Effect of Etanercept Concentration on Human Islet Integrity.

BACKGROUND: Etanercept is widely used as an antiinflammatory drug to improve engraftment after intraportal islet transplantation. In contrast to other immunosuppressive agents, very little is known about detrimental effects of etanercept on islets. The aim of this pilot study was to define the toxic range of etanercept. METHODS: Human islets isolated from 8 donors were cultured for 4-5 days at 37°C in culture medium supplemented with etanercept at concentrations from 2.5 to 40 µg/mL, corresponding to potential in vivo levels within the portal vein. After culture, islet equivalent (IEQ) yield, fragmentation index (islet number/IEQ), purity, viability, and stimulated insulin release (2 vs 20 mmol/L) were assessed and normalized to islets before culture. RESULTS: Yield (73 ± 8%) and viability (91 ± 4%) were highest with 5 µg/mL etanercept. Islet loss was evident when etanercept was ≥10 µg/mL (55 ± 7%; P < .05 vs control). Fragmentation (154 ± 34%; P < .05) was markedly increased and viability (81 ± 4%, P < .05) markedly decreased with etanercept >10 µg/mL. The accumulation of cell debris at concentrations ≥20 µg/mL resulted in a significant reduction of islet purity (84 ± 3%; P < .05). Etanercept did not interfere with stimulated insulin secretion at concentrations ≤10 µg/mL. The maximum stimulation index was noted at 2.5 µg/mL (1.8 ± 0.1). CONCLUSIONS: Etanercept is tolerated by isolated human islets at concentrations <10 µg/mL. Our data suggest that the tight range between benefit and toxicity should be considered for dosage and administration of etanercept.

Hypoxia-Induced Damage in Human Islets Is Reduced With the Use of Mesenchymal Stem Cell-Preconditioned Medium.

BACKGROUND: Mesenchymal stem cells (MSCs) are protective for islets when cotransplanted in a hypoxic environment. However, the risk of neoplasia is increased when MSCs are transplanted into immunosuppressed patients. This initial study aimed to investigate whether the production of protective factors from MSC can be stimulated by different culture conditions to benefit human islets cultured in hypoxia. METHODS: MSC were isolated from human adipose tissue and cultured for 2 days in supplemented Minimum Essential Media α (MEMα) and 21% (21%-MEMα) or 1% oxygen (1%-MEMα). Native MEMα served as control. After MSC harvesting, cell-depleted media were frozen at -20°C until use for human islet culture in 2% oxygen for 72-96 hours before islet characterization. Data were normalized to control islets cultured in native MEMα and 2% oxygen (mean ± SEM). RESULTS: After culture in 21%- or 1%-MEMα, islet recovery increased to 117 ± 12% (NS) and 138 ± 12% (P < .05), respectively. Viability did not change after culture in native MEMα (59 ± 2%), 21%-MEMα (59 ± 3%), or 1%-MEMα (61 ± 3%). Compared with control samples, the glucose stimulation index was increased after culture in 21%-MEMα (P < .05) or 1%-MEMα (P < .05). Overall survival was higher in 1%-MEMα (143 ± 14%) than in 21%-MEMα (119 ± 14%; NS) or native MEMα (P < .05). CONCLUSIONS: This study demonstrates that MSC-preconditioned MEMα increases survival and in vitro function of hypoxic human islets. These findings indicate that hypoxic MSCs seem to produce factors that improve survival of islets suffering from hypoxia.

Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model.

