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James McKelvie, Dipika Patel, Trevor Sherwin, Charles McGhee; The Role Of Cell Aggregation And Division And The Expression Of Extracellular Matrix During Human Keratocyte Sphere Formation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1094.
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Human keratocytes and progenitor cells can form spheres in serum-free culture conditions and maintain the expression of keratocyte-specific markers. The aim of this study was to determine the role of cell migration/aggregation and division in keratocyte sphere formation and the associated gene, protein and glycoprotein expression.
Stromal cells were isolated from human corneal rims and cultured in a serum-free sphere-forming culture system. The mechanisms and temporal sequence of sphere formation were investigated using live-cell, darkfield, scanning electron and confocal imaging. Live keratocytes were labelled and tracked using quantum dots, azito labelled sugars and nucleotides and investigated using immunocytochemistry to monitor collagen subtype and glycoprotein expression in conjunction with cell division during sphere formation. Isolated cells were sorted using fluorescence activated cell sorting before and after sphere formation and gene expression was analysed using quantitative microfluidic arrays and real time PCR.
Serum-free culture of keratocytes demonstrated early sphere formation by day 7. Spheres formed predominantly by cell migration and aggregation with less than 10% of cells dividing by day 7, although, as sphere formation progressed cell proliferation played an increasing contribution in late sphere development with 30% of cells dividing by day 10. Primary spheroid cultures remained stable for up to 6 months and demonstrated up-regulated expression of extracellular matrix genes including the stromal collagen subtypes and proteoglycans including keratocan. Immunohistochemistry confirmed expression of collagen subtypes and keratocan in cultured spheres and deposition of azito labelled glycoproteins were detected during early and late sphere formation.
Human keratocytes can be isolated and cultured using a sphere-based serum-free culture system that maintains the keratocyte molecular phenotype. Early sphere formation is predominantly due to cell migration/aggregation and late sphere formation is associated with increased cell division and glycoprotein expression.
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