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James McKelvie, Trevor Sherwin, Dipika Patel, Charles McGhee; Characterization Of Human Keratocyte And Progenitor Cell Migration And Cell Division In Two And Three Dimensional Collagen Matrices Using Live Cell Imaging. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3423.
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© ARVO (1962-2015); The Authors (2016-present)
The sphere forming assay produces spheres of keratocytes and progenitor cells. It is not known if sphere formation is a result of keratocyte/progenitor cell division or cell migration. This study aims to characterise the mechanism of sphere formation and the stimulus and extent of cell migration and division of transplanted keratocyte and progenitor cell spheres.
Human corneal stroma was enzymatically digested to isolate stromal cells which were subsequently cultured using a sphere forming assay and labelled using fluorescent cytoplasmic Qdot nanocrystals. Labelled spheres were investigated using confocal and fluorescent microscopy. Spheres were placed on collagen coated culture dishes and ex vivo corneal stromal tissue and observed with fluorescent time-lapse photography to monitor cell migration. Migrating cells were incubated with Click-iT EdU to confirm cell division. Gene and protein expression of migrating cells, cultured spheres, and freshly digested non-cultured stromal cells were compared using TaqMan quantitative gene array cards and immunohistochemistry.
Digested stroma yielded 7x10E5 cells/gm. Isolated stromal cells efficiently endocytosed Qdot nanocrystals and were visible with fluorescent and confocal microscopy. Cultured cells aggregated without cell division to form spheres containing several different coloured Qdots indicating that spheres primarily form from migration rather than division of cultured cells. Spheres transplanted onto collagen matrix and ex vivo corneal stroma triggered radial cell migration away from spheres at speeds of up to 16um/h with concomitant cell division as observed on time-lapse photography and positive EDU staining. Immunohistochemistry of cultured spheres demonstrated extracellular matrix deposition including collagen subtypes in similar proportions to that seen in corneal stroma.
It is possible to isolate and culture keratocyte/progenitor cell spheres from human corneal stroma. Cultured cells can be stimulated to migrate and divide with the addition of collagen. These results imply that using cell-based transplants, in the form of cultured spheres, may be possible to treat corneal diseases such as keratoconus.
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