Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets

Abstract

One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.

Keywords: Clostridium histolyticum; class I; class II; collagen degradation activity; collagenase; islet isolation; porcine islet; tissue dissociation.

Figure 1.

Impact of Molecular Form of Recombinant Class I Collagenase [i.e. intact (rC1_116kDa) vs truncated protein (rC1_{100 kDa})]on Digestion of the Porcine Pancreas. Split pancreas experiment, n = 6. CDA activity was targeted at 25,000 CDA U/gram in a standardized Ricordi procedure for both molecular forms of recombinant C1. Wunsch activity was provided by recombinant C2 and maintained at 7.5 Units/gram. a, b, c, respectively, % undigested tissue, packed tissue volume (PTV) and average islet yield (IE/g) were equivalent between the two molecular forms of C1. d To match CDA activity of the rC1, approximately 13-fold higher protein concentrations were required for the C1_{100 kDa}, * P < .001