A method for isolation of islets of Langerhans from the human pancreas

A method has been developed for the isolation of islets of Langerhans from the human pancreas. The average number of islets isolated was 1011 islets per gram of pancreas (SD 475, range 752-2111), and the purity of the preparation as defined by histologic examination and specific staining for insulin varied from 10% to 40%. Islet structure was well preserved and the islets were shown to be viable by supravital staining, demonstration of insulin response to glucose, and by transplantation of isolated islets beneath the renal capsule of nude mice. The essential features of this technique for isolation of human islets include injection of a high concentration of collagenase (6 mg/ml) into the pancreatic duct under pressure, followed by a short incubation (23 min) at 39 degrees C. The gland is then dispersed by a process of teasing and shaking, and the islets are separated by a two-stage process of filtration on a nylon mesh to remove the larger islets and centrifugation on a preformed Ficoll density gradient to separate the small islets.     

Isolation of islands of Langerhans from the human adult pancreas

Nine of 38 islet isolation experiments, using the duct collagenase technique, were selected for quality checks on isolated islet tissue. Pancreas was harvested, following aortic multi-organ perfusion. The total number of islets isolated amounted to 112,461 +/- 11,828 in 13.7 ml of suspension on average. In vitro secretion of beta cells was increased by a factor of 3.8 in response to glucose stimulation. Isolated islets in morphologically intact condition were detected by histological investigations. A new viability test (MTT, Sigma) for isolated pancreas islets worked well, in that it provided very soon information on islet survival in the wake of collagenase preparation. These results produced evidence to an improvement of technical conditions for clinical use of adult islet transplantation in cases of type I diabetes.     

Preparation of islets of Langerhans from the hamster pancreas

We describe a method for the preparation of viable islets from the pancreas of normal 8-week-old female Syrian golden hamster, based on the injection of the pancreatic duct with collagenase and on mechanical dissociation which liberates islets that maintain their normal morphological appearance and physiologic function. In a series of 11 animals, we examined the dose response of intraductal collagenase on islet yield. The mean number of isolated islets was 423 with a range from 130 to 873 per pancreas. Islet yield was most dependent on the concentration of collagenase solution used to inject the duct. The optimal concentration was determined to be 3.5 mg/ml when a total volume of 3.0 ml was injected. Islets responded to glucose stimulus in a normal biphasic pattern. Mesh filtration, rather than Ficoll, can be performed rapidly and results in a high yield of functional islets with minimal contamination by acinar tissue.     

Isolation of human and porcine islets of Langerhans and islet transplantation in pigs

Using a modification of the collagenase digestion, Ficoll gradient separation technique, we isolated the islets of Langerhans from fresh human pancreata removed from braindead cadavers. Comparison of the tissue insulin/amylase ratios between whole pancreas and isolated material revealed a significant purification of islet from acinar tissue. Also, islets isolated by this technique, when incubated in vitro, incorporated radiolabeled amino acid precursors into acid-alcohol-soluble islet proteins, thus indicating their viability and protein hormonal synthesis capabilities.Pig pancreata were processed by a similar technique, and the isolated material was transplanted to pigs with diabetes induced by total pancreatectomy. The mean survival time of the 11 pancreatectomized pigs that received an islet transplant was 15.3 days, while 13 pancreatectomized control pigs had a mean survival time of 6.0 days. Control pigs had no detectable circulating insulin 2 days after pancreatectomy. The pancreatectomized pigs that received an islet transplant, although remaining hyperglycemic, had insulin levels ranging from 5 to 14 μU/ml for up to 25 days after transplantation.The present studies demonstrate the feasibility of an approach designed for our ultimate goal—the large scale isolation and transplantation of islets in man.     

Fractions from commercial collagenase preparations: Use in enzymic isolation of the islets of Langerhans from porcine pancreas

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the liberation of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.     

