Distribution of collagens type I, type III and type V in the pancreas of rat, dog, pig and man.

The presence of collagens type I, type III and type V was determined immunohistochemically in pancreatic tissue of rat, pig, dog and man. The reaction to anti-collagen type I is weak (pig, dog) or moderate (rat, man) in the peri-insular region and in the lobar, lobular and acinar septa, whereas the reaction to anti-collagen type III is well developed. In rat and dog, the latter reaction deposit on the lobar and acinar septa is prominent. These elements only show a moderate reaction intensity in pig and man. The peri-insular region displays a weak (rat, dog, man) or very weak (pig) reaction against collagen type III. Anti-collagen type V reacts moderately (rat, dog, man) or weakly (pig) in the lobar and lobular septa. The acinar septa show a moderate (rat, dog, man) or very weak (pig) reaction. This information regarding the types and distribution of the collagenous compounds in pancreatic extracellular matrix could lead to differentiated enzymatic pancreas dissociation and, ultimately, increased islet yield and improved reproducibility of pancreatic islet isolation procedures for transplantation purposes.

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.

Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig, and man.

The presence and distribution in the peri-insular region of extracellular matrix, and in particular basement membrane, was investigated in a comparative study comprising pancreata of rat, dog, pig, and man. Basement membrane markers, collagen type-IV and laminin, were determined immunohistochemically. Additional information pertaining to the structural relationships between endocrine and exocrine pancreas, in particular cell-to-cell and cell-to-matrix contacts, was obtained by electron microscopy. In pig, very little peri-insular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas almost exclusively depends on cell-to-cell adhesion. In the canine pancreas, the islets are almost completely encapsulated with very little direct exocrine-to-endocrine cell-to-cell contact. In rat and man, the situation is intermediate with a tendency towards predominance of cell-to-matrix adhesion. The intra-insular adhesion mechanisms depend largely on cell-to-cell adhesion in all four species. The ultrastructural results suggest that collagenase preparations employed in islet isolation procedures should be of high purity as to preserve the protease-sensitive intra-islet cell-to-cell adhesion. Under these conditions, however, the endocrine-to-exocrine cell-to-cell contacts will be conserved also, resulting in an exocrine-tissue contamination of the islets of Langerhans. Consequently, additional steps for the effective removal of exocrine tissue and the purification of islets are required.

Factors influencing the isolation process of islets of Langerhans.

Determination of collagenase and proteolytic activities and slabgel electrophoresis of collagenase preparations obtained from different suppliers reveals a highly variable content of collagenase, proteolytic activities and other components. The electrophoresis pattern can help to select collagenase preparations with a low content of impurities suitable for islet isolation. During islet isolation the exocrine tissue releases large amounts of proteolytic activities into the digestion medium, which decreases islet yield. Addition of 10% albumin to the digestion medium strongly suppresses the release of proteolytic activity and increases the number of islets obtained. Addition of both albumin and trypsin inhibitors further increases not only the number of islets, but also the total volume of islet tissue obtained. The islet isolation process is not critically dependent on the pH in the range 6.2 to 8.0 and only marginally dependent on the calcium concentration.