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. 2007 Oct;20(4):357–362. doi: 10.1080/08998280.2007.11928323

Improvement of pancreatic islet cell isolation for transplantation

Shinichi Matsumoto 1,✉, Hirofumi Noguchi 1, Bashoo Naziruddin 1, Nicolas Onaca 1, Andrew Jackson 1, Hatanaka Nobuyo 1, Okitsu Teru 1, Kobayashi Naoya 1, Göran Klintmalm 1, Marlon Levy 1
PMCID: PMC2014805  PMID: 17948109

Abstract

Pancreatic islet transplantation is a promising treatment for diabetes but still faces several challenges. Poor islet isolation efficiency and poor long-term insulin independence are currently two major issues, although donor shortage and the need for immunosuppressants also need to be addressed. We established the Kyoto islet isolation method (KIIM), which has enabled us to isolate and transplant islets even from non–heart-beating donors. KIIM involves 1) cooling the donor pancreas in situ, 2) preserving the ducts with modified Kyoto solution, 3) using a modified two-layer pancreas preservation method, and 4) adjusting the density of the density gradient centrifugation and using an iodixanol-based solution for purification. KIIM has enabled us to transplant 17 islet preparations out of 21 isolations (an 81% success rate). All transplanted islets functioned, and all transplanted patients had improved glycemic control without hypoglycemic unawareness. Recently, we used KIIM for islet isolation from a brain-dead donor at Baylor, which resulted in a very high islet yield (789,984 IE) with high viability (100% by fluorescein diacetate/propidium iodide staining and a stimulation index of 4.7). This preliminary evidence suggests that KIIM may also be promising for islet isolation from brain-dead donors. In addition, to assess engrafted islet mass, we developed a secretory unit of islet transplant objects (SUITO) index: fasting C-peptide (ng/dL) / [fasting blood glucose (mg/dL) – 63] × 1500. This simple index has enabled us to monitor the engrafted islet mass. This index should be useful when deciding whether to perform additional islet transplantations to maintain insulin independence. Poor islet isolation efficacy and poor long-term results could be resolved with ongoing research.

Type 1 diabetes still represents a therapeutic challenge and remains a substantial burden for patients and their supporters. The Diabetes Control and Complications Trial showed that intensive insulin therapy improved glycated hemoglobin A1c and protected against diabetic triopathy (1), but the penalty was a thrice-increased risk of serious hypoglycemic events, including recurrent seizures and coma (2). Whole-pancreas transplantation can make those patients insulin independent, but the morbidity of that procedure is too high to advocate it for most patients (3).

An attractive alternative is islet transplantation. The islet transplantation procedure doesn't involve major surgery, general anesthesia, or the complications related to exocrine enzymes. Since the first human islet allograft transplant was done in 1974 (4), this treatment has continuously improved, and a dramatic improvement was achieved with the Edmonton protocol in 2000 (5). Shapiro et al demonstrated that seven out of seven preuremic type 1 diabetic patients who received islet transplants became insulin independent, with a dramatic decrease in the frequency of hypoglycemic unawareness at 1 year posttransplantation.

Key elements of the Edmonton protocol are 1) avoidance of corticosteroids with combined sirolimus, tacrolimus, and anti-interleukin-2-receptor antibody therapy to protect against rejection and autoimmunity and 2) the use of two or more fresh islet preparations (within 3 to 4 hours after isolation) processed by the Edmonton islet isolation protocol. The Edmonton islet isolation protocol includes 1) procurement of the donor pancreas from a brain-dead donor and organ preservation in cold University of Wisconsin solution with a minimal storage period, 2) collagenase infusion via the main pancreatic duct using a pressure-controlled method, 3) pancreas digestion using the Ricordi system, 4) islet purification by continuous density gradient using Ficoll with a chilled COBE 2991 cell processor, and 5) removal of all xenoproteins from the islet isolation process (5). Isolated islets were transplanted immediately by simple gravity infusion.

