Culture of the islets of Langerhans for transplantation

The isolated islets of Langerhans are the most available donors for transplantation. As the preservation of the isolated islets is difficult, we attempted to keep these tissues viable by use of an organ culture. Islets of Langerhans from adult Wistar rats were isolated by a collagenase technique and cultured in air-CO2 (95-5%) incubator at 37 degrees C. Insulin contents of the culture media which was changed every 3 days ranged from 1097 to 1434 microunits/ml during the 80 days' culture period. Transplantation of these islets into the portal vein of streptozotocin-induced diabetic rats resulted in a good recovery from the diabetic state. These studies indicate that cultured islets do preserve their original biological abilities.     

Transplantation of preserved pancreatic islets into the portal vein of rats.

Langerhans islets were isolated from the exocrine pancreata of Wistar rats by the improved collagenase-digestion method. The isolated islets were preserved in a tissue culture medium for seven days. Transplantation of these preserved islets into the portal vein of streptozotocin-induced diabetic rats resulted in a significant reduction of hyperglycemia, polyuria and glucosuria, and a restoration of weight gain. It was found that these effects could be maintained for 16 weeks. In order to normalize the K-values and plasma insulin levels, at least 600 islets had to be transplanted into each diabetic rat.     

Xenotransplantation of microencapsulated fetal rat islets

Fetal pancreatic islets were isolated from 21-day pregnant Wistar rats and enclosed in semipermeable alginate-polylysine-alginate capsules. Encapsulated islets that had been previously cultured for eight days in vitro were shown to secrete insulin in response to glucose challenge: low-glucose, high-glucose, and high-glucose + 3-isobutyl-1-methyl-xanthine (IBMX). Transplants of 800-1000 encapsulated cultured fetal islets into the peritoneal cavities of BALB/c mice with streptozotocin-induced diabetes restored normoglycemia for up to 171 days without immunosuppression. When the capsules were removed from 2 of the recipients they both quickly regressed to a diabetic state. Control groups of diabetic mice received unencapsulated, uncultured islets or empty capsules. The mortality rate among these animals was high and none experienced relief from hyperglycemia for longer than 6 days. These results demonstrate that cultured microencapsulated fetal rat islets of Langerhans can release insulin in response to an in vitro glucose challenge, and that transplants of these islets into diabetic mice can restore normoglycemia without the need for immunosuppressive therapy.     

Perfusion preservation of cadaver rat pancreas: II. Culture after perfusion and successful transplantation

We designed a new process for culturing pancreatic islets and applied this method to cultures of rat pancreatic islets which had degenerated during preservation by the perfusion-method for 6 hours under the condition of hypothermia and oxygenation. The objective was to determine the extent of the original function. Transplantation of these sotreated islets was also attempted. When pancreatic islets isolated from the ancreas after 6-hour-perfusion were cultured, morphological restoration was apparent within the first 3–4 days. Insulin contents of the culture media renewed every 3 days, ranged from 851 to 1,134 μU/ml/two islets during culture period of 21 days. In the glucose-loading test, insulin secretion of the islets was the same as that of islets in the control experiments. Transplantation of these islets into the portal vein of streptozotocin-induced diabetic rats resulted in a good recovery from the diabetic state.     

Perfusion preservation of cadaver rat pancreas: II. Culture after perfusion and successful transplantation

We designed a new process for culturing pancreatic islets and applied this method to cultures of rat pancreatic islets which had degenerated during preservation by the perfusion-method for 6 hours under the condition of hypothermia and oxygenation. The objective was to determine the extent of the original function. Transplantation of these so-treated islets was also attempted. When pancreatic islets isolated from the pancreas after 6-hour-perfusion were cultured, morphological restoration was apparent within the first 3-4 days. Insulin contents of the culture media renewed every 3 days, ranged from 851 to 1,134 microU/ml/two islets during culture period of 21 days. In the glucose-loading test, insulin secretion of the islets was the same as that of islets in the control experiments. Transplantation of these islets into the portal vein of streptozotocin-induced diabetic rats resulted in a good recovery from the diabetic state.     

