Abstract
Purpose :
It has been shown that human corneal endothelial cells (HCEnCs) are difficult to grow, especially from old aged donor corneas. Several attempts have been made to increase the propensity of the HCEnCs with only one successful clinical study. It is known that HCEnCs generate their own extracellular matrix (ECM). However, the role of ECM for culturing HCEnCs is unknown, as most of the cultures are dependent on pre-FNC coating. We have therefore attempted to study the importance of ECM coating on the culture capacity of HCEnCs.
Methods :
Descemet membrane-endothelial complex from 13 research grade human donor corneas were peeled and the endothelial cells were isolated using Collagenase Type 1. After discarding the supernatant, the cells were trypsinised for 10 minutes and centrifuged at 1000 rpm for 5 mins. The isolated cells were re-suspended in the culture media supplemented with ROCK inhibitor. Simultaneously, HCEC-12 cell lines were cultured on 13 wells of 0.7cm2 lab-tek chamber slide for 5 days at 37oC with 5%CO2. The cells were sacrificed leaving the secreted ECM from HCEC-12 on the lab-tek slides. ECM was analysed using scanning electron microscope (SEM). Isolated primary HCEnCs from each donor cornea were equally distributed and plated on FNC (control) and ECM simultaneously, and cultured till confluence. Confluence rate, expression of ZO-1 (hexagonality, polymorphism and cell area) and Na/K-ATPase, Annexin V staining (live/apoptotic/necrotic cell) was performed at the end of culture period. Wilcoxon and Student's t-test for paired data was used for statistical analysis.
Results :
ECM showed long collagen fibres on the lab-tek slides. Cells from both the groups reached full confluence with significantly high proliferation rate observed at day 5 and day 7 (p<0.05) in ECM group. Endothelial cell density at confluence was significantly higher (p<0.05) in the ECM group. Cells from ECM group showed significantly (p<0.05) higher number of hexagonal cells, lower number of polymorphic cells and less cell area compared with cells on FNC. Cell viability, necrosis or apoptosis did not differ in either groups.
Conclusions :
Culturing human corneal endothelial cells from old aged donor tissues on the endothelial ECM allows better proliferation and morphological maintenance of the cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.