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R. Tandon, S. Sharma, S. Sen, S. Mohanty; Culture of Corneal Limbal Epithelial Stem Cells (CLESC) Using Suspension Culture Technique. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6060.
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To established a method of isolating and culturing CLESC by using enzymatic method in order to get a better yield of cells and reduce the expansion time
10 cadaveric limbal rims from eye donors (median 67.5 yrs, range 25-80) were used. CLESC were harvested under sterile conditions. Harvested tissue was dissected into 2 mm length pieces and washed with antibiotic solution. The tissue was then treated with trypsin EDTA at 37°C for 45 mins. After 45 mins of incubation, 1-2 ml of growth medium were added to the cell suspension. Cell viability and count was assessed with trypan blue. The viable cell yield was noted and CLESC suspension was seeded over polystyrene six well culture plates. Cell suspension was incubated at 37° C with 5% of CO2 upto 15 days and media changed twice a week. After 14 days, cells were collected from the culture and processed for immunocytochemistry and RT PCR for p63, ABCG2 and K3.
Limbal rims used in this study were processed within 1-4 days after enucleation and all were preserved in McCarey-Kaufman (MK) medium. The average yield of cells from a single rim was 0.94 X 104 cells/ml (range .4x106 - 1.7 x106) with average viability of 65% (range 35.67%-90.05%) (Table1). Successful cultures were maintained in 6 samples upto 2 weeks, 2 were contaminated on day 2 and 4. Phase contrast microscopy showed small, compact and polygonal cells which were able to form a honey comb like structure in culture. The phenotypes and genotypes of cultivated cells were described by immunocytochemical staining and RT PCR for p63, ABCG2 and K3 after 14. We observed expression of both stem cell associated markers (p63, ABCG2) and differentiation marker (K3).Table 1
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