Challenges in improving cell density of tissue-engineered corneal endothelium

摘要

Introduction: Comeal endothelial dysfunctions cause blindness and are the main indications for allogeneic comeal transplantation. Limited availability of donor corneas has motivated the development of tissue-engineered posterior corneas from cultured corneal endothelial cells (CECs). However, expansion of CECs in vitro 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/mm~2 required by eye banks. Materiel and Method: CECs were isolated form eye bank corneas and cultured as described in Zhu & Joyce. Different approaches were tested in order to increase ECD: 1) Cells were seeded at increasing cell densities (375 to 12 000 cells/mm~2) 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 (1.059 g/ml) or cell strainers (10 μm - 15 μm). Cell size was analysed using a Coulter counter. 4) Cells were seeded at 1 000 cells/mm2 on a tissue engineered stromal substitute. After 2 weeks of culture, the engineered tissue was released from its anchor, allowing for contraction of the tissue. Macroscopic pictures were used to calculate de % of contraction. Calculation of ECD was either performed by counting the number of nucleus (Hoechst staining) using Cellproftler software (n=4 pictures/condition) or by morphometric analysis of alizarin red-stained endothelia. Results and Discussion: 1) ECD increased with the augmentation of cell seeding density. At 375 cells/mm~2, the monolayer reached 500 cells/mm2 while at 6000 cells/mm~2, the monolayer was 1400 cells/mm~2. However, when seeded at 12000 cells/mm~2, 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/mm~2, still under the minimal density for grafting. 3) The two methods tested to select the smallest cells failed to separate the small from the bigger cells. The final size of the cells separated by the cell strainers were 11.5 μm and 12.8 μm for the 10 and 15 μm cell strainer respectively (controls = 16.0 μm), while CEC of high and low density had respectively 14.8 μm and 16.5 μm in diameter. 4) The presence of endothelial cells reduced the tissue sheet contraction from 40% without CECs to 30% with CECs. The monolayers of CECs did not form proper cell-cell junction preventing the identification of cell borders following alizarin red staining and therefore morphometric analysis. Conclusion: 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.