Pancreas transportation between donor center and islet production facility is frequently associated with prolonged ischemia impairing islet isolation and transplantation outcomes. It is foreseeable that shipment of pig pancreases from distant centralized biosecure breeding facilities to institutes that have a long-term experience in porcine islet isolation is essentially required in future clinical islet xenotransplantation. Previously, we demonstrated that perfluorohexyloctan (F6H8) is significantly more efficient to protect rat and human pancreata from ischemically induced damage compared to perfluorodecalin (PFD). To evaluate the effect of F6H8 on long-term stored pig pancreases in a prospective study, we utilized the split lobe model to minimize donor variability. Retrieved pancreases were dissected into the connecting and splenic lobe, intraductally flushed with UW solution and immersed alternately in either preoxygenated F6H8 or PFD for 8-10 h. Prior to pancreas digestion, the intrapancreatic pO2 and the ratio of ATP-to-inorganic phosphate was compared utilizing 31P-NMR spectroscopy. Isolated islets were cultured for 2-3 days at 37°C and subjected to quality assessment. Pancreatic lobes stored in preoxygenated F6H8 had a significantly higher intrapancreatic pO2 compared to pancreata in oxygen-precharged PFD (10.11 ± 3.87 vs. 1.64 ± 1.13 mmHg, p < 0.05). This correlated with a higher ATP-to-inorganic phosphate ratio (0.30 ± 0.04 vs. 0.14 ± 0.01). No effect was observed concerning yield and purity of freshly isolated islets. Nevertheless, a significantly improved glucose-stimulated insulin response, increased viability and postculture survival (57.2 ± 5.7 vs. 39.3 ± 6.4%, p < 0.01) was measured in islets isolated from F6H8-preserved pancreata. The present data suggest that F6H8 does not increase islet yield but improves quality of pig islets isolated after prolonged cold ischemia.

Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation.

For more than a decade Liberase HI was commonly used as the standard enzyme blend for clinical human islet isolation until enforced replacement by collagenase NB1 (NB1). This change resulted initially in a reduction in islet isolation outcome and transplant activities worldwide. This retrospective study was initiated to compare the efficiency of NB1 premium grade with Liberase in 197 human islet isolations. All pancreata were processed between January 2006 and June 2008 utilizing the same procedures for isolation and quality assessment except the administration of preselected lots of either Liberase (n = 101) or NB1 (n = 96). Utilizing Liberase, significantly more digested tissue and purified islet yield was produced compared to NB1. In contrast, the use of NB1 was associated with significantly higher purity and glucose stimulation index during dynamic perifusion. The expression of proinflammatory markers was almost identical except tissue factor expression, which was higher after utilization of Liberase. No difference was found in the percentage of pancreata fulfilling the criteria for clinical islet transplantation. The results suggest that Liberase is more efficient for pancreas dissociation than collagenase NB1 but seems to be more harmful to exocrine cells and islet tissue.

Pig pancreas oxygenation at 20 degrees C increases islet ATP generation but deteriorates islet function.

Successful pancreas preservation during storage in oxygenated perfluorodecalin (PFD) is mainly related to oxidative ATP generation during storage. Increasing the storage temperature would accelerate this process essential for resuscitation of ischemically damaged pancreatic tissue. The present study aimed at comparing islet isolation outcome from adult pig pancreata preserved in oxygenated PFD by means of a one-layer method during storage on ice or at 20 degrees C. Resected pancreata were intraductally flushed with cold UW solution and promptly processed (n = 6) or stored for 3 h in continuously oxygenated PFD at 4 degrees C (n = 5) or 20 degrees C (n = 7). Prior to digestion-filtration pancreata were intraductally injected with UW supplemented with Serva collagenase NB8 and neutral protease. Islet quality assessment determined viability, glucose stimulation index, mitochondrial activity, intracellular ATP content, and transplant function in diabetic nude mice. Pancreata oxygenated for 3 h at 20 degrees C yielded islet numbers similar to organs oxygenated at 4 degrees C. Compared to a storage temperature of 20 degrees C, preservation at 4 degrees C reduced islet ATP content (p < 0.05) as well as islet viability (p < 0.05). Nevertheless, PFD storage at 20 degrees C decreased insulin response to glucose compared to unstored pancreata (p < 0.05). In contrast to unstored pancreata or cold-stored organs, transplantation of islets isolated after oxygenation at 20 degrees C was characterized by an early loss of transplant function in 50% of recipients (p < 0.05). The present study demonstrates that PFD storage at 20 degrees C enhances islet ATP synthesis within a short period of oxygenation but deteriorates islet function. We conclude that the present data reflect an equilibration between reduced depression of metabolic activity resulting in damage of islets and temperature-stimulated acceleration of ATP synthesis. Future studies are required to adjust the optimum storage temperature for pancreas oxygenation in different species.

Degraded collagenase deteriorates islet viability.

