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Victoria E Tovell, Julie T Daniels; Functional limbal epithelial cells can be successfully isolated from organ cultured rims following long-term storage. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5169.
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Due to a shortage of fresh corneal rims for research it was of interest to investigate the potential of successfully growing human limbal epithelial cells from long-term storage organ culture rims.
Superficial segments (approx. 500 μm deep) of corneal limbus were dissected and digested using collagenase (2mg/ml, 16hrs at 37°C). Cell suspensions were separated into 4 different growing conditions (Corneal epithelial cell media, CECM; CECM + 3T3; Corneal stromal stem cell (CSSC) media or CSSC media + 3T3). Growth conditions were assessed by observing at colony size and quantifying colony number, total cell count and cell density. Colonies were counted and classified into viable and aborted colonies by following previously published methods. Epithelial integrity was determined by observing the potential to form a multilayered stratified epithelium using our Real Architecture For 3D Tissue (RAFT) model of corneal tissue.
Human limbal epithelial cells can be successfully grown from corneal rims that have been stored in organ culture media for 1 month or more. This isolation technique was reproducible with an 88% success rate out of 25 corneal rims. CSSC media, which was originally designed for the isolation of CSSCs, was found to be a valuable growth condition for limbal epithelial cells. CSSC media + 3T3s provided optimal growth conditions following collagenase digestion, with cultures displaying large uniform colonies after 24hrs. Colony number, total cell number and cell density were all significantly higher at day 7 in cultures with CSSC media + 3T3 compared with other growth conditions. Cells maintained in CSSC media compared with CECM were found to produce a more stable and higher quality epithelium made up of basal columnar cells and squamous superficial cells.
Human corneal rims stored in organ culture media can be a valuable source for isolation of human limbal epithelial cells. Using a combination of media designed for CSSC isolation and tissue that might have otherwise been discarded, a high number of good quality limbal epithelial cells can be obtained using this new combination.
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