Engelmann K, Böhnke M
Fortschr Ophthalmol. 1989;86(1):72-5.
Enzyme treatment of the corneal endothelium with collagenase was employed in a new method of isolating human corneal endothelial cells (HCEC) from the corneas of adult donors (30-70 years). It was possible to isolate 5-10 X 10(4) endothelial cells from each cornea, and primary cultures were established. During the first 3-5 passages, the growth of contaminating fibroblast like cells was inhibited by the use of an L-valine-free selective culture medium. Proliferation of the cells was stimulated by supplementation of the culture medium with fibroblast growth factor and also by coating the culture dishes with a mixture of laminin and chondroitin sulfate. After 3-6 passages, the cell number had increased 150-200 times. During the passage the cells changed with regard to their typical morphology. This change depended on the amounts of serum and mitogen, respectively, in the culture medium, as well as on the in vitro age of the cells.
采用胶原酶对角膜内皮进行酶处理，这是一种从成年供体（30至70岁）角膜中分离人角膜内皮细胞（HCEC）的新方法。从每个角膜中能够分离出5 - 10×10⁴个内皮细胞，并建立了原代培养。在最初的3 - 5代培养过程中，通过使用无L - 缬氨酸的选择性培养基抑制了污染的成纤维细胞样细胞的生长。通过在培养基中添加成纤维细胞生长因子以及用层粘连蛋白和硫酸软骨素的混合物包被培养皿来刺激细胞增殖。经过3 - 6代培养后，细胞数量增加了150 - 200倍。在传代过程中，细胞的典型形态发生了变化。这种变化分别取决于培养基中血清和促细胞分裂剂的量，以及细胞的体外培养时间。
