Fine Structure and Collagen Synthesis Activity of Monolayer Cultures of Rabbit Corneal Endothelium

The corneal endothelium is a single layer of polygonal cells covering the posterior surface of the cornea. The apical free surface of these cells faces the anterior chamber and the basal surface rests on an unusually thick basement lamina, Descemet's membrane. This membrane is collage-nous (1, 6, 11, 12) and is believed to be secreted by the endothelial cells (6, 13, 18). These cells form the primary barrier across which metabolic sub-strates enter and waste products leave the cornea (19, 23), and are believed to be responsible for the active transport processes by which the adult cornea maintains its normal hydration state (8, 14, 15). Recently, a technique for mass culture of pure rabbit endothelial cells has been described (16). The present study was undertaken to ascertain by electron microscopy if these are pure endothelial cultures and to describe their fine structure. Further studies were undertaken to correlate biochemical evidence of collagen synthesis and fine structure in sister cultures. Corneas were excised from adult male albino rabbits sacrificed with 5 ml intravenous pentobarbital. Under sterile conditions in a complete culture medium (20), Descemet's membrane and endothelium were dissected from the underlying stroma of the corneas of 96 rabbits (21). Descemet's membrane-endothelium layers from six pairs of eyes were explanted into each of 16 30-ml Falcon tissue culture flasks) containing 3 ml of culture medium per flask. Cultures were incubated at 35°C in a mixture of 5% CO,-95% air. At the same time Descemet's membrane-endothelium layers from three pairs of eyes were fixed immediately after dissection in 2% glutaraldehyde in Sorensen's phosphate buffer, post-fixed with 2% OsO~ in Sorensen's phosphate buffer, and embedded in Epon for light and electron microscope evaluation of the possible contamination of the dissected layer with stromal fibroblasts. When initial outgrowth (Fig. 1) from the explant was about 25 cell diameters, approximately 2-3 wk, the culture flasks were rinsed with 0.2% EDTA in sterile buffered saline and suspended by incubation for I h with 0.4 ml of 0.5% trypsin plus 0.4% EDTA in buffered saline. The suspended cells from each initial culture were transferred to a new flask with 3 ml of complete medium and the remnants of the original Descemet's membrane were discarded. 7 10 days after trypsinization, a conflu-ent monolayer of endothelium (Fig. 2) was obtained which could be used for experimental or analytical purposes or subculture. Confluent cultures intended for chemical analysis of