Collagenase H is Crucial for Isolation of Rat Pancreatic Islets

Abstract

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by <i>Clostridium histolyticum</i>

“…This conclusion was also confirmed in our study, where the best results were achieved with a batch from Group 3 containing intact and fragmented collagenase in combination with neutral protease (NP) and clostripain. Previously published studies [11] [13] showed that NP and clostripain can compensate reduced C1 activity; however the results of our study revealed successful islet isolations with batches without the presence of collagenase. Potentially deteriorate influence of NP on islet viability was not fully confirmed in our study except decreased vitality and lover glucose stimulated insulin secretion 24 hours after isolation in Group 2 but not in Group 3.…”

“…Clostridium collagenases C1 and C2 hydrolyze collagen chains sequentially from the ends into mixture of small peptides [9]. It was reported that C2 was more efficient in collagen degradation [10] whereas C1 played only supporting role in islet isolations [11]. Study by Brandhorst et al [12] showed that neither C1 nor C2 alone were able to dissociate pancreatic tissue.…”

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

“…Pancreatic islet transplantation is currently regarded as one of the treatment options for severe diabetes. 26 Considering that the clinical applications of HTx remain limited, 2,7 a number of experiences originating from islet transplantation, including cell isolation methods, 21,27,28 culture systems, 21,29 cell preservation, 18,21,22 cell graft evaluation, 30-32 and engraftment 33-35 could be applicable for improving the outcomes of HTx. Per the above-noted concepts, in the present study, the TD was applied to hepatocytes and was found to be effective for maintaining the viability and function of hepatocytes during a short-term preservation period.…”

Evolution of Enzyme Requirements for Human Islet Isolation

“…Reports on the three-dimensional structure of collagenase focused primarily on C1 collagenase since there is minimal information on the role C2 collagenase plays in the tissue dissociation process. C2 collagenase has been shown to be more effective in rodent islet isolation than C1 collagenase [72,73] but this same effect was not observed with porcine islet isolation [74]. Moreover, biochemical studies using purified natural C1 and C2 collagenase to degrade collagen fibrils [45] and a recent report using a 30:70 C1:C2 recombinant collagenase-BP Protease enzyme mixtures [54] have shown the effectiveness of C2 in the degradation of collagen fibrils and human islet release, respectively.…”