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N. Szentmary, K. Barabás, A. Stündl, K. Demeter, J. Németh, I. Süveges, E. Madarász; Expansion of Limbal Progenitors on Type IV Collagen. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1785.
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© ARVO (1962-2015); The Authors (2016-present)
To determine immunocythochemical properties and proliferative potential of limbal stem-like cells maintained in vitro, on collagen.
Limbal pieces were harvested from multiorgan donors and cultured on collagen IV using culture media conditioned by embryonic mouse fibroblasts (E16). Immunocytochemical properties were determined by p63, cytokeratin 19 (CK19), ssea-3 and bcrp (ABCG-2) stainings, cells with active metabolism by MTT assay (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromid -> formazan) (0.25mg/ml) and the percentage of proliferating cells by propidium iodid uptake (FACS).
24-48 hours following explantation onto type IV collagen, spheroid cells grew out of the explants. After 48 hours, migrating bipolar and epitheloid cells appeared. After 3-4 days, the center of the explants was necrotized. 80-90% of the cells in the outgrowth zone were viable and about 7 % of non-attached cells was in S or G2 phase. A number of outgrowing cells showed p63, CK19, ssea-3 and bcrp immune-reactivity.
Cells growing out from limbal explants on collagen, show immunocytochemical characteristics of limbal stem cells. Collagen-like surfaces may be the appropriate substrates for ex vivo expansion and for a future in vivo implantation of limbal stem cells.
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