OBJECTIVE: The utilization of purified enzyme blends consisting of collagenase class I (CI) and II (CII) and neutral protease is an essential step for clinical islet isolation. Previous studies suggested that the use of enzyme lots containing degraded CI reduced islet release from human pancreata. The present study sought to assess the effect of degraded collagenase on islet function in vitro and posttransplantation. MATERIALS AND METHODS: Crude collagenase was chromatographically separated into CI, CII, and a mixture of degraded CI and CII isomers. Subsequently, classes were recombined to obtain a CII/CI ratio of 0.5. Rat islets were isolated utilizing neutral protease and 20 units of recombined collagenase containing either intact (Ci) or degraded isomers (Cd). RESULTS: Digestion time was reduced utilizing Cd (P < .001). The highest islet yield and lowest islet fragmentation were obtained with Ci (P < .01). Utilization of Cd corresponded to a reduction in viability and in vitro function (NS). Islet transplantation reversed hyperglycemia in diabetic nude mice, but revealed an absence of weight gain in recipients receiving islets isolated using Cd (P < .01). CONCLUSION: This study suggested that islet function posttransplantation is affected by degraded collagenase isomers. This finding has to be considered for the purification process of collagenase.

New class of oxygen carriers improves islet isolation from long-term stored rat pancreata.

OBJECTIVE: Pancreas shipment is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome. The present study investigated the efficiency of perfluorohexyloctane (F6H8), a hyperoxygen carrier characterized by low specific density (1.33 g/cm3) and lipophilic qualities, to facilitate islet isolation from long-term stored rat pancreata. MATERIALS AND METHODS: Prior to islet isolation, pancreata were intraductally flushed in situ with Kyoto solution (KS) and stored for 24 hours in KS, oxygenated PFD, or F6H8. RESULTS: Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 in PFD- and F6H8-incubated pancreata was almost the same. In correspondence, the ATP content and viability of isolated islets were similar as well. In contrast, islet yield and in vitro function were significantly reduced after storage in PFD compared with F6H8. CONCLUSION: This study suggested that islet isolation performed after long-term pancreas preservation can be significantly improved utilizing semifluorinated alkanes as oxygen carriers.

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.

Effect of pretransplant preconditioning by whole body hyperthermia on islet graft survival.

Previous observations in heat-shocked pig islets revealed the ambivalent character of the stress response simultaneously inducing processes of protection and apoptosis. To clarify whether the proapoptotic character of the stress response is reduced in heat-exposed islets still embedded in their native environment, hyperthermia was performed in the present study either as whole body hyperthermia (WBH) prior to pancreas resection or as in vitro heat shock (HS) after isolation. HS (42 degrees C/45 min) was induced in donors 12 h before isolation (WBH, n = 32) or in freshly isolated islets prior to 12 h of culture at 37 degrees C (in vitro HS, n = 25). Islets continuously incubated at 37 degrees C served as controls (n = 34). Proinflammatory treatment was performed with H2O2, DETA-NO, or a combination of IL-1beta, TNF-alpha, and IFN-gamma. Quality assessment included islet yield, viability staining, static glucose incubation, and nude mouse transplantation. WBH was significantly less effective than in vitro HS to induce HSP70 overexpression and to increase islet resistance against inflammatory mediators. Although characterized by an unaltered Bax to Bcl-2 ratio, islets subjected to WBH partially failed to restore sustained normoglycemia in diabetic nude mice. The inflammatory response observed in the pancreas of WBH-treated rats was associated with significantly reduced viability that seems to have a higher predictive value for posttransplant outcome compared to islet in vitro function or mitochondrial activity. In contrast, in vitro HS significantly decreased transcript levels of Bcl-2, but did not affect posttransplant function compared to sham-treated islets. These findings suggest that WBH is primarily associated with increased necrosis as a secondary tissue type-specific effect of pancreas damage while in vitro HS mainly induces apoptosis.

Ischaemia is linked to inflammation and induction of angiogenesis in pancreatic islets.

Beta-cell replacement is the only way to restore euglycaemia in patients with type-1 diabetes. Pancreatic tissue, processed for subsequent clinical islet transplantation, is exposed to ischaemia causing injury and death in a large number of islets before and after transplantation. In this review we summarize what is known on the sources of environmental stress for pancreatic islets, such as insufficient oxygen supply during pancreas procurement and in culture prior to intraportal transplantation, nutritional and oxygen deprivation during the isolation process, and the consequences of hyperglycaemia. An increasingly recognized role in the modulation of beta-cell function and these environmental stress factors plays the vascular network of the pancreatic islets. Islet revascularization by angiogenesis is relevant for the survival of the graft subsequent to transplantation. Potential strategies offered by therapeutic induction of revascularization to ameliorate the detrimental impact of these factors on the quality of islet transplants are discussed.

