morphology at confluence, positive staining for von Willebrand factor, and uptake of Di-I-Ac-low density lipoprotein. Because rat and other small animal aortic endothelial cells are resistant to isolation by enzymatic treatment, this technique provides a simple means to obtain large numbers of this cell type. Further, the method permits study of endothelial cell functions in vitro, and the roles which the extracellular matrix may play in these processes.