H. Brandhorst

Significant progress in porcine islet mass isolation utilizing Liberase HI for enzymatic low-temperature pancreas digestion

BACKGROUNDnFrequent success in human islet isolation is prevented by the large variability of scarce organ donors; this favors the future utilization of pigs as donors for clinical islet xenotransplantation. Porcine-specific difficulties of islet isolation are attributed to the intrinsic fragility of islets during pancreas digestion.nnnMETHODSnTo preserve islet integrity during efficient pancreas dissociation, porcine pancreata (n=48) were distended after cold storage with cold University of Wisconsin solution containing Liberase HI and digested at 24-28 degrees C using digestion-filtration. Pancreata distended with University of Wisconsin solution containing well-proven crude collagenase and digested at 32-34 degrees C served as controls (n=46). Monolayer Ficolldiatrizoate gradient purification was performed in a Cobe 2991.nnnRESULTSnPurified yield of islet equivalents per pancreas (mean+/-SEM) was almost doubled by Liberase HI compared with crude collagenase (526,480+/-46,560 vs. 270,270+/-19,420; P < 0.0001) and also significantly increased comparing islet equivalents per gram of pancreas (4,210+/-320 vs. 2,640+/-245; P=0.0004). Islet integrity was better preserved during Liberase HI digestion compared with crude collagenase digestion as indicated by isolation index (2.1+/-0.1 vs. 1.4+/-0.1; P<0.0001). Purity, viability, and in vitro function of islets did not differ between experimental groups. Preserved in vivo function of islets isolated by Liberase HI was demonstrated after subcapsular transplantation into 16 diabetic nude rats.nnnCONCLUSIONSnIf the problems related to xenograft rejection and xenosis could be solved, low-temperature digestion of porcine pancreata using Liberase HI could serve as an essential prerequisite for successful 1:1 xenotransplantation of pig islets into type 1 diabetic human recipients.

Assessment of intracellular insulin content during all steps of human islet isolation procedure.

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

Transduction of non-dividing adult human pancreatic beta cells by an integrating lentiviral vector

Summary Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 ± 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation. [Diabetalogia (1998) 41: 736–739]

Improved glucose counterregulation and autonomic symptoms after intraportal islet transplants alone in patients with long-standing type I diabetes mellitus.

BACKGROUNDnDefective glucose counterregulation and hypoglycemia unawareness are both well-recognized risk factors for recurrent episodes of severe hypoglycemia in patients with type I diabetes. At present, no conventional therapy is available to routinely overcome these acquired impairments in long-standing diabetes.nnnMETHODSnTo test the hypothesis that successful intraportal islet transplantation could improve this syndrome, hormonal counterregulatory responses and symptoms were studied during stepped hypoglycemic clamp tests before and after intraportal islet transplantation in three patients with type I diabetes who were prone to severe hypoglycemia.nnnRESULTSnAs compared with matched nondiabetic control subjects, before islet transplantation, glucagon responses were absent while epinephrine and cortisol responses were either markedly decreased or absent in all diabetic subjects. One patient also had decreased norepinephrine and growth hormone responses. Autonomic warning symptoms were absent in all patients during hypoglycemia. One month after successful islet transplantation, there was no improvement in the glucagon response. However, glycemic thresholds and/or peak incremental responses of epinephrine, norepinephrine, and cortisol improved in all patients. Moreover, all patients had developed autonomic warning symptoms so that glycemic thresholds were detectable within the examined range.nnnCONCLUSIONnWe conclude that intraportal islet transplantation does not restore hypoglycemia-induced glucagon secretion, but it improves the responses of most counterregulatory hormones and hypoglycemic warning symptoms even in long-standing type I diabetes.

Long-chain CoA esters activate human pancreatic beta-cell KATP channels: potential role in Type 2 diabetes.