Adjustment of the ratio between collagenase class II and I improves islet isolation outcome.

BACKGROUND: Previous studies have clarified the distinct roles of collagenase class I (ccI) and class II (ccII) in enzymatic release of islets from pancreatic tissue. The present study sought to enhance the limited knowledge about the optimal ratio between collagenase classes. METHODS: Rat islets were isolated utilizing 0.4 DMC-U of neutral protease and 20 PZ-U of fractionated NB-1 collagenase recombined to obtain a ccII/I ratio of 0.5, 1.0, and 1.5. Quality control included assessment of yield (islet equivalents), trypan-blue exclusion, insulin release during static glucose incubation, and transplant function in diabetic nude mice. Data are expressed as mean values +/- SEM. RESULTS: Digestion time was only minimally influenced by different ccII/I ratios. The highest islet yield (P < .05) was obtained using a ccII/I ratio of 1.0. Purity and glucose stimulation index were only marginally affected by different ccII/I ratios. A significant loss of islet viability after 24-hour culture (P < .05) was observed only in islets isolated by means of a ccII/I ratio of 0.5 and 1.5 but not 1.0. Transplantation into diabetic nude mice revealed sustained islet graft function in all experimental groups. CONCLUSIONS: The present study indicates that the ratio between ccII and ccI is of significant relevance for optimizing islet yield and viability.

Effect of stable glutamine compounds on porcine islet culture.

BACKGROUND: Pig islets are characterized by significant fragility, preventing successful islet culture prior to xenotransplantation. To improve outcome after culture, we compared the effects of glutamine supplementation on survival and viability of isolated pig islets during culture. METHODS: Pig islets were suspended in CMRL 1066 supplemented either with 2.5 mmol/L N-acetyl-L-alanyl-L-glutamine (NALG), a stable compound of L-glutamine, or with 2.5 or 5.0 mmol/L of free L-glutamine (L-Glu). After 24 hours of preincubation, islets were stressed for additional 48 hours with H2O2, DETA, or a cytokine mix. RESULTS: Twenty-four-hour survival of unstressed controls precultured with 2.5 mmol/L NALG was significantly decreased compared with islets pretreated with 2.5 or 5.0 mmol/L L-Glu (P < .01). Fresh islets, viability decreased significantly after NALG preincubation, but was maintained after preincubation in 2.5 or 5.0 mmol/L L-Glu (not significant vs fresh; P < .05 vs NALG). Compared with NALG pretreatment L-Glu did not significantly ameliorate the relative survival (related to cultured controls) of islets during proinflammatory treatment. Nevertheless, the beneficial effect of L-Glu preculture on absolute survival (related to freshly isolated islets) of stressed islets was still present in contrast to NALG pretreatment (P < .01). Viability of stressed islets was significantly protected by L-Glu but not by NALG. CONCLUSIONS: Pig islet culture is significantly improved if L-glutamine is administered in an unbound form compared with the stable compound NALG. Stress resistance of pig islets seems to be increased by free L-glutamine as well.

The ratio between class II and class I collagenase determines the amount of neutral protease activity required for efficient islet release from the rat pancreas.

UNLABELLED: Previous investigations clearly showed that the successful release of islets from the pancreas is mediated by both neutral protease (NP) and collagenase, consisting of subclasses I and II showing different capacities to cleave islets from the pancreas. Since no informations about the optimal ratio between class II and class I collagenase (II/I-ratio) are available yet, the present study sought to evaluate the efficient range for the II/I-ratio. METHODS: Following intraductal pancreas collagenase distension, rat islets were isolated utilizing 20 PZ-U Serva collagenase NB 1 and 1.0 or 0.4 DMC-U NP. After purification we determined the islet yield (IEQ), viability (trypan-blue exclusion) and function in diabetic nude mice. RESULTS: At 1.0 DMC-U NP, a II/I-ratio of 2.6, 1.5 or 0.7 yielded 2200 +/- 280, 2185 +/- 420, and 2205 +/-90 IEQ, respectively (ns). Viability varied between 70% and 80% (ns). Digestion time was significantly lowest (P < .05) using a II/I-ratio of 0.7. Utilization of 0.4 DMC-U NP resulted in a viability of >98% among all experimental groups (P < .001 vs 1.0 DMC-U). Islet yield decreased at a II/I-ratio of 2.6 (1520 +/- 120 IEQ, P < .05) and 1.5 (1780 +/- 130 IEQ, ns), but not at 0.7 (2310 +/- 160 IEQ, ns). Again, digestion time was lowest (P < .001) using a II/I- ratio of 0.7. Transplantation into diabetic nude mice demonstrated islet function in all experimental groups. CONCLUSIONS: NP significantly affects islet viability. This study indicates that the minimal amount of NP required for efficient islet cleavage depends on the II/I-ratio.

