Optimisation of protocols for ex vivo expansion of limbal stem cells and their enrichment

Abstract

The corneal epithelial cells are constantly replaced by the stem cells located at the limbus, the peripheral edge of the cornea, therefore known as limbal stem cells (LSCs). LSCs can be destroyed by numerous factors which results in the condition called limbal stem cell deficiency (LSCD). Ex vivo expansion of LSCs is a well-established technique used successfully to cure patients with LSCD. Therapeutic use of LSCs must be performed in compliance with good manufacturing practice (GMP) as a quality assurance system. However, traditional culture media for ex vivo expansion of LSCs contains a number of ingredients derived from animal sources which may compromise its safety profile for human transplantation. The first aim of the study was to define new GMP grade medium for cultivation and maintenance of LSCs in vitro. Formulation of new GMP compliant media resulted in equal growth to non-GMP grade media. Strick regulations for cell therapy promote centralization of culture units, therefore definition of reliable and practical transportation strategies is vitally important. The second aim of this study was to optimise the transport conditions for limbal biopsies (LBs) and cultured limbal epithelial cells (LECs). Transport of LBs at room temperature proved to be significantly superior to 4°C transport. We also showed that cultured LECs may be stored in serumfree media and transported up to 7 days at 23°C without any negative effect on cell number, viability, colony forming efficiency or gene expression profile. Due to the absence of specific LSC markers, identification and isolation of putative LSCs is a complicated task. The third and final aim of this study was to identify novel cell surface markers for LSCs. We reported herein the identification of a new cell surface marker for LSCs (CD200) as well as a cell surface marker for proliferating progenitor cells (CD109).