Ippei Matsumoto

Transplantation of Cultured Islets from Two-Layer Preserved Pancreases in Type 1 Diabetes with Anti-CD3 Antibody

We sought to determine whether or not optimizing pancreas preservation, islet processing, and induction immunosuppression would facilitate sustained diabetes reversal after single‐donor islet transplants. Islets were isolated from two‐layer preserved pancreata, purified, cultured for 2 days; and transplanted into six C‐peptide‐negative, nonuremic, type 1 diabetic patients with hypoglycemia unawareness. Induction immunosuppression, which began 2 days pretransplant, included the Fc receptor nonbinding humanized anti‐CD3 monoclonal antibody hOKT3γ1 (Ala‐Ala) and sirolimus. Immunosuppression was maintained with sirolimus and reduced‐dose tacrolimus. Of our six recipients, four achieved and maintained insulin independence with normal HbA1c levels and freedom from hypoglycemia; one had partial islet graft function; and one lost islet graft function 2 weeks post‐transplant. The four insulin‐independent patients showed prolonged CD4+ T‐cell lymphocytopenia; inverted CD4:CD8 ratios; and increases in the percentage of CD4+CD25+ T cells. These cells suppressed the in‐vitro proliferative response to donor cells and, to a lesser extent, to third‐party cells. Severe adverse events were limited to a transient rash in one recipient and to temporary neutropenia in three. Our preliminary results thus suggest that a combination of maximized viable islet yield, pretransplant islet culture, and preemptive immunosuppression can result in successful single‐donor islet transplants.

Effect of Donor Age on Function of Isolated Human Islets

This study intended to evaluate the impact of donor age on the function of isolated islets. Analysis of human islets from cadaveric donors (age 16–70 years) was performed using glucose-stimulated insulin release (GSIR) (n = 93), islet ATP content (n = 27), diabetic nude mouse bioassay (n = 72), and the insulin secretory function after single-donor clinical islet allotransplantation (n = 7). The GSIR index was significantly higher in younger donors (age ≤40 years) than in older donors and negatively correlated with the donor age (r = −0.535). Islet ATP was higher in younger donors (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/μg DNA). The diabetes reversal rate of mice with 2,000 IE was significantly higher in younger donors (96 vs. 68%). C-peptide increment to glucose during intravenous glucose tolerance test at days 90–120 after clinical transplantation showed negative correlation with donor age (r = −0.872) and positive correlation with the islet mass (r = 0.832). On the other hand, acute insulin response to arginine only showed correlation with the islet mass and not with donor age. These results show that insulin secretory response to glucose deteriorates with increasing age and that it may be related to changes in ATP generation in β-cells.

Improvement in islet yield from obese donors for human islet transplants.

Background. The feasibility of human islet transplantations has been firmly established. To increase the number of islet transplants, the suitability of pancreases from organ donors considered inappropriate for pancreas transplantations must be evaluated. Methods. We isolated islets from 114 human cadaver donor pancreases by the automated Ricordi method, followed by purification using continuous-density gradients. We divided the pancreases into two groups by donor body mass index (BMI)—group 1: n=51, BMI of 30 or more; group 2: n=63, BMI of less than 30. We compared the results of human islet isolation, in vitro potency assays, and a nude mouse bioassay. Results. In group 1 (vs. group 2), we found a significantly higher mean pancreas weight (109.5±30.7 vs. 90.6±24.0 g; P=0.0002); higher mean islet equivalents/pancreas, after digestion (442,565±238,741 vs. 289,860±158,995; P<0.0001) and after purification (319,129±164,002 vs. 215,753±126,089; P=0.0002); and a higher islet isolation success rate—defined as isolations yielding more than 300,000 islet equivalents/pancreas, with purities of more than 50% (37.3% [19 of 51 pancreases] vs. 15.9% [10 of 63]; P=0.009). Our in vitro potency assays and bioassay uncovered no differences between the two groups. Notably, all except one of the donor BMIs for the successful isolations in group 2 exceeded 26; the mean donor BMI for the successful isolations (27.3±3.0, n=10) was significantly higher than for the unsuccessful isolations (24.8±3.3, n=53) (P=0.03). Conclusions. Pancreases from both overweight (BMI ≥26 but <30) and obese (BMI ≥30) cadaver donors are suitable for islet isolation and transplantations. Their use could increase the size of the islet donor pool.