Timing of hyperthermic preconditioning affects islet resistance against inflammation.

BACKGROUND: Heat exposure of isolated islets enhances resistance against inflammation but decreases islet graft function. In contrast, donor preconditioning by whole-body hyperthermia increases islet ischemic tolerance and improves viability of pancreatic isografts. This study aimed to compare yield, viability, and inflammatory resistance of rat islets subjected to heat shock prior to (pre-HS) or after isolation (post-HS). METHODS: Islets were isolated as previously described. HS (42 degrees C/45 min) was induced 12 hours before islet isolation (pre-HS, n = 31) or in freshly isolated islets prior to 12 hours of recovery at 37 degrees C (post-HS, n = 12). Islets continuously incubated at 37 degrees C served as controls (n = 33). Proinflammatory treatment included incubation with 0.05 mmol/L H(2)O(2), 1.0 mmol/L DETA-NO or cytokines (interleukin-1beta + tumor necrosis factoralpha + interferongamma). RESULTS: Purified islet yield was 1200 +/- 80 IEQ in unconditioned donors (n = 45) and 980 +/- 80 IEQ after pre-HS (ns). Islet viability was not affected by post-HS, but the glucose stimulation index (P < 0.001, P < 0.01) and formazan production (P < 0.05) were significantly lower compared to pre-HS or sham treatment. The expression of heat shock protein HSP70 in pre-HS islets was slightly higher compared to controls (ns) but lower compared to post-HS islets (P < 0.05), correlating with the resistance against H(2)O(2) and DETA-NO compared to post-HS islets (P < 0.05) or controls (ns). Cytokines did not affect mitochondrial formazan production. CONCLUSIONS: The findings indicate that hyperthermic islet treatment is less harmful if performed in the native pancreatic environment. This beneficial effect is associated with a decreased HSP70 expression resulting in a reduced resistance against inflammatory mechanisms.

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation.

BACKGROUND: Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10). METHODS: Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM. RESULTS: Compared to unstored (429200 +/- 86700 IEQ) and OLM-stored pancreases (338600 +/- 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 +/- 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 +/- 0.4) was significantly decreased after TLM storage (1.8 +/- 0.2, P < .05) but not after OLM storage (2.3 +/- 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups. CONCLUSIONS: This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.

Influence of neutral protease activity on human islet isolation outcome.

UNLABELLED: Observations in rat pancreata have revealed that enzymatic islet release is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. Since no information is available about the effect of NP activity on islet release from the human pancreas, the present study evaluated the effect of various NP concentrations on the outcome of human islet isolation. METHODS: Following intraductal collagenase distension, pancreata obtained from adult multiorgan donors were digested using 2000 PZ-U of purified Serva collagenase NB 1 supplemented with 2.6 (n = 10) or 4.5% (DMC-U/PZ-U) (n = 10) of NP. RESULTS: Increasing NP from 2.6% to 4.5% reduced the amount of undigested tissue from 22 +/- 2 to 17 +/- 2 g (P < .05) while simultaneously increasing the volume of digested tissue (26 +/- 2 vs 40 +/- 3 mL, P < .01). Increased NP concentrations increased the islet yield prepurification (459,800 +/- 22,900 vs 587,600 +/- 69,000 IEQ, P < .05), but simultaneously affected islet purification, resulting in equal islet yields (345,700 +/- 31,200 vs 391,500 +/- 35,400 IEQ, NS) and less purity (70 +/- 6 vs 49% +/- 5%, P < .01). A NP concentration of 4.5% reduced the stimulation index (4.7 +/- 1.2 vs 2.0 +/- 0.5, P < .01) and viability (100 +/- 1 vs 95% +/- 3%, P < .05). CONCLUSIONS: Although increased NP activity seems to improve islet release from adult human pancreata, it significantly affects islet viability and function. The reduction in purity reflected damage to acinar tissue by increased NP activity presumably affecting islet integrity.

