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S. Schrader, M. Notara, M. Beaconsfield, S. Tuft, G. Geerling, J. T. Daniels; The Effect of Long Term Culture and Cryopreservation on Conjunctival Stem Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2608.
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Severe ocular surface disorders such as ocular mucous membrane pemphigoid, Stevens-Johnson syndrome, toxic epidermal necrolysis, recurrent pterygia, and chemical/thermal burns can result in complete ocular surface stem cell failure up to ankyloblepharon and complete keratinisation of the cornea. Conjunctival stem cells taken early in the disease or from an unaffected eye could be ideal for conjunctival surface reconstruction. The aim of this study was to evaluate the effect of long term in vitro culture and cryopreservation on conjunctival epithelial cell survival and stem cell like characteristics.
Human conjunctival cells from bulbar biopsies were isolated and expanded on a growth arrested 3T3 feeder layer. The cells were evaluated for cytokeratin (CK4/CK19) expression by immunostaining. An aliquot of the cells from the initial culture was frozen for 14 days and both cryopreserved and non-cryopreserved cells were then cultured for several passages. For each passage the colony forming efficiency was evaluated and expression of putative stem cell markers P63 and ABCG2 were assessed by immunostaining and RT-PCR.
Both non-cryopreserved and cryopreserved cells showed a high proliferative capacity by means of colony forming efficiency, which decreased during the passages. Strong expression of P63 was found in both groups, whereas ABCG2 showed weaker expression. The presence of P63 and ABCG2 was also confirmed by RT-PCR.
We were able to show, that conjunctival epithelial cells with stem cell characteristics can be cryopreserved and maintain their function in-vitro over several passages. The possibility of cryopreservation and subsequent in-vitro expansion is advantageous when cells have to be cultivated for clinical transplantation.
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