Assessment of Attachment Factors for Primary Cultured Human Corneal Endothelial Cells

Purpose: To assess the ability of various attachment factors to promote attachment of primary cultured human corneal endothelial cells. Materials and Methods: Primary cultured human corneal endothelial cells (HCEC) were incubated for 2 hours in 24-well plates. Wells had been precoated with commercially available cell attachment improvement media (FNC coating mix), human collagen 1, human fibronectin, fibronectin/collagen 1, or poly-D-lysine. Ratios of cell count before and after rinsing with culture medium and ratios of cells showing morphological signs of spreading to total cells were calculated to measure effectiveness of attachment factors. Results: Incubation of HCEC for 2 hours in wells without pre-coating of attachment factors led to a rate of 41 +/- 16% (mean +/- SD) of cells showing signs of spreading. FNC coating Mix, collagen 1, and fibronectin/collagen I increased significantly the percentage of cells showing morphological features of attachment at 2 hours. Total cell loss was highest with poly-d-lysine and no pretreatment with attachment factor. Without the use of any attachment factor, 67 +/- 19% of cells remained after rinsing. The lowest cell loss was observed with FNC coating mix where 108 +/- 5% of cells remained after rinsing. Conclusion: Collagen I, collagen I/fibronectin, and FNC coating mix significantly enhance the spreading of human corneal endothelial cells to tissue culture plates after 2 hours. FNC coating mix significantly reduces cell loss due to rinsing. Without the use of any attachment factor, 67% of the cells remained in situ after rinsing.