Characterization of an Important Enzymatic Component in Collagenase that is Essential for the Effective Digestion of the Human and Porcine Pancreas

Abstract

Recent clinical results from Edmonton have demonstrated the feasibility of achieving normoglycemia in type I diabetic patients by islet transplantation. One of the key issues in obtaining this success was transplanting sufficient numbers of islets by sequential transplants. Although the development of semipurified endotoxinfree Clostridium histolyticum-derived collagenase (Liberase) has improved islet yields from the human pancreas, batch-to-batch variation and loss of activity with time still hampers progress in obtaining consistent islet preparations. In order to define key components of crude collagenase, a panel of monoclonal antibodies (McAbs) was raised against crude collagenase. Monoclonal antibodies were generated by fusions between splenocytes of BALB/c mice immunized with Boheringer P collagenase and the myeloma cell line NS-0. These monoclonal antibodies were used as probes to study molecular differences between effective and ineffective collagenase batches using Western blotting. Two monoclonal antibodies (LDS71 and LDS81) were raised and characterized as recognizing separate epitopes on a 125-kDa component. Western blotting indicated that the 125-kDa band was rapidly broken down by storage or by dialysis in the presence of dithiothreitol. However, this breakdown could be prevented by the addition of leupeptin (a protease inhibitor) to the dialysis buffer. On testing fractions at 5-min intervals from the "Ricordi" digestion circuit during porcine and human pancreas digestion, the 125-kDa component was rapidly broken down in relatively ineffective collagenase batches but in effective batches was present throughout the digestion process. The correlation between the presence of the 125-kDa band and effectiveness of pancreas digestion suggests that this may be a key component in the formulation of C. histolyticum collagenase.

Flexible Management of Enzymatic Digestion Improves Human Islet Isolation Outcome from Sub‐Optimal Donor Pancreata

“…Reasons for early release of islets are potentially numerous and not mutually exclusive. It has been reported that lot-to-lot differences in Liberase TM activity can account for variations in digestion time (27). In damaged organs, possible isolation factors combine their effects with the longer manipulation required to clamp the pancreatic cuts prior to enzyme injection.…”

Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture

“…The interaction between the two mechanical and two enzymatic digestion steps was crucial for optimal tissue penetration of digesting enzymes on the one hand and enzymatic tissue softening for optimal mechanical effects on the other hand. The most effective enzyme mix comprised collagenase XI, DNAse I and dispase II with efficient proteolytic activity towards interstromal cell connections, collagen type I and hyaluronidase V to hydrolyze glycosidic linkages in hyaluronic acid and elastase to address elastin fibers which are associated with lysyl oxidase-like 2 mediated tumor migration [27][28][29][30][31]. Cell culture media and added growth and differentiation factors play a pivotal role in successful cell culture initiation and should reflect in vivo conditions as realistic as possible [32].…”

Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture

“…This harmful effect acts in a time‐dependent manner and can be due to the direct effect of crude collagenase on cells and not some degraded components produced during the enzymatic digestion of connective tissue because the same pattern of cell death was observed in in vitro established dermal fibroblasts, which revealed that extracellular matrix is not only the target of crude collagenase but may also directly influence some molecules such as membranous collagen present on the cell surface, thus leading to a higher rate of death among cells with a longer time of incubation with this enzyme. Also, regarding the presence of other components in crude collagenase (nonspecific proteases, clostripain, neutral protease, aminopeptidase activities, and trypsin), it is possible that some of them, such as proteases, have a detrimental effect on cell survival 24–29. However, as protease accelerates tissue dissociation, it should be considered as a double‐edged sword that can damage cells subsequent to prolonged exposure to crude collagenase 30.…”