Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis

Total pancreatectomy with islet autotransplantation is performed to treat chronic pancreatitis in children. Successful islet isolation must address the challenges of severe pancreatic fibrosis and young donor age. We have progressively introduced modifications to optimize enzymatic and mechanical dissociation of the pancreas during islet isolation. We evaluated 2 islet isolation metrics in 138 children—digest islet equivalents per gram pancreas tissue (IEQ/g) and digest IEQ per kilogram body weight (IEQ/kg), using multiple regression to adjust for key disease and patient features. Islet yield at digest had an average 4569 (standard deviation 2949) islet equivalent (IEQ)/g and 4946 (4009) IEQ/kg, with 59.1% embedded in exocrine tissue. Cases with very low yield (<2000 IEQ/g or IEQ/kg) have decreased substantially over time, 6.8% and 9.1%, respectively, in the most recent tertile of time compared to 19.2% and 23.4% in the middle and 34.1% and 36.4% in the oldest tertile. IEQ/g and IEQ/kg adjusted for patient and disease factors improved in consistency and yield in the modern era. Minimal mechanical disruption during digestion, warm enzymatic digestion using enzyme collagenase:NP activity ratio < 10:1, coupled with extended distension and trimming time during islet isolation of younger and fibrotic pediatric pancreases, gave increased islet yield with improved patient outcomes.     

Total pancreatectomy and autologous islet cell transplantation as a means to treat severe chronic pancreatitis

Autologous islet cell transplantation after near-total or total pancreatic resection can alleviate pain in patients with severe chronic pancreatitis and preserve endocrine function. From February 2000 to February 2003, a total of 22 patients, whose median age was 38 years, underwent pancreatectomy and autologous islet cell transplantation. Postoperative complications, metabolic studies, insulin usage, pain scores, and quality of life were recorded for all of these patients. The average number of islet cells harvested was 245,457 (range 20,850 to 607,466). Operative data revealed a mean estimated blood loss of 635 ml, an average operative time of 9 hours, and a mean length of hospital stay of 15 days. Sixty-eight percent of the patients had either a minor or major complication. Major complications included acute respiratory distress syndrome (n = 2), intra-abdominal abscess (n = 1), and pulmonary embolism (n = 1). There were no deaths in our series. All patients demonstrated C-peptide and insulin production indicating graft function. Forty-one percent are insulin independent, and 27% required minimal amount of insulin or a sliding scale. All patients had preoperative pain and had been taking opioid analgesics; 82% no longer required analgesics postoperatively. Pancreatectomy with autologous islet cell transplantation can alleviate pain for patients with chronic pancreatitis and preserve endocrine function. Presented at the Presidential Plenary Session, at the Forty-Fourth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Florida, May 18–21, 2003 (oral presentation).     

Experimental pancreatic preservation prior to islet isolation and transplantation

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved human pancreatic islet isolation for a prospective cohort study of islet transplantation vs best medical therapy in type 1 diabetes mellitus

HYPOTHESIS: A local multiorgan donor pancreas procurement program can provide a source for optimized isolation of purified viable islets for transplantation into patients with type 1 diabetes mellitus receiving best medical therapy. DESIGN: Prospective before-after cohort study. SETTING: Tertiary referral center. PATIENTS: Glycemic control was assessed in 10 patients with diabetes-induced renal dysfunction who were enrolled in a best medical therapy program and then crossed over to islet transplantation. INTERVENTIONS: Thirty human pancreata were retrieved from local multiorgan donors and consecutively processed with intraductal collagenase perfusion, continuous digestion, and density gradient purification (group 1, n = 9) or similarly processed but impure tissue fractions cultured in vitro and then repurified to retrieve additional islets (group 2, n = 21). Islets were implanted by percutaneous portal embolization, providing more than 10 000 islet equivalents (IE) per kilogram of body weight (infusions from 1-3 donors per patient) under cover of antithymocyte globulin, sirolimus, or mycophenolate mofetil and tacrolimus. MAIN OUTCOME MEASURES: Islet yields, purity, and cell viability (caspase 3, terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine 5-triphosphate nick-end labeling stain, and insulin secretion in vitro) were compared. In patients, monitored metabolic parameters were C-peptide secretion, insulin requirements, glycemic excursion, and hemoglobin A(1c) (HbA(1c)). RESULTS: For group 1 vs group 2, no differences were observed in pancreas age (43 vs 44 years), cold storage (5 vs 4 hours), or weight (73 vs 82 g). Group 2 yielded 453 690 IE vs 214 109 IE in group 1 (P = .002). Grafts contained 50% or more endocrine cells in both groups. No difference occurred in cell viability or insulin secretion. Islets from 90% of group 2 pancreata met release criteria for transplantation. C-peptide secretion was detected in all recipients and persisted with a median follow-up to 12 months (range, 6-21 months) after full islet transplantation. Daily insulin dependence was reversed in all patients for at least 3 months. Five patients resumed small insulin doses. Compared with the best care program, all patients had improved metabolic stability. The mean +/- SE HbA(1c) level at entry into the study was 7.8% +/- 0.5%, and this decreased to 6.9% +/- 0.2% after best care (P = .38) and further to 6.2% +/- 0.2% at 6 months after transplantation (P = .002 vs entry; P = .15 vs best care; analysis of variance). CONCLUSIONS: Local pancreas donor retrieval with islet isolation and culture conditioning enabled an offer of islets for transplantation for 90% of consecutively processed pancreata. Isolated islets secreted insulin during prolonged follow-up after implantation into patients, yielding metabolic control comparable with that achieved by best medical therapy.     

