Tissue dissociation enzyme (TDE) products have not been improved for over 50 years. "Crude collagenase" products contain collagenases and other proteases used to free cells from collagen rich, extracellular matrix. They are derived from minimally purified culture supernatants of Clostridium histolyticum. Collagenases are the key enzymes; they alone initiate the degradation of collagen. Once initiated, other proteases in the product help it rapidly degrade collagen. TDE products can be improved significantly if the key enzymes responsible for collagen degradation from C. histolyticum are identified, purified, and optimized to maximize the yield and quality of specific cells recovered from tissue. This approach led to the development of Liberase HITM, which was shown to be significantly superior to crude collagenase in recovering islets from human pancreata. This improvement contributed to the success of the "Edmonton protocol", a breakthrough in using islet transplantation to treat adult, Type I, brittle diabetic patients. Further improvements are required in the development, manufacture, and application of purified, islet specific; TDE's to increase islet recovery from human pancreata. Currently, most centers use 2-4 organs to recover a sufficient number of islets for transplantation. This ratio must be reduced to 1:1 organ donor to recipient ratio to move this procedure from research to routine clinical practice. The 3 aims of my Grant address this problem. 1) I will prepare a purified, islet specific TDE mixture that meets specifications used in clinical islet transplantation protocols. Higher quality collagenase will be used in this preparation based on improvements in the raw material, purification, and characterization of the purified enzymes. This TDE preparation will be compared to similar products for its stability, enzyme activity, and ability to recover islets from human pancreata. 2) I will develop a robust assay to assess collagenase in mixtures containing other proteolytic enzymes. This assay will assess the quality and stability of current TDE products used to isolate islets. It will also be used to monitor changes in collagenase during the course of human pancreas digestion. 3) I will identify those enzymes, which efficiently degrade native collagen. This fills a gap in the scientific literature since highly purified collagenase and other proteases will be used in these experiments. This knowledge offers the potential to use fewer, more effective enzymes in TDE mixtures, leading to increases in the yield and quality of cells recovered from any mammalian organ.
Thesaurus Terms: Clostridium, collagenase, enzyme activity, pancreatic islet, protein degradation, protein purification, technology /technique development enzyme mechanism, extracellular matrix, isozyme, protein denaturation, protein isoform biotechnology, clinical research, human tissue, mass tissue /cell culture, microorganism culture
