Abstract
Purpose :
In vitro expansion of human corneal endothelial cells (CECs) induces cell enlargement and results in the formation a monolayer with low endothelial cell density (ECD). Our objective was to increase the ECD over the minimum density of 2000 cells/mm2 required by eye banks.
Methods :
CECs were isolated form eye bank corneas and cultured as described in Zhu & Joyce (2004). Different approaches were tested in order to increase ECD: 1) Cells were seeded at increasing cell densities (375 to 12 000 cells/mm2) then cultured for 9 days before analysis. 2) Cells were seeded at 6 000 cells/mm2 then immediately centrifuged (300 g), cultured for 9 days, then analysed. Controls consisted of omitting the centrifugation step. 3) Small cells were selected by centrifugation on a Ficoll density gradient (1.038 to 1.080 g/ml) or cell strainers (10 µm - 15 µm). Cell size was analysed using a Coulter counter. ECD was calculated by counting the number of nucleus (Hoechst staining) using Cellprofiler software (n=4 pictures/condition).
Results :
1) ECD increased with the augmentation of cell seeding density. At 375 cells/mm2, the monolayer reached 500 cells/mm2 while at 6000 cells/cm2, the monolayer was 1400 cells/mm2. However, when seeded at 12000 cells/mm2, CECs aggregated and failed to adhere to the culture plate. 2) Centrifugation slightly increased ECD of cells seeded at 6000 cells/mm2 from 1400 to 1750 cells/mm2, still under the minimal density for grafting. 3) The selection of the smallest cells using the cell strainers resulted in a modest reduction in cell size. Cells that passed through the 10 and 15 µm cell strainers had a mean diameter of 11.5 µm and 12.8 µm respectively (controls = 16.0 µm, n = 1). The diameter of CECs separated on the Ficoll density gradient was similar between fractions, varying from 13.3 to 16.7 µm. The cells in the pellet contained 28 ± 12 % of input cells with a mean diameter of 15.4 ± 0.4 µm (controls = 16.1 ± 0.6 µm, n=6).
Conclusions :
Centrifugation at the time of cell seeding was the most promising technique to increased ECD. A combination of multiple approaches could be necessary to reach an acceptable ECD. Future experiments should also be aimed at maintaining the small cell size during the in vitro expansion process, which would generate engineered endothelia of high ECD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.