Ryan U S
Environ Health Perspect. 1984 Jun;56:103-14. doi: 10.1289/ehp.8456103.
Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.
肺内皮细胞的分离、鉴定和培养方法现已成为常规操作。过去，分离方法使用蛋白水解酶来分离细胞；此后，传统的细胞传代方法使用胰蛋白酶/乙二胺四乙酸（EDTA）混合物。使用蛋白水解酶分离和传代的细胞在该领域的建立以及某些内皮细胞特性的验证中发挥了作用。然而，人们越来越意识到内皮细胞在处理血管活性物质、对激素和其他激动剂作出反应以及与血管壁的其他细胞类型、血细胞和细胞产物进行细胞间相互作用中的作用。因此，对体外培养的细胞产生了新的要求，即这些细胞要保持其体内对应细胞的分化特性。细胞培养中常用的胰蛋白酶和其他蛋白水解酶对内皮细胞表面结构（如酶、受体和连接蛋白）以及细胞外层（如糖萼或“内皮绒毛”）的有害影响，促使人们开发出在分离步骤和后续传代培养过程中都避免使用蛋白水解酶的方法。本章介绍了分离肺内皮细胞的传统方法，但重点强调了使用机械收获和利用微载体进行扩大培养的新方法。这些新方法能够维持细胞的长期大规模培养，使细胞保留完整的表面特性，保持鹅卵石样单层形态和分化的功能特性。因此，分离细胞的鉴定方法也被视为在体外培养期间验证培养物的方法。
