Advanced stem cell delivery systems for the treatment of corneal epithelial limbal stem cell deficiency

Abstract

Limbal stem cell deficiency (LSCD) can be treated successfully using ex vivo limbal epithelial stem cells (LESC) derived from cadaveric donor tissue. However, shortages in such tissues and graft rejection, resulting from inflammation, are persistent issues. The purpose of this study was to optimize current culturing techniques used for LESC transplant tissue, considering expansion and cryopreservation issues surrounding the establishment of a stem cell bank. In addition, a novel anti-inflammatory biomimetic peptide was investigated to address issues surrounding amnion and steroid use in LESC transplantation, inflammation and transplant rejection. Cell cultures derived from Optisol and organ culture stored tissues were examined for optimum growth, characterized by an ability to grow to 70 % to 80 % confluence while maintaining epithelial cell morphology and the presence of positive and negative LESC markers (K3, K19, p63 and PAX-6) as identified by immunocytochemical staining and QRT-PCR. Furthermore, the effect of culture expansion and cryopreservation on stem cell characteristics was examined. A short chain IL-1 receptor antagonist peptide was synthesized and characterized using mass spectroscopy (MS), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Antiinflammatory properties were investigated using ELISA detection of IL-8, IL-6 and IL-1β in keratocytes and LESC following stimulation with either lipopolysaccharide or recombinant human IL-1β. Feasible delivery of cells and peptide on a contact lens was investigated to assess the possibility of an “all in one” graft.

Date of Award	2014
Original language	English
Awarding Institution	University of Brighton