Ascorbic acid enhances the cold preservation period of human adipose tissue-derived mesenchymal stromal cells.

Introduction: We previously developed 3% trehalose-added lactated Ringer's solution (LR-3T) and 3% trehalose- and 5% dextran-40-added lactated Ringer's solution (LR-3T-5D), which can be used to preserve adipose-derived mesenchymal stem cells (hADSCs) for 24 h at 5 and 25 °C. However, it is necessary to further extend the storage duration of cells to expand transportation zones and ensure time for quality control testing of final cell products. Therefore, we attempted to prolong the preservation duration of hADSCs by adding supplements to LR-3T-5D. We focused on ascorbic acid as an antioxidant because it is widely clinically as a nutrient. Methods: We added the antioxidant ascorbic acid to LR-3T-5D and evaluated the viability, colony formation rate, proliferative capacity, and surface markers of hADSCs before and after preservation at 5 °C. Results: Analysis of the concentration of ascorbic acid added to LR-3T-5D indicated that 1000 mg/L was the optimal concentration for maintaining the viability of hADSCs after 72 h of cold preservation. No changes were observed in the expression of specific cell surface markers or in the potential of hADSCs to differentiate into adipocytes, osteoblasts, or chondrocytes before and after cold preservation. Discussion: These results suggest that cold preservation of hADSCs in LR-3T-5D supplemented with ascorbic acid helps maintain the quality of cells for use in cell therapy.

Preventive effect of aripiprazole once monthly on rehospitalization for bipolar disorder: A multicenter 1-year retrospective mirror image study.

AIM: We conducted a 1-year retrospective mirror-image study to investigate the effect of aripiprazole once monthly (AOM) on rehospitalization for bipolar disorder. METHODS: Participants were recruited from psychiatric emergency and acute care hospitals in western Japan. We included 39 participants with bipolar disorder who had been administered AOM for at least 1 year with no missing medical records during the observational period. The primary outcomes were rehospitalization rate, number of rehospitalizations, total hospitalization days, and time to rehospitalization in the context of overall psychiatric readmissions. The significance level was set at p < 0.05. RESULTS: AOM significantly reduced the rehospitalization rate from 23/39 (59%) to 7/39 (18%) (p = 0.001). The number of rehospitalizations decreased significantly from a mean of 0.85 per person-year to 0.41 per person-year (p = 0.048). The total hospitalization days significantly decreased from a mean of 34.9 days to 14.4 days (p = 0.008). AOM significantly prolonged the time to rehospitalization (p < 0.001). CONCLUSION: This study found that AOM reduces overall psychiatric rehospitalization for bipolar disorder based on data from 1 year before and after AOM administration in the real-world setting. Future studies should examine the robustness and persistence of the rehospitalization preventive effect of AOM with larger sample sizes and longer observation periods beyond 1 year.

Relationship between oxygen partial pressure and inhibition of cell aggregation of human adipose tissue-derived mesenchymal stem cells stored in cell preservation solutions.

Introduction: This study investigated the storage conditions under which cell aggregation occurs and the conditions that inhibit cell aggregation when human adipose tissue-derived mesenchymal stem cells (hADSCs) are stored in lactated Ringer's solution (LR) supplemented with 3% trehalose and 5% dextran 40 (LR-3T-5D). Methods: We first examined the effects of storage temperature and time on the aggregation and viability of hADSCs stored in LR and LR-3T-5D. The cells were stored at 5 °C or 25 °C for various times up to 24 h. We then evaluated the effects of storage volume (250-2,000 µL), cell density (2.5-20 × 105 cells/mL), and nitrogen gas replacement on aggregation, oxygen partial pressure (pO2), and viability of hADSCs stored for 24 h at 25 °C in LR-3T-5D. Results: When stored in LR-3T-5D, viability did not change under either condition compared with pre-storage, but the cell aggregation rate increased significantly with storage at 25 °C for 24 h (p<0.001). In LR, the aggregation rate did not change under either condition, but cell viability decreased significantly after 24 h at both 5 °C and 25 °C (p < 0.05). The cell aggregation rates and pO2 tended to decrease with increasing solution volume and cell density. Nitrogen gas replacement significantly decreased the cell aggregation rate and pO2 (p < 0.05). However, there were no differences in viability among cells stored under conditions of different storage volumes, densities, and nitrogen gas replacement. Conclusions: Aggregation of cells after storage at 25 °C in LR-3T-5D may be suppressed by increasing the storage volume and cell density as well as by incorporating nitrogen replacement, which lowers the pO2 in the solution.

