Abstract
Purpose :
Predicting in vivo endothelial cell migration after surgical removal of central diseased endothelium is challenging since the capacity of endothelial cells (EC) to migrate can vary. In this study, outer corneal graft rims were cultured in a temperature-reversible hydrogel matrix to simulate in vitro the EC migration after Descemet stripping only (DSO) with and without ROCK-inhibitor.
Methods :
Twenty-one outer rims with the trabecular meshwork still attached were prepared from Descemet membrane-endothelial cell (DM-EC) sheets (from 12 research-grade donors (mean age 76±5 years)) by a 6.5-mm trepanation. For the initial proof-of-concept, 7 outer rims were cultured in a thermo-reversible hydrogel matrix for up to 45 days. To assess the effect of ROCK-inhibitor on the in vitro cell migration, 7 paired outer rims were cultured either with or without ROCK-inhibitor. At the end of culture, tissue was retrieved from the polymer matrix to examine cell viability and expression markers (ZO-1, Na+/K+-ATPase, NCAM, glypican and vimentin).
Results :
All cultured outer rims remained viable and displayed single regions (n=5/21) or collective (n=16/21) cell migration, regardless of the presence or absence of ROCK-inhibitor. Collective migration started on average after 4±2 days and continued for at least 29 days. Migrated cells showed a more regular cell morphology when cultured in the presence of ROCK-inhibitor than not. Furthermore, 7 outer rims additionally demonstrated a phenotypically distinct late-onset, but fast growing cell population emerging from the far periphery of the endothelium. This occurred after 3 weeks of culture and displayed characteristics for undifferentiated cells. Immunostaining showed that migrated EC maintained the expression patterns of endothelial cell markers.
Conclusions :
Using a 3D-culture system, we observed the migration of two morphologically distinct cell populations. The first type was triggered by a broken physical barrier, consistent with disruption of contact inhibition. The second, late-onset type showed a higher proliferative capacity, which appears to be mediated by other stimuli next to cell exposure to free space and ROCK-inhibitor. Further exploration of the differences between these cell types may assist in learning how to selectively stimulate cell migration from DM-EC areas that harbor the latter cell type and may ultimately help to improve DSO outcomes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.