There has been an exponential increase in clinical islet transplantation activity, with 471 patients transplanted at 43 international institutions (6). The University of Alberta, the University of Minnesota, and the University of Miami demonstrated that 82% of 118 recipients of completed transplants were insulin independent within the first year after transplantation (6). The University of Alberta further demonstrated progressive loss of insulin independence over time, leaving approximately 10% of patients still insulin independent at 5 years (7). However, more than 80% of patients continued to demonstrate persistent islet function at 5 years with effective prevention of recurrent hypoglycemia or severe lability combined with correction of hemoglobin A1c (7). In addition, patients’ quality of life was significantly improved by islet transplantation (8). Therefore, it should be reasonable to consider islet transplantation an option for the treatment of unstable type 1 diabetes.

The most difficult part of islet transplantation is islet isolation from a donated pancreas. Indeed, even in the leading centers, a transplantable yield of isolated islets is obtained in <50% of processed pancreata (9–11). In Japan, there is an extremely low number of brain-dead donors, and it has been almost impossible to utilize such donors for islet isolation. Therefore, alternative resources for islet isolation were sought, including islet isolations from non–heart-beating donors (NHBDs). The quality of the donor pancreas has a substantial impact on islet isolation. Therefore, using NHBDs for islet isolation is very challenging (12, 13).

In order to utilize pancreata from NHBDs for islet isolation, numerous protocols have been tested. Ultimately, an islet isolation method was established that enabled islet transplantation using pancreata from NHBDs (14–16). Very recently, this isolation method was applied to a regular brain-dead donor with promising preliminary data. In this article, we describe our endeavor to develop a new islet isolation method and our current effort to adapt this method to brain-dead donors.

THE PROCESS OF PANCREATIC ISLET ISOLATION

Pancreatic islet cell isolation starts at the point of donor selection and pancreas preservation (Figure) When a pancreatic graft arrives at the islet isolation facility, the pancreas is distended with collagenase and digested in a Ricordi chamber. The final step is purification of isolated islets from exocrine tissues. After purification, islets are washed and put into a transplantation bag or culture medium before transplantation.

Figure.

Figure

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Donor selection and pancreas procurement is the first step for islte isolation. The procured pancreas is typically preserved by the two-layer method. When a pancreatic graft arrives at the islet isolation facility, the pancreas is distended with collagenase and digested in a Ricordi chamber. The final step is purification of isolated islets from exocrine tissues. A COBE 2991 cell processor is used for purification.

Donor selection

The quality of the donor pancreas is an important factor in successful islet isolation (17–20). However, pancreata are offered as whole organs first and only next for islet transplantation since whole-pancreas transplantation is considered an established treatment and islet transplantation is considered experimental.

Pancreata from obese donors give higher islet yield than those from lean donors (11, 17). Recently, researchers at the University of Minnesota demonstrated that the average islet yield from a pancreas donor with a body mass index (BMI) >30 was 319,129 IE and from a donor with a BMI <30 was 215,753 (P = 0.0002) (11). In addition, the donor with a BMI >30 had a higher islet isolation success rate—defined as isolations yielding >300,000 islet equivalents per pancreas, with purities of >50% (37.3% vs 15.9%; P = 0.009). This study showed that successful islet isolation can be difficult with low-BMI donors.

Previously, older donors were considered more suitable for islet isolation than younger donors (17). However, investigators have recently confirmed the advantages of islets from young donors, both in vitro in terms of insulin secretory function (21) and in vivo after transplanting islets in diabetic mice (22). In addition, we discovered that high islet yields could be obtained from younger donors with a modified islet isolation and purification method (18). The main obstacle to gaining high postpurification islet yields from young donors lies in the higher percentage of mantled islets embedded in acinar tissue. To recover mantled islets, we individualized the density of the high-density purification solution for each islet preparation (18). We believe that if we can recover mantled islets from young donors, the islet preparations should be of high quality, both in function and in islet number.

The stability of the donor during brain-dead status is another important factor. Based on the Edmonton protocol, many islet centers, including ours, have several exclusion criteria related to stability. The first concerns circulation and blood pressure: prolonged hypotensive episodes are exclusion criteria when they have caused significant biochemical abnormalities (e.g., elevation of serum creatinine levels by >50% of the initial value or elevation of transaminases to levels >2 times the normal values). Cardiac arrest is an exclusion criterion when constantly stable circulation cannot be achieved in the 2 days following the event or when significant biochemical abnormalities have occurred, as described above. A third exclusion criterion relates to vasopressors, specifically norepinephrine, if required for maintenance of stable circulation. However, we have recently developed an islet isolation method for marginal donors, especially NHBDs. With this method, we have successfully isolated islets from marginal donors, and we have changed the criteria from absolute contraindications to relative contraindications. This change will significantly increase the islet isolation number, and we estimate that about 50% of donor pancreata will be used for islet isolation (Matsumoto et al, manuscript in preparation).

