A study of the factors influencing the collagenase digestion phase of human and porcine islet isolation
thesisposted on 15.12.2014, 10:29 by Paul Robert Vellacott. Johnson
Human pancreatic islet transplantation offers a potential cure for type 1 diabetes mellitus by restoring normoglycaemia at an early stage of the disease. The crucial stage of the islet isolation process is the collagenase-digestion phase. However, this process is extremely variable and also poorly understood. The aim of this thesis therefore, was to investigate the collagenase-digestion phase of human and porcine islet isolation. The first three chapters consist of reviews of islet transplantation and cell isolation and outline the problems to be addressed. Chapter 4 investigates different aspects of the variability of different batches of crude collagenase. Twelve different batches of crude collagenase were tested on both human and porcine pancreata. Clear differences were noted between collagenase digestions by the different batches and also between the two species. Chapter 5 describes a 'simple in vitro' method that was developed for testing different batches of collagenase or collagenase components on any one mammalian pancreas, thereby controlling for inter-pancreatic variability. Chapter 6 outlines the optimisation of biochemical assays for different components of crude collagenase. Chapter 7 investigates the stability of four different batches of crude collagenase under eight different storage conditions over a six-month period using biochemical assays. Chapter 8 describes a series of experiments investigating the inhibition of crude collagenase by University of Wisconsin solution. Chapter 9 evaluates the influence of different administration pressures on the distribution of collagenase within the porcine pancreas using novel methods with anti-collagenase monoclonal antibodies. Finally, the overall conclusions and suggestions for future research are discussed in Chapter 10.