Influence of Different Ratios of Purified Class I and Class II Recombinant C. histolyticum Collagenase on Human Pancreas Digestion and Islet Yields When Using with Purified P. polymyxa Protease Enzyme Mixtures.

A major factor that influences the success of human islet isolation is the consistency of the purified Clostridium histolyticum collagenase and bacterial protease enzymes used for the islet isolation procedure. The current study was designed to determine the impact of different ratios of recombinant (rec) C. histolyticum class I (C1) and class II (C2) collagenase on human islet yields isolated using a fixed amount of P. polymyxa protease. Human islets were isolated from split pancreas experiments using research pancreases (n=12). Purified natural (Vitacyte CIzymeTM Collagenase HA) and recombinant collagenase C1 and C2 (Vitacyte), and purified P. polymyxa protease (CIzymeTM BP Protease) and C. histolyticum neutral protease (SERVA-CHNP) were used. In all experiments, the pancreas was split into head and tail sections with each side digested with a collagenase-protease enzyme mixture and free islets counted prior to and after density gradient purification. The same amount of neutral protease activity was used for head or tail islet isolations with CHNP and P. polymyxa protease used in the head and tail, respectively. Different doses of rec C1 collagenase collagen degradation activity (CDA) units and rec C2 collagenase Wunsch units (WU) were used in the tail to assess the influence of each collagenase isoform on the islet recovery. Each head, islet isolation used a standard dose of natural collagenase (CIzymeTM) with CHNP to assess the suitability of each organ for islet recovery. Islets were subjected to standard quality control assays.The results (mean ± std dev) are summarized in the following table. Three independent islet isolations were performed for each combination of enzymes.Table: No Caption available.Purified P. polymyxa protease in combination with recombinant collagenase was successfully used to recover human islets. The ratio of recombinant C1 and rec C2 used for these experiments did not have a significant effect on recovery of islets from the organ. Normal pancreas digestion profile with >5000 IEQ/g pancreas was obtained with this enzyme blend. High islet yield can be obtained from sub-optimal pancreas utilizing different ratio of C1 and C2 collagenase. DISCLOSURES:Green, M.: Employee, Supplying enzymes. Breite, A.: Employee, Supplying enzymes. Dwulet, F.: Employee, Supplying enzymes. McCarthy, R.: Employee, Supplying enzymes.