Improved islet yield and function with ductal injection of University of Wisconsin solution before pancreas preservation

Background. Ensuring sufficient islet yield from preserved pancreases is a critical step in clinical islet transplantation. The aim of this study was to investigate whether pancreatic ductal injection, performed at procurement, using a small volume of preservation solution before cold storage (ductal preservation method) would improve islet yield and function from rat pancreases preserved for 6 and 24 hr. Materials and Methods. Islets were isolated from Lewis rats. Pancreases were classified into five groups: fresh (group 1); preserved for 6 hr in University of Wisconsin solution without and with ductal preservation (groups 2 and 3); and preserved for 24 hr in University of Wisconsin solution without and with ductal preservation (groups 4 and 5). We assessed islet yield, function, and viability of pancreatic ductal cells. Results. Islet yields per pancreas in groups 1 to 5 were 2010±774, 674±450, 1418±528, 527±263, and 1655±618 (islet equivalent) (±SD), respectively. Stimulation indices in groups 1 to 5 were 11.97±3.17, 6.48±4.04, 12.44±5.65, 2.56±2.03, and 5.55±2.71. Functional success rates in groups 1 to 5 were 100%, 0%, 100%, 0%, and 66.7%. Percentages of nonviable pancreatic duct cells in groups 1 to 5 were 3.8±2.7%, 59.7±4.4%, 19.5±7.3%, 64.7±4.5%, and 17.2±2.6%. In all experiments, the differences were significant between the groups without versus the groups with ductal preservation (P <0.05, group 2 vs. group 3 and group 4 vs. group 5). Conclusions. Ductal preservation improved islet yield and function after 6 and 24 hr of preservation. Well-preserved pancreatic ducts maintained good distribution of collagenase solution.

Caspase-3 Inhibitor Prevents Apoptosis of Human Islets Immediately After Isolation and Improves Islet Graft Function

Objectives: Apoptosis appears in islets after isolation, and it has a detrimental effect on the islet function. To improve the outcome of clinical islet transplantation, it is crucial to protect islets from apoptosis. The aim of this study was to determine whether a caspase-3 inhibitor (Z-DEVD-FMK) added to culture media protects islets from apoptosis and to compare the effects of fetal bovine serum (FBS) with human serum albumin (HSA) as a protein supplement in culture. Methods: Isolated human islets were cultured under 4 different conditions: 0.5% HSA (control), 0.5% HSA + 25 μmol/L Z-DEVD-FMK, 0.5% HSA + 100 μmol/L Z-DEVD-FMK and 10% FBS for 2 days. Next, 1000 IEQ islets precultured with 0.5% HSA and with or without 100 μmol/L Z-DEVD-FMK were transplanted to diabetic nude mice. Results: The islet yields were higher in Z-DEVD-FMK–treated groups, and the inhibitor prevented apoptosis dose dependently. The yield and insulin release were higher in FBS-treated group than in the control group, but FBS did not affect apoptosis. All 6 mice transplanted with islets pretreated with Z-DEVD-FMK, and 3 of 8 mice with control islets became normoglycemic posttransplantation. Conclusion: Z-DEVD-FMK prevented apoptosis of isolated human islets and improved its function. FBS (10%) improved the islet yield and insulin secretion more than 0.5% HSA.

Effect of short‐term culture on functional and stress‐related parameters in isolated human islets

The Edmonton protocol for islet transplantation utilizes fresh islet grafts but other protocols increasingly transplant short‐term cultured grafts mainly for practical reasons. To improve our understanding of the impact of culture pretreatment of human islets, we assessed post‐transplant function by nude mouse bioassay, islet ATP, activity of stress‐activated MAP kinases, and expression of stress‐related genes by focused cDNA array in freshly isolated and cultured islets. Mean blood glucose levels over 4 weeks after transplantation (2000 IE) of (i) freshly isolated, (ii) cultured and preculture counted (recovery rate; 78 ± 6%), and (iii) cultured and postculture counted islets in diabetic mice were 330 ± 40, 277 ± 65, and 256 ± 52 mg/dl (i versus ii, P = 0.004; i versus iii, P = 0.002). During culture, islet ATP/DNA and ATP/ADP increased; JNK and p38 MAPK activities decreased. Among 96 genes studied, mRNA expression of heat shock protein 70 genes decreased >twofold during culture in all four pairs; expression of cyclooxygenase‐2, superoxide dismutase‐2, interleukin‐6 and cytochromes P450 1A1 genes increased. Our results show that culturing human islets before transplantation is not disadvantageous in regard of functional recovery from changes induced by nonphysiologic stimuli during islet isolation. The increase in expression of several stress‐related genes during culture also shows that improving culture conditions may further enhance post‐transplant islet function.