Successful reversal of diabetes in nude rats by transplantation of isolated adult human islets of Langerhans

A method is described in which the viability of isolated adult human islets of Langerhans can be assessed in vivo. The Rowett nude rat, made diabetic with streptozocin (STZ), has been used as the islet recipient in these studies. Although these animals are athymic and are able to accept xenogeneic grafts for prolonged periods, they are very susceptible to dehydration and infection once made diabetic. Therefore, a considerably shortened diabetes induction period was used. The basis of the study was to prepare pure adult human pancreatic islets that were cultured for 48 h. Nude rats were given 80 mg/kg i.v. STZ during islet isolation and were transplanted with 800-1000 islets under the renal capsule at 48 h. To monitor islet function, animals were bled regularly for random blood glucose measurements and were given a glucose tolerance test at day 20. The kidney containing the graft was removed on day 21 to allow histological assessment of the graft and to confirm that glucose control was due to the transplanted islets and was not secondary to reversion of the animal's own islets. Seven rats were transplanted, and five were deemed to have received viable human islets. Two rats that received islets from the same donor did not reverse their diabetes and were found by histology to have vacuolated islet structures with scant insulin-staining tissue under the kidney capsule. This method allows a definitive judgment of the ability of isolated adult human islets to reverse diabetes.     

Transplantation of preserved pancreatic islets into the portal vein of rats

Langerhans islets were isolated from the exocrine pancreata of Wistar rats by the improved collagenase-digestion method. The isolated islets were preserved in a tissue culture medium for seven days. Transplantation of these preserved islets into the portal vein of streptozotocin-induced diabetic rats resulted in a significant reduction of hyperglycemia, polyuria and glucosuria, and a restoration of weight gain. It was found that these effects could be maintained for 16 weeks. In order to normalize the K-values and plasma insulin levels, at least 600 islets had to be transplanted into each diabetic rat.     

Experimental pancreatic islet transplantation.

Transplantation of isolated islets of Langerhans has been suggested as a treatment of certain forms of diabetes mellitus. Injection of 200-400 syngeneic pancreatic islets isolated by collagenase digestion into the pancreas or submandibular gland of diabetic rats rendered most of the hosts nearly normoglycaemic. Blood glucose determinations were monitored for 2 months after islet transplantation. Although intrapancreatic and intrasubmandibular implantation reduced hyperglycaemia and polyuria in these animals, consistent normal values were rarely achieved.     

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.     

Effects of epicatechin on rat islets of Langerhans

Plants containing epicatechin (a flavonoid) have been used to treat diabetes mellitus in Indian medicine. The present study reports effects of this compound on isolated islets of Langerhans. The flavonoid (1 mM) was found to increase insulin secretion from isolated rat islets of Langerhans in the presence of either 2 or 20 mM glucose, in static incubations, or in perifusion. The increase in insulin secretion mediated by epicatechin was both ATP- and temperature-dependent. Ultrastructural studies showed no deleterious changes in the structure of the B-cells after 5 days of exposure to the compound. Intraperitoneal injection of 30 mg/kg body wt of epicatechin twice daily for 4 days increased the islet insulin content by 30%. Secretion of insulin from islets isolated from epicatechin-injected rats was significantly increased when exposed to 20 mM glucose in comparison with water-injected controls. Furthermore, islets of adult rats cultured with 5.5 mM glucose for 4 days showed a significant increase in DNA synthesis in the presence of 0.05 mM epicatechin. These results suggest direct effects of epicatechin on various aspects of islet function.     

Isolation of porcine pancreatic islets for xenotransplantation studies: Effects of low collagenase digestion temperatures

Abstract: Islets of Langerhans were isolated from porcine pancreata by a modification of our previously described method. The modification involved the use of a low temperature of collagenase digestion (30°C) during the process of islet isolation. The resulting islets were then evaluated in vitro and in vivo and compared to islets isolated at the regular 37°C temperature.
The islets produced at the low temperature were more compact compared to the control islets. In the dextran density gradient these islets were deposited at the interface of the 1.060 and 1.068 g/ml density bands as compared to 1.050 and 1.060 g/ml for the control islets. In addition, the experimental islets contained a higher proportion of compact, unfragmented islets (68%) compared to the regular islets (55%), and their uptake of the dithizone stain was considerably slower than with the control islets. All ten batches of freshly isolated microencapsulated islets produced at both temperatures responded to the glucose stimulation. After 4 weeks of in vitro culture the islets of both groups microencapsulated in alginate-polylysine-alginate (APA) microcapsules still retained glucose responsiveness, with the experimental islets demonstrating significantly higher responsiveness to the high glucose (16.7 mM) and 0.1 mM IB MX stimulation. The morphology of unencapsulated islets in the experimental group following 4 weeks of in vitro culture indicates much firmer islet structure compared to the control islets. In addition, the unencapsulated experimental islets following the 4 week culture were still found to have secreted insulin when exposed to glucose. In transplantation studies both the experimental and the control islets normalized diabetic hyperglycemia in diabetic mice in a comparable fashion. In general, the low temperature digestion results in superior islets in terms of their morphology, viability, and physiological function.     

