An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation

Abstract

Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.

Harmful Delayed Effects of Exogenous Isolation Enzymes on Isolated Human Islets: Relevance to Clinical Transplantation

“…During dissociation, the free tissue, including free islets, is collected over a period of 1-2 h in which digestive enzymes are present in the milieu, although in progressively lower concentrations. A prolonged exposure of islets and acinar cells to digestive enzymes unquestionably causes cell destruction (7,20). To limit its occurrence, it is necessary to maintain a tight control on the digestion process (21-23).…”

“…Reduction in temperature and extensive dilution, all common procedures adopted to inactivate digestion enzymes, may not be sufficient to reduce exposure and overdigestion. It is evident that prolonging digestion of the pancreatic tissue leads to exacerbated islet damage (7). However, less emphasis has been traditionally given to the delayed effects of exogenous enzyme exposure on the islets that survive 'the isolation phase'.…”

Assessment of six different collagenase-based methods to isolate feline pancreatic islets

“…Accutase, in addition to collagenase, contains a mixture of other proteases that contribute to enhance tissue dissociation (Wolters et al, 1992).…”

Bioartificial Pancreas: Evaluation of Crucial Barriers to Clinical Application

“…In rodent and human, the situation is intermediate with a tendency towards predominance of cell-matrix adhesion. The presence of protease in the collagenase preparations has been reported to reduce the yield and quality of isolated islets in rats (Vos-Scheperkeuter et al, 1997), however it is more efficient for the isolation of pig islets (Deijnen et al, 1992). Figure 2 shows the effect of transplanted islet mass on short-and long-term glycemic normalization in a rat.…”