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Andreas Gießl, Naresh Polisetti, Shen Li, Friedrich E Kruse, Ursula Schlötzer-Schrehardt; Effective isolation and expansion of human limbal niche cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1200.
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The limbal stem cell niche harbours several niche cell populations including melanocytes and mesenchymal stromal cells, which have been shown to support maintenance and function of limbal epithelial stem/progenitor cells (LEPC) in vivo and in vitro. The aim of this study was to establish improved methods for isolation, purification and cultivation of limbal melanocytes for purposes of analysing their biological roles and generating advanced corneal epithelial grafts.
LEPC, limbal melanocytes, and limbal mesenchymal stromal cells (LMSC) were isolated either by collagenase or dispase digestion from human corneoscleral rims. Contaminating cell types, such as epithelial cells or fibroblasts, were eliminated either by a selective cytotoxic agent, geneticin, or by magnetic activated cell sorting (MACS) using cell type-specific antibodies in stepwise purification protocols. Purified cell cultures were maintained in serum-free medium and characterized by qPCR, Western blotting, flow cytometry and immunocytochemistry. The effect of conditioned media, soluble factors and matrix substrates on proliferation of limbal melanocytes was investigated using BrdU proliferation assays.
All enzymatic digestion and purification methods provided pure populations of melanocytes derived from both the limbal epithelium and the limbal stroma. The MACS purification using negative selection with anti-EpCAM and anti-fibroblast antibodies as well as positive selection with anti-CD117 (c-Kit) antibodies was superior to geneticin treatment regarding duration and gentleness resulting in vital cells of lower passages. Proliferation and expansion of melanocytes could be stimulated by laminin-511 and soluble factors, such as FGF-2 (fibroblast growth factor-2) and TPA (12-O-Tetradecanoylphorbol-13-acetate), but was most significantly enhanced by LMSC conditioned medium. Proliferating melanocyte cultures were maintained for up to 12 months.
This effective isolation and cultivation technique allows for relatively rapid generation of pure populations of human limbal melanocytes with good viability, growth rate and long-term maintenance, which could be useful for tissue engineering of biomimetic grafts in cultivated limbal stem cell transplantation.
This is a 2020 ARVO Annual Meeting abstract.
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