![We want to be a resource for your research: Honorbridge]({"type":"image" , "src" : "/sda/112420/JournalAd-leaderboard-kisl-honorbridge.jpg"})
Abstract
The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.
Introduction
Islet transplantation is a promising treatment for type 1 diabetic patients.Citation1-Citation3 Despite significant methodological progress in human islet isolation and purification, it is still difficult to recover a sufficient number of islets from a single donor pancreas. Although the islet number in an adult human pancreas is estimated to be over one million, average islet isolation generally yields only about 50% of those islets.Citation4 The purification process is one of the most difficult procedures in islet isolation. There has been solid progress in islet purification since the introduction of the semiautomated computerized purification method.Citation5,Citation6 Currently, a continuous density gradient purification method with top loading using COBE 2991 cell processor (COBE) is considered the gold standard method. This procedure reduces the volume of tissue infused into the patients, which can minimize the risk of increased portal pressure and thrombosis.Citation7 On the other hand, the purification procedure often results in a diminution of the number of recovered islets. In addition, islets are exposed to various stresses during the purification procedure that may cause cellular damage and functional impairment and eventually lead to an overall reduction of the viable islet mass that engrafts and to poor clinical outcomes.Citation8-Citation11
Recently, it was reported that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution damaged islets mechanically by high shear force.Citation12 The unique design and shape of a COBE bag with its narrow segment produced strong shear forces that made the islets come apart.Citation12 To minimize such stresses during purification, we recently established an effective purification method using large hard cylindrical plastic bottles with lower viscosity gradient solution, which led to substantial improvement of porcine islet isolation.Citation13 The method has some advantages; the shear force can be minimized because the bottles have a simple shape without a narrow segment and no centrifugation is required during the loading and collecting processes. In addition, we introduced a top-loading method with continuous density gradients instead of the standard bottom-loading method with discontinuous density gradients.Citation14 In this study, we performed experiments for head-to-head comparison of COBE vs. bottle method (top or bottom loading) for human islet purification.
Results
Stability of the density gradient in the bottle purification methods
After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods as described in the Materials and Methods section and in . After making continuous density gradients with the digested tissue, centrifugation and deceleration with the brakes, the solution was collected into 10 tubes. The density of each tube was measured, and we confirmed that the continuous density gradients were maintained after centrifugation in both the top and bottom groups (; the high density was 1.095, n = 3).
Figure 1. Bottle purification method. (A) The schema of the continuous gradient solution for the bottle purification method. (B) A “tip-bent” candy cane–shaped stainless pipe used for making the gradient (left panel). The pipe was useful to load the tissue into the top layer in the Top loading method (right panel). (C) Pictures of the bottles after loading and centrifugation. Left: Top loading. Right: Bottom loading. Islets exist in the upper layer in both methods (arrows).
Figure 2. Continuous density gradient was maintained in both the top- and bottom-loading methods during the purification. After centrifugation, the solution, including the digested tissue, was collected into 10 tubes, and the density of each tube was measured. The density of the low-density solution was 1.075, while that of the high density was 1.095. The experiments were performed three times independently.
Evaluation before and after purification
Nine human pancreata were used in the current study. The islet isolation variables before purification are shown in , and the variables after purification are shown in . The islet purity in the bottom group was higher than in the other two groups, but it did not reach statistical significance. The percentage of the embedded islets after purification decreased in all groups, but there was no significant difference among the three groups. The average islet viability in all groups was > 96%. The stimulation index in the top group was higher than in the other two groups, although there was no statistically significant difference. After 24 h of culture, the islet viability was examined. There was no significant difference among the three groups. The final tissue volume rate was not significantly different among the 3 groups, although the tissue was most reduced in the bottom group. We estimated the sterility of the final products of each group. The endotoxin concentration was under the detectable limit, and no microbe growth was detected in any of the cases.
