Isolation of porcine pancreatic islets: low trypsin activity during the isolation procedure guarantees reproducible high islet yields.
During the past few years, interest in xenotransplantation of porcine islets of Langerhans for the future therapy of type I diabetes has increased markedly. Therefore, we established a semiautomated digestion method for isolating islets from the porcine pancreas. However, although the isolation technique was standardized and collagenase of controlled quality was used, we were unable to attain high islet yields with a satisfactory degree of reproducibility. One hypothesis was that varying degrees of interference by donor pancreatic enzymes were responsible for this failure. The aim of this study was to examine the kinetics of four types of enzymatic activity during the isolation procedure, as well as their effects on islet yield: collagenase, trypsin, neutral protease, and clostripain. Our results indicate that while exogenous collagenase activity decreases slightly during the isolation procedure, the activity of the pancreas enzymes neutral protease and trypsin increases. In some cases, trypsin activity increases very strongly. A strong increase in trypsin activity correlates with poor islet yield, whereas low trypsin activity always correlates with high islet yield. Addition of the protease inhibitor Pefabloc to the isolation medium results in low trypsin activity and reproducible high islet yields.
Heiser
A
A
Institute of Immunology, Medical School, University of Kiel, Germany.
Ulrichs
K
K
Müller-Ruchholtz
W
W
D000818:Animals; D005260:Female; D007515:Islets of Langerhans / Q000201:enzymology*; D016381:Islets of Langerhans Transplantation; D013450:Sulfones / Q000494:pharmacology; D013552:Swine; D014357:Trypsin / Q000032:analysis*
D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D014357:Trypsin