Assessment of Islet Isolation Efficacy in Dogs

Abstract

The outcome of islet isolation is considered uncertain because of the large variability of islet and insulin yield, but comparison of the isolated and native islet population has not been attempted. We therefore addressed the efficacy of collagenase digestion, and density gradient purification of islets from the splenic dog pancreas (n = 31) by morphometry of the islet volume and size distribution, and by extraction of insulin and amylase, in samples from the pancreas, the digest, and gradient fractions. In contrast to a ~90% recovery of pancreatic insulin and amylase after digestion, islet yield amounted to 50% of the islet content of the pancreas. After density separation, islets were mainly found in the purified fractions, while half of the recovered insulin was located in the acinar fraction of the gradients — indicating a substantial proportion of islets entrapped in acinar fragments. The islet and insulin content of the pancreas correlated well with islet and insulin yield after digestion (r = 0.7, p < .0001). The insulin content of digest suspensions did neither correlate with islet nor insulin recovery in the purified fraction of the gradients (r = 0.4) as opposed to the islet content of digest suspensions, which correlated with both (r = 0.7, p < .0001). After density separation near 100% purity was obtained, and no loss of insulin from isolated islets was demonstrated by extraction and microscopy. Size distributions of native and isolated islets demonstrated no fragmentation. We conclude that the variability of isolation outcome may be attributed to a large extent to the variability of the native endocrine pancreas. Isolation efficacy was best documented by morphometry, because insulin extraction did not discriminate between free and entrapped islets. However, assessment by both morphometry and extraction allowed the quantitation of entrapped islets, and demonstrated preservation of β-cell granulation. Similar studies should facilitate the analysis of other factors affecting islet isolation in man.

A Comparison of Cross Sectional Surface Area Densities Between Adult and Juvenile Porcine Islets of Langerhans*

“…However, a previously unreported finding from this study were the significant differences (p = 0.001) between the mean CSSA density between pancreata of adult pigs of approximately the same age and species but not between juveniles (p=0.055), and one can assume that this would have a profound influence upon islet isolation and can thus explain some of the extreme variabilities seen in islet yield. Similar significant differences in the insulin content of pancreata have been reported in both human [21] and dog [22] by using insulin extraction. Futhermore, the percentage CSSA density showed an increasing trend in juvenile pigs (2.13%) compared to that of adults (1.83%).…”

Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation

“…This inconsistency is well known, especially for porcine isolation (Burghen and Murrel 1989). The quantity of islets harvested depends on the initial number of islets contained in the pancreas (Van der Burg et al 1994), age, race , and nutritional factors (Ernst et al 1993;Kirchhof et al 1994). In our work, discrepancies are frequently noticed between the seemingly intact LM appearance and the abnormal morphological features of the same islets in EM.…”

Pancreatic Islets

“…Pigs are beneficial in several aspects, most profoundly owing to their relatively small size as far as farming considerations are regarded (minipigs are as small as 10-30 kg), and the advances in genetic manipulation allow researchers to obtain pig tissues with low immunogenicity 22 . Similar approaches are employed for the isolation and culture of dog islets 25 , as well as sheep 26 , goat 27 , monkey 28 and cow islets 29 . Technically, pig islets have gained the reputation of being particularly difficult with respect to achieving consistency across isolation sessions 23 .…”