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A. Solomon, E. Walter, N. Lanxner; Human Limbal Epithelial Cells Expansion in Long-Term Repeated Explant Cultures From Cornescleral Rims. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3744.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the effects of repeated sequential primary culturing of human limbal explants on the properties of the cultured limbal epithelial cells.
Fresh human corneoscleral rims remaining after corneal transplantation were cut into 12 separate explants and placed on plastic culture dishes, and cultured in SHEM medium. After a period of 14 days each explant was removed from the culture dish and placed in a new dish for repeated culturing. The remaining cells were fixed and immunostained with antibodies against p63 and ABCG2. Explants were used for repeated cultures for sequential two-week periods, until no cellular growth was evident. The first culture was named P0, and consecutive cultures were named from P1 through P6 respectively. The percentage of positive cells for p63 and ABCG2 was recorded in the different periods from several fields and in 3 different distances from each explant (which were named near, middle and edge).
Positive limbal epithelial cells for p63 and ABCG2 were recorded through P5 (10 weeks culture) and P6 (12 weeks culture), respectively. There was a gradual decline of the positive cells from P0 through P6 and from the near fields to the distant fields in relation to the explant location. The percentage of p63 positive cells was 52.8% ± 20.9% at P0, 34.9% ± 20.8% at P2, 26.9% ± 22.3% at P4, and 0% at P6 (p=0.035, Kruskal-Wallis Test). The p63 positive cells were more abundant near the explant compared to fields at the culture edge (26.9% ± 22.3% vs. 1.7% ± 2.9% at P4, p=0.07, Mann-Whitney Test). The percentage of ABCG2 positive cells was 12.8% ± 6.1% at P0, 10.7% ± 3.3% at P5, and 2.1% ± 2.9% at P6 (p=0.16, Kruskal-Wallis Test). Significant reduction of ABCG2 positive cells was recorded in fields that were more remote from the explant location (10.7% ± 3.3% for fields near the explant compared with 1.1% ± 2.0% at the culture edge, p= 0.07, Mann-Whitney Test).
Limbal epithelial progenitor cells with typical stem cell markers can be generated from repeated sequential explant cultures. This culture system may be used to expand large and viable populations of progenitor epithelial cells for the purpose of cell transplantation in total limbal stem cell deficiency.
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