Harris, Anna Rachael; (2010) Optimisation and characterisation of the culture of limbal epithelial stem cells. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Limbal epithelial stem cells (LESCs) are adult stem cells in the eye responsible for maintenance and repair of the corneal surface. Cultured LESC therapy aims to deliver stem cells to patients with a deficiency of these LESCs due to injury or disease and has been shown to be to be effective for restoring the corneal surface. The aim of this thesis was to characterise the culture of LESCs and improve the current method used to produce these LESCs for therapeutic use. Since human tissue for research is in very short supply, models of human LESC culture and the human LESC niche are required to identify mechanisms involved in LESC regulation. In this study rabbit limbal tissue and cultured cells were evaluated for this purpose. At present the 3T3 co-culture system seems to be optimal for the expansion of LESCs, and is approved for clinical use. NIBSC 3T3 J2s have recently (2006) been banked under GMP and this study showed that the GMP scale-up process had not affected the ability of these feeder cells to support the expansion of LESCs. As human limbal epithelial cells typically only survive for a few passages in vitro before they senesce, conditions in the in vivo stem cell niche were mimicked in an attempt to improve the in vitro culture of LESCs. Sub-atmospheric oxygen and ascorbic acid supplementation were investigated and found to be beneficial for maintenance and expansion of LESC progenitors in vitro. Microfluidic technology was investigated as a possible method for sorting LESCs, and was found to show promise as a tool for the identification of LESCs when used in conjunction with methods such as photoluminescence and fourier transform infrared spectroscopy.

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