Aims/hypothesisThe ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches.MethodsIn this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique.ResultsHuman KATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7±0.1xa0µmol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1xa0mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1xa0µmol/l LC-CoA ester. The presence of 1xa0µmol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5±0.2xa0nS to 2.5±1.3xa0nS.Conclusion/interpretationTaken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate KATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.

Long-term survival, morphology and in vitro function of isolated pig islets under different culture conditions

BACKGROUNDnIslet culture aims to optimize islet survival and to reduce islet immunogenicity. To achieve these objectives, culture periods at 37 degrees C and 22-24 degrees C are mainly used.nnnMETHODSnThis study compares the influence of donor age (juvenile vs. adult), temperature (22 degrees C vs. 37 degrees C), and serum supplementation (10% newborn calf serum [NCS] with 10% pig serum) on morphological integrity and in vitro function of porcine islets during long-term culture (LTC).nnnRESULTSnAfter 21 days at 22 degrees C, the survival rate of cultured islets isolated from juvenile donors was lower than of adult islets (23+/-0.9% vs. 88+/-2.8%, P<0.001). Compared with 37 degrees C, LTC at 22 degrees C increased survival of adult islets and DNA recovery (92+/-2.5% vs. 45+/-4.8%, P<0.001; 72+/-4.1% vs. 30+/-5.1%, P<0.001) and reduced viability (62+/-8% vs. 89+/-5%, P<0.05). LTC at 22 degrees C was associated with a reduction of insulin content (85+/-9 vs. 152+/-10 microU/islet equivalents [IEQ], P<0.01), 24 hr-insulin secretion (82+/-7 vs. 552+/-91 microU/ day/IEQ, P<0.001), and integrated dynamic insulin response to glucose (1093+/-124 vs. 3074+/-708 microU/60 min/100 IEQ, P<0.05), compared with 37 degrees C LTC. Histologic analysis revealed disintegration of islet periphery after 22 degrees C, whereas smoothly shaped islets were present after 37 degrees C LTC. Integrity after 14 days at 37 degrees C was significantly better preserved when medium CMRL 1066 was supplemented with 10% porcine serum, compared with 10% NCS (40+/-2.3% vs. 21+/-6.7%, P<0.05), contrasting with 22 degrees C (52+/-4.0% vs. 59+/-3.7%, not significant).nnnCONCLUSIONSnThis study demonstrates that survival of cultured porcine islets is increased at 22 degrees C, whereas in vitro function and viability are better preserved at 37 degrees C. Survival at 37 degrees C can be improved by adding homologous serum to the medium.

Leptin inhibition of insulin secretion from isolated human islets

Abstract Leptin is a hormone produced and secreted from the adipose tissue. Its physiological actions include the regulation of satiety, food intake and energy balance. The production of leptin is increased by high insulin levels. Here, we demonstrate that leptin acts as an inhibitor of glucose-induced (20 mM) insulin secretion from isolated human islets. No effect was observed in the presence of lower glucose levels (2.8 and 10 mM glucose). The pancreatic β-cell might represent a target of a direct physiological action of leptin. We suggest the presence of an “adipo-insular axis” in which leptin mediates negative feedback from the adipose tissue to the endocrine pancreas.

Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4±30.1×106) compared with empty capsules (41.4±19.7×106) and non-encapsulated porcine islets (18.1±3.3×106). The percentage of CD 3 positive T-lymphocytes rose to 44.5±11.5% in case of microencapsulated xenografts compared with 19.2±8.2% for non-encapsulated xenografts and 4.9±2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2±8.9% vs 15.2±7.0% for free islets and 4.9±1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.

Human Islet Separation Utilizing a Closed Automated Purification System

A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 ± 1.1% vs. 98.2 ± 2.0%, p < 0.05). Islet yield (120,468 ± 15,970 vs. 114,570 ± 15,313 IE, NS) and purity (51.7 ± 4.8% vs. 54.4 ± 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.

Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets

In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft [1]. With improved isolation methods based on the original procedure published by Ricordi et al. [2] yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation [3, 4]. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.