Dimethyl sulfoxide-free cryopreservation solution containing trehalose, dextran 40, and propylene glycol for therapy with human adipose tissue-derived mesenchymal stromal cells.

We evaluated a dimethyl sulfoxide (Me2SO)-free cryopreservation solution to freeze human adipose-derived mesenchymal stromal cells (hADSCs). In the first experiment, we compared the combined effects of 3% trehalose (3 T) and 5% dextran (5D) in lactated Ringer's solution (LR) as a cryopreservation base solution containing 10% propylene glycol (PG). The cell viability of hADSCs immediately after thawing was significantly higher (p < 0.05) in LR supplemented with 3 T (LR-3 T) and with 3 T and 5D (LR-3 T-5D) than in LR. In the second experiment, we compared the cell characteristics of hADSCs freeze-thawed in LR-3 T-5D containing either 10% Me2SO or 10% PG. The cell viability, annexin V-positive ratio, colony-forming capacity, cell proliferation, cell surface antigen positivity, adipogenic differentiation, osteogenic differentiation, and genetic response to cytokine stimulation of hADSCs immediately after thawing were similar between the LR-3 T-5D containing 10% Me2SO and 10% PG. In the third experiment, we examined various concentrations of PG on the cell proliferative capacity of freeze-thawed hADSCs. The cell proliferative capacity of hADSCs frozen with LR-3 T-5D containing 2.5% to 5% PG was significantly higher (p < 0.05) than LR-3 T-5D containing 10% PG. Furthermore, the cell proliferative capacity of hADSCs frozen with LR-3 T-5D containing 4% PG was similar to that of fresh hADSCs. These results indicate that the combination of 3 T-5D in an LR solution as a basic solution is effective for post-thaw cell viability, and that the optimal concentration of PG to maintain the cell characteristics of hADSCs frozen with LR-3 T-5D is 2.5% to 5%, which is promising for cell therapy applications.

Protein-free solution containing trehalose and dextran 40 for cryopreservation of human adipose tissue-derived mesenchymal stromal cells.

We tested the efficacy of lactated Ringer's solution with 3% trehalose and 5% dextran 40 (LR-3T-5D) as a vehicle solution for cryopreservation using human adipose-derived mesenchymal stromal cells (hADSCs) with dimethyl sulfoxide (Me2SO). We also tested the effect of the Me2SO concentration in the cryopreservation solution, and the effect of washing with lactated Ringer's solution with 3% trehalose (LR-3T) and replacement with LR-3T or LR-3T-5D. LR-3T-5D was more effective for cell viability, viable cell recovery ratio, annexin V-positive ratio, and colony-forming capacity as a vehicle solution for cryopreservation with 10% Me2SO than LR. The additive effects as cryoprotectants of trehalose and dextran 40 were confirmed to be dose dependent. The cell viability, cell proliferation ability, cell differentiation ability, and the ratio of cell surface positive/negative markers of hADSCs were well maintained after cryopreservation with LR-3T-5D containing 10% Me2SO in liquid nitrogen or in a -80 °C freezer. The cell viability and the proliferation curve in LR-3T-5D with 5% Me2SO were comparable to those with 10% Me2SO. LR-3T-5D was superior to LR-3T as a replacement solution in terms of viability and annexin V positivity. Our data showed that LR-3T-5D is effective as a vehicle solution for cryopreservation. Reducing Me2SO concentration to 5%, and washing and replacement with fresh LR-3T and LR-3T-5D after thawing, are feasible approaches to maintain cryopreservation efficacy.

