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Sheyla Gonzalez, Hua Mei, Jonathan Rodriguez, Elfren Baclagon, Rebecca Gentry, Sophie X. Deng; Influence of the Quality of Donor Human Corneas on the Limbal Stem Cell Culture. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3859.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate parameters related to the donor corneal tissues that could affect the efficiency of the limbal stem cell (LSC) expansion in vitro.
LSCs were cultured from 2x2 mm limbal explants (n=550) on denuded amniotic membrane using xenobiotic-free modified SHEM (mSHEM). The percentage of p63bright cells was calculated, and the cultures were divided into two groups: those containing a high percentage (>3%, high p63 group) and those containing a low percentage (≤3%, low p63 group). The parameters related to the tissue quality were as follows: death to cooling/procurement/experiment (DTC, DTP and DTE), donor age and sex, and location of explant biopsy (palisades P, close to palisades CP, far from palisades FP). The percentage of p63bright cells, small cells (≤12 µm), and cells expressing cytokeratin (K)14, K12, pancytokeratin (PanK), and vimentin (Vim) were analyzed in each culture. Statistical analysis was performed by using Chi-Square, Kruskal-Wallis and Wilcoxon tests.
mSHEM supported LSC growth with small and cuboidal limbal epithelial-like morphology in 99.4% of cultures. The cultivated LSCs (cLSCs) from the low p63 group contained a mean value of 4.6% ± 5.3% small cells, 95.3% ± 4.3% K14+, 1.0% ± 1.3% K12+, 96.8% ± 3.6% PanK+ and 1.1% ± 1.4% Vim+ cells. The cLSCs from the high p63 group contained 5.6% ± 6.7% small cells, 94.3% ± 4.2% K14+, 1.0% ± 1.7% K12+, 96.9% ± 2.9% PanK+ and 1.2% ± 1.7% Vim+ cells. No significant differences were found for any of the markers tested between both p63 groups (p>0.05). DTC, DTP, and donor age and sex did not differ in the high and low p63 groups (p=0.06, p=0.14, p=0.62 and p=0.43, respectively). However, DTE had a significant effect (p=0.01); corneal tissues with a DTE >10 days had poor or no cell growth. The percentage of p63bright cells was not affected by the location of the explant tissue (p=0.34).
Donor quality could affect the amount of stem cells expanded in culture.
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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