Abstract
Purpose:
The aim of our ongoing study is to establish a carrier for HCECs for therapeutic purposes. In the present study we investigate the feasibility of using nonsynthetic, biological carrier for cultivated HCECs.
Methods:
Descemet’s membrane with the attached endothelial cells was carefully dissected from human corneas in small strips. One part harvested as non-cultured cells and the other cultivated in corneal endothelial cell growth medium for 6 weeks at 37 degree Celsius with 5% CO2 in a humidified atmosphere and the medium was changed every 2-3 days. The cultivated HCECs were seeded on acellular, nonsynthetic carrier and cultivated for further 3 weeks in corneal endothelial cell expansion medium that was changed every 2-3 days. Cultivated HCECs on nonsynthetic carrier and non-cultured HCEC were comparatively analyzed by qRT-PCR, electron microscopy (EM) and immunohistochemistry.
Results:
In our study the cultivated HCECs seeded on nonsynthetic carrier formed a stable monolayer. Our results show that the cultivated HCECs seeded on nonsynthetic carrier and the non- cultured HCECs are functional and express stem cell markers when analyzed by qRT-PCR. The expression levels of markers associated with neural crest, stem cells and corneal endothelial function (SNAI1, SNAI2, SOX9, NES, ZO-1, CX43, VADC2 and VADC3) are higher in cultivated HCECs seeded on nonsynthetic carrier compared to non-cultured HCECs. The structure of cultivated HCECs seeded on nonsynthetic carrier on transmission EM is very similar compared to ex vivo HCECs.
Conclusions:
The preliminary results show that nonsynthetic carrier used in this study can potentially be used in therapeutic purposes in the future.
Keywords: 481 cornea: endothelium