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas.

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Can the density of native pancreatic tissue slices predict human islet isolation and purification outcome?

INTRODUCTION: With currently available technology, the outcomes of human islet isolation and purification are still inconsistent, in part due to a lack of control of the pancreas donor and the procurement conditions. Using a single donor pancreas, the critical islet mass for establishing insulin independence of approximately 5000 engrafted islet equivalents (IEQ)/kg of recipient weight can only be retrieved from about one third of isolations. The purpose of this study was to analyze whether successful islet isolation and purification outcomes might be predicted from the density of native pancreatic tissue. METHODS: Tissue slices (TS) were obtained from the neck of 9 nondistended human donor pancreata. The density of the TS was determined using gravity sedimentation in continuous density gradients under either iso-osmolar or hyperosmolar conditions. Correlation coefficients were calculated with regard to the density of isolated exocrine and endocrine tissue, donor age, body mass index (BMI), cold ischemia time (CIT), IEQ prepurification and postpurification, IEQ recovery, and purity. RESULTS: (1) There was no change in density over time for TS in 300 mOsm/kg (mean, 1.079 +/- 0.0019 g/cm(3)) (2) In 500 mOsm/kg, there was a significant increase in density from 1.086 +/- 0.0021 g/cm(3) to 1.092 +/- 0.0021 g/cm(3) over time. (3) Density of isolated exocrine and endocrine became more distinct with lower density of TS (r = -0.776; P < .05). (4) Donor age, BMI, recovery of IEQ from gradients, and number of IEQ after purification did not correlate significantly with TS density. (5) In contrast, a significant inverse correlation existed betwen TS and CIT (r = -0.829; P < .05), and between TS versus IEQ number prior to purification (r = -0.867; P < .05). CONCLUSION: No homogeneous distribution of pancreas tissue density was seen among 9 consecutive human organs. Taken together, the density of native pancreas TS is not a suitable sole predictor for successful islet isolation and purification.

Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.

INTRODUCTION: According to previous estimates from large animals and man, a minimum of approximately 5000 to 6000 engrafted islet equivalents (IEQ)/kg recipient weight is critical to establish insulin independence. Utilizing a single donor, this threshold yield of purified islets can be retrieved from approximately one third of all isolations. The aim of this study was to improve human islet purification by optimization of the osmolality and the density range of the continuous Ficoll-sodium-diatrizoate (FSD) gradient to facilitate consistent purities >80% of human islet preparations without considerable loss of islet yield. METHODS: Aliquots of human pancreatic digests were placed on continuous density gradients. After centrifugation, sequential aliquots were extracted for amylase and insulin to determine the relative and cumulative density distribution of endocrine and exocrine tissue. We addressed the impact of two factors: (1) osmolalities (300 to 600 mosm/kg) in the gradient of FSD covering a density range of 1.070 to 1.100 g/cm(3); and (2) density (FSD 500/1.070 to 1.100) versus density-osmolarity gradient (DO-FSD 400-530/1.080 to 1.113). RESULTS: The density of exocrine and endocrine tissue increased with rising osmolality. Differences in density of both tissues were highest at 450 and lowest at 300 and 600 mOsmol/kg. Purity and recovery were highest at 450 versus 400 or 500 mOsm/kg (NS). Exocrine but not endocrine tissue was more dense in DO-FSD than in FSD gradient (P < .05). The differences in density were 0.004 versus 0.013 g/cm(3) (P < .01), resulting in an increased islet purity and recovery. CONCLUSION: The best osmolality for the FSD 1.070 to 1.100 g/cm(3) is at 450 mOsm/kg. Using the DO-FSD may improve human islet purification allowing successful clinical islet transplantation.

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

AIMS/HYPOTHESIS: The 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. METHODS: In 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. RESULTS: Human 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+/-0.1 micromol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 micro mol/l LC-CoA ester. The presence of 1 micro mol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5+/-0.2 nS to 2.5+/-1.3 nS. CONCLUSION/INTERPRETATION: Taken 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.