Design of a simple, in vitro method for evaluating the efficiency of crude Clostridium histolyticum collagenase and its components for porcine islet isolation

Abstract

Abstract: The wide variability of different batches of crude collagenase is one of the major factors influencing the isolation of porcine islets of Langerhans. In order to enable the production of reproducible collagenase batches, however, it is first necessary to determine which of the many components of crude collagenase are required for porcine islet isolation. For this to be done, experiments must control for the inter‐pancreatic variation seen with porcine pancreata, and this requires methodology that enables many different crude collagenases or collagenase components to be tested on any one pancreas. The aim of this study was to assess four different in vitro methods for evaluating different batches of crude collagenase. Each of the methods involved placing blocks of either distended or undistended pancreas in centrifuge tubes, adding crude collagenase in various concentrations dissolved in Hanks' and incubating the tubes in a waterbath at 35°C. The remainder of the pancreas was simultaneously processed using the semi‐automated method. Every 10 min, samples were taken from both the semi‐automated circuit and the relevant centrifuge tubes and assessed for number of cleaved islets, cleavage index, degree of fragmentation and quality of exocrine digestion. A correlation was made between each in vitro method and the “gold standard” semi‐automated method. We conclude that it is possible to use an in vitro method to evaluate crude collagenase that correlates well with the digestion occurring in the Ricordi chamber and that enables the testing of many batches or components of crude collagenase on any one porcine pancreas.