Application of the two-layer method on pancreas digestion results in improved islet yield and maintained viability of isolated islets

Background. Oxygenation of the pancreas during preservation by the two-layer method (TLM) has shown beneficial effects in islet transplantation. Here, we apply this concept (oxygenation) to the isolation process. Methods. Rat pancreases were digested using four different methods. Pancreases were digested with preoxygenated perfluorocarbon (PFC) in group 2 and without it in group 1. Additionally, adenosine was included in the collagenase solution in subgroups B but not in subgroups A. Islet yields and viability were compared between groups. Results. Tissue oxygen tension in group 1 was essentially zero during digestion, but rapidly reached around 300 mm Hg and was maintained in group 2. The tissue adenosine triphosphate (ATP) level in rat pancreas just after laparotomy (control) was 4.2±0.7 &mgr;mol/g dry weight; after digestion, it was 0.12±0.03 &mgr;mol/g, 0.70±0.10 &mgr;mol/g, 0.30±0.18 &mgr;mol/g, and 2.90±0.80 &mgr;mol/g in groups 1A, 1B, 2A, and 2B, respectively. No significant differences were observed between group 2B and control (P=0.19). Islet yields (IEQ/pancreas) were 1600±400, 1400±400, 1300±400, and 2400±100 in groups 1A, 1B, 2A, and 2B, respectively. The islet yield of group 2B was significantly higher than other groups (P<0.05). The cure rate after transplanting 200 islets into athymic nude mice did not differ (80% in all groups). The stimulation indices in the four groups were also the same. Conclusions. Tissue ATP levels after digestion were well maintained using TLM with adenosine digestion method. Consequently, greater numbers of islets could be retrieved. The new method was at least equivalent to islet function isolated by conventional method. Clinical study is therefore warranted.

Effect of Oxygenated Perfluorocarbon on Isolated Islets During Transportation

BACKGROUND Previous studies demonstrated the efficacy of the two-layer method (TLM) using oxygenated perfluorochemicals (PFC) for pancreas preservation. The current study investigated the effect of oxygenated PFC on isolated islets during transportation. MATERIALS AND METHODS Purified rat islets were stored in an airtight conical tube for 24h in RPMI culture medium at 22 degrees C or University of Wisconsin solution (UW) at 4 degrees C, either with or without oxygenated PFC. After storage, the islets were assessed for in vitro viability by static incubation (SI), FDA/PI staining, and energy status (ATP, energy charge, and ADP/ATP ratio) and for in vivo viability by a transplantation study. RESULTS UW at 4 degrees C and RPMI medium at 22 degrees C maintained islet quality almost equally in both in vitro and in vivo assessments. The ATP levels and energy status in the groups with PFC were significantly lower than those without PFC. The groups with PFC showed a significantly higher ADP/ATP ratio than those without PFC. In the transplantation study, blood glucose levels and AUC in the UW+PFC group were significantly higher than those in UW group. CONCLUSIONS UW at 4 degrees C and RPMI medium at 22 degrees C maintained islet quality equally under the conditions for islet transportation. The addition of oxygenated PFC, while advantageous for pancreas preservation, is not useful for islet transportation.

Improved quantity and in vivo function of islets isolated by reduced pressure-controlled injection of collagenase in a rat model.

In islet transplantation, insufficient yield is a major obstacle to one-donor/one-recipient transplant. Collagenase, which is injected via a pancreatic duct to separate islets from acini, can so easily distribute into the islet core that it may result in disruption of islets. The purpose of this study was to evaluate the superiority of reduced pressure-controlled collagenase injection (RPCI) at 80 mmHg on islet isolation to injection at 180 mmHg by examining in vivo transplant experiments besides the yield and the glucose stimulation test in a rat model. Lewis rat pancreases were distended with collagenase solution at 80 mmHg pressure as the RPCI group (group 1) and at 180 mmHg (group 2), followed by isolation. The yield in group 1 (1100 ± 160 islets with 2750 ± 530 IEQ) was significantly higher than that in group 2 (900 ± 130 islets with 1570 ± 350 IEQ, p < 0.01) due to the significant difference of the number of islets sized >150 μm in diameter, although the purity was not significantly different between the two groups. Stimulation indices in the glucose stimulation tests were 2.88 ± 1.12 in group 1 and 1.93 ± 0.62 in group 2 (p < 0.05). The cure rate by transplantation of 100 islets to diabetic nude mice in group 1 (8/10) was significantly higher than that in group 2 (3/10, p < 0.05). In a syngenic transplant model of 90% of islets isolated from one donor, the cure rates were 100% and 67% in groups 1 and 2, respectively (NS). The area under the curve on the graph of IPGTT on postoperative day 28 in group 1 was significantly smaller than that in group 2 (p < 0.05). In conclusion, our data show that RPCI at 80 mmHg could contribute to consistently high islet yield and in vivo function in a rat model. It was suggested that the current human protocol should be reviewed from this viewpoint.