Evaluation of energy state of islet independent of size using a newly developed ATP bioluminescence assay

ATP content or energy charge (EC) of islets may be a good parameter to assess viability. In this study, we examined adenine nucleotides and EC of freshly isolated or 24-hour cultured rat islets of various diameters using a novel bioluminescent enzymatic cycling assay system. For freshly isolated islets, ATP content and islet diameter showed a high correlation (r = 0.842, P < .001), but a significant correlation was not observed for cultured islets (r = 0.284) when all islets were included for the analysis. When only the cultured islets with a diameter <350 microm were included for analysis, a significant correlation was observed (r = 0.719). EC of freshly isolated islets fluctuated widely irrespective of diameter, in contrast with results of 24-hour cultured islets, which showed a stable, high EC, regardless of diameter. These data suggest that the ATP content of islets correlates with the islet size and that EC of islets widely fluctuates following isolation, indicating a significant role of monitoring ATP and EC of islets before transplantation.     

An Isolated Venous Sac as a Novel Site for Cell Therapy in Diabetes Mellitus

BACKGROUND: Transplanting pancreatic islets is of significant interest for type 1 diabetes mellitus. After intraportal injection of islets, inferior engraftment and eventual loss of transplanted islets constitute major limitations. Therefore, alternative approaches will be helpful. Here, we evaluated in animals whether an isolated venous sac would support survival of transplanted islets, along with correction of hyperglycemia. METHODS: Pancreatic islets isolated from adult Lewis rats were transplanted either into an isolated venous sac made from lumbar vein or into the portal vein of syngeneic rats. The integrity and vascular organization of the venous sac was determined by studies of the local microcirculation. The engraftment, survival, and function of transplanted islets were analyzed by histology, including endocrine function in situ and by glycemic control in rats with streptozotocin-induced diabetes. RESULTS: Transplanted islets showed normal morphology with insulin expression in isolated venous sac during the long term. Transplanted islets received blood supply from vasa vasorum and had access to drainage through venous tributaries in the venous sac. This resulted in restoration of euglycemia in diabetic rats. Removal of islet graft-bearing venous sac in diabetic rats led to recurrence of hyperglycemia. By contrast, euglycemia was not restored in rats treated by intraportal transplantation of islets. CONCLUSIONS: We demonstrated that pancreatic islets successfully engrafted and functioned in the isolated venous sac with ability to restore euglycemia in diabetic rats. Therefore, the isolated venous sac offers a new site for transplantation of pancreatic islets. This would be clinically beneficial as an alternative to intrahepatic islet transplantation.     

大量人胰岛分离技术的改进及相应胰岛功能的检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能。方法采用经典及改良技术分离、纯化28例人胰岛。胰岛收获量以胰岛当量(IEQ)表示。胰岛功能测定分别为体外测定胰岛素/DNA比率、静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内功能鉴定并行糖耐量试验,连续测血糖水平及其体内C肽浓度。结果 前13例采用经典方法,平均每个胰腺收获49123 IEQs,平均846 IEQs/g胰腺组织,平均纯度为87％。改进技术后15例的分离结果则分别为:501813 IEQs/胰腺,7003 IEQ/g胰腺组织及纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的基础。     

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.     

大鼠胰岛移植物制备与异种移植

增加胰岛收获量，提高胰岛纯度一直是胰岛移植中面临的重要问题，本实验经胰管注射胶原酶，胰静止消化分离成年大鼠胰岛纯度一直是胰岛葡聚糖离心纯化。纯化后胰岛收获量为６１０－８２０个／胰纯度达９２％；胰岛形态结构完整，内分泌细胞超微结构保持良好，对葡萄糖刺激反应胰岛素释放量是基本分泌水平的８倍；异种移植可逆转实验性糖尿病小鼠的高血糖达一周。     

Repeated isologous transplantation of islets of Langerhans in the diabetic rat.

The metabolic response to repeated isologous transplantation of isolated pancreatic islets was studied in 11 streptozotocin-diabetic AGUS rats. The islets were isolated with collagenase and transplanted intra-portally in batches previously found to be quantitatively insufficient for reversal of the diabetic state. Primary transplantation caused a slight improvement whereas retransplantation with the same number of islets three weeks later was followed by normalization of blood glucose, plasma insulin, urine volume and urine glucose per 24 hours during a three-month observation period. Repeated isologous transplantation of isolated pancreatic islets has an additive effect on the metabolism in that two insufficient tissue doses correct streptozotocin induced diabetes in the rats.     

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.     

