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B. Shaharuddin, R. Megaw, C. Osei-Bempong, S. Ahmad; Repeat Trypsinisation of Human Limbal Tissue Enriches for Limbal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3742.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal stem cells (LSCs) can be found in the basal layers of human limbal epithelium. Trypsinisation of limbal tissue is used to release epithelial cells for culture. The purpose of this study was to determine whether repeat trypsinisation of limbal tissue enriches for LSCs.
Human limbal tissue, donated for research and obtained from the UK Eye Bank Service was trypsinised for 20 minutes at 37°C. After this time, epithelial cells released by the trypsin were isolated by centrifugation. Some pieces of limbal tissue were also removed at this stage for histological analysis. More trypsin was added to the remaining pieces incubating for a further 20 minutes. Released epithelial cells were again isolated and pieces of limbal tissue were removed for histology. This process was repeated for a total of 4 cycles of trypsinisation. Cells released from each cycle were counted and analysed using CFE assays and real time PCR for putative LSC markers p63 and ABCG2 and differentiated corneal epithelial markers CK3 and CK12. Limbal pieces collected from each cycle were cryosectioned and stained using haematoxylin and eosin.
Cell counts from the 4 cycles of trypsinisation revealed higher cell counts during the second and third cycles with the lowest being in the first cycle (p<0.05). CFE increased significantly (p<0.05) in a gradient with the lowest being from cells in cycle 1 (2.4-3.6%) and highest from cells in cycle 4 (6.2-8.4%). Real time PCR data revealed increasing expression of both p63 and ABCG2 (p<0.01) decreasing expression of CK3 and CK12 (p<0.05) in cells from cycles 1 to 4. Haematoxylin and eosin staining of limbal tissue sections from each cycle revealed sequential removal of epithelial layers from cycles 1 to 4 with no remaining epithelium in samples from cycle 4.
Results from this study show that sequential trypsinisation of human limbal tissue progressively enriches for LSCs. As shown by the histological data, progressive removal of epithelial layers of the limbal epithelium occurs with each trypsinisation cycle. The CFE data and the real time PCR data indicate that cells from cycle 4 have the strongest expression of LSC properties. This is not surprising since LSCs can be found in the basal limbal epithelial layers. In conclusion therefore, this study shows that the final cycle of trypsinisation enriches for LSCs.
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