Comparison between Human Corneal Endothelial Cells (HCEC) expansion with corneas preserved in intermedium-term and long-term corneal storage media.

Abstract

Purpose : There is a worldwide shortage of donor corneas for transplantation. Several attempts of HCEC expansion have been proposed, the gold standard for this technique is yet to be established. Donor corneas preserved in CorneaMax are still viable for transplantation after 30 days. We hypothesized that maintaining the donor corneas in this medium prior to expansion would enhance stability, expansion and cell density of cultured HCECs.

Methods : For the CorneaMax group, 18 corneas obtained from the Barcelona Tissue Bank (BST-Barcelona, Spain) were preserved in CorneaMax medium at 31° with a maximum culture time of 30 days and cultured.with dual culture medium, consisting of a M4 medium which promotes proliferation and M5 medium, Rho-kinase (ROCK) inhibitor (Y-27632) was added to the culture medium (10µM). For the Fresh group, 16 corneas were obtained from the São Paulo Hospital Eye Bank (BOHSP, São Paulo, Brazil) were preserved in Eusol for 3-5 day and then cultivated on Dulbecco’s modified Eagles’s (DMEM) culture medium and ROCK inhibitor was also added. All HCECs were cultured for 3-4 weeks, until the first passage. HCEC morphology of the cells was assessed with contrast microscopy; cell density was performed with a Neubauer chamber before the first passage.

Results : After 3-4 weeks it was possible to see the confluence among cell clusters all over the wells. Morphology of the HCEC expanded cells during the culture procedure resembled to normal HCEC until the second passage in both groups. A homonogeneous monolayer, with relatively small cell size and hexagonal shaped cells was identified at the end of every week. After the second passage there were changes in HCEC phenotype and morphology, with loss of hexagonality and homogeneity. There was no statistically significant difference in cell density after expansion between both groups (p>0.05)

Conclusions : Expanding HCEC from corneas preserved in the CorneaMax medium seemed feasible, with maintenance of HCEC morphology and phenotype for at least 3-4 weeks before the first passage.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

HCEC morphology 3 weeks after culture of Eusol preserved corneas. Phase contrast microscopy with HE staining, 200x magnification microphotography .

 

HCEC morphology 3 weeks after culture of CorneaMax preserved corneas. Phase contrast microscopy, 600x magnification microphotography .