The effectiveness of very slow switching to aripiprazole in schizophrenia patients with dopamine supersensitivity psychosis: a case series from an open study.

Dopamine supersensitivity psychosis (DSP) in patients with schizophrenia is induced by treatment with a high dosage of antipsychotics for a long time period, and it is characterized by unstable psychotic symptoms. The upregulation of dopamine D2 receptor (DRD2) provoked by antipsychotics underlies DSP. Aripiprazole does not cause an excessive blockade of DRD2 and is less likely to upregulate DRD2 by aripiprazole's dopamine partial agonistic profile. Aripiprazole; however, has a potential risk of inducing severe rebound psychosis in patients who have already developed dopamine supersensitivity. Recently, an animal model study suggested that aripiprazole could attenuate established dopamine supersensitivity. The present study was conducted to examine whether very slowly switching to aripiprazole could help patients with schizophrenia with dopamine supersensitivity while avoiding rebound psychosis. This study was a single-armed and open-labeled study in which patients were observed over a period of 2 years. Only 11 patients were ultimately recruited. Five patients were successfully switched to a sufficient dose of aripiprazole and completed the study protocol. These five patients did not present with severe DSP over the study period, but only one patient showed a large improvement in psychopathology. Five patients dropped out of the study, and one of these five showed a severe worsening of psychosis. The present study indicated that the introduction of aripiprazole in patients with DSP was difficult, but suggested that aripiprazole could contribute to attaining a stable state in psychosis if it was applied with careful observation.

A pair of cell preservation solutions for therapy with human adipose tissue-derived mesenchymal stromal cells.

INTRODUCTION: Stem cells for therapy are often suspended in a preservation solution, such as normal saline or lactated Ringer's solution, for a short time before intravenous infusion. However, these solutions are not necessarily ideal for maintaining cell viability and preventing the sedimentation of cells during storage and infusion. In this study, we attempted to optimize the compositions of preservation solutions, which could affect the efficacy and safety of stem cell therapy. METHODS: We determined the characteristics of a preservation solution that would optimize cell viability and the percentage of cells in the supernatant using human adipose-derived mesenchymal stromal cells (hADSCs). We compared solutions that differed by electrolytes (e.g., normal saline and Ringer's solution) and the concentrations of dextran 40 and trehalose. The effects of the solutions on hADSCs were evaluated by assessing cell surface markers, colony-forming capacity, differentiation potential, and cell concentrations in the infusion line. RESULTS: Optimized preservation solutions consisted of lactated Ringer's solution with 3% trehalose without or with 5% dextran 40 (LR-3T and LR-3T-5D, respectively). The cell viabilities after 24 h of storage at 5 °C in LR-3T and LR-3T-5D were 94.9% ± 2.4% and 97.6% ± 2.4%, respectively. The percentage of cells in the supernatant after 1 h of storage at room temperature in LR-3T-5D was 83.5% ± 7.6%. These solutions preserved the percentage of cell surface marker-positive cells, the colony-forming capacity, and the adipogenic and osteogenic differentiation ability in hADSCs for at least 24 h after preservation at 5 °C and 25 °C. DISCUSSION: We determined the optimal composition of preservation solutions for hADSCs and confirmed the effects of these solutions on cell viability and the stability of cell characteristics in vitro. Our results suggest that LR-3T and LR-3T-5D can help maintain the quality of stem cells for therapy during preservation and infusion. However, further in vivo research is needed on the efficacy and safety of the solutions in different therapeutic cell lines before clinical use.

The pharmacokinetics of porcine C-peptide after intraperitoneal injection.

