The effect of air, SF6 and C3F8 on immortalized human corneal endothelial cells

Abstract

Purpose: While anterior chamber air bubbles aid attachment during posterior lamellar surgery only for few days, these periods can be prolonged with gases in non-expanding concentrations. To test the effects of different gas compositions on immortalized human corneal endothelial cells (HCEC-12), we utilized Transwell inserts with semipermeable membranes as an artificial anterior chamber model. Methods: Human corneal endothelial cells (HCEC-12) were cultured on Transwell inserts for 24 hr, then flipped, burdened and sunk with titanium rings in medium (M1), as well as filled with 2 ml of air (A), 20% sulphur hexafluoride (SF6) (S), or 12% C3F8 (C). After gas exposition for 24, 48 and 120 hr, cells were evaluated by live/dead staining, cell viability assay and Ki67 immunohistochemistry. Results: Proliferation was significantly reduced (Ki67-positive fraction; M1, 14.8 ± 2.0%; A, 7.9 ± 1.4%; S, 8.1 ± 1.3%; C, 9.9 ± 2.3%; p-values; A, S, C versus M1 < 0.01), the total cell number decreased and the percentage of dead cells increased under gas exposition, independently of the type of gas (120 hr cell count/2.25 cm2: M1 = 660.8 ± 57.0 cells; A = 125.5 ± 17.4 cells, S = 123.5 ± 17.0 cells, C = 118.8 ± 16.6 cells; p-value: M versus A/S/C < 0.001; 120 hr dead cells: M = 2.6 ± 1.0%, A = 8.4 ± 2.7%, S = 9.5 ± 3.2%, C = 11.3 ± 3.1%; p-value: M1 versus A/S/C < 0.01). Medium (M1)-control also proved significantly higher cell viability values in comparison with the gases, which did not differ significantly among them (120 hr luminescence: M1 = 1752.2 ± 91.4, A = 433.0 ± 30.3, S = 507.8 ± 23.3, C = 523.8 ± 20.3; p-value: M1 versus A/S/C < 0.01). Conclusions: Gas exposition led to a reduction in proliferation and an increase in cell death in HCEC-12, independently of the gas composition.