实验大鼠胰岛分离移植技术方法的比较分析

目的 探索高效的大鼠胰岛分离移植技术方法.方法 应用Wistai-Furth大鼠,于体内或体外胶原酶经胰管灌注膨化胰腺,联合不同密度Ficoll液或Histopaque液纯化胰岛细胞,评估胰岛的数量、纯度、胰岛当量以及肾被膜下胰岛移植的有效性.结果 体外经胰管灌注膨化胰腺结合Histopaque液纯化提取胰岛的数量、纯度和胰岛当量值均显著高于体内灌注组各数值(P＜0.01),其提取时间无显著差别.1000个胰岛细胞移植进入左肾被膜下,有效的逆转了糖尿病大鼠高血糖,其远期糖耐受结果优于500和800个胰岛细胞移植组.结论 体外灌注膨化消化胰腺结合Histopaque液纯化胰岛的分离方法是一种满意的分离技术.1000个胰岛细胞是保证肾被膜下胰岛移植成功的最低有效数量.     

Technique of pancreatic procurement for pancreatic islet isolation

AIM OF THE STUDY: The allograft of pancreatic islets represents a potential alternative to insulin therapy in patients suffering from the most severe forms of Type 1 diabetes. Here we report our experience of pancreatic procurement for isolation and islet allograft. MATERIALS AND METHODS: Pancreata were procured in brain-dead donors. The islets were isolated using techniques developed and validated in pigs and men. Injection of a given preparation was decided after quantitative and qualitative controls. Islets were transplanted in Type 1 diabetic patients already grafted with a kidney or suffering from severe and/or unstable diabetes, after percutaneous or surgical settlement of an intra-portal catheter. Patients received an "Edmonton-like" immunosuppressive protocol. Grafts were repeated once or twice until a total quantity of 10,000 transplanted islet-equivalents was obtained. RESULTS: Twenty-nine pancreata were procured and 14 preparations were grafted to 7 patients. Eleven graftings were done percutaneously and three were surgical. The initial function of the 14 transplants was confirmed by secretion of C-peptide and decrease of insulin doses. Insulin therapy was completely interrupted in the 5 patients having received at least two grafts. CONCLUSION: These preliminary clinical results confirmed that the isolation technique of human islets and the technique of pancreas procurement are mastered by our team. If the results of this assay (assessment one year after graft) confirm our hopes, we will be able to offer islet allografts to an increasing number of patients with severe Type 1 diabetes.     

Canine pancreatic islet transplantation: a comparison of two isolation techniques

A new method (type B) for isolation of canine islets was developed, utilizing intraductal collagenase perfusion, stationary digestion, filtration and tissue separation by means of a dextran density gradient. The results of this technique were compared with those of a previously established method (type A). Islets were autotransplanted either via the splenic or the portal vein. Lasting normoglycemia was obtained in 5/8 intrasplenic type B transplants (63%), 6/8 intrahepatic type B transplants (75%), 5/6 intrasplenic type A transplants (83%) and 0/3 intrahepatic type A transplants. No difference was found in metabolic parameters 2 weeks after successful transplantation following type A or B islet isolation. Islet yield was higher with type A and purification better with type B isolation. Both techniques are relatively simple and inexpensive. Because of its higher purification rate and the success of intrahepatic islet transplantation method B has replaced method A as the routine procedure in our laboratory.     

A simple method of canine pancreatic islet isolation and intrahepatic transplantation

Clinical pancreatic islet transplantation has been impeded by the inability to isolate an adequate mass of functional tissue that will ameliorate diabetes. A simplified method of canine islet isolation was developed that allowed for either intrasplenic or intrahepatic transplantation. Following total pancreatectomy, parenchymal digestion was accomplished by intraductal collagenase perfusion and stationary incubation. The digested tissue was dispersed by filtration through a steel mesh (400 microns), washed, and separated on a discontinuous dextran density gradient. Enhanced islet tissue (2-4 ml) was recovered from the uppermost interface of the gradient and autotransplanted. The islet isolation procedure was tested in two series of dogs undergoing either intrasplenic or intrahepatic engraftment. Immediate and sustained normoglycemia (plasma glucose less than 200 mg%) was obtained in 5 of 8 dogs (63%) in the intrasplenic group and 6 of 8 dogs (75%) in the intrahepatic group. The mean fasting plasma glucose concentration 2 weeks after transplantation was 102.8 +/- 6.4 mg% in the intrasplenic group and 103.3 +/- 8.4 mg% in the intraportal group. The mean IVGTT K-values 2 weeks after transplantation were -1.41 +/- 0.35% and -1.21 +/- 0.13%, respectively. On the basis of insulin content, the islet yield was 33.0 +/- 3.7% of the total pancreas in the intrasplenic group and 33.0 +/- 3.1% in the intrahepatic group. Islet mass was enhanced 10.2 +/- 1.5 and 20.0 +/- 6.2 fold, respectively, on the basis of insulin/amylase ratios. The success rate in this canine model compared favorably with previously published results from other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of adverse pancreatic injury at surgical procurement upon islet isolation outcome