BACKGROUND: Previously, we have demonstrated that there were very low C-Peptide concentrations and normal blood glucose levels when we transplanted encapsulated islets in the abdominal cavity of diabetic nude mice. In addition, the C-peptide concentration in the ascites fluid of the peritoneal cavity was 40 times higher than in the peripheral blood. In this study, we investigated the pharmacokinetics of intraperitoneal porcine C-peptide. METHODS: To assess the pharmacokinetics of porcine C-peptide, a synthesized porcine C-peptide solution was injected into the peripheral circulation through the tail vein or into the peritoneal cavity in rats at low or high doses of either 200 or 2000 pmol/kg, respectively. Arterial blood samples were collected at time intervals of 1-120 minutes after injection to calculate the terminal elimination half-life (t1/2 ) and area under the time-concentration curve (AUC0-t ). RESULTS: After intraperitoneal C-peptide injection, the highest porcine C-peptide concentration in peripheral blood was only one-fortieth compared to after intravenous injection. The AUC0-t for the intraperitoneal injection was 78% at the low dose and only 39% at the high dose compared to the intravenous injection. This finding indicates that C-peptide remains in the abdominal cavity when intraperitoneally transplanted islets release C-peptide via high glucose stimulation. CONCLUSIONS: Porcine C-peptide injected into a peritoneal cavity slowly and incompletely entered peripheral circulation, which resulted in very low concentration in peripheral blood.

Development and characterization of novel clinical grade neonatal porcine bone marrow-derived mesenchymal stem cells.

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow-derived MSCs (npBM-MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM-MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM-MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow-derived MSCs (hBM-MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM-MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM-MSCs was significantly shorter than that of hBM-MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM-MSCs was also significantly smaller than that of hBM-MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 µm, P < 0.001). The npBM-MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM-MSCs secreted VEGF-A, VEGF-C, and TGF-ß1. We have established npBM-MSCs which show super-rapid growth, small size, and robust proliferation profile. The np-MSCs might be able to solve the donor issues for MSC therapy.

Interpreting the deposition and vertical migration characteristics of 137Cs in forest soil after the Fukushima Dai-ichi Nuclear Power Plant accident.

We investigated the deposition and depth distributions of radiocesium in the Takizawa Research Forest, Iwate Prefecture, in order to understand the behavior of radionuclides released from the Fukushima Dai-ichi Nuclear Power Plant. The deposition distribution and vertical depth distribution of radiocesium in the soil were compared between topographically distinct parts of the forest where two different tree species grow. The results for all investigated locations show that almost 85% of the radiocesium has accumulated in the region of soil from the topmost organic layer to a soil depth of 0-4 cm. However, no activity was detected at depths greater than 20 cm. Analysis of the radiocesium deposition patterns in forest locations dominated by either coniferous or deciduous tree species suggests that radiocesium was sequestered and retained in higher concentrations in coniferous areas. The deposition data showed large spatial variability, reflecting the differences in tree species and topography. The variations in the measured 137Cs concentrations reflected the variability in the characteristics of the forest floor environment and the heterogeneity of the initial ground-deposition of the Fukushima fallout. Sequential extraction experiments showed that most of the 137Cs was present in an un-exchangeable form with weak mobility. Nevertheless, the post-vertical distribution of 137Cs is expected to be governed by the percentage of exchangeable 137Cs in the organic layer and the organic-rich upper soil horizons.

Effects of encapsulated porcine islets on glucose and C-peptide concentrations in diabetic nude mice 6 months after intraperitoneal transplantation.

