Successful long-term cryopreservation of highly purified canine islets.

This study evaluated the ability of cryopreservation to store large quantities of canine islets for transplantation studies. Islets were isolated by automated screen methods and purified by Euro-Ficoll gradients. After overnight 37 degrees C incubation, islets were equilibrated with 2 M dimethylsulfoxide, cryopreserved at a cooling rate of 0.25 degree C/min and subjected to long-term storage in liquid nitrogen. Six months later, some islets were thawed at a rate of 180 degrees C/min. The viability of cryothawed islets was determined in vitro by comparing islet count, total insulin content, morphology and perifusion studies in both control and cryothawed islets, and in vivo by transplantation into diabetic nude mice. The percentages of recovery of both islet count and total insulin content were 74.17 +/- 4.46 and 70.66 +/- 14.08, respectively. Islet morphology after cryothaw by light and electron microscopy revealed structurally intact islets with varying degrees of granulation. The results of perifusion indicated that there was no significant difference (p > 0.1) in both total stimulated insulin secretion and stimulation index response to high-dose glucose plus 3-isobutyl-1-methylxanthine and carbachol of isolated islets as compared to those of cryothawed islets. Transplantation into nude mice proved that grafted islets can successfully reverse diabetes. In conclusion, these findings indicate that the majority of purified canine islets can survive the frozen-thawed insult while maintaining their secretory function and permitting mass storage of canine islets for further transplant studies.

cAMP-enhancing agents "permit" stimulus-secretion coupling in canine pancreatic islet beta-cells.

Isolated canine islets of Langerhans differ from isolated islets of other species (including rodents and man) in that elevated glucose concentrations are unable to stimulate insulin secretion. Here we demonstrate that addition to the perifusate of isobutylmethylxanthine (IBMX), forskolin or 8-CPT-cAMP, all of which enhance cytosolic cAMP, permits insulin secretion in response to glucose, leucine or tolbutamide. These cAMP enhancers increase secretogogue-induced electrical activity in beta-cells and restore depolarization-induced, Ca(2+)-dependent granule exocytosis measured as stepwise increases in membrane capacitance. We propose that the primary permissive action of cAMP is to tightly link Ca2+ entry to insulin granule release, while a secondary action is to tighten the link between glucose metabolism and cell depolarization.

Xenotransplantation of microencapsulated canine islets into diabetic rats.

Islets of Langerhans were isolated in high yields from canine pancreata. In the procedure, the pancreata were perfused and digested with collagenase, and the islets were then purified on histopaque density gradients. As many as 60,000 islets were isolated from a single pancreas. Islets were encapsulated in alginate-polylysine-alginate membranes with the aid of an air-jet droplet generator. In vitro studies demonstrated that the isolated and encapsulated islets secreted insulin in response to glucose and IBMX challenge for at least 9 weeks. In in vivo studies 6 diabetic Wistar rats were transplanted with 5,000 to 8,000 encapsulated islets each. The diabetic condition was reversed in all recipients for up to 112 days. In control animals, which received free, unencapsulated islets, the xenografts remained functional for fewer than 21 days. Microcapsules retrieved from normoglycemic transplant recipients 1 and 2 months posttransplantation were shown to contain viable islet tissue, and no cellular overgrowth was observed on capsular surfaces. The results of the study indicate a considerable clinical potential of microencapsulated canine islet xenografts.

Mass isolation of highly purified canine islets using an automated method and Histopaque Ficoll gradients.

Eighteen pancreata from adult mongrel dogs were used for the study of islet isolation. The pancreas was distended with collagenase in Hanks' solution. The automated screen method and Histopaque Ficoll gradients were used to isolate and purify the canine islets. In vitro, the viability of isolated islets was assessed by both histology and perifusion studies. In vivo, the islet function was evaluated by using a nude mice xenograft model. Fair to good isolation and purification was found in 12 experiments. Before and after purification, the isolated islet count was 4767.1 +/- 560.1 and 3637.7 +/- 333.4 islet equivalence (I.E.)/gm pancreatic tissue. The purity was above 90%. Aldehyde Fuchsin stain disclosed islets with copious beta granules. The stimulation index of islets responding to 16.7 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine (IBMX) versus 1.67 mM glucose was 12.93 +/- 4.75. Normoglycemia was restored and maintained for up to 2 weeks in 7 of 10 and up to 3 weeks in 5 of 10 diabetic nude mice transplanted with canine islets. In conclusion, the automated screen method and Histopaque Ficoll gradients afford a good yield of highly purified canine islets, and functional viability was verified both in vitro and in vivo. This will be an ideal model for isolation of human islets.

