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Che J. Connon, Bo Chen; Location and Structure of Amniotic Membrane Affects the Success of Limbal Stem Cell Expansion. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3940.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic Membrane (AM) is commonly used as a substrate for limbal stem cell expansion. Previously we have shown structural variations in different parts of AM (1). Here we describe the effect of these differences on the subsequent expansion of limbal stem cells.
AM samples were collected from areas adjacent to the placental disc (proximal AM) and approximately 10cm from placental disc (distal AM) (n>10), all AM samples were thoroughly washed and frozen at -80 prior to use. Suspended limbal epithelial cells were isolated from fresh bovine limbal epithelial sheet by treating with collagenase (overnight) and trypsine (10mins). Limbal epithelial cells were cultured for 15 days in basal medium. Expression of K3 was compared by immunohistochemistry (IHC) between bovine limbal epithelial cells cultured on proximal and distal AM (n=6). K3, antibody (Millipore, U.K.) was used at concentrations of 1:100 for IHC. Secondary antibodies were incubated overnight at concentrations of 1:100 (donkey anti mouse) for IHC. Scanning electron microscopy was used to examine the proximal and distal AM surface (n=3). Col IV and Laminin proteins were detected in both proximal and distal AM prior bovine cell expansion by IHC (n=3).
Limbal epithelial cells cultured on intact proximal AM showed well stratified (layer number>6) and undifferentiated cells. Limbal epithelial cells cultured on AM in the distal region, however, showed a poorly stratified (layer number <3) and highly differentiated cell population. SEM pictures showed a rough surface on the distal region, but in proximal AM, epithelium showed a healthy condition. Collagen IV showed no clear differences underneath the proximal and distal AM native epithelial cell layer. Laminin expression in proximal AM showed strong and sustained, whereas in the distal region, laminin expression was slightly weaker and transient.
Our findings are the first to explore the effect of structural variations between proximal and distal AM on limbal stem cell expansion, and we highlighted that the differences within AM can alter cell micro- environment and change the phenotype and genotype of cultured cells.1. CJ Connon et al. The biomechanics of amnion rupture: An x-ray diffraction study. PLoS ONE 2(11): e1147 doi:10.1371/journal.pone.0001147
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