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C. J. Connon, B. Chen; An Assessment of Bovine Limbal Stem Cells as an Experimental Model of ex vivo Limbal Stem Cell Expansion. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1784.
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The procurement of fresh healthy human corneas for experimental use can be costly and/or sporadic. Bovine corneas may form a representative and reliable alternative to human corneas when studying the biology of ex vivo expanded limbal stem cells.
Bovine eyes were collected from a local abattoir and processed within 4 hours of death. Limbal tissue was excised and cultivated upon intact or denuded human amniotic membrane (denuded following incubation in 1%EDTA for 120mins). Limbal pieces (5mm2) and suspensions of limbal epithelial cells (established by incubating limbal pieces in collagenase overnight, followed by trypsin, 10 min) were cultivated under standard limbal stem cell conditions (50µg/ml antibiotics, 5% FBS, 0.5% dimethyl sulfoxide, 2ng/ml human Epidermal Growth Factor, 5µg/ml insulin, B27 supplement medium at 37°C, 5% CO2) for 2 weeks followed by 1 week under ‘air-lifting’ conditions. The number of cell layers and the number of cells differentiated (K3 positive) or undifferentiated (K14 positive) were quantified by light microscopy and immunohistochemistry before and after air-lifting of the samples.
Bovine limbal epithelial cells once expanded upon amniotic membrane formed stratified layers (4-6 layers) of differentiated corneal epithelial (n=6). Limbal epithelial cells added as a suspension resulted in an increased number of cell layers (20%). Furthermore, cells added as a suspension retained a significantly higher proportion in an undifferentiated state following airlifting (45%) compared to cells cultured from explants (<5%). Bovine limbal epithelial cells expanded upon intact amniotic membrane had a greater proportion of differentiated cells (60%) than cells expanded upon denuded amniotic membrane (<5%). However both intact and denuded supported a similar level of undifferentiated cells following airlifting and no difference was observed in the number of cell layers.
Bovine limbal epithelial expanded ex vivo can form a well stratified and differentiated corneal epithelial construct similar to that formed by expanded human limbal epithelia and can therefore be considered as an alternative source of limbal stem cells to improve ex vivo expansion conditions. Furthermore, we have found that for bovine limbal epithelia, the cell suspension method produces a more stratified construct and that denuding the amniotic membrane can influence limbal stem cell differentiation.
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