Table 1. Human islet isolation variables before purification
Table 2. Postpurification variables based on purification method: COBE method, bottle method with top loading, or bottle method with bottom loading
Islet yield and size after purification
Islet yield per pancreas weight (IE/g) after purification was significantly higher in the top group than in the COBE and bottom groups (COBE, 8,155 ± 826; top, 11,861 ± 1,313; bottom, 5,983 ± 825; p < 0.025 in top vs. COBE and bottom groups, respectively; ). The percentage of postpurification recovery was significantly higher in the top group than in the other two groups (COBE, 76.6 ± 8.6; top, 108.6 ± 9.4; bottom, 56.6 ± 8.8; p < 0.025 in top vs. COBE and bottom groups, respectively; ). The average diameter of purified islets in the top group was significantly larger than in the COBE group (COBE, 169 ± 6 μm; top, 184 ± 5 μm; bottom, 180 ± 13 μm; p < 0.025 in top vs. COBE group; ).
Figure 3. Postpurification islet yield and size of islets in groups with different purification methods: COBE method, bottle method with top loading, or bottle method with bottom loading. (A) Postpurification Islet yield per pancreas weight (islet equivalent [IE]/g). (B) Postpurification recovery rate (postpurification IE/prepurification IE × 100). (C) Average islet diameter. *p < 0.025.
![Figure 3. Postpurification islet yield and size of islets in groups with different purification methods: COBE method, bottle method with top loading, or bottle method with bottom loading. (A) Postpurification Islet yield per pancreas weight (islet equivalent [IE]/g). (B) Postpurification recovery rate (postpurification IE/prepurification IE × 100). (C) Average islet diameter. *p < 0.025.](/cms/asset/599b6bb9-6343-4289-b31c-0c6bf63352f4/kisl_a_10923008_f0003.gif)
Assessment of islet function in vivo
To assess the islet graft function in vivo, 2,500 IE islets, considered a marginal number, were transplanted under the kidney capsule of streptozotocin-induced diabetic nude mice. For this experiment, we conducted the comparison between the COBE and top groups, without the bottom group. The bottom method showed a poor islet recovery. Since islet yield is the most important factor for clinical outcomes of islet transplantation, the purification method leading to low islet yield will not be clinically applied. Therefore we omitted the bottom group from in vivo assay. The blood glucose levels in 8 of 19 mice (42%) in the COBE group and 13 of 18 mice (72%) in the top group reached normoglycemia. The top group had a significantly higher cure rate (p < 0.05; ).
Figure 4. Normoglycemic rate of streptozotocin-induced diabetic nude mice after islet transplantation. COBE indicates COBE purification method (n = 19); top, bottle purification method with top loading (n = 18). *p < 0.05.
Discussion
In the current study with human pancreata, we showed that the top-loading bottle method significantly improved both the quantity and quality of purified islets. The shear force on the islets during the centrifugation for purificationCitation12 could be reduced in the bottle method, because the bottle had a simple shape and smooth inner surface. Furthermore, the bottle method requires a shorter centrifugation period since the loading process does not require centrifugation. Direct loading of the digested pancreas using a unique bended cane onto the static surface of the gradient solution also reduces the shear stress. As a result, the islets could be less fragmented and their size and viability could be maintained. Indeed, our data indicated that the size of the purified islets in the top group was significantly larger than in the COBE group although they had the similar purity and tissue volume. This may be the main reason for the higher islet recovery and the better islet quality, according to the in vivo evaluation. Of note the cure rates of our in vivo study seemed low compared with previous publications.Citation15,Citation16 However, the marginal number of islets to cure diabetic mice varies among institutes.Citation17-Citation19 In addition, since our group has been aiming for maximizing the islet recovery, the islet purity was relatively low. This relative low purity might hinder the cure rate in our model. Nonetheless, the in vivo model also supported the benefit of our bottle purification method.
The average recovery rate in the top group was more than 100%. In the digested pancreatic tissue, more than 30% of islets were embedded in the exocrine tissues. We speculated that some of the embedded islets might not have been counted due to the difficulty of dithizone staining. During the purification process, the digested pancreatic tissue was immersed in UW solution and washed. This process can release some of the embedded islets from exocrine tissue,Citation20 and the islets could become detected and counted. This might be the main reason why the recovery rate after purification was more than 100% in the top-loading group. There is another advantage of the bottle method. Many embedded islets are often lost during purification process due to the similarity of the densities between embedded islets and exocrine tissues. In fact, it is common that more than a few islets are left in the COBE bag after the COBE purification. However, in the bottle purification method, we can collect all tissues from the bottle because there is no dead space in the bottle. Actually, the embedded islet rate in the top group was higher than the COBE group, indicating that many embedded islets were recovered in the top group.
