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Kazunari Higa, Hideyuki Miyashita, Jun Shimazaki, Kazuo Tsubota, Shigeto Shimmura, ; Spheroidal cultivation of human limbal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):502.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cells have a specialized microenvironment for maintaining self-renewal and multipotent capacities. It is believed that a cornea epithelial stem cell niche exists in the limbus. We presented that aquaporin 1 positive (AQP1+) cells immediately beneath epithelial basement membrane may be mesenchymal niche cells that directly interact with N-cadherin positive (N-cad+) limbal basal epithelial cells (2010 ARVO). To establish a cultivation method the limbal epithelial stem cells niche in vitro, we performed spheroidal cultivation from the human limbus using Matirgel.
N-cad+ cells and mesenchymal niche-like cells were prepared from donor corneal sclera used after cornea transplantation. After treatment with collagenase or dispase II, limbal epithelial cells and surrounding cells were scraped from limbal tissue, and spread on Matrigel. Cells sere cultivated using medium with KGF and the Rho kinase inhibitor, Y27632, or medium with EGF. After 1 month, we attempted to engineer corneal epithelial sheet from a single spheroid. Frozen sections of spheroids and epithelial sheets were observed by histochemical analysis.
Spheroids tended to be uniformly round with the treatment of collagenase compared to dispase II. Extension of limbal epithelial cells from the spheroid was observed in standard medium with EGF, but was rarely observed in medium with KGF+Y27632 maintaining phenotype of limbal epithelial cells. Keratin (K) 15 and p63 were expressed in the spheroid after 1-month cultivation, as well as in epithelial sheets engineered form a single spheroid.
The limbal niche can be maintained in spheroids for up to 1 month in culture and can give rise to epithelial cell sheets with a limbal phenotype
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