Experimental studies on cryopreservation combined with the cultural process of isolated rat pancreatic islets

Recently many investigators have attempted transplantation of isolated islets of Langerhans in an expectation of therapeutic effects in patients with diabetes. In the course of time, preservation of the islets was found to be a prerequisite for successful transplantation. In the present studies, we used our newly designed method of preserving islets, i.e., cryopreservation combined with the cultural process before and after freezing, and we examined the insulin-releasing activity of the islets and attempted transplantation of the islets so-obtained. As to the insulin-releasing activity, the islets cryopreserved after short-term culture increased insulin output as compared with those cryopreserved without culture. Damage to Langerhans’ islets resulting from isolation and cryopreservation was overcome during the culture. Transplantation of Langerhans’ islets cultured for 3–4 days before and after the cryopreserving process produced favorable results.     

Xenotransplantation of Microencapsulated Canine Islets into Diabetic Rats

Abstract: Islets of Langerhans were isolated in high yields from canine pancreata. In the procedure, the pan-creata 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-polylysinealginate 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.     

Superiority of Fresh Islets Compared With Cultured Islets

Introduction

It has recently been reported that the outcomes of islet transplantation with short periods of culture are comparable with those of freshly isolated islets. To clarify the influence of culture, fresh islets were compared with cultured islets in terms of quality.

Materials and Methods

The quality of freshly isolated islets was compared with that of cultured islets with CMRL 1066 including 10% allogeneic serum, CMRL 1066 including 0.5% human serum albumin, or Miami medium. We evaluated static glucose stimulation tests, insulin/DNA contents, ADP/ATP ratios, and an intraportal transplantation model into syngeneic diabetic rats. The expression of inflammatory mediators in the islets was examined using Western blotting for tissue factor (TF), which is the initiator of detrimental instant, blood-mediated, inflammatory reactions (IBMIR).

Results

Although the survival rate was similar in all groups, the stimulation index upon glucose challenge and the insulin/DNA ratio were significantly higher among fresh islets. Most importantly, the expression of TF on islets was significantly lower in fresh islets, suggesting that culture enhanced TF-dependent IBMIR after transplantation. In an in vivo transplantation model, the curative rate and insulin production by the recipient liver was considerably greater in the fresh islet group.

Conclusions

Isolated islets without prior culture showed results superior to cultured islets.     

Fresh islets are more effective for islet transplantation than cultured islets

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.     

Experimental studies on cryopreservation combined with the cultural process of isolated rat pancreatic islets

Recently many investigators have attempted transplantation of isolated islets of Langerhans in an expectation of therapeutic effects in patients with diabetes. In the course of time, preservation of the islets was found to be a prerequisite for successful transplantation. In the present studies, we used our newly designed method of preserving islets, i.e., cryopreservation combined with the cultural process before and after freezing, and we examined the insulin-releasing activity of the islets and attempted transplantation of the islets so-obtained. As to the insulin-releasing activity, the islets cryopreserved after short-term culture increased insulin output as compared with those cryopreserved without culture. Damage to Langerhans' islets resulting from isolation and cryopreservation was overcome during the culture. Transplantation of Langerhans' islets cultured for 3-4 days before and after the cryopreserving process produced favorable results.     

Allotransplantation of isolated pancreatic islets in diabetic rats. Effect of culture on islet rejection

Reduction of the immunogenicity of the tissue by tissue culture has been a controversial concept in transplantation immunology. In the present study, isolated islets were cultured prior to allotransplantation. The obtained results were compared in comparison with the results of the non-cultured islet allotransplantation group. Two kinds of rats differing from each other in histocompatibility antigen were used. WKA rats ( RT1k ) were donors, and LEJ rats ( RT1j ) recipients. No immunosuppressive agents were given. About 500 isolated islets of the donors were transplanted to the spleen of the recipients with streptozotocin induced diabetes mellitus. Although the mean survival time of non-cultured islet was 12.2 +/- 4.3 days, that of 7-day cultured islet increased significantly to 26.4 +/- 4.8 days. The response of the lymphocytes to the islets was also examined by mixed lymphocyte-islet culture. Response of lymphocytes to non-cultured islet was compared with that of lymphocytes to 7-day cultured islets. The response of lymphocytes to the 7-day cultured islets was significantly decreased. These results suggested that the cultured islet prolongs survival time and islet culture diminish the concentration or availability of stimulatory antigens on the cell surface membranes.     

