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Procurement of the Human Pancreas for Pancreatic Islet Transplantation from Marginal Cadaver Donors

Nagata, Hideo1,6; Matsumoto, Shinichi2; Okitsu, Teru1; Iwanaga, Yasuhiro1; Noguchi, Hirofumi1; Yonekawa, Yukihide1; Kinukawa, Tsuneo3; Shimizu, Tomohiro4; Miyakawa, Shuichi4; Shiraki, Ryoichi5; Hoshinaga, Kiyotaka5; Tanaka, Koichi1,2

doi: 10.1097/01.tp.0000228886.15985.62
Original Articles: Cell Therapy and Islet Transplantation
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Background. Recent advances in pancreatic islet transplantation (PIT) have contributed significantly to the treatment of patients with type 1 diabetes. The specific aim of this study was to develop an effective technique for the procurement of pancreas for PIT from nonheart-beating-donor (NHBDs).

Methods. Between January 2004 and August 2004, eight human pancreata were procured and processed for isolation of islets at a cell processing center. After confirmation of brain death status, a double balloon catheter was inserted to prevent warm ischemic damage to the donor pancreas by using an in situ regional organ cooling system that was originally developed for procurement of kidneys. The catheter position of the cooling system was modified specifically for the pancreas and kidney. Furthermore, we worked in cooperation with a kidney procurement team to protect the pancreas during kidney procurement.

Results. Warm ischemic time could be controlled with the modified in situ regional cooling system at 3.0±0.8 min (mean±SE). The operations for procurement of the kidneys and pancreata lasted 45.6±3.6 min and 10.6±1.8 min, respectively. Islet yield per isolation was 444,426±35,172 IE (islet equivalent). All eight cases met the criteria for PIT based on the Edmonton protocol.

Conclusion. We developed a novel procurement technique in cooperation with our kidney procurement team. This protocol for the procurement of pancreas and kidney from a NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.

1Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

2Transplantation Unit, Kyoto University Hospital, Kyoto, Japan.

3Department of Urology, Chukyo Hospital, Nagoya, Japan.

4Department of Surgery, Fujita Health University, Toyoake, Japan.

5Department of Urology, Fujita Health University, Toyoake, Japan.

This work was supported in part by the Ministry of Education, Science, and Culture, the Ministry of Health, Labour and Welfare, and the 21st Century Center of Excellence Program, Japan.

6Address correspondence to: Hideo Nagata, M.D., Ph.D., Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawara-cho Shogoin, Sakyo-ku, Kyoto, Japan 606-8507.

E-mail: hnagata@kuhp.kyoto-u.ac.jp

Received 29 March 2005.

Accepted 31 May 2005.

Islet transplantation has been proven to be highly effective as a therapy for type 1 diabetes mellitus (1, 2). Because the number of cadaver donors was inadequate to treat the type 1 diabetic patient, the utilizing marginal donor pancreas for islet isolation would alleviate the shortage of donor. Currently, only a few clinical studies have reported that islet transplantation from nonheart-beating donors (NHBDs) was possible to cure type 1 diabetic patients (3, 4). Such studies are particularly important for the countries like Japan where the isolation of islets from heart beating brain death donor pancreata is prohibited by law. To overcome the resultant limited availability of donor pancreas for pancreatic islet transplantation (PIT), we have identified various conditionally marginal NHBDs who could be used as a source of cells for this purpose. In this report, we performed eight human islet isolations from non-heart beating donor pancreata that could be procured after kidney procurement. We developed this novel procurement technique in collaboration with the kidney procurement team and achieved successful islet isolation in all cases.

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MATERIALS AND METHODS

Procurement of Human Pancreas

Eight human pancreata were acquired from NHBDs after informed consent had been obtained from their relatives. We procured all eight pancreata through the Japan Organ Transplantation Network in the mid-Japan region (Aichi Prefecture, Japan) from January 17 to August 4, 2004. Donor characteristics and clinical data are shown in Table 1.

TABLE 1

TABLE 1

Islet transplantation was categorized as tissue transplantation in Japan. Transplant tissues have to be procured after the procurement of transplant organs such as kidneys.