Porcine Pancreatic Islets: Isolation, Microencapsulation, and Xenotransplantation

Abstract: To provide a plentiful source of pancreatic islets for future clinical transplants into diabetic patients, we have developed a simple and reliable method to isolate porcine islets of a high degree of purity. Porcine pancreata were perfused and digested with collagenase, and the islets were then purified on dextran density gradients. In order to avoid any damage to the islets, no mechanical devices nor any strenuous treatment was employed. As many as 5 times 10⁵ islets were isolated from a single porcine pancreas. Islets were encapsulated in alginate-polylysine-alginate membranes with the aid of an electrostatic droplet generator. In vitro studies demonstrated that the isolated islets secreted insulin in response to glucose and 3-isobutyl-L-methylxanthine (IBMX) challenge for at least 4 weeks. Perifusion studies showed that the kinetics of insulin release from the encapsulated islets was similar to that exhibited by free islets. In in vivo studies, 18 diabetic BALB-c mice were transplanted with 1,500-2,500 encapsulated islets each. In 13 recipients, the diabetic condition was reversed for at least 85 days. When capsules were removed from 2 transplant recipients, their diabetic condition quickly recurred.     

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.     

Effect of human somatotropin hormone on cultured rat islets

Because growth hormone (GH) improves the insulin secretion capacity of isolated human fetal islets in vitro, we sought to show that it positively influences isolated rat islets. Islets isolated from Wistar albino rats by a modified automated system were cultured in media containing 87% RPMI 1640, 10% FCS, 2% antibiotic-antimycotic, and 1% L-glutamine for 12 +/- 2 days. The cultured islets were divided into two groups: growth hormone negative (Group I) and growth hormone positive (Group II). On the 5th day we observed a decrease in the islet cell counts in both groups (Group I 28% versus Group II 45%). On the 10th day, the decrease continued in the GH-negative group (59%), while the count remained stable in the GH-positive group. The viability of rat islets was determined by fluorescein diacetate (FDA) plus propidium iodide (PI) staining. In comparison to the peripheral green, central orange-red staining pattern of Group I islets upon fluorescent microscopy, Group II showed more compact islets. Cultured islets seemed to be brighter than those without GH in the cultured islets. In conclusion, we observed that 2 weeks of incubation in the presence of GH acts positively on cultured rat islets for both their amount and their viability.     

Studies on insulin secreted by isolated islets of the monkey, Macaca radiata radiata.

The effects of various experimental conditions during the isolation of monkey islets by the collagenase method on the insulinogenic response of the isolated islets to glucose have been studied and compared with rat islets isolated under similar conditions. The monkey islets gave a normal response for at least 120 min. The results are compared with available studies on primate islets.     

Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets

One of the most important factors in clinical islet transplantation is isolation of a great number of islets with good viability. According to viability assessments of isolated islets, the static incubation test and the perifusion test of islets, which are used retrospectively, take much time and need various apparatus. But viability assessments of isolated islets for clinical islet transplantation require a simple, rapid, sensitive, and prospective method. We have developed a microfluorometric viability assay for isolated human, porcine, and dog islets of pancreata using fluorescein diacetate and ethidium bromide (FDAEB). Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation (490 nm) and ethidium bromide (EB) causes dead cells to fluoresce red. In this study, we investigated the applicability of FDAEB staining to quality assessment of isolated islets for clinical use by correlation with the counting method with insulin secretion of islets. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDAEB staining. The proportion of living islets within isolated canine islets, as measured by microfluorometric counting, was found to correlate highly significantly on low-temperature (24 degrees C) culture (R = 0.831, p < 0.001) and on 37 degrees C culture (R = 0.553, p < 0.05) with the insulin contents of the same islets. Therefore, it is possible to differentiate degrees of viability, and a scoring system is described for this purpose. The FDAEB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation.     

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.     

A model to evaluate toxic factors influencing islets during collagenase digestion: the role of serine protease inhibitor in the protection of islets

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.     

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.     

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.     

Heat shock preconditioning impairs revascularization of freely transplanted pancreatic islets