On the other hand, the islet recovery in the bottom-loading method was lower than with other methods, presumably because many islets were entrapped by exocrine tissues at the bottom of the bottle. Embedded islets or islets that are not completely free are easily entrapped by surrounding exocrine tissues. This may explain why, among the three groups, the postpurification purity was the highest and the final tissue volume was the lowest in the bottom group. Therefore, we concluded that the top-loading method should be adopted for the bottle purification method. Traditionally, the bottom loading method has been used for the bottle purification due to the difficulty of loading digestive tissue into the top of the gradient solution (light layer).Citation14 Our unique bended cane and recipe of mixing digestive tissue with the low density solution enabled us to load the digestive pancreatic tissue into the top layer of the gradient solution. Another advantage of top loading over bottom loading is that we can start to create the density gradient while washing the digested pancreatic tissue, which can reduce the procedure time. Furthermore, the procedure time can be further shortened by centrifuging multiple bottles at once. This study shows that our new top loading method could be superior to the traditional bottom-loading bottle method and even better than the COBE purification.
Concerns may be raised about the sterility of the bottle method. All processes in the current study were conducted following good manufacturing practice procedures. Moreover, we measured the endotoxin levels and microbial contamination of the final islet products, and all samples were negative. There might be another issue that the dilution factor may affect on the efficacy of purification between the COBE and bottle methods, because the half digest was used for the COBE group and the quarter digest was used for the bottle methods. In this study, when the digest tissue volume was large, we performed the COBE purification twice using the quarter of the tissue for each, which was the same condition as the bottle purification. We summarized the data in this condition and the results showed the same tendency. Islet yield per pancreas weight (IE/g) after purification was higher in the top group than the COBE group (COBE, 9,421 ± 1,096; top, 14,171 ± 1,076; bottom, 6,705 ± 1,514; p = 0.06 in top vs. COBE group, n = 3). Therefore, the dilution factor was not a significant factor in the condition of this study. Another potential issue is that we always use pancreatic ductal injection method and two-layer pancreas preservation before islet isolation that might affect the purification efficacy. In fact, the two-layer method increases density of tissues and ductal injection might change the tissue density as well. To overcome this issue, we always measure the tissue density then adjust the density of purification solution.
Another important feature of this study is that ET-Kyoto solution and Iodixanol were used for the density gradient solution. We have reported that islet purification using ET-Kyoto and Iodixanol can be superior to the Ficoll gradient, which has been used in many institutes.Citation27 Therefore, we have not examined the bottle purification method with Ficoll. However, since Ficoll is more viscous than ET-Kyoto solution, the sheering force could have stronger influence in the COBE bag. Therefore, the benefit of the bottle method to reduce the shearing force might be more apparent when the Ficoll gradient is used.
Of note, this simple bottle purification method can eliminate the cost of and requirement for experience with a COBE 2991 cell processor and its accessories, which may allow more institutes to offer islet isolation both for clinical and basic research.
In conclusion, use of our novel bottle purification method with top loading improved the efficacy of human islet purification. We plan to apply this method to human islet isolation for clinical transplantation.