Morphological changes of porcine islets of Langerhans after collagenase and HBSS infusion of the pancreas

Hilling DE, Rijkelijkhuizen JK, Marang‐van de Mheen PJ, Töns A, Terpstra OT, Bouwman E. Morphological changes of porcine islets of Langerhans after collagenase and HBSS infusion of the pancreas. Xenotransplantation 2010; 17: 413–417. © 2010 John Wiley & Sons A/S. Abstract: Background: A remarkable change in porcine islet morphology was observed after infusion of the pancreas with collagenase. The aim of the present study was to quantify these morphological changes and to assess whether these changes were due to the volume expansion caused by the collagenase entering the islet or the result of its digestive effects. Methods: This study was performed in pancreata of 28 crossbred pigs. First, eight pancreata were intraductally injected with collagenase by a continuous controlled pressure of 180 mmHg. Pancreas samples before collagenase infusion were used as controls. All tissue samples, both before and after infusion, were stained with anti‐insulin. To quantify the morphological change of the islets, the mean beta cell/endocrine content ratio of the infused and not‐infused tissue samples was compared. In a second experiment, 20 pancreata were similarly assessed after intraductal injection with Hank’s balanced salt solution (HBSS). Results: In both the collagenase‐ and HBSS‐infused groups, mean beta cell/endocrine content ratio was lower than in the control samples. The observed decline in the beta cell/endocrine content ratio was not significantly different between collagenase‐ and HBSS‐infused pancreata. This suggests that the lower beta cell/endocrine content ratio and thus the morphological change in the infused tissue samples is caused by volume expansion of the fluid entering the islet and that the digestive effect of collagenase plays no or only a minor role. Conclusion: Morphological changes of islets are observed after infusion of pancreata with collagenase and HBSS, most likely caused by volume expansion due to fluid entering the islets.     

Studies of the isolation and viability of human islets of Langerhans

Pancreas obtained from 34 adult human cadaver organ donors was divided into proximal and distal segments, and the duct to each segment was cannulated. Collagenase was injected into the proximal duct of 7 glands and into the distal duct of 7 others; the duct of the opposite segment was perfused with collagenase. The pancreas was then dispersed by teasing, trituration, and passage through filters. Perfused proximal and distal segments released 1461 +/- 287 and 2728 +/- 797 islets/g (+/- SEM) versus 710 +/- 149 (P less than 0.05) and 1950 +/- 636 after injection. Twenty other pancreases were perfused with collagenase warmed rapidly to 39 degrees C (n = 4) or warmed slowly to 37 degrees C (n = 6) or 39 degrees C (n = 10): the yield was 1625 +/- 632, 1320 +/- 116, and 2009 +/- 277 islets/g respectively. Total yields from the latter were 76 X 10(3) large (greater than 100 microns) and 85 X 10(3) small (less than 100 microns) islets with recoveries of 61% and 42%, respectively, after Ficoll density gradient purification. Histology showed highly purified islets. Perifusion with glucose elicited a biphasic release of insulin with the mean response (microU/islet/min) rising to a first peak of 0.5 and constant second phase secretion of 0.25, followed by a return to baseline. Reduced response was observed for islets from pancreas stored greater than 6 hr and tissue obtained from multiple centers. Less insulin was produced by freshly isolated islets, islets less than 100 microns, and after Ficoll separation. Secretion was similar for islets derived from proximal or distal segments. Perfusion of collagenase via the ducts of human pancreas improves islet isolation and Ficoll gradient separation yields highly purified islets. Important factors influencing insulin secretion are the source of donor tissue, cold storage of pancreas, Ficoll purification, islet size, and tissue culture.     

Histomorphological characteristics of the porcine pancreas as a basis for the isolation of islets of Langerhans

