歯学ジャーナル
オープンアクセス

概要

Standardization of Human Corneal Endothelial Cell Isolation and the Use of Denuded Human Amniotic Membrane as a Scaffold for Human Corneal Endothelial cells

Kalpana Suresh, Alan Mathew Punnoose, Sarah Kuruvilla and Tanvi Khanna

Objectives: To standardize the isolation of human corneal endothelial cells (HCECs) and to use the denuded human amniotic membrane (HAM) as a scaffold for isolated HCECs.
Methods: Human amniotic membrane was denuded using 1.2 units/ml of Dispase II at 37°C for 60 minutes followed by mechanical scraping. Corneal endothelial and Descemet’s membrane sheets were peeled from human donor cadaveric eyes unfit for surgical use and enzymatically digested with 2 mg/ml of collagenase II solution at 37°C and 5% CO2 for 2 hrs. Isolated cells were resuspended in culture medium with supplements and plated onto uncoated cultureware for four hours to eliminate fibroblasts which adhere more rapidly than endothelial cells. After preplating, the non-adherent cells were seeded onto gelatin coated dishes or onto denuded amniotic membrane in OptiMEM media supplemented with growth factors. The cells were analyzed by microscopy for adherence and polygonal morphology.
Results: Microscopy of the denuded amniotic membrane showed no epithelial cell remnants. Enzymatic digestion of cornea left behind acellular Descemet’s sheets with the endothelial cells floating individually or in clusters with preplating aiding in a more fibroblast free endothelial cell isolation. Few isolated cells managed to scaffold onto the amniotic membrane and retain that adhesion during subsequent media replacements.
Conclusion: Prolonged Treatment of HAM using the mild enzyme Dispase-II results in denuding the membrane, which serves as a successful scaffold for harvested corneal endothelial cells. This approach may be further explored as a cost effective alternative for endothelial cell proliferation and as an in vitro model for corneal tissue engineering studies.