A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans

Noel, Jack; Rabinovitch, Alex; Olson, Les; Kyriakides, George; Miller, Joshua; Mintz, Daniel H.

doi:10.1016/0026-0495(82)90133-0

Cited by 49 publications

(19 citation statements)

References 16 publications

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“…Horaguchi et al 9 rendered three out of five recipients normoglycemic by injection of the tissue into the portal vein. Alejandro et al 24 have also developed a method of islet preparation in dogs that allows injection into the portal vein with a high success rate. Their method is quite different from both the Mirkovitch and Horaguchi techniques, but it does rely on intraductal collagenase injection before pancreatic tissue dispersal.…”

Section: Discussionmentioning

confidence: 98%

Comparison of Two Methods of Islet Preparation and Transplantation in Dogs

et al. 1986

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Seventy-nine mongrel dogs underwent total pancreatectomy. Fifteen dogs served as apancreatic controls and died 7.0 +/- 4.2 days later (mean +/- SD). The pancreases of 44 dogs (group 1) were intraductally distended by manual injection of Hanks' balanced salt solution (HBSS). Thereafter each organ was mechanically disrupted and subjected to collagenase digestion as described by Mirkovitch et al. The pancreases of 20 dogs (group 2) were intraductally distended and subsequently perfused with collagenase by a roller pump. The organs were then mechanically disrupted and filtered through screens as described by Horaguchi et al. The resulting tissue suspensions were injected into the spleens of the dogs as autotransplants in both groups, by direct punction of the splenic capsule in group 1 and by retrograde infusion via a splenic vein tributary in group 2. The functional outcome was better in group 2 than in group 1, as assessed by the number of animals that became normoglycemic after transplantation [15/20 (75%) vs. 13/44 (30%); P = .0025]. The degree of islet purification, as measured by an increase in the tissue insulin/amylase ratio, was higher in group 2, and in both groups it was higher in normoglycemic than in hyperglycemic animals. The percent engraftment [i.e., amount of insulin recovered from spleen as percent of tissue transplanted (mean, 15.4% in group 1 and 14.5% in group 2) or as percent of original pancreas (mean, 4.9% in group 1 and 4.4% in group 2)] was low in both groups but again was higher in normoglycemic than in hyperglycemic animals within each group. In conclusion, both the degree of engraftment and purification and the route of implantation influenced the functional outcome after dispersed pancreatic islet autotransplantation to the spleen of totally pancreatectomized dogs, with purified tissue injected retrogradely functioning better than unpurified tissue injected directly.

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Section: Discussionmentioning

confidence: 98%

Comparison of Two Methods of Islet Preparation and Transplantation in Dogs

et al. 1986

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“…Numerous studies have been conducted using such preparations of islets in dogs after either direct injections of the splenic pulp (2, [4][5][6][7][8] or after retrograde reflux into splenic venous tributaries (9,10). Although there have been a few citations (7)(8)(9) of long-term functional survival of islet autografts prepared by these techniques, continuing islet autograft function has not yet been studied in detail.…”

Section: Introductionmentioning

confidence: 99%

“…Although there have been a few citations (7)(8)(9) of long-term functional survival of islet autografts prepared by these techniques, continuing islet autograft function has not yet been studied in detail. Also, islet preparations by these techniques have not usually been transplanted into the portal venous system due to the threat of inducing portal hypertension or other complications associated with exocrine secretions (1 [1][2][3][4][5][6][7][8][9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Natural history of intrahepatic canine islet cell autografts.

Alejandro¹,

Cutfield²,

Shienvold³

et al. 1986

J. Clin. Invest.

143

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We have serially followed the function of intrahepatic canine islet autografts in 15 beagle dogs for up to 24 mo. Of these, only 20% sustained normal levels of fasting blood glucose for >15 mo posttransplant. Failure of autograft function was accompanied by a preferential loss of well-granulated beta cells in the engrafted islets. The chronic stimulation of an initially marginal intrahepatic beta-cell mass ultimately resulted in metabolic deterioration and loss of beta cells below the minimal threshold required to maintain normal fasting blood glucose levels. It is possible that transplantation of a larger mass of islets would result in indefinite graft function in dogs. However, it remains to be demonstrated in larger mammals, including humans, whether an islet cell mass that is initially adequate in a heterotopic site such as the liver can remain functionally competent over a prolonged period.

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“…The tissue was weighed and resuspended at 4°C to a total volume of 120 ml in medium 199 with 25 mM HEPES (Gibco), 10% (vol/ vol) fetal calf serum (FCS; Gibco, Grand Island, NY), and 100 U/ml penicillin with 100 |xg/ml streptomycin (PS). Aliquots of 4 ml were removed to 50-ml tubes, suspended in 4.3 ml 5X medium 199 and 16.7 ml Ficoll (density 1.125; Sigma), and overlaid with 5 ml each of Ficoll with densities 1.085, 1.075, and 1.045 (3,9). They were centrifuged at 550 x g for 25 min at 22°C.…”

Section: Methodsmentioning

confidence: 99%

“…In these studies, we combined the principles of collagenase perfusion via the ducts (7), gentle dissociation of tissue (8), and density-gradient separation (9) to separate purified canine islets. Islet mass was quantified by counts of islets and determination of islet diameter.…”

mentioning

confidence: 99%

Critical Mass of Purified Islets That Induce Normoglycemia After Implantation Into Dogs

Warnock

Rajotte²

1988

Diabetes

110

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Twenty grafts of highly purified islets of Langerhans (mean +/- SE vol 0.98 +/- 0.3 ml, islet diam 122 +/- 5 micron) were autoimplanted into the spleen or liver of totally pancreatectomized dogs. Portal venous pressure did not change significantly. Incremental doses of islets of 1000-3000 (n = 3), 3000-4000 (n = 6), 4000-5000 (n = 5), 5000-6000 (n = 3), and 6000-8000 (n = 3) per kilogram body weight resulted in corresponding fasting plasma glucose (PG) of 258 +/- 18, 163 +/- 13, 158 +/- 17, 138 +/- 15, and 108 +/- 6 mg/dl. In 3 apancreatic control dogs, PG was 338 +/- 9 mg/dl. One (normoglycemic) dog died of wound complications, and follow-up PG at 1 mo was 89 +/- 5 mg/dl in 6 of 10 dogs that received 3000-5000 islets/kg and 91 +/- 6 mg/dl in all 6 that received greater than 5000 islets/kg. K values 1 mo after surgery during glucose tolerance tests were 1.8 +/- 0.3 for 6 spleen dogs and 1.6 +/- 0.3 for 6 liver dogs. Six months after splenic implantation, PG was 75 +/- 4 mg/dl and rose to greater than 350 mg/dl after splenectomy. These data define the critical number of purified dog islets of known size that is necessary to induce prolonged normoglycemia. Sufficient pure islets can be collected from 1 dog pancreas to correct diabetes after autoimplantation into the liver or spleen.

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A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans

Cited by 49 publications

References 16 publications

Comparison of Two Methods of Islet Preparation and Transplantation in Dogs

Comparison of Two Methods of Islet Preparation and Transplantation in Dogs

Natural history of intrahepatic canine islet cell autografts.

Critical Mass of Purified Islets That Induce Normoglycemia After Implantation Into Dogs

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