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Ricardo F Frausto, Vinay S Swamy, Doug D Chung, Anthony J Aldave; Factors Affecting Primary Human Corneal Endothelial Cell Adhesion. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1373. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the factors affecting primary corneal endothelial cell (pCEnC) adhesion.
Primary CEnC were prepared from age-matched donor corneas using two isolation/growth protocols, Tryp-LN-F99 or ColA-ColIV-M4M5. To measure cell adhesion we used 8W10E+ electric cell-substrate impedance sensing (ECIS) disposable electrode arrays connected to the ECIS Zθ instrument to measure electric impedance. Cell-cell and cell-substrate adhesion were modeled from the electric impedance data obtained at 4000 Hz. Statistical significance was defined as p-value≤0.05 (n=3-6; 2-way ANOVA, Bonferroni post-test). Primary CEnC were collected and total RNA was isolated and processed for RNA sequencing. Sequencing reads were aligned to the hg38 genome and transcripts were quantified using Kallisto. Sleuth was used for differential gene expression (false discovery rate adjusted p-value≤0.05), with a focus on genes associated with the cell adhesion and cell junction gene ontology terms.
The ColA-ColIV-M4M5 protocol induced greater cell-cell and cell-substrate adhesion function than Tryp-LN-F99 (p<0.05). No marked difference was observed between dissociation enzymes in cell-cell adhesion. Cells in ColIV-M4M5 achieved greater cell-cell and cell-substrate adhesion than cells in LN-F99 (p<0.05). While the cells in Tryp-ColIV-M4M5 achieved greater cell-substrate adhesion than the Tryp- or ColA-LN-F99 groups (p<0.05), we observed greater cell-substrate adhesion after 60 hours for cells in ColA-ColIV-M4M5 compared with Tryp-ColIV-M4M5. The extracellular matrix (ECM) protein ColIV facilitated the establishment of robust cell-cell adhesion after 60 hours compared with LN (p<0.05). After incubation in M4M5 for 60 hours ColIV coating conferred greater cell-substrate adhesion compared with LN (p<0.05), but no difference was observed in F99. Compared with F99, M4M5 induced an increase in the expression of cell adhesion-associated genes (e.g., CDH3, GJB2).
Herein, we show that pCEnC adhesion function in vitro is highly dependent on the growth medium (M4M5>F99), with some dependence on ECM (ColIV>LN), but little dependence on the dissociation enzyme. In addition, robust adhesion observed with M4M5 may be a consequence of increased expression of genes associated with adhesion. These data support the use of the M4M5 dual-medium for the establishment of pCEnC cultures for use in the management of corneal endothelial dysfunction.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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