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Payton Boere, Jessica Wu, Ricardo F Frausto, Anthony J Aldave; Low mitogenic conditions maintain the corneal endothelial cell phenotype in vitro. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5413. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In vitro methods for the culture of corneal endothelial cells (CEnC) have been developed for generating therapeutically viable CEnCs. The purpose of this study was to compare the effect of two distinct culturing methods on barrier function, and on the expression of transporter-, barrier- and senescence-associated genes.
Primary CEnC cultures were prepared from age-matched donor corneas using one of two isolation/expansion protocols: Trypsin/Laminin/[RF1] F99 and Collagenase/Collagen IV/[RF2] M5. F99 medium (high mitogenic medium) contains higher concentrations of mitogens compared with M5 medium (low mitogenic medium). Cells were passaged four times to create 5 passage groups (P0-P4). Cells at P0 were used for assessing barrier function. Cell barrier function was assessed using electric substrate impedance sensing. Expression was assessed using quantitative PCR (qPCR). Total RNA was isolated and mRNAs were reverse transcribed with poly(dT) primers to generate DNA templates for use in qPCR. Relative gene expression was obtained by comparison to the housekeeping gene RAB7 and was calculated by the comparative Ct (2-delta(Ct)) method. Changes in the expression of barrier- and transporter-associated genes were assessed in P0 cells, while changes in the expression of senescence-associated genes (CDKN1A (p21CIP1), CDKN2A (p16INK4) and LMNB1) were assessed in P3 cells compared with P0 cells.
Cells cultured in M5 demonstrated significantly greater barrier function compared with cells cultured in F99 (p<0.05). Twelve (12/19, 63%) of the barrier-associated genes showed significantly higher expression in cells cultured in M5 compared with cells in F99. Five (5/6, 83%) of the transporter genes showed significantly (p<0.05) higher expression in pCEnC cultured in M5 compared with cells in F99. Significant changes were observed in P3 cells for two senescence-associated genes, CDKN2A and LMNB1 (p<0.05).
We demonstrate that CEnC-associated function and gene expression profiles are better maintained by culture in M5 versus F99. While senescence develops in both M5 and F99, our study identifies potential gene targets that can be manipulated to moderate the progression of senescence. Ultimately, use of animal models will establish in vitro culture of CEnC as a viable option for the management of CEnC disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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