After confirmation of brain death status, a double balloon catheter was inserted to prevent ischemic damage to the pancreas by using an in situ-regional organ cooling (ISRC) system that was originally developed for procurement of the kidney (5). Before cardiac arrest, a tip of the double balloon catheter was placed above the celiac axis in the aorta via the femoral artery, and only a few centimeters above the location used for an ordinary nephrectomy for procurement from a NHBD. Ultrasound was used to confirm whether the celiac axis and renal arteries were placed between the two balloons (Fig. 1). A venous catheter was also placed in the inferior vena cava via the femoral vein for drainage of the perfusate and blood.

FIGURE 1.

FIGURE 1.

ISRC for the pancreas and kidney (ISRC-PK) was achieved by pump or drip infusion of a hypothermic lactated ringer solution beginning immediately after cardiac arrest and continuing until the end of the nephrectomy and pancreatectomy. The drip speed was adjusted to 20 mL/min. The pressure of the catheter inside was not measured. After laparotomy, the lesser sac was opened by dividing the gastrocolic and gastrohepatic ligaments to determine whether the pancreas had uniform perfusion efficacy by means of ISRC-PK. The perfusion of the pancreas was evaluated by changing color of pancreas uniformly, regardless of the specific accident during the perfusion (like stopping the pump), and the coldness of the pancreas surface after laparotomy. We did not check a temperature of the pancreas surface, but we adjusted the temperature of the perfusate at 4°C. After a visual check of the pancreas, 500 ml of sterile crushed ice was placed on it to avoid warm ischemic injury. Our team and the kidney-procurement-team (KPT) cooperated to avoid warm ischemic and mechanical damage during procurement of the organs. One surgeon from our team paid special attention to the pancreas and checked the pressure differential between arteries and veins in the cooling system while the KPT procured the kidney.

To expose the left kidney, the splenic flexure and the attachments of the descending colon to the lateral peritoneum were divided. When it was totally exposed, the splenic flexure of the colon was mobilized toward the middle until the renal artery could be entirely separated from the aorta. The left ureter was then exposed by careful dissection, ligation and division. Then, the right kidney was also exposed from dissection in the avascular plane between Gerota’s fascia and mesocolon. Mobilization of the duodenum was started to separate the right renal artery and the right renal veins. While the duodenum mobilized, the proper hepatic artery (PHA) was ligated and divided to avoid the perfusate inflow in to the liver. The gastroduodenal artery (GDA) and the superior mesenteric artery (SMA) were left as intact as possible to maximize perfusion of the pancreas during the excision of both kidneys. The care was taken to avoid any injury to the pancreas when the procedure involved raising the pancreas from retro-peritoneal cavity. The aorta and inferior vena cava were dissected and both kidneys were harvested en bloc. After ligation of the major vessels, perfusion was terminated. While the pancreas was largely free on the retro-peritoneal side, duodenum to jejunum around of the pancreas was intact. We then divided the first and fourth portions of the duodenum with a proximate 55 mm linear cutter (Ethicon; Johnson and Johnson K.K., Suture Div, Tokyo, Japan.). After division of the duodenum, the attachment between retro peritoneal and body of the pancreas was dissected toward the spleen. The superior surface of the pancreas was divided toward the spleen and the short gastric arteries and vein dissected until the stomach was separated from the spleen. The pancreas was then rapidly excised en bloc together with the spleen. The spleen was subsequently removed on the back table before the pancreas was placed in the two-layer solution (6).

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Human Islet Isolation

Our center is qualified as a good manufacturing practice (GMP) facility and is called the Center for Cell and Molecular Therapy (CCMT).

The procured pancreas was isolated with a modification of the Ricordi method by our islet isolation team (7). Briefly, the main pancreatic duct was perfused in a controlled fashion with a cold enzyme of Liberase HI (Roche Molecular Biochemicals, Indianapolis, IN). Recirculating the enzyme solution through the Ricordi chamber at 37°C resulted in digestion of the pancreas. Islets for the first two cases were purified using a continuous density gradient of high osmolality Ficoll in an apheresis system (COBE 2991 cell processor; Gambro Laboratories, Denver, CO) with refrigeration unit (Walker Centrifugal Systems LLC, Loveland, CO) according to the Edmonton protocol. For the other six cases, islets were purified with a continuous density gradient of an iodixanol-based solution in an apheresis system (7–9). We employed an optiprep-based solution for islet isolation in present cases.

Isolated islets were evaluated according to the Edmonton protocol (1, 