Microencapsulated pancreatic islets: a pathologic study.

Dog pancreatic islets isolated by an enzymatic digestion method were encapsulated in an alginate-poly L-lysine-alginate membrane. These microencapsulated pancreatic islets were cultured in vitro to study their ability of insulin secretion. Portions of these in vitro-cultured microencapsulated pancreatic islets were taken out for a viability dye exclusion study as well as for pathologic studies to correlate them with insulin secretion ability. We found that there was a strong correlation between them. Good insulin-secreting microcapsules showed well-preserved cell membranes and beta-cell granules. An in vitro culture for one to two days in RPMI-1640 made the islets more stable, the cellular surface became smoother and the beta-granules were in better shape. The microencapsulated pancreatic islets were also injected into the peritoneum of streptozotocin-induced diabetic CDF1 mice. Blood glucose levels dropped and stayed low for up to 60 days. But, when non-encapsulated dog pancreatic islets were used, the blood glucose levels remained low for only about 14 days. A small portion of the injected microcapsules were washed out at specific times for pathologic study. Up to 28 days after injection, only a few of the injected microcapsules showed pericapsular cellular infiltrate. However, after 56 days, most of the microcapsules showed dense pericapsular cellular infiltrate. Immunohistochemical analysis of these infiltrates showed that the majority of cells were fibroblasts and macrophages. Most of the cells located in the inner portion of the infiltrate were fibroblasts, while the macrophages were located mainly on the outer portion. Both scanning and transmission electron microscopy showed that the surface of the microcapsule outer wall was much smoother than the inner wall. The size of the microcapsules was approximately 0.6-0.8 mm and the thickness of the wall measured around 10 nm. The smaller the microcapsule is, the less chance there is of rupture with release of the xenographic islets. Once the wall of the transplanted microcapsules was ruptured, the inner surface showed more increased inflammatory cell and fibroblast infiltration than the outer surface.

Morphometric studies of parietal cells in cats before and after lesser-curvature seromyotomy.

A modified, highly selective vagotomy-seromyotomy of the lesser curvature of the stomach was performed on five groups of cats. The horseradish peroxidase (HRP) tract-tracing method was used to detect the regeneration or reinnervation of vagal nerve branches. Morphological changes to the parietal cells and to the gastric mucosa were also examined by light and electron microscopy. Following surgery, the cats were sacrificed at the fourth, eighth, twelfth, sixteenth and the twentieth week. At the sixteenth week, partial regeneration of vagal nerve branches was found. Between the fourth and the twelfth week there was a significant increase in the number of parietal cells per 0.1 mm-wide of mucosa column and in the volume fraction of the mucosa made up of parietal cells. Of the four types of parietal cells, "stimulated", "partially stimulated", "returning" and "resting", the resting type was predominant after seromyotomy, especially between the fourth and the twelfth week. Based on the above observation, we concluded that the modified lesser-curvature seromyotomy depresses the function and responsiveness of the parietal cells despite an increase in their number and in their volume fraction.

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum.

The effects of bovine parathyroid hormone (bPTH1-84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 nM bPTH1-84, there was an approximately 2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 nM bPTH1-84 to tissues pretreated with IBMX resulted in an approximately 3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 nM bPTH1-84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH1-84 but no change in the medium [cAMP]. In tissues pretreated with 1 mM IBMX the response to 10 nM bPTH1-84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 nM bPTH1-84. These results suggest the presence of an adenylate cyclase that can be activated by a mammalian bPTH1-84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.