AbstractInsulin producing cells are vulnerable to damage at several key stages during clinical islet transplantation. This damage is caused by a number of factors; enzymatic-digestion during islet isolation, post-transplant exposure to hypoxic condition, and toxicity from immunosuppressant therapy. The enzymatic isolation process disrupts islet cell membranes, damages cell-to-cell interactions and inhibits vital functions. Extracellular Matrix (ECM) is a complex 3D-network of proteins and polypeptides and an essential component of the islet cell basement membrane (BM). We have developed a novel bioreactor, rotary cell culture system (RCCS) in order to study the molecular remodelling of Min6-Pseudoislets (PIs) following enzymatic digestion. The initial aim was to study the biology of PIs within our controlled cell culture environment RCCS and then to investigate the effects of digestive enzymes on PIs-ECM contacts and to determine if the novel 3D- microgravity cell culture system could restore and enhance ECM expression. Thus, PIs-ECM expression, localization and remodelling was analysed pre-and post-enzymatic digestion using qRT-PCR (for gene expression), western blotting (for protein expression) and immunocytochemistry (for cellular localization) techniques. Results showed that our RCCS system had a beneficial effect on PIs structure and function compared to standard culture conditions. Also, qRT-PCR gene analysis and immunoblotting of protein content of PIs detected alterations in ECM expression levels. Observed changes included increased expression of Fibronectin, Collagen IV, and Laminin V in PIs cultured in the RCCS system. PIs digestion with collagenase-accutase enzyme was optimized such that cell viability was not affected but cellular stress and subsequent remodelling of ECM expression was achieved. Insulin gene expression and insulin release in PIs were significantly increased in response to high glucose. These responses were significantly decreased following enzymatic digestion but recovered following subsequent culture in the RCCS-bioreactor system. This recovery of insulin secretory function was directly associated with increased ECM expression as demonstrated by enhanced cytoplasmic staining in the remodelled RCCS-cultured PIs. Our results confirmed that a controlled microenvironment optimized to promote cell survival and recovery is able to control the detrimental effects of enzymatic and mechanical stress. The ECM is a very good indicator of cellular integrity and function in PIs and it has a vital role in sustaining glucose-responsive insulin release. Our RCCS-bioreactor system has the potential to restore the lost ECM expression in PIs following enzymatic digestion. We believe that ECM interaction is an accurate biomarker for functional islets integrity and by maintaining high levels of its expression we hope to be able to improve islet cells graft function for current clinical islet transplantation programmes.
|Date of Award||2016|
Cell Culture on Extracellular Matrix Expression, Islet Remodelling, and Insulin Secretory Function Following Enzyme Digestion
AL-Hasawi, N. (Author). 2016
Student thesis: Doctoral Thesis