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Karl David Brown, Mark Daniell; Culture of corneal endothelial cells from resident progenitor cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2258.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that in the absence of cell contact inhibition from primary CEC, purified CEPC have enhanced proliferation. Also to determine the proportion of corneal endothelial progenitor cells (CEPC) in the corneal endothelial cell (CEC) population.
CEC suspensions were prepared from scavenged sheep cadavers1. To produce CEPC spheres cell suspensions were seeded into low-attachment plates in DMEM:F12 with L-glutamine, bFGF, and EGF.To determine the proportion of CEPC in the CEC population 5000 cells/well were seeded in 23 wells/animal. After 10 d, spheres with a diameter of >50 μm were counted to determine the proportion of sphere-forming CEPC. A 24th well was a control.To determine the minimum primary cells to achieve CEC confluence within 7 d, two-fold serial dilutions primary CEC were seeded onto 13mm plastic cover slips with fresh corneal medium and cultured for 7 d. Days to 100% confluence recorded.To determine the minimum primary cells to achieve CEC confluence within 7 d from sphere seeding, spheres were prepared from two-fold serial dilutions of primary CEC were seeded into ultra-low-attachment plates and cultured for 14 d. The resulting spheres were then seeded onto tissue culture plastic cover slips and cultured for 7 d. Days to 100% confluence was recorded.Two-way ANOVA was performed to determine the significance of any effects of seeding type (primary cells or spheres) and initial primary cell number.
There was one sphere >50 µm per 1934 primary CEC (n=4, SEM 277). The ovine corneal endothelium in vivo has a cell density of 3150 CEC/mm2 1 this equates to a mean CEPC density of 1 CEPC/1.6 mm2. Spheres are likely to be descended from single cells because of the low seeding density used.There was no effect of cell seeding type (primary cells or spheres) on the time from seeding of the surface to CEC confluence in days (F(1,20)=0.17, p=0.7). The initial number of primary cells (5.0 x 104, 2.5 x 104, or 1.25 x 104cells) did not have a significant effect on time to confluence (F(2,20)=1.86, p=0.2).
The proportion of progenitor cells is 1/1934 primary ovine CEC. CEPC do not proliferate more rapidly than primary CEC. The null hypothesis was accepted. Future work will investigate dissociated spheres.1. Ozcelik et al. Adv Healthc Mater 2014;3:1496-15072. Mimura et al. Invest Ophthalmol Vis Sci 2005;46:3637-3644
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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