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Hannah J Levis, Julie T Daniels; Tissue engineering the human limbal crypts: further characterisation of an in vitro model. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2197.
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Design and recreation of elements of various stem cells niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. The limbal epithelial stem cell (LESC) niche houses the cells responsible for the maintenance of a healthy corneal epithelium and is composed of a vast array of cellular, physical and matrix components. We have developed a method to rapidly and reliably create bioengineered limbal crypts (BLCs) that mimic the 3D architecture seen in the limbal niche in human limbal fibroblast (hLF) populated RAFT tissue equivalents (RTEs). This study further characterises our niche model that incorporates two aspects of the LESC niche.
Type 1 collagen hydrogels containing hLFs were cast in a 24-well plate. Liquid was removed using hydrophilic porous absorbers (HPAs) with either flat bases or bases with custom moulded micro-ridges to produce BLCs. The resulting RTEs were then seeded with human limbal epithelial cells (hLEs) and maintained in submerged culture for 2 weeks before airlifting for 1-3 weeks. The transparency of RTEs was measured over time using a spectrophotometer. RTEs were fixed for immunochemical analysis or cryosectioning. Orientation of hLFs in RTEs was compared using ImageJ after staining with propidium iodide and phalloidin. Sections and wholemount RTEs were stained with antibodies to limbal specific basement membrane proteins (laminin β1, laminin α2, laminin γ3) and putative LESC markers (ΔNP63α and Bmi-1).
hLEs multi-layered on RTEs and filled BLCs. There was no significant difference in transparency between BLC+ and BLC- RTEs after 8 weeks in culture (p>0.05). hLFs immediately below the epithelial layer preferentially aligned in BLC+ RTEs. All antigens used in this study were highly expressed in the limbus compared with central cornea. Expression of Bmi1, ΔNP63α, laminin β1 and γ3 was detected in cells grown on BLC+ RTEs.
Recreation of a stem cell niche in vitro is a useful tool to study cell-cell and cell-matrix interactions. We have developed a method to rapidly and simply combine two of these elements for the culture of hLEs. We have shown that hLEs grown on BLC+ RTEs express markers that are associated with limbal niche cells and that BLCs can influence the alignment of hLFs within the construct. This is one further step towards creation of our ultimate goal of a LESC “niche in a dish”.
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