We have found that histology-proven chronic pancreatitis has led to the worst islet isolations (18). The duration of pancreatic digestion was significantly longer, and the undigested tissue volume was significantly larger with chronic pancreatitis. In addition, the purity of isolated islets was significantly lower with chronic pancreatitis. This finding suggests that fibrotic pancreata are resistant to collagenase digestion, resulting in poor islet isolation.

Pancreas preservation

Traditionally, the pancreas is preserved in University of Wisconsin solution. However, even for a short duration, oxygenated perfluorocarbon (PFC) provides the best method for pancreas storage (23–25). With the oxygenated PFC, pancreas grafts are directly oxygenated and continuously generate ATP (26), and the viability of endothelial cells is maintained (27, 28). Because of these effects, oxygenated PFC seems to be the most suitable substance for preservation before pancreas transplantation and islet isolation.

When PFC is added to the University of Wisconsin solution or another solution—the “two-layer method”—it is necessary to oxygenate the PFC just before pancreas preservation, ensure adequate oxygenation of PFC just before storage, and ensure sufficient attachment of PFC to the pancreata. With inadequate oxygenation to the pancreas, ATP production was low, and the benefit of the two-layer method was lost (29).

Complete immersion of the pancreas into oxygenated PFC—the “one-layer method”—seems to be better than the two-layer method (30). Even for the one-layer method, the top layer is necessary to keep oxygen from escaping from the surface of the PFC.

Pancreatic ductal preservation seems important because collagenase is delivered through the pancreatic duct (31). Sawada et al demonstrated that a small amount of University of Wisconsin solution perfused into the pancreatic duct significantly improved the results of islet isolation in a rodent model (31), and a European group introduced the method in humans (32). We have demonstrated that ductal injection of a large amount of modified Kyoto solution into the main pancreatic duct significantly reduced apoptotic cell death of both exocrine tissue and islet cells (Noguchi et al, Cell Transplantation, in press). We avoided University of Wisconsin solution for ductal injection since it inhibits collagenase activity, which is essential for pancreas digestion and islet isolation (33). We preferred a large ductal injection because the solution protects not only pancreatic ducts but also exocrine tissues.

Collagenase selection and delivery

Selection of collagenase is important for successful islet isolation, and currently Liberase is used exclusively (34). Liberase is considered to be the best collagenase, but lot-to-lot variation has been a concern. Kin et al demonstrated that optimization of thermolysin dosage based on caseinase unit per gram of pancreas contributed to the islet isolation outcome, but the collagenase dosage provided by the manufacturer (Wünsch unit per gram of pancreas) was not a major determinant of islet isolation outcome (9). In addition, they pointed out that the lot-to-lot inconsistency of the enzyme's performance was explained not by the activity values provided by the manufacturer but rather by the proportion of class I collagenase and class II collagenase, as determined by an in-house assay (9). Specifically, the odds of successful isolation were 8.67 times higher when a vial with a class II:class I ratio of <0.204 was used than when a vial with a ratio of ≥0.204 was used.

Collagenase delivery with pressure monitoring is the current standard (35). It is widely believed that during the infusion of collagenase into the pancreas, the goal is excellent distension with minimum leakage. An important modification that we have made is the use of only one cannula, inserted from the duodenal orifice of the main pancreatic duct (one-cannula method) (25). For collagenase delivery, usually a pancreas is cut and cannulas are inserted into two or three pancreatic ducts; this has been done since if the pancreatic duct is not adequately preserved, one cannula cannot deliver collagenase through the pancreas. However, this technique inevitably causes collagenase leakage. The one-cannula method, which requires the pancreas to be preserved intact and not cut, has resulted in minimal collagenase leakage with excellent distension.

