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Louise Morgan, Julie T Daniels; A simple method to culture limbal epithelial stem cells from cryopreserved corneal limbal explants. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5164.
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Transplants of cultured epithelial cells and stem cells grown from donated corneal limbal tissue provide therapy for corneal surface deterioration due to limbal stem cell deficiency. Limbal epithelial stem cells can be expanded in culture for these treatments, however isolated limbal epithelial stem cells require xeno-feeder cells for survival (for example mouse fibroblastic cell lines), conditions that are not optimal for human transplantation. Explant cultures of corneal limbal tissue have the advantage that xeno-feeder cells are not necessary as the explant itself has auto-feeder properties that maintain progenitor cell characteristics. Here we describe the development and validation of a reliable cell culture model using cryopreserved, liquid nitrogen-stored, limbal tissue explants.
Strips of limbal tissue were collected from Optisol-stored human and from fresh porcine corneo-scleral rims, cut into small pieces, cryopreserved and stored in liquid nitrogen. Thawed tissue was then attached to plastic compressed collagen sheets and explants maintained in medium optimised for culture of corneal epithelial cells. Cellular outgrowth from the explants was followed and cultures were characterised for expression of corneal epithelial and stem cell-associated markers.
Thawed porcine explants attached well to the collagen substrate and robust outgrowth was seen over the first few days in culture. Cultures achieved confluence and maintained a stable monolayer for many days. Thawed human tissue showed variability in explant survival and in the rate of cellular outgrowth, this variation reflected the situation in cultures from the same tissues before freezing. Expression of corneal epithelial and stem cell-associated markers was similar to not-frozen explants for both human and porcine tissue.
Corneal limbal explants can be frozen and stored in liquid nitrogen with no apparent loss of viability. Stored supplies of viable limbal tissue will provide a valuable tool to facilitate cell and molecular biology research and in vitro drug development studies.
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