A robust approach to assess cellular yield from simple non-cultured endothelial cells (SNEC) harvesting technique for corneal endothelial cell-injection therapy

Abstract

Purpose : The aim of the study is to establish a robust approach in determining the corneal endothelial cell yield following a recently described simple non-cultured endothelial cells (SNEC) harvesting approach for cell-injection therapy.

Methods : Pairs of donor corneas deemed unsuitable for transplantation were procured for this study. Optimization studies were initially performed using primary human corneal endothelial cells (CECs) isolated and cultured to the second or third passage using a dual media approach. Propagated CECs (n=3) were dissociated where 2 X 10⁶ cells were obtained, before being serially diluted into 4 concentrations, where a manual cell count together with two automated cell counts using 2 different cell counters (BioRad TC-20 & NucleoCounter NC-250) were compared. Remaining cells from each dilution were seeded, stabilized (48hrs) and assessed morphologically. Subsequent validation was performed on CECs isolated from independent donor pairs (n=3) via the SNEC harvesting approach as previously described. The isolated CECs were strain-filtered, spun down and resuspended in 1mL where 50uL were used for cell counting and the rest seeded onto FNC-coated WillCo-Dish, and stabilized for 48hrs before morphometric analysis were performed.

Results : Initial optimization experiments using cultivated primary CECs showed that the consistency of the cell counts by the two cell counters were comparable to that of manual cell counting across the various dilutions. These results could be corroborated by the respective morphological assessments. More importantly, these automated cell counts were accurate down to the range of approximately 1 x 10⁵ cells, which was the estimated cell yield from the SNEC harvesting technique from a single donor cornea. Subsequent validation studies using 3 sets of paired donor corneas, and 1 single donor corneas showed consistency in overall cell yield for the paired corneas of 2.7±0.4 X 10⁵, 3.1±0.1 X 10⁵, 3.2±0.3 X 10⁵; and for the single donor: 1.7±0.1 X 10⁵.

Conclusions : With regulatory consideration in mind, the capacity to robustly and accurately determine the cellular yield of isolated single-celled CECs from SNEC harvesting technique for cell-injection therapy - as shown in this study, is essential for its translation towards clinical practice.

This is a 2021 ARVO Annual Meeting abstract.