Porcine Islet Isolation, Cellular Composition and Secretory Response

Abstract

Porcine islets were isolated by infusion of a warm collagenase solution into whole pancreata followed by static incubation at 37 degrees C for 15 minutes. The pancreata were then chopped into small pieces and the free islets purified by filtration and centrifugation over a ficoll gradient. The insulin:amylase ratio of the islets compared to that in the intact pancreas was determined in 19 pancreata and indicates that the isolated islets were of a high degree of purity. The distribution of insulin, glucagon, somatostatin and pancreatic polypeptide containing cells in pig pancreas sections was compared with that in rat. Porcine islets were much smaller and less well defined than rat islets with infiltration of acinar material even into the islet core. The levels of insulin, glucagon and somatostatin in porcine pancreas and isolated porcine islets were measured using conventional radioimmunoassay techniques. The ratio of these hormones in the pancreas was 105.1:5.8:1 respectively, and in the islets 105.1:0.68:0.087 respectively. Fragmentation of the islets during the isolation may have led to the loss of glucagon and somatostatin-containing cells. Islets cultured overnight and tested with a range of glucose concentrations for one hour did not show a significant stimulation of insulin secretion in the presence of 8.3 mM or 16.7 mM glucose compared to that in 2.8 mM glucose. However freshly isolated islets challenged with 8.3 mM, 13.9 mM and 22.2 mM glucose showed a 1.8 fold, 2.0 fold and 2.3 fold response respectively, over that in 2.8 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological and insulin secretory studies on isolated porcine islets of Langerhans

“…We found that porcine islets with low basal percentage secretion rates (0-7 + 0-04%) still gave less than a twofold increase in insulin secretion in the presence of 16-7 mmol glucose/1 (1-3 + 0-13%). We have previously reported that por¬ cine islets lose a large proportion of their glucagon content during the isolation procedure (Crowther et al 1989). This hormone is thought to play an import¬ ant role in maintaining cyclic AMP within the cell at a level which allows the cell to respond fully to stimuli for insulin secretion (Schuit & Pipeleers, 1985).…”

“…Porcine islets were isolated using a modified version of the technique described by Crowther et al (1989). Briefly, warm collagenase was injected into the pancreas via the pancreatic duct.…”

“…However, they have a lower secretory response to these compounds compared with that reported for human islets. Monoclonal antibody and human sera binding studies show that rat and porcine islets share some cell surface antigens but it remains to be seen whether the inability of porcine islets to discriminate between diabetic and non-diabetic sera is an advantage for transplantation, indicating less like- INTRODUCTION Porcine islets represent the best alternative to human islets for the possible treatment of human insulindependent diabetes mellitus by transplantation, since there is easy availability of porcine pancreata, poten¬ tially high yields of islets per pancreas (Crowther, Gotfredsen, Moody & Green, 1989), and a close similarity between porcine insulin and the human hormone. Earlier papers have given details of porcine islet isolation protocols (Ricordi, Finke & Lacy, 1986; Marchetti, Zapella, Giannarelli et al 1988), the most detailed being from Crowther et al (1989).…”

Insulin-releasing pituitary cells as a model for somatic cell gene therapy in diabetes mellitus

“…Sections were also ex¬ amined for insulin-like immunoreactive fluorescence us¬ ing guinea-pig anti-bovine insulin antibody (1:300 at 4°C overnight; raised at the University of Sussex by I C G) and fluorescein-conjugated anti-guinea-pig antibody from goat (1:16 at 37°C for 1 h). Non-specific binding was blocked by prior exposure of the sections to 10% goat serum for 10 min (Crowther et al 1989). …”

Pig islets for clinical islet xenotransplantation

“…In contrast to foetal and neonate cells, islets from adult pigs are able to respond to hyperglycaemia within hours after transplantation [36][37][38][39]. Several studies, however, reported low insulin secretion from isolated adult pig islets [29,36,[40][41][42][43][44][45][46][47].…”