An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation.
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.
Wolters
G H
GH
Department of Surgery, University of Groningen, The Netherlands.
Vos-Scheperkeuter
G H
GH
van Deijnen
J H
JH
van Schilfgaarde
R
R
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007202:Indicators and Reagents; D007515:Islets of Langerhans / Q000166:cytology*; D007700:Kinetics; D008297:Male; D003012:Microbial Collagenase / Q000302:isolation & purification / Q000378:metabolism*; D010179:Pancreas / Q000166:cytology*; D051381:Rats; D011919:Rats, Inbred Strains
D007202:Indicators and Reagents; D010450:Endopeptidases; D003012:Microbial Collagenase