BACKGROUND: Revascularization of freely transplanted pancreatic islets is essential for appropriate graft function and survival. During the first days after transplantation, however, islet transplants are avascular, and successful engraftment is believed to be markedly hampered by hypoxia-induced tissue injury. Because heat shock has been shown to induce cell resistance against hypoxia, it seems reasonable to stress pancreatic islets by heat before transplantation. In contrast, hypoxia is a major stimulus for angiogenesis, and thus heat shock preconditioning-induced resistance against hypoxia may decrease stimulation of angiogenesis. The authors therefore studied in vivo whether heat shock preconditioning of isolated islets affects angiogenesis and revascularization after free transplantation. METHODS: After collagenase isolation, heat shock-preconditioned islets (42 degrees C for 30 min) were transplanted syngeneically into nontreated skinfold chambers of Syrian hamsters. In a second group of animals, nontreated islets were transplanted into heat shock-preconditioned chambers. Nontreated islets transplanted into nontreated chambers served as controls. Islet angiogenesis and revascularization were quantitatively analyzed during 14 days after transplantation using intravital fluorescence microscopy. Expression of heat shock proteins (HSP) was confirmed by immunohistochemistry and Western blotting. RESULTS: Immunohistochemistry revealed expression of HSP32 (heme oxygenase [HO]-1), HSP72, and also intracellular insulin in isolated and transplanted pancreatic islets. Western blot analysis showed enhanced HSP32 but slightly decreased HSP72 expression in heat shock-preconditioned islets when compared with controls. Intravital microscopy revealed appropriate vascularization of control islets within 14 days after transplantation. Heat shock preconditioning of the host tissue (i.e., the skinfold chambers) did not affect islet vascularization when compared with controls. In contrast, heat shock preconditioning of the isolated islets resulted in a significantly (P < 0.05) impaired take rate, a reduced (P < 0.05) size of the newly formed microvascular network, and thus a smaller area (P < 0.05) of microvascularly perfused endocrine tissue. CONCLUSION: These data suggest that heat shock preconditioning of isolated pancreatic islets before transplantation impairs the process of graft angiogenesis and revascularization. Therefore, transient exposure of isolated islets to heat may not be considered a promising tool to improve the outcome of islet transplantation.     

两种酶对猪胰岛消化分离效果的对比

目的　研究单纯酶 (胶原酶Ⅳ )与复合酶 (由胶原酶Ⅰ、Ⅳ、弹性蛋白酶、纤维素酶组成 )对成年猪胰岛的分离效果。方法　采用经胰管灌注消化酶的方法分离胰岛 ,用双硫腙染色做胰岛计数 ,台盼蓝染色判定胰岛活化水平 ,胰岛培养第 2d、第 4d、第 6d测定胰岛素含量 ,培养第 6d做胰岛活性评价 ,电镜观察胰岛的形态结构。结果　经复合酶消化法制备的胰岛数量为 (4 915±l0 42 )个胰岛 /克胰腺 ;而经单纯酶消化的胰岛数量为 (30 12± 989)个胰岛 /克胰腺。两组比较差异显著 (P <0 .0 5 )。结论　复合酶消化法是一种优于单纯酶消化法的成年猪胰岛分离方法。     

成人胰岛大容量分离技术的改进及胰岛功能检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能,为利用同种异体胰岛移植治疗1型和部分2型糖尿病提供理论依据和技术基础。方法 采用改良的自动分离技术连续分离28例人胰岛,然后用连续性密度样度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量(islet equivalent,IEQ)表示。胰岛功能的测定分别为体外测定胰岛的胰岛素／DNA比率;静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内胰岛功能鉴定并随后进行腹腔糖耐量试验,连续测定移植鼠血糖水平及其体内C肽浓度。结果 28例成人胰腺分离的胰岛收获量为5000～1030000IEQ／胰腺,平均为291635IEQ／胰腺,前13例平均每个胰腺收获49123IEQ,平均每克组织收获846IEQ。平均纯度为87％,随着技术的改进后15例的分离结果则分别为：平均每个胰腺501813IEQ,平均每克组织7003IEQ,平均纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常鼠。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的实验基础。     

Hormone release, islet yield, and transplantation of fetal and neonatal rat dorsal and ventral pancreatic islets

Fetal (20-21 day gestation) and neonatal (less than 5-day-old) rat islets were isolated from the glucagon-rich (dorsal) and glucagon-poor (ventral) pancreatic regions. After 1 or 2 wk in culture, groups of islets from each region were transferred to culture dishes containing Krebs-Ringer bicarbonate buffer with low (2.4 mM) and high (16.7 mM) glucose plus aminophylline. After 2 wk in culture, insulin released into medium was higher than after 1 wk, and more so if the islets originated from dorsal tissue (P less than .01). Glucagon release in response to alanine (10 mM) stimulation was also significantly higher in dorsal than in ventral islets (6.38 +/- 1.98 vs. 1.49 +/- 0.89 pg X islet-1 X h-1, respectively; P less than .02). Coincident with higher insulin and glucagon release, islet yield was greater in tissue from the dorsal neonatal pancreas than from that obtained in the fetal and neonatal ventral pancreas [range: dorsal, 131-180 (median, 153), vs. ventral, 53-127 (median, 84), islets obtained on day 5 of culture]. Neonatal islets of dorsal origin were transplanted intrasplenically (500-3000 islets) to streptozocin-induced diabetic inbred Lewis rats. Only rats receiving greater than 2500 islets were cured by transplantation. These experiments show that dorsal fetal islets secrete more insulin than do ventral islets and that islet yield is higher when islets are isolated from dorsal rather than from ventral perinatal pancreatic tissue. Despite their in vitro behavior, more neonatal dorsal islets are required to cure experimental diabetes than are reported with adult islets.