Abstract: Isolation of porcine islets of Langerhans for future clinical xenotransplantation in type I diabetic patients is limited by the difficulty to isolate sufficiently functioning islets and by their particular fragility. Seven domestic pig races and the wild boar were investigated histologically to obtain detailed information on islet profiles, numbers, sizes, and volume density of the endocrine tissue. Theoretically calculated islet numbers were correlated with those obtained after islet isolation. Results: 1) Porcine islets are round, oval, dumbbell-like and triangular, and, as expected, occur in all intermediate profiles. Round and oval islets predominate. These profiles are also detected in crude islet preparations, which indicates that they can withstand the enzymatic digestion process. 2) The total number of islets varies greatly in all eight pig races, with, e.g., the wild boar showing twice as many islets (approx. 500/cm² tissue section) as the minipig (approx. 250/ cm²). 3) The tail of the pancreas, which is usually used for islet isolation, accounts for about 50% of the pancreas mass. 4) The majority of islets, 64.3% (range 59.8–68.9%), of the seven domestic races are 50–100 μm in diameter. With its 86% the wild boar is a remarkable exception. German landrace pigs show the greatest number (2%) of large islets (250 to >300 μm. 5) With 3.4%, German landrace pigs reveal the greatest islet volume density of all pig races (range 2.6–4.2%). 6) Only 25% of all islets are surrounded by a collagen “capsule” that covers >75% of the islet surface. Roughly 33% of all islets are “capsulated” by collagen type I, III, and IV fibers to an extent of <25%. 7) In young pure bred pigs (7–12 month old) only 3.0–10.6% of all theoretically available islets can be isolated during the enzymatic digestion. However, much better islet yields (30.1%) can be obtained from adult hybrid pigs (3 years old) much better islet yields (30.1%) can be obtained than from young hybrid pigs (7.6%). It may be concluded from our results that histological parameters-besides those already known (pH of the isolation medium, trypsin inhibition by Pefabloc-may have a greater influence on porcine pancreatic islet isolation than was assumed so far.     

Isolation of viable human pancreatic islets

This article reports a method for the isolation of viable pancreatic islets from the human pancreas. Isolated islets were obtained from human pancreata of cadavers, patients undergoing surgical operations, and fetuses, using a freehand microdissection procedure. Viability was assessed by light microscopy of sections stained with aldehyde fuchsin and by measuring the insulin output of islets in response to a glucose stimulus in vitro using a perifusion system. Ten pieces of cadaver pancreas were studied. Islets were isolated from 6 specimens and in 5 of these were shown to respond to a glucose stimulus in vitro. Histologically the islets showed minimal damage with slight degranulation of the beta cells. Five pieces of pancreas removed at operation were studied as well. Islets were isolated in all cases, but only 2 showed a response to a glucose stimulus. Pancreata from a 26-week and 34-week human fetus were also studied. It was not possible to microdissect islets from either case, but small pieces of pancreas from the 26-week fetus were shown to respond to a glucose stimulus by producing a significant increase in insulin output.

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Résumé Cet article décrit une méthode pour isoler des ilÔts de Langérhans viables à partir de pancréas humain. Une technique de microdissection manuelle a permis d'isoler ces ilÔts à partir de pancréas humains obtenus chez des patients opérés, chez des donneurs cadavériques et chez des foetus. Leur viabilité a été établie par l'examen en microscopie optique de sections tissulaires préparés à la fuchsine aldehyde et par mesure de la sécrétion d'insuline en réponse à une stimulation au glucose au moyen d'un système de périfusion in vitro. Dix échantillons de pancréas cadavériques ont été étudiés. On a réussi à isoler des ilÔts chez six d'entre eux et cinq ont répondu au test de stimulation au glucose in vitro. à l'examen histologique, les ilÔts ne présentaient que peu de modification et une légère dégranulation des cellules bÊta. Cinq spécimens de pancréas obtenus chirurgiculement ont été étudiés. On a pu isoler des ilÔts dans tous les cas, mais seulement deux ont répondu à la stimulation au glucose. Finalement on a aussi étudié des échantillons pancréatiques obtenus chez deux foetus humains de 24 et 36 semaines respectivement. Chez ni l'un ni l'autre il n'a été possible d'isoler des ilÔts, mais de petites tranches de tissus pancréatiques préparés chez le foetus de 26 semaines et soumis au test de stimulation au glucose ont augmenté significativement leur sécrétion d'insuline.

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Part of the cost of this project was defrayed by a grant from the Wellcome Trust.     