Materials and Methods
Human islet isolation
This study and the islet isolation protocol were approved by the institutional review board of Baylor Research Institute. Donors were selected based on the Edmonton protocol for a clinical-grade pancreas;Citation2 however, all pancreata were declined for clinical use due to various reasons. Nine pancreata from brain-dead donors were procured through either Southwest Transplant Alliance or LifeGift between September 2008 and October 2011. All pancreata were procured using a standardized technique to minimize warm ischemia. University of Wisconsin (UW) solution (ViaSpan, DuPont Pharmaceuticals) or SPS-1 (Organ Recovery System) was used for general organ perfusion through the aorta. After the pancreas was procured, we immediately removed the duodenum and spleen from the pancreas and inserted a cannula into the pancreas through the main pancreatic duct from the direction of the pancreatic head at the procurement site. Approximately 1 mL/g pancreas weight of ET-Kyoto solution (Otsuka Pharmaceutical Factory Inc.) or Cold Storage/Purification Stock Solution (Mediatech, Inc.) was administered intraductally.Citation21 All pancreata were preserved by the oxygen-charged static two-layer (oxygenated perfluorocarbon/preservation solution) method for less than 6 h.Citation22,Citation23
Islets were isolated according to the Ricordi method with our modifications.Citation24-Citation26 In brief, after decontamination of the pancreas, the ducts were perfused in a controlled fashion with cold Collagenase NB with neutral proteases (Serva Electrophoresis GmbH, lot #080157, 080421, 080793) or cold Liberase MTF (Roche Molecular Biochemicals, lot #14850700, 14862700, 12436700, 13171900). The distended pancreas was then cut into seven to nine pieces, placed in a Ricordi chamber, and shaken gently. While the pancreas was being digested by recirculating the enzyme solution through the Ricordi chamber at 37°C, we monitored the extent of digestion with dithizone staining (3 mg/mL, Sigma Chemical Co.) by taking small samples from the system. Once digestion was confirmed to be complete, dilution solution (Mediatech) was introduced into the system, and then the system was cooled to stop further digestive activity. The digested tissue was collected in tubes and washed with fresh medium to remove the enzyme and incubated in UW solution prior to purification.
Islet purification
All purification methods were conducted with a continuous density gradient of iodixanol (Optiprep, Sigma-Aldrich)-ET-Kyoto solution as previously reported.Citation27 Iodixanol has a low viscosity so that the centrifugation for dispersion can be slower than it is for Ficoll. For the continuous density solution, low-density (density: 1.075) and high-density (density: 1.085–1.120) iodixanol-ET-Kyoto solutions were produced by changing the volumetric ratio of iodixanol and ET-Kyoto solution. The high-density solution was determined as reported previously.Citation27 The digested tissue was separated into two halves. One half was purified using a COBE 2991 cell processor (COBE group) (CaridianBCT) as described previously.Citation22,Citation27 In the COBE bag, the gradient consisted of about 150 mL of high-density solution (the volume varied depending on the tissue density and the volume of iodixanol) in the most external layer during centrifuge, about 300 mL of continuous density gradient in the second layer, 100 mL of UW solution with the digest tissue in the third layer, and 50 mL of wash solution (Mediatech) in the most internal layer as a cap solution. When the volume of the digest for COBE was > 25 mL, the half digest was divided into quarters and two purifications were performed with COBE. The other half was separated into two parts again, and one quarter was purified with the “top-loading” bottle method (top group) and the other quarter was purified with the “bottom-loading” bottle method (bottom group), as described previouslyCitation13 and below.
For the bottle purification, we used two 500-mL flat bottom plastic cylindrical bottles, one for top loading and the other for bottom loading. The schematic of the loading solutions in the bottle is shown in . The gradient consisted of 50 mL of high-density solution in the bottom layer, 300 mL of continuous density gradient in the second layer, 50 mL of low-density solution in the third layer, and 50 mL of wash solution (Mediatech) on the top as a cap solution. The continuous gradient for the bottle methods was produced with a gradient maker in the same way of the COBE group.Citation13 The gradient maker drew high-density solution and low-density solution into a mixing chamber. After mixing, the solution was dripped into the bottle. At first, mostly high-density solution was drawn into the mixing chamber, so the density was near the bottom of the bottle. Over time, more and more of low-density solution (and less and less of high-density solution) was drawn into the mixing chamber. Therefore, the solution dripped into the bottle gradually changes from high-density solution to low-density solution. For both bottle methods, we used a “tip-bent” candy cane–shaped stainless pipe (Umihira Works Co., ) to pour the solution into the bottle. That form can minimize the convection current and hold the gradient. There were differences of the volume and composition of the layers between COBE and the bottle methods, because the sizes of the COBE bag and the bottle are different and it is necessary for the digest tissue to be mixed in the low density gradient solution to prevent it from aggregating and sticking in the top bottle method. However, the volume of the continuous gradient layer was the same between groups so that the efficacy to separate tissues could be the same. Immediately before loading, the pancreatic digest was mixed into the low-density solution in the third layer for the top method, and it was mixed into the high-density solution in the bottom layer for the bottom method. All systems were centrifuged at 1000 rpm for 5 min at 4°C. For the bottle method, the brake was used to decelerate. After centrifugation, islets were seen in the upper layer of the bottles (). In the bottom group, some tissue remained in the bottom of the bottle (, right panel).
