VitaCyte LLC is working to improve tissue dissociation enzymes (TDE) required for the isolation of insulin producing pancreatic islet cells from both human and porcine organs. Transplantation of islets into Type 1 Diabetic patients represents a promising therapy as means to replace administration of insulin by external factors. The development of the Edmonton Protocol demonstrated insulin independence in patients transfused with purified pancreatic islets. Insufficient islet yields arising from inconsistencies in TDE manufacturing have plagued continuing attempts to transfer this promising therapy to routine clinical practice. Improved biochemical assays have been used to correlate collagenase molecular forms with tissue dissociation. These data indicate the importance of each molecular form for different tissue types. The goal of this project is to develop a robust and reproducible manufacturing process using recombinant TDE that will improve the quality and commercial availability for both human allotransplantation and porcine xenotransplantation. Specifically, three recombinant constructs of Clostridia histolyticum collagenases: intact Class 2 (C2intact), intact Class 1 with two collagen binding domains (C1intact), and degraded C1 with a single collagen binding domain (C1100kDa) will be generated using standard molecular biology techniques. A large scale bacterial fermentation and expression production protocol will be developed by adopting current industry practices for production scale manufacturing to optimize expression yields. A column chromatography purification protocol will be developed using modifications to existing purification methods currently used in natural collagenase production. This process will be scaled for production of highly pure recombinant enzymes for use in islet isolation procedures. Finally, an assessment of the different molecular forms and their role in islet isolations will be determined in both human and porcine tissue both internally and at leading islet isolation centers. Using a standard modified Ricordi islet isolation procedure, the specific molecular form will be correlated to successful islet yield using design of experiment statistical control methods. PUBLIC HEALTH RELEVANCE: Pancreatic islet transplantation represents a promising therapy for Type 1 Diabetic patients as a replacement for traditional insulin therapies. Hurdles to moving this therapy to routine clinical practice lies in achieving greater islet yields for either human allotransplantation or porcine xenotransplantation. VitaCyte is working to develop a robust and reproducible manufacturing process using recombinant tissue dissociation enzymes to improve islet isolation procedure yields.