Amniotic Membrane Preparation Affects Its Ability to Serve as an ex vivo Niche for Corneal Limbal Epithelial Stem Cells

Abstract

Purpose: : Human amniotic membrane (AM) is used as a substrate to culture human limbal epithelial stem cells for transplantation. Many methods of AM preparation exist but it is unclear whether any one method is superior for stem cell expansion.

Methods: : Primary human limbal epithelial cells were cultured submerged in serum containing culture medium for 14 to 21 days on a variety of AM preparations. Firstly, three preparations were assessed to determine the effect of partial, total or non decellularisation on limbal epithelial expansion. A second series of experiments compared DMEM/Glycerol (50:50) cryopreservation with amnion frozen in HBSS alone at -80ºC. Confluence of epithelial growth was assessed by toluidine blue staining, digital imaging and image analysis. Cell density,morphology and wholemount immunofluorescence for ABCG2 and p63 alpha were assessed by confocal microscopy.

Results: : At 21 days after seeding of cells, the decellularised AM had the highest mean % confluence at 97.2±1.2% (mean±SEM)(n=4). This was significantly higher than the mean confluence for intact (66.7±4.1%) and partially decellularised (77.5±6.5%) preparations (p<0.05). After 14 days of culture the confluence of amnion frozen in Hank's balanced salt solution (HBSS) (88.5±3.66%) was significantly greater than intact Glycerol:DMEM cryopreserved AM (21.8±3.9%) (n=4, p<0.05). AM frozen in HBSS gave rise to the most limbal basal cell like morphology and the highest cell density (1528±70 cells/mm² )(n=4, p=0.05). The density of cells cultured on both intact (800±16 cells/mm²) and partially decellularised AM (901±66 cells/mm²) were significantly higher than decellularised AM (387±22 cells/mm²(n=4, p<0.05). AM frozen in HBSS had the highest percentage of cells expressing p63alpha (68.4±4.14%) and ABCG2 (61.7±3.2%). For all other preparations these values were less than 6% and 8% respectively (p<0.05).

Conclusions: : Decellularisation of amniotic membrane results in a reduction in the time taken to reach confluence and a reduction in cell density, suggesting that cells migrate more rapidly rather than proliferate more rapidly. Cryopreservation of amniotic membrane in DMEM/Glycerol results in significantly lower cell densities, increased time to confluence and reduced expression of p63alpha and ABCG2 when compared with AM frozen in HBSS.

Keywords: cornea: epithelium • cornea: basic science • anterior segment