Culture of Human Corneal Endothelial Cells (HCECs) for therapeutic purposes

Abstract

Purpose: : In the present study we characterize selected phenotypic and genotypic parameters of HCECs in vivo and also in cell culture. The aim of our ongoing study is to establish optimal culture conditions in order to increase proliferative capacity and maintain morphology and function of HCECs for therapeutic purposes.

Methods: : Descemet’s membrane with the attached endothelium was carefully dissected from human corneas in small strips. One part harvested as non-cultured cells and the other cultured on Descemet's membrane or in monolayer on a biological substrate. Cultures were incubated for different time points at 37º C with 5% CO2 in a humidified atmosphere and medium was changed every 2-3 days. Cultured and non-cultured HCEC were analyzed by immunohistochemistry, electron microscopy (EM) and qRT-PCR.

Results: : Our results show that non-cultured HCECs are resting cells when analyzed by qRT-PCR and EM. In contrast, HCECs maintained on Descemet's membrane in monolayers on biological substrates and in our optimized culture medium are proliferating cells. When cells cultured on biological substrates reach confluence, the proliferation is reduced and cells show an in vivo endothelial morphology. HCECs cultured on substrate maintain their cell integrity for more than one year in culture. To examine proliferation and cell-cell interaction, Ki67, N- Cadherin and Occludin were analyzed and expression of the junctional markers was similar in all conditions.

Conclusions: : HCECs may be proliferated ex vivo and generate a monolayer of cells. When expanded in an optimized medium and on a biological substrate, the cells present several of the in vivo characteristics of the endothelium. The present culture system is compatible with a long term stability of these characteristics, and could represent an improvement for tissue engineering and treatment of corneal endothelial disorders.

Keywords: cornea: endothelium • cornea: basic science • gene screening