BACKGROUND: In patients with type 1 diabetes, allogeneic islet transplantation can provide normal HbA1c concentrations, but it requires immunosuppression. Transplanting encapsulated islets into the peritoneal cavity could reduce or eliminate the need for immunosuppression. One of the uncertain features of intraperitoneal islet transplantation is the difficulty of measuring C-peptide concentrations in peripheral blood, which is often used for the marker of islet function. We hypothesized that secreted C-peptide from intraperitoneally transplanted islets was mostly consumed in the peritoneal cavity, which resulted in low C-peptide concentrations in peripheral blood. METHODS: In each of two experiments, encapsulated neonatal porcine islets were intraperitoneally transplanted into four nude mice with streptozotocin-induced diabetes. Three diabetic nude mice without transplanted islets were used as diabetic controls, and three untreated healthy nude mice were used as normal controls. Islet functions were monitored for 2 months in the first experiment and 6 months in the second experiment. Encapsulated islets were retrieved after each experiment and evaluated by fluorescein diacetate/propidium iodide tests for the viability and static glucose-stimulated insulin release tests for the function. C-peptide concentrations from the blood and from the intraperitoneal cavity at 6 months were compared. RESULTS: In both experiments, diabetes was reversed in all transplanted mice, and oral glucose tolerance test showed improved profiles. In general, retrieved islets were viable and functional. However, blood porcine C-peptide concentrations were low at both 2 and 6 months, and concentrations in the ascites of peritoneal cavity were 40 times as high as those in blood. CONCLUSIONS: The peripheral blood sampling for c-peptide, though highly informative in vascularized grafts, may not be the primary tool for monitoring the health and function of encapsulated products when transplanted into intraperitoneal cavity. Our results might explain the clinical feature of the low C-peptide blood concentrations after successful intraperitoneal encapsulated islet transplantation.

Factors associated with the risk of relapse in schizophrenic patients after a response to electroconvulsive therapy: a retrospective study.

PURPOSE: Electroconvulsive therapy (ECT) is an effective treatment for depression and schizophrenia. However, there is a high rate of relapse after an initial response to ECT, even with antidepressant or antipsychotic maintenance therapy. This study was carried out to examine the factors that influence the risk of relapse in schizophrenic patients after a response to ECT. PATIENTS AND METHODS: We retrospectively reviewed the records of 43 patients with schizophrenia who received and responded to an acute ECT course. We analyzed the associated clinical variables and relapse after response to the acute ECT. Relapse was defined as a Clinical Global Impressions Improvement score ≥6 or a psychiatric rehospitalization. RESULTS: All patients were treated with neuroleptic medication after the acute ECT course. The relapse-free rate of all 43 patients at 1 year was 57.3%, and the median relapse-free period was 21.5 months. Multivariate analysis showed that the number of ECT sessions was associated with a significant increase in the risk of relapse (hazard ratio: 1.159; P=0.033). Patients who were treated with adjunctive mood stabilizers as maintenance pharmacotherapy after the response to the acute ECT course were at a lower risk of relapse than were those treated without mood stabilizers (hazard ratio: 0.257; P=0.047). CONCLUSION: Our study on the recurrence of schizophrenia after a response to an acute ECT course suggests that the number of ECT sessions might be related to the risk of relapse and that adjunctive mood stabilizers might be effective in preventing relapse.

Improvement of porcine islet isolation by inhibition of trypsin activity during pancreas preservation and digestion using α1-antitrypsin.

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Comparison of ulinastatin, gabexate mesilate, and nafamostat mesilate in preservation solution for islet isolation.

For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

Evaluation of osmolality of density gradient for human islet purification.

For pancreatic islet transplantation, the most common method of islet purification is density gradient centrifugation because of the differences in density between islets and acinar tissue. The density of islets/acinar tissue depends on several conditions, such as osmolality of purification solution. In this study, we evaluated the osmolality of iodixanol-controlled density gradients (400, 450, and 500 mOsm/kg) on the islet purification step. The density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto solutions; IK solutions). The osmolality of density gradients was controlled by addition of 10× Hanks balanced salt solution (HBSS) solution. Density of both islets and acinar tissue increased relative to increase of the osmolality of purification solutions. There were no significant differences among the three groups on islet yield after density-adjusted purification and the rate of postpurification recovery. In vitro and in vivo assays suggest that the quality of islets was similar among the three groups. Our data suggest that efficacy of purification and quality of isolated islets is similar when the osmolality of purification solutions is between 400 and 500 mOsm/kg and density adjustment is applied. Since the density of islet and acinar tissue is changed according to osmolality, the density adjustment is important when using several osmolality solutions.

