Purified enzymes In pig pancreas dissociation for islet Isolation: Preliminary observations

Abstract: We have previously shown that high islet yields are obtained by digesting the rat pancreas with a defined mixture of purified collagenase and protease. In this study we investigate whether a similar approach may be applied to the pig pancreas. We have used a method for the in vitro evaluation of different enzymes on a single pancreas that was recently developed in our laboratories. Blocks of pig pancreas were placed in centrifuge tubes and incubated in a waterbath at 35°C in Hanks containing either i) crude collagenase, ii) purified collagenase obtained from the crude collagenase by chromatography or iii) purified collagenase and protease, at comparable collagenolytic and proteolytic activities. Samples were assessed for number of clean islets, cleavage index, islet integrity, and degree of exocrine dissociation. The experiments were performed on five pig pancreata. The results do not indicate a clearcut advantage of one of the enzyme formulas. There is considerable variation in results of different experiments with the same enzyme formula. The parameters mainly indicate that a substantial number of pig pancreatic islets may be neatly liberated from the exocrine tissue without adherent exocrine tissue. They also indicate, however, that these islets are readily subject to fragmentation with any of the three enzyme formulas tested. In conclusion, the pig pancreas can be dissociated with purified enzymes, which warrants further investigation since the variable composition of crude collagenase contributes to the poor reproducibility of islet isolation procedures. © 1995 Munksgaard