Development of a method for isolation of islets from human fetal pancreas

Three methods for the preparation of islets from human fetal pancreas (17.4 +/- 1.2 wk gestational age) were compared. In each method, the pancreases were minced and followed by 1) no collagenase digestion, 2) 5 min of collagenase digestion, or 3) 14 min of collagenase digestion. The culture conditions prevented adherence of the fragments. Culture for 6-7 wk of minced fetal pancreas without collagenase digestion resulted in fragments that were a mixture of cells positive for insulin or glucagon, ducts, necrotic debris, and other unidentified cells with complete degeneration of the acinar cells. Culture of minced pancreas digested for 5 min with collagenase resulted in fragments that superficially appeared to be islets but did not have the size characteristics of human fetal islets and contained fibrous and duct elements not seen in islets. Culture of minced pancreas digested for 14 min with collagenase resulted in islets that were released into the medium and harvested by picking. These islets were morphologically similar to islets of the intact human fetal pancreas and isolated islets from rat neonatal pancreas. These islets and fragments were viable for at least 7-8 wk in culture.     

Survival of cryopreserved isolated adult human pancreatic islets of Langerhans

Human islets of Langerhans were isolated from the pancreata of 13 adult organ donors, cultured for 24 h, cryopreserved, stored in liquid nitrogen at -196 degrees C for 6-88 days, thawed, and then cultured again. The number of islets recovered after this procedure was 80% of that present at the beginning. The viability of cultured/cryopreserved islets was then compared with that of islets from the same donor cultured for 24 hr, and was assessed by supravital staining, insulin response to glucose, and survival after implantation under the kidney capsule of nude rats. Supravital staining showed more nonviable cells in cryopreserved islets than in their cultured counterparts. Significant response to glucose was seen before and after cryopreservation in 2 of 4 sets of islets. Xenografts of 200 cultured islets (from 13 donors) were implanted in 15 nude rats under the kidney capsule, and a further 15 rats had cryopreserved islets (from the same 13 donors) similarly implanted beneath the kidney capsule. Two weeks later tissue was visible at the site of implantation in all 30 rats. Histological examination of both groups showed the tissue to have the morphology of islets, confirmed by immunohistochemical chemical localization of insulin. The insulin content of kidneys bearing 200 cultured islets was 7.88 +/- 1.6 mU (n = 13) versus 6.84 +/- 1.43 mU (n = 13) for kidneys bearing cryopreserved islets. Thus this technique for cryopreservation of isolated adult human islets enables a high recovery of endocrine tissue that survives after transplantation to nude rats, but some evidence of damage was apparent from insulin secretion studies and electron-microscopic studies.     

Long-term cryogenic storage of purified adult human islets of Langerhans

Reliable high-recovery human islet storage would facilitate tissue matching, organ sharing, and immune manipulation of donor islets and prospective diabetic recipients. Collagenase-isolated, Ficoll-purified pancreatic islets (median 21,000, 15% of total islet yield) from eight cadaver pancreases were cultured in vitro for 24 h, equilibrated in three steps with dimethyl sulfoxide (DMSO) to a 2-M concentration, supercooled, nucleated, and cooled at 0.25 degree C/min to -40 degrees C before storage at -196 degrees C for 44.25 +/- 8.75 days. Rewarming at 200 degrees C/min and removal of DMSO with 0.75 M sucrose preceded 48 h of culture and retesting. Recovery postthaw by microscope count on duplicate aliquots was 94.2 +/- 3.5% of prefreeze counts and by triplicate assay of extractable insulin was 90.0 +/- 22.3% on day 0 and 74.1 +/- 12.6% after a 48-h culture. Nonfrozen islets increased basal insulin secretion 7.7 +/- 2.8 times after stimulation with 300 mg/dl glucose in perifusion, whereas islets frozen-thawed and cultured 48 h increased 6.2 +/- 0.8 times (NS). Peak stimulated insulin release was 0.92 +/- 0.14 microU.islet-1.min-1 before storage and 0.73 +/- 0.14 microU.islet-1.min-1 (79% of control, NS) after freeze-thaw and a 48-h culture. Total insulin secretion (area under curve) was 66% of prefreeze values at 48 h. Immunocytochemical stains revealed preservation of islet morphology postthaw. Electron microscopy showed intact cellular and nuclear membranes and intracellular organelles. Frozen-thawed islets harvested 14 days after renal subcapsular xenografting in nude mice were revascularized and well granulated. Cryopreservation can achieve prolonged storage of large numbers of human islets with high recovery numerically and functionally, making this a feasible approach for future trials of human islet transplantation.