After centrifugation, the gradient solution was collected into 10 tubes from the top to the bottomCitation13 and examined for purity. To confirm the stability of continuous density gradient, we measured the density of each of the 10 fractions. In the COBE method, approximately 80 mL of the solution was left in the COBE bag structurally after collection. On the other hand, the whole solution was collected in the bottle method, which can prevent the loss of islets.
Islet evaluation
Islet preparations were evaluated for yield, purity, and morphology by using dithizone staining. The evaluation was conducted by two independent investigators who did not know which group each sample was taken from. The crude number of islets in each diameter class was determined by counting islets using an optical graticule. The crude number of islets was then converted to the standard number of islet equivalents (IE; diameter standardizing to 150 µm).Citation28 The islet recovery was defined as the percentage of IE recovered after purification divided by the IE before purification.
Islet viability after purification was assessed using double fluorescein diacetate/propidium iodide staining to visualize living and dead islet cells simultaneously. Fifty islets were inspected, and their individual viability was determined visually, followed by calculation of their average viability.Citation2,Citation23,Citation28 The final tissue volume rate was calculated by determining the ratio of the postpurification tissue volume to the prepurification tissue volume in each method.
In vitro islet function was assessed by monitoring the insulin secretory response of the islets during glucose stimulation as described before.Citation2 Briefly, after culture at 37°C for 24 h, 1,000 IE were incubated with either 2.8 mM or 25 mM glucose in RPMI 1640 for 2 h at 37°C in a 5% CO2 atmosphere. The supernatant was collected, and insulin levels were determined using an enzyme-linked immunosorbent assay kit (ALPCO Diagnostics). The stimulation index was calculated by determining the ratio of insulin released from islets in the high glucose concentration to the insulin released from islets in the low concentration.
Sterility and contaminant testing
The islet cell suspension samples were taken from the final islet products of each group. The endotoxin concentration was measured using Endosafe PTS (Charles River Laboratories International, Inc.). The Gram stain test and the sterility culture were conducted for detecting the contamination of bacteria or fungi.
In vivo assessment
Animal studies were approved by the institutional animal care and use committee (IACUC) of Baylor Health Care System. Athymic nude mice (Harlan) rendered diabetic by a single injection of streptozotocin at a dose of 160 mg/kg were used. When the nonfasting blood glucose level exceeded 350 mg/dL for two consecutive days, the mice were considered to be diabetic. The 2,500 IE islets obtained from each group were transplanted into the renal subcapsular space of the left kidney of a diabetic nude mouse after a 2-d culture. During the 30-d posttransplantation period, the nonfasting blood glucose levels were measured three times per week. Normoglycemia was defined as two consecutive blood glucose level measurements < 200 mg/dL. No statistical differences in either pretransplantation blood glucose levels or body weight were observed among the groups.
Statistical analysis
All results were expressed as mean ± SE. The differences between each group were considered significant if the p value was < 0.05 using the Student’s t-test or the Kaplan-Meier log-rank test. Differences among three groups were analyzed by ANOVA followed by Student’s t-test with Bonferroni correction.