The consequence of a pancreas injury during the procurement for islet isolation purpose is unknown. The goal of this work was to assess the injuries of the pancreata procured for islet isolation, and to determine their effect on the islet yield. Between January 2007 and October 2013, we prospectively documented every injury of the pancreata processed in our centre for islet isolation. Injuries involving the main duct were classified as major, the others as minor. Donors’ characteristics and islet yields were compared between the groups of injuries. A pancreas injury was identified in 42 of 452 pancreata received for islet isolation (9.3%). In 15 cases, the injury was major (3.3% of all pancreata). Although a minor injury did not affect the islet yield, a major injury was significantly associated with unfavourable outcomes (postpurification mean islet equivalent of 364 ± 181, 405 ± 190 and 230 ± 115 × 10³ for absence of injury, minor injury and major injury, respectively). A major injury was significantly more prevalent in lean and short donors. We recommend assessing the quality of the pancreas in the islet isolation centre before starting the isolation procedure. Each centre should determine its own policy based on its financial resources and on the wait list.     

The standardization of pancreatic donors for islet isolation

INTRODUCTION: Islet transplantation has proven to be a successful treatment for insulin-dependent diabetes mellitus (IDDM). The aim of this study was to establish an algorithm by which the combination of the donor quality and pancreas quality was given a numerical score from 0 to 100 for use in determining the quality of a pancreas for islet isolation. METHODS: In this study we retrospectively analyzed 326 pancreata and the outcomes of their respective isolations. Specific donor variables and physical characteristics were identified and weighted according to their influence on the success of the isolation. For each variable, ranges and point weightings were established based on our laboratory experience and literature review. RESULTS: Analysis of the data showed a strong association of the donor point with isolation outcome. Pancreata with lower donor point scores had lower transplant success rates, while higher donor point scores in turn produced higher transplant rates. CONCLUSION: This scoring system has proven to be effective in assessing the potential of pancreata for a favorable isolation outcome. By analyzing the final score of the pancreas, a standardized decision can be made on whether to accept or decline the pancreas. Another benefit of the scoring system is that it is a quick and efficient way to trend the quality of donor organs.     

Transaminitis after pancreatic islet transplantation

BACKGROUND: An asymptomatic, self-limited transaminitis uniformly follows pancreatic islet transplantation (PIT) performed through portal vein (PV) infusion. The underlying cause and significance of this transaminitis is unclear. STUDY DESIGN: Records of all patients with insulin-dependent diabetes mellitus who had undergone PIT at our institution were reviewed. All PITs were performed in conjunction with a remote pancreatic islet isolation center and done through percutaneous transhepatic PV infusion. Alanine aminotransferase (ALT) levels, serum glucose concentrations, insulin requirements, and color-flow Doppler ultrasonography examinations of the right upper quadrant were assessed before and after PIT. RESULTS: Eleven patients have undergone a total of 26 PITs. An elevated ALT level occurred in all 11 patients (100%) after the first PIT, with the median post-PIT peak ALT level reaching 187 IU/L. Transaminitis was less frequent and less marked after the second PIT. A negative correlation between viability of the pancreatic islets transplanted (r = -0.44, p = 0.03) and a positive correlation between the ratio of maximum to initial PV pressure (r = 0.41, p = 0.04) were seen with the subsequent ALT peak. Color-flow Doppler ultrasonography examinations showed no occurrences of PV thrombosis or intrahepatic hematoma. Finally, the degree of transaminitis did not correlate with post-PIT insulin requirements, indicating that post-PIT transaminitis cannot be used to measure allograft rejection or function. CONCLUSIONS: Transaminitis after PIT is common and self-limited. Post-PIT transaminitis does not signal acute rejection or serious procedure-related complications such as PV thrombosis or intrahepatic hematoma.     

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.     

Experience in xenografts of pancreatic islet cell cultures after surgical treatment of chronic pancreatitis

Xenotransplantation of the pancreatic islet cells was performed in 8 patients with chronic pancreatitis and concomitant diabetes mellitus, operated on the pancreas. In 2 patients, the rejection of the cells transplanted occurred. The causes of failure and possibilities for improving the technique are analysed.