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C. Osei-Bempong, R. D. Megaw, J. Armitage, F. C. Figueiredo, M. Lako, S. Ahmad; Investigation of Cryopreservation Media for Cultured Human Limbal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3763.
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© ARVO (1962-2015); The Authors (2016-present)
Cultured human limbal stem cells (LSCs) can be used to successfully treat LSC deficiency. Use of this technique in centres without suitable tissue culture facilities will require optimisation of cultured LSC transport. Cryopreservation provides a method of transporting cultured LSCs. This study aimed to determine which medium can be used to optimally cryopreserve LSCs.
Limbal epithelial cells isolated from limbal tissue, donated for research, were plated on mitotically inactivated 3T3 mouse fibroblasts. Limbal epithelial cells were cultured for 7 days. These cultured limbal epithelial cells were trypsinised and placed in 5 different cryopreservation media (1ml each in polypropylene tubes): 100% limbal epithelial medium (LEM), 90% LEM/10% DMSO, 45% LEM/45% foetal calf serum (FCS)/10% DMSO, 90% FCS/10% DMSO, and 100% FCS. Cells in the cryovials were then passively cooled in an insulated container at -80°C for 24 hours. After 24 hours, the cells were thawed in a 37°C water bath and analysed using the trypan blue assay, colony forming efficiencies (CFEs) of the intact cells were performed, and RNA was extracted for reverse transcription and real time PCR for putative LSC markers p63 and ABCG2. These tests were also performed on non-cryopreserved cultured limbal epithelial cells as a control.
24 hour cryopreservation resulted in a significant decline in intact cells as shown by trypan blue staining (45 to 93%; p<0.05). DMSO however resulted in maintenance of cell integrity. The CFE assays of intact cells showed that CFE in comparison to LEM (4.1%) was maintained in all groups, with higher CFE in the FCS containing media (4.7 to 5.7%) and lower in the LEM containing media (3.4 to 4.6%). Real time PCR for p63 and ABCG2 showed that their expression was significantly higher (p<0.01) in the FCS containing groups and lower in those containing LEM.
Three main conclusions can be drawn from this study. Firstly, 24 hour cryopreservation for cultured human limbal stem cells is possible. Secondly, cell integrity declines with all media, with DMSO maintaining integrity. Thirdly, the LSC properties of cultured cells was highest in media containing FCS, as indicated by CFE and real time PCR data. These results would suggest that 90% FCS/10% DMSO is the optimal cryopreservation medium for cultured human LSCs.
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