| Abbreviations: | ||
| ANOVA | = | analysis of variance |
| UW solution | = | University of Wisconsin solution |
| IE | = | islet equivalents |
Acknowledgments
This study was partially supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (1R21DK090513-019) (to S.M.) and Roche Diagnostics Corp. (to S.M.). The authors wish to thank Ms Yoshiko Tamura, Ms Ana M. Rahman, and Mr Greg Olsen (Annette C. and Harold C. Simmons Transplant Institute) for their technical support and Ms Cynthia Orticio (Baylor Research Institute) for professional editing.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
References
- Matsumoto S, Okitsu T, Iwanaga Y, Noguchi H, Nagata H, Yonekawa Y, et al. Insulin independence after living-donor distal pancreatectomy and islet allotransplantation. Lancet 2005; 365:1642 - 4; http://dx.doi.org/10.1016/S0140-6736(05)66383-0; PMID: 15889479
- Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230 - 8; http://dx.doi.org/10.1056/NEJM200007273430401; PMID: 10911004
- Shapiro AM, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson RP, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 355:1318 - 30; http://dx.doi.org/10.1056/NEJMoa061267; PMID: 17005949
- Weir GC, Bonner-Weir S. Islets of Langerhans: the puzzle of intraislet interactions and their relevance to diabetes. J Clin Invest 1990; 85:983 - 7; http://dx.doi.org/10.1172/JCI114574; PMID: 1969425
- Alejandro R, Strasser S, Zucker PF, Mintz DH. Isolation of pancreatic islets from dogs. Semiautomated purification on albumin gradients. Transplantation 1990; 50:207 - 9; http://dx.doi.org/10.1097/00007890-199008000-00007; PMID: 1696406
- Lake SP, Bassett PD, Larkins A, Revell J, Walczak K, Chamberlain J, et al. Large-scale purification of human islets utilizing discontinuous albumin gradient on IBM 2991 cell separator. Diabetes 1989; 38:Suppl 1 143 - 5; PMID: 2642839
- Casey JJ, Lakey JR, Ryan EA, Paty BW, Owen R, O’Kelly K, et al. Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 2002; 74:913 - 5; http://dx.doi.org/10.1097/00007890-200210150-00002; PMID: 12394830
- Abdelli S, Ansite J, Roduit R, Borsello T, Matsumoto I, Sawada T, et al. Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure. Diabetes 2004; 53:2815 - 23; http://dx.doi.org/10.2337/diabetes.53.11.2815; PMID: 15504961
- Aikin R, Maysinger D, Rosenberg L. Cross-talk between phosphatidylinositol 3-kinase/AKT and c-jun NH2-terminal kinase mediates survival of isolated human islets. Endocrinology 2004; 145:4522 - 31; http://dx.doi.org/10.1210/en.2004-0488; PMID: 15242986
- Bottino R, Balamurugan AN, Tse H, Thirunavukkarasu C, Ge XH, Profozich J, et al. Response of human islets to isolation stress and the effect of antioxidant treatment. Diabetes 2004; 53:2559 - 68; http://dx.doi.org/10.2337/diabetes.53.10.2559; PMID: 15448084
- Noguchi H, Matsumoto S, Onaca N, Naziruddin B, Jackson A, Ikemoto T, et al. Ductal injection of JNK inhibitors before pancreas preservation prevents islet apoptosis and improves islet graft function. Hum Gene Ther 2009; 20:73 - 85; http://dx.doi.org/10.1089/hum.2008.125; PMID: 18925829
- Shintaku H, Okitsu T, Kawano S, Matsumoto S, Suzuki T, Kanno I, et al. Effects of fluid dynamic stress on fracturing of cell-aggregated tissue during purification for islets of Langerhans transplantation. J Physics D Applied Physics 2008; 41 115507 (9pp)
- Shimoda M, Noguchi H, Fujita Y, Takita M, Ikemoto T, Chujo D, et al. Islet purification method using large bottles effectively achieves high islet yield from pig pancreas. Cell Transplant 2012; 21:501 - 8; http://dx.doi.org/10.3727/096368911X605411; PMID: 22793058
- van der Burg MPM, Graham JM. Iodixanol density gradient preparation in university of wisconsin solution for porcine islet purification. ScientificWorldJournal 2003; 3:1154 - 9; http://dx.doi.org/10.1100/tsw.2003.107; PMID: 14646009