Fresh islets are more effective for islet transplantation than cultured islets.

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.

Islet purification method using large bottles effectively achieves high islet yield from pig pancreas.

Porcine islets are a promising resource for xenotransplantation. However, low efficacy of islet isolation because of their marked fragility remains a problem. Recently we found that the standard purification method using COBE 2991 cell processor (COBE) with Ficoll density gradient solution damaged islets mechanically by high shearing force. In this study, we evaluated our new purification method using large plastic bottles for the efficacy of islet purification. Ten porcine pancreata were used. The average warm ischemic time was over 40 min; therefore, these pancreata were considered to be in a marginal condition. After digestion, the digested tissue was divided into three groups. Each group was purified using either top loading method with bottle (top group) or bottom loading method with bottle (bottom group) or standard COBE method (COBE group). Islet yield per pancreas weight (IEQ/g) and the rate of postpurification recovery in the top group were significantly higher than the COBE group (top: 8060 ± 1652 IEQ/g, bottom: 4572 ± 614 IE/g, COBE: 3900 ± 734 IE/g. p < 0.02 in top vs. COBE; top percentage of recovery: 99.3 ± 12.3%, bottom: 62.6 ± 8.8%, COBE: 49.5 ± 6.7%, p < 0.02 in top vs. bottom and COBE). The average sizes of purified islets in the top and bottom groups were significantly larger than COBE group (Average diameter top: 156 ± 8 µm, bottom: 147 ± 6 µm, COBE: 119 ± 6 µm, p < 0.01 in top vs. COBE and in bottom vs. COBE), which indicated that bottle method can reduce shear force during purification. Our new purification using top loading bottle method enabled us to obtain a high yield of porcine islets from marginal pancreata.

Improved pancreatic islet isolation outcome in autologous transplantation for chronic pancreatitis.

Total or partial pancreatectomy followed by autologous islet transplantation is a therapeutic option for the treatment of refractory chronic pancreatitis (CP). Maximization of islet yields from fibrotic and inflamed organs is crucial for prevention of posttransplant diabetes. We adapted technical advancements developed for islet allotransplantation toward islet autotransplantation. Eight patients (two men, six women; ages 24-58 years) underwent total (n = 7) or partial (n = 1) pancreatectomy for the treatment of CP refractory to maximal medical management. Pancreata were preserved in UW solution (UW group) in initial three cases and the last five pancreata were preserved with pancreatic ductal injection followed by ET-Kyoto/oxygenated PFC solutions (DI+TLM group). Islets were isolated by modified Ricordi method and were purified only in one case. All islet infusions were performed under general anesthesia via direct vein injection into the portal venous system with pressure monitoring. Total islet yields (129,314 ± 51,627 vs. 572,841 ± 116,934 IEQ, p < 0.04), islet yield/pancreas weight (1,233 ± 359 vs. 6,848 ± 847 IEQ/g, p < 0.003), and islet yield/patient body weight (1,951 ± 762 vs. 7,305 ± 1,531 IEQ/kg, p < 0.05) were significantly higher in the DI+TLM group when compared to the UW group. Pellet size was also higher (5.3 ± 0.3 vs. 13.5 ± 3.4 ml) in the DI+TLM group, suggesting that this method of preservation effectively protected pancreatic tissue against autolysis. First month posttransplant basal C-peptide and the secretory unit of islet transplant objects (SUITO) index were also higher in the DI+TLM group when compared to the UW group (2.0 ± 0.3 vs. 1.4 ± 0.4 ng/ml and 42.6 ± 12.7 vs. 14.6 ± 5.6, respectively). There were no technical complications related to the infusion. Our results suggest that higher islet yields can be achieved even from chronically inflamed and fibrotic organs using DI+TLM. The techniques applied for islet isolations from normal pancreata are showing promise for fibrotic pancreata from CP patients.