Pancreas digestion

The Ricordi method is a standard for pancreas digestion in clinical islet transplantation (36). The key component of this method is a special Ricordi chamber for pancreas digestion and the effective collection of digested pancreatic tissue (36). The Ricordi chamber is designed for effective pancreas digestion with meticulous temperature control and is useful for effective dilution and collection of digested pancreatic tissue with a large volume of solution. Besides the Ricordi method, other static digestion methods have been effective for pancreas digestion (37, 38); however, those methods may not be effective for dilution. Since islets are sensitive to overdigestion, effective dilution may be important. Therefore, dilution, temperature control, and neutralization of digestive enzymes are all important, and the Ricordi method is the best to provide these conditions.

Trypsin inhibitors may help to avoid overdigestion (38). Previously, we have shown that the use of the trypsin inhibitor Pefabloc during islet isolation using the simple open-pan islet isolation method improved islet yield in nonhuman primate and human models (38). The University of Alberta also demonstrated that human islet isolation was improved with Pefabloc when pancreata were preserved for extended time periods (39). However, trypsin inhibition had no effect on improved islet isolation when pancreata were procured from brain-dead heart-beating donors using the Ricordi islet isolation method (40, 41). In addition, when collagenase activity is not strong enough, trypsin may actually help to digest a pancreas. Therefore, trypsin inhibition during islet isolation might not be important when an optimal pancreas is processed with the Ricordi islet isolation method.

Islet purification

Purification of islets from exocrine tissue is a critical step for maintaining high islet yields. The common method of islet purification is density gradient centrifugation. Ficoll is widely used for density gradients (42) with a COBE 2991 cell processor (43). However, an iodixanol-based solution has contributed to increased islet yield, especially for porcine islet isolation (44–47). Iodixanol has low viscosity and, therefore, it needs less force during centrifugation. Iodixanol-based purifications were clinically applied by others as well as by our own group with promising results (15, 16, 48, 49). We diluted iodixanol with ET-Kyoto solution. However, other solutions, such as culture media or other preservation solutions, could be examined.

THE KYOTO ISLET ISOLATION METHOD FOR NHBDS

In Japan, it is difficult to use brain-dead heart-beating donors for islet isolation and transplantation. Therefore, we pursued islet transplantation from NHBDs (14, 15) or living donors (50–52). To initiate islet transplantation using NHBDs, we established several criteria that resulted in the Kyoto islet isolation method (KIIM) (14–16).

First, we inserted a double-balloon catheter before cessation of heart beating to chill the pancreas immediately after cardiac arrest (53). This technique enabled us to minimize warm ischemic time. It was demonstrated that islet yield and function deteriorated after 30 minutes of warm ischemia in rat and dog models (54). As a matter of fact, without this technique, warm ischemic time is >30 minutes and results in unsuccessful islet isolation. Second, we introduced ductal injection immediately after procurement, as described in the pancreas preservation section. Third, we modified the two-layer (modified Kyoto solution and oxygenated PFC) method of pancreas preservation and recently switched to a one-layer method. Fourth, we used ulinastatin for trypsin inhibition during islet isolation. Ulinastatin is not only a trypsin inhibitor but also an antiinflammatory drug (55). Therefore, this drug might be useful for an ideal donor with the Ricordi method. Finally, we performed density measurements on exocrine tissue since acinar tissue density could decrease during warm ischemia. Adjusting the density of the gradient solution enabled us to recover embedded islets. In addition, we used iodixanol instead of Ficoll for islet purification because iodixanol has low endotoxin activity and low viscosity, which should be less harmful for islets.

We have isolated 21 human pancreata using KIIM from NHBDs. Double-balloon catheters inserted before cardiac arrest were combined with kidney retrieval in 18 cases (18). The average transplanted islet yield was 382,945 ± 44,146 IE (4,589 ± 504 IE/g), and the purity was 46.8 ± 3.3%. The viability of transplanted islets, as assessed by acridine orange/propidium iodide, was 96.2 ± 0.7%, and all of the samples were above 85%. The average insulin stimulation index was 4.2 ± 1.8. Islet preparations from 17 cases (16 cases with a double balloon and one case without a balloon) met transplantation criteria. These islet preparations were transplanted into eight type 1 diabetic patients. In all cases after islet transplantation, hemoglobin A1c levels were improved and there was no hypoglycemic unawareness. Thus, 17 out of 21 islet preparations (81%) achieved success. Compared with results of leading institutes, KIIM provided a very high success rate of transplantation, even using NHBDs (9–11).