- Brandhorst H, Asif S, Andersson K, Theisinger B, Andersson HH, Felldin M, et al. A new oxygen carrier for improved long-term storage of human pancreata before islet isolation. Transplantation 2010; 89:155 - 60; http://dx.doi.org/10.1097/TP.0b013e3181c9266c; PMID: 20098277
- Ihm SH, Matsumoto I, Sawada T, Nakano M, Zhang HJ, Ansite JD, et al. Effect of donor age on function of isolated human islets. Diabetes 2006; 55:1361 - 8; http://dx.doi.org/10.2337/db05-1333; PMID: 16644693
- Balamurugan AN, Breite AG, Anazawa T, Loganathan G, Wilhelm JJ, Papas KK, et al. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation 2010; 89:954 - 61; http://dx.doi.org/10.1097/TP.0b013e3181d21e9a; PMID: 20300051
- Fornoni A, Pileggi A, Molano RD, Sanabria NY, Tejada T, Gonzalez-Quintana J, et al. Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation. Diabetologia 2008; 51:298 - 308; http://dx.doi.org/10.1007/s00125-007-0889-4; PMID: 18066521
- Rush BT, Fraga DW, Kotb MY, Sabek OM, Lo A, Gaber LW, et al. Preservation of human pancreatic islet in vivo function after 6-month culture in serum-free media. Transplantation 2004; 77:1147 - 54; http://dx.doi.org/10.1097/01.TP.0000116769.94299.F4; PMID: 15114076
- Shimoda M, Itoh T, Sugimoto K, Takita M, Chujo D, Iwahashi S, et al. An effective method to release human islets from surrounding acinar cells with agitation in high osmolality solution. Transplant Proc 2011; 43:3161 - 6; http://dx.doi.org/10.1016/j.transproceed.2011.09.098; PMID: 22099746
- Noguchi H, Ueda M, Hayashi S, Kobayashi N, Okitsu T, Iwanaga Y, et al. Ductal injection of preservation solution increases islet yields in islet isolation and improves islet graft function. Cell Transplant 2008; 17:69 - 81; http://dx.doi.org/10.3727/000000008783907062; PMID: 18468237
- Matsumoto S, Okitsu T, Iwanaga Y, Noguchi H, Nagata H, Yonekawa Y, et al. Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method. Transplantation 2006; 82:460 - 5; http://dx.doi.org/10.1097/01.tp.0000231710.37981.64; PMID: 16926588
- Matsumoto S, Qualley SA, Goel S, Hagman DK, Sweet IR, Poitout V, et al. Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol. Transplantation 2002; 74:1414 - 9; http://dx.doi.org/10.1097/00007890-200211270-00013; PMID: 12451242
- Matsumoto S, Noguichi H, Shimoda M, Ikemoto T, Naziruddin B, Jackson A, et al. Seven consecutive successful clinical islet isolations with pancreatic ductal injection. Cell Transplant 2010; 19:291 - 7; http://dx.doi.org/10.3727/096368909X481773; PMID: 19995483
- Matsumoto S, Takita M, Chaussabel D, Noguchi H, Shimoda M, Sugimoto K, et al. Improving efficacy of clinical islet transplantation with iodixanol based islet purification, thymoglobulin induction and blockage of IL-1 beta and TNF alpha. Cell Transplant 2011; 20:1641 - 7; http://dx.doi.org/10.3727/096368910X564058; PMID: 21396171
- Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988; 37:413 - 20; http://dx.doi.org/10.2337/diabetes.37.4.413; PMID: 3288530
- Noguchi H, Ikemoto T, Naziruddin B, Jackson A, Shimoda M, Fujita Y, et al. Iodixanol-controlled density gradient during islet purification improves recovery rate in human islet isolation. Transplantation 2009; 87:1629 - 35; http://dx.doi.org/10.1097/TP.0b013e3181a5515c; PMID: 19502953
- Ricordi C, Gray DW, Hering BJ, Kaufman DB, Warnock GL, Kneteman NM, et al. Islet isolation assessment in man and large animals. Acta Diabetol Lat 1990; 27:185 - 95; http://dx.doi.org/10.1007/BF02581331; PMID: 2075782
![QR code for Honorbridge. Scan the QR code or visit https://partners.honorbridge.org/research/ .]({"type":"image" , "src" : "/sda/112423/JournalAd-mpu-kisl-honorbridge.jpg"})
![QR code for Honorbridge: We might have what your research needs. Click the QR code or visit https://partners.honorbridge.org/research/ to learn more.]({"type":"image" , "src" : "/sda/112422/JournalAd-skyscraper-kisl-honorbridge.jpg"})