Abstract
Purpose:
A tissue engineering technique for treating corneal endothelial dysfunction has been anticipated as an alternative therapy to conventional corneal transplantation. As cultivation of human corneal endothelial cells (HCECs) is difficult and cell density is rapidly decreased after culture passage, an efficient culture protocol is needed. We previously reported the culture of laminin-511 (LM-511) enhanced HCECs (ARVO, 2014). The purpose of this study was to evaluate the effect of LM511-E8 (the minimal functional form of LM-511) on the culture of HCECs for clinical application.
Methods:
Corneal endothelium was isolated from 3 independent donor corneas, and then seeded on an LM511-E8 (1.0 μg/cm2)-coated culture dish and an FNC Coating Mix® (Athena Enzyme Systems) (a widely used culture substrate)-coated culture dish. Cell density was evaluated by Image J software after reaching confluency at 4 weeks of cultivation. Next, HCECs were seeded on an LM511-E8-coated culture dish or on a non-coated culture dish with culture medium supplemented with LM511-E8 (2.1nM). The numbers of adhered HCECs were then evaluated by CellTiter-Glo® Luminescent Cell Viability Assay after 24 hours. The effect of functional blocking of integrin α3β1 and α6β1 by neutralizing antibody were then evaluated by Cell Titer-Glo® and western blotting.
Results:
The average cell density of the HCEC culture on LM511-E8 was 2397.1±264.8 cells/mm2, while that on FNC Coating Mix® was 1203.3±97.9 cells/mm2 (p<0.01). The cell adhesion of HCECs cultured on the LM511-E8-coated culture dish was significantly enhanced by 1.3 fold compared with that the non-coating dish (p<0.01). In addition, cell adhesion was also enhanced by 1.3 fold when HCECs were seeded with the culture medium supplemented with LM511-E8 (p<0.01). However, the neutralizing antibody of integrin α3β1 and α6β1 suppressed the cell adhesion enhanced by LM511-E8. Phosphorylation of focal adhesion kinase of HCECs was promoted by LM511-E8, while it was suppressed by the neutralizing antibody of integrin α3β1 and α6β1.
Conclusions:
Our findings indicate that LM511-E8 modulates the adhesion of HCECs by activating focal adhesion proteins via binding to integrin α3β1 and α6β1, and enables efficient in vitro expansion of HCECs with maintaining high cell density for regenerative medicine for corneal endothelial dysfunction.