SECRETORY UNIT OF ISLET TRANSPLANT OBJECTS INDEX

To assess engrafted islet mass, we developed the secretory unit of islet transplant objects (SUITO) index (56, 57). The formula of the SUITO index is as follows: fasting C-peptide (ng/dL) / [fasting blood glucose (mg/dL) – 63] × 1500. A SUITO index of 100 reflects 100% pancreatic beta-cell function in a healthy person. If the fasting C-peptide level is 0.8 ng/dL and blood glucose is 103, the SUITO index will be 0.8 / (103–63) × 1500 = 30.

Previously, we have shown that a SUITO index of >25 is necessary for insulin-independent status (56). The SUITO index of islet-transplanted patients with cultured islets from NHBDs was significantly lower than that of patients with fresh islets from NHBDs (56). In addition, living-donor islet-transplanted patients showed the highest SUITO index with insulin-inde-pendent status (56).

Recently at Baylor, we performed islet transplantation from a brain-dead donor without culturing the islets. After a single islet infusion, the average SUITO index from day 3 to day 30 was 29.7 ± 10.4. The patient's glycemic control improved substantially without hypoglycemic unawareness. The insulin dosage has been substantially reduced, and the patient is expected to be insulin independent. After single islet infusion with NHBDs, the average SUITO index from day 3 to day 30 was approximately 12. Therefore, islets from brain-dead donors seem to be of higher quality than those from NHBDs.

APPLICATION OF KIIM FOR ISLET ISOLATION FROM A BRAIN-DEAD DONOR

Recently, we applied KIIM for islet isolation from a brain-dead donor (except for the double-balloon technique, which is necessary only for NHBDs). In this case, the islet team joined the Baylor University Medical Center donor team for the pancreas procurement. The donor was a 52-year-old woman with a BMI of 39.1 and a pancreas weight of 98 g. After the pancreas was retrieved, the attached spleen, duodenum, and fat tissue were immediately removed. A single cannula was inserted from the duodenal orifice of the main pancreatic duct. Approximately 100 mL of modified Kyoto solution was infused. An accessory pancreatic duct was identified and was ligated with a hemoclip. The pancreas graft was preserved by the two-layer method (modified Kyoto and oxygenated PFC) and transported to the islet isolation laboratory at the Baylor Institute for Immunology Research. The cold storage period was approximately 3 hours. Upon arrival at the islet isolation laboratory, the pancreas was immediately immersed in the decontamination solution since it had already been trimmed. Chilled collagenase solution (Serva collagenase with neutral protease) was infused using a single cannula with pressure control. The pancreas distended excellently with minimum collagenase leakage. The distended pancreas was cut into nine pieces and put into the Ricordi chamber for digestion. Islets were purified using a density-adjusted Kyoto-iodixanol continuous density gradient with a COBE 2991 cell processor.

Islet yield after digestion and before purification was 803,467 IE. After purification, the islet yield was 789,984 IE with approximately 40% purity. A viability assay with fluorescein diacetate/propidium iodide showed that 100% of the islets were viable after purification, and the stimulation index with static glucose challenge was 4.7. Thus, the first trial of KIIM for islet isolation from a brain-dead donor is promising.

CONCLUSIONS

At this time, islet transplantation is the most promising method to cure diabetes with minimum risks—although the success rate for islet isolation is still relatively poor. However, continuous improvements in islet isolation are occurring. Single-donor islet transplantation for insulin independence should be established with an advanced islet isolation technique. The SUITO index is a powerful tool to estimate engrafted islet mass (56). We should supply islets to maintain a SUITO index of >30;this practice should result in patients who remain insulin independent for the long term. Current major concerns of poor islet isolation efficacy and long-term results could be resolved with ongoing research.

Acknowledgments

This study is partially supported by All Saints Health Foundation and Otsuka Pharmaceutical Company (Tokushima, Japan). We thank Dr. Carson Harrod for his careful review of this manuscript.

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