| Summary: | 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-l receptor antagonist peptide was synthesized and characterized using mass spectroscopy (MS), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Anti-inflammatory properties were investigated using ELISA detection of IL-8, IL-6 and IL- l ~ in keratocytes and LESC following stimulation with either lipopolysaccharide or recombinant human IL- l~. Feasible delivery of cells and peptide on a contact lens was investigated to assess the possibility of an "all in one" graft. Results showed that organ culture stored tissues can provide 100 % successful cell cultures using current techniques in terms of reaching confluence and maintaining LESC morphology and phenotype. Sub-culturing and cryopreservation of cultures however did not produce confluent cell sheets, as required for clinical application. The anti-inflammatory peptide was shown to effectively suppress production of key inflammatory cytokines in LESC and keratocytes by acting as an IL- l receptor antagonist and interrupting the IL- l inflammatory pathway. Binding of the peptide to the contact lens was shown to be possible. Such a scaffold also supported expansion of LESC. However, the 2.7 nmol of peptide bound to the lens did not significantly lower cytokine production. Findings suggest that it is possible to culture adequate numbers of LESC for the initiation of a stem cell bank using current techniques. However, modifications to culturing methods are needed to ensure successful sub-culturing and cryopreservation. The peptide has been shown to be effective in reducing inflammatory cytokine production, providing a possible alternative to steroids. An a11-in-one graft could provide a key development in treating LSCD. However further work is required to optimize the peptide concentration to allow effective inflammation control.
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