Encapsulation of islets in rough surface, hydroxymethylated polysulfone capillaries stimulates VEGF release and promotes vascularization after transplantation

The transplantation of encapsulated islets of Langerhans is one approach to treat type 1 diabetes without the need of lifelong immunosuppression. Capillaries have been used for macroencapsulation because they have a favorable surface-to-volume ratio and because they can be refilled. It is unclear at present whether the outer surface of such capillaries should be smooth to prevent, or rough to promote, cell adhesions. In this study we tested a new capillary made of modified polysulfone (MWCO: 50 kDa) with a rough, open-porous outer surface for islet transplantation. Compared with free-floating islets, encapsulation of freshly isolated rat islets affected neither the kinetics nor the efficiency of glucose-induced insulin release in perifusion experiments. Free-floating islets maintained insulin secretion during cell culture but encapsulated islets gradually lost their glucose responsiveness and released VEGF. This indicated hypoxia in the capillary lumen. Transplantation of encapsulated rat islets into diabetic rats significantly reduced blood glucose concentrations from the first week of implantation. This hypoglycaemic effect persisted until explantation 4 weeks later. Transplantation of encapsulated porcine islets into diabetic rats reduced blood glucose concentrations depending on the islet purity. With semipurified islets a transient reduction of blood glucose concentrations was observed (2, 8, 18, 18 days) whereas with highly purified islets a sustained normoglycaemia was achieved (more than 28 days). Explanted capillaries containing rat islets were covered with blood vessels. Vascularization was also observed on capillaries containing porcine islets that were explanted from normoglycaemic rats. In contrast, on capillaries containing porcine islets that were explanted from hyperglycemic rats a fibrous capsule and lymphocyte accumulations were observed. No vascularization on the surface of transplanted capillaries was observed in the absence of islets. In conclusion, encapsulated islets can release VEGF, which appears to be an important signal for the vascularization of the capillary material. The rough, open-porous outer surface of the polysulfone capillary provides a site well suited for vascular tissue formation and may allow a prolonged islet function after transplantation.     

Multiple transplantation of islets of Langerhans.

In 6 diabetic rats about 1,000 isolated islets of Langerhans were transplanted into the liver with little effect on the uninsulinism and hyperglycemia. Subsequently, a second transplantation resulted in a decrease of blood sugar to normal and in a reversal of the loss in body weight. Finally, in animals who had reversed to diabetes again, a third islet transplantation was performed. These rats with a total of 3,100 islets showed a decrease in the glucose levels from 250-305 to 110 +/- 27 (X +/- SEM) mg/100 ml. The insulin level after glucose stimulations was measured at 27 less than X less than 35 micromicron/ml.     

Transplantation of pancreatic islets from hypothalamic obese rats corrects hyperglycemia of diabetic rats

Pancreatic islets isolated from adult obese rats, obtained by neonatal treatment with monosodium L-glutamate (MSG), oversecrete insulin stimulated by glucose concentration. Whereas adult MSG obese rats are hyperinsulinemic, their pancreatic islets still secrete insulin after high glucose demand. This is crucial so that the animals do not become hyperglycemic. Islets from MSG obese rats were implanted in diabetic donor rats so that the capacity of islets in regulating blood glucose concentration could be evaluated. Hyperglycemic (glucose 22 to 34 mmol/L) rats obtained with streptozotocin (STZ) treatment were used as recipients. Islet donors consisted of control adult and MSG obese rats. Only 600 islets were transplanted via the portal vein to diabetic rats. During 4 days after the transplant, fed blood glucose was monitored. After 12 hours of fasting the rats were killed; their blood samples were used to measure glucose and insulin concentration; retroperitoneal fat pads were isolated and weighed to estimate body fat. Transplanted islets from MSG obese rats decreased of fed glucose levels by 34% in diabetic rats (P < .05); however, glucose levels still remained twofold higher than those of intact controls (P < .05). Similar to MSG islets, islets grafts from control rats provoked the same effects in diabetic rats. High fasting blood glucose and low insulin levels of diabetic rats were corrected by islet grafts. Transplantations were able to recover 40% of fat in diabetic rats. The results demonstrated that islets from MSG obese rats may regulate blood glucose concentrations in diabetic rats, and suggesting that their function was not permanently altered by the onset of obesity.