Standardization of human corneal endothelial cell isolation and the use of denuded amniotic membrane as a scaffold for human corneal endothelial cells

Abstract

Purpose: To identify the best technique for complete denudation of amniotic membrane. To standardize the isolation of human corneal endothelial cells. To use the denuded amniotic membrane as a scaffold for isolated human corneal endothelial cells.

Methods: Human amniotic membrane denudation was carried out using 1.2 units/ml of Dispase II at 37degree C for 60 minutes followed by mechanical scraping and microscopic examination. This was followed by isolation of corneal endothelial cells using human donor cadaveric eyes unfit for surgical usage. Corneal endothelial and descemet’s membrane sheets were peeled in a manner similar to capsulorrhexis after corneoscleral button excision and enzymatically digested with 2mg/ml of collagenase II solution at 37 degree C and 5 % CO2 for 2 hrs. Pre plating was done onto an uncoated culture ware to separate any attached fibroblasts from the endothelial cells which were then seeded onto denuded amniotic membrane in OptiMEM media supplemented with human epidermal growth factor, fibroblast growth nerve growth factor and bovine pituitary extract. The cells were analyzed microscopically to assess if they maintained their polygonal morphology and subjected to RT-PCR analysis for Keratin 3, neuron specific enolase, Vimentin and collagen VIII mRNA markers.

Results: Microscopic examination of the denuded amniotic membrane showed no epithelial cell remnants and an underlying exposed stromal collagen. Peeling of the corneal endothelial and descemet’s membrane gave sheets of ideal thickness exhibiting typical cobblestone morphology. Enzymatic digestion of the harvested corneal tissue left behind acellular descemet’s sheets with the endothelial cells seen floating individually or in tightly packed clusters with preplating aiding in a more fibroblast free endothelial cell isolation. Microscopic evaluation showed that a few isolated cells managed to scaffold onto the amniotic membrane and that they manage to retain that adhesion during subsequent media replacements.

Conclusions: Usage of Dispase-II for enzymatic digestion of amniotic membrane yielded a complete denudation which acted as a successful scaffold for harvested corneal endothelial cells. Further studies can be done for endothelial cell proliferation serving as an in vitro model for corneal tissue engineering studies.

Keywords: 481 cornea: endothelium • 480 cornea: basic science