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Sharon L. Mason, Carl Sheridan, Eric Austin, Paul Rooney, Stephen Kaye; Expansion Of Cells From Bovine Limbal Explants On Extracellular Matrix Proteins And Other Substrates. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5131.
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The recent past has seen an application of cell-based techniques to regenerate the corneal surface by ex-vivo expanding autologous or heterologous limbal cells. The success of this procedure depends on the substrate the cells are cultured on - typically a mouse 3T3 feeder layer. Here we evaluate the suitability of several substrates (in the absence of 3T3 cells) for the culture of bovine limbal cells, assessing their proliferation, morphology and protein expression.
Explants were taken from fresh bovine eyes and placed into tissue culture plates coated with laminin, gelatin, fibronectin, fetal calf serum (FCS), or uncoated. Proliferation was assessed by measuring the distance from the explants to the leading edge of the cells at 4 equally spaced points around the explants. Morphology was assessed under the phase contrast microscope. After 10 days, cells were fixed in methanol and immunofluorescently stained with putative limbal stem cell markers such as Hsp70 and P63 as well as mature corneal markers including cytokeratin 3.
Limbal cells expanded from explants on all substrates stained strongly with Hsp70, and P63 thus providing evidence for their stem cell identity. The highest rate of expansion occurred on laminin and the lowest on FCS and gelatin. The difference in proliferation between laminin and FCS/Gelatin was found to be significant. There was no discernable difference between the cells for Hsp70 or P63 staining as they grew away from the explants, although cytokeratin 3 staining was higher furthest away from the explants. The cells had the correct epithelial morphology across all the substrates used, showing closely packed cuboidal cells characteristic of epithelium, with a well defined leading edge.
All substrates supported good epithelial cell morphology and limbal stem cell staining; but laminin coated plates induced a higher rate of proliferation. This aids successful growth of ex-vivo limbal cells and their ultimate transplantation into the patient, removing the need for a mouse 3T3 feeder layer.
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