Bowman P D, Betz A L, Goldstein G W
In Vitro. 1982 Jul;18(7):626-32. doi: 10.1007/BF02796395.
To provide an in vitro system for studying retinal capillary function we have developed methods for isolation and culture of microvascular endothelial cells from retina. Retinal microvessels were isolated by homogenization of the retina and collection of the microvessels onto nylon mesh. Treatment of the isolated microvessels with collagenase and dispase followed by Percoll gradient centrifugation yielded endothelial cells that were largely free of pericytes. A homogeneous population of endothelial cells that were capable of at least six population doublings was obtained by plating onto a fibronectin coated substrate in plasma derived serum. The endothelial origin of these cells was confirmed by the presence of Factor VIII antigen, angiotensin converting enzyme activity, numerous tight junctions, and a cell surface that did not bind platelets. A second cell type, which did not exhibit these cell markers and which is presumably the intramural pericyte, was obtained when the isolated microvessels were plated on tissue culture grade plastic in fetal bovine serum.
为了提供一个用于研究视网膜毛细血管功能的体外系统，我们开发了从视网膜分离和培养微血管内皮细胞的方法。通过将视网膜匀浆并将微血管收集到尼龙网上来分离视网膜微血管。用胶原酶和分散酶处理分离的微血管，然后进行Percoll梯度离心，得到的内皮细胞基本不含周细胞。通过接种到含有血浆来源血清的纤连蛋白包被的底物上，获得了能够至少进行六次群体倍增的均匀内皮细胞群体。这些细胞的内皮来源通过以下特征得以证实：存在因子VIII抗原、血管紧张素转换酶活性、大量紧密连接以及不结合血小板的细胞表面。当将分离的微血管接种到含有胎牛血清的组织培养级塑料上时，获得了第二种细胞类型，该细胞类型不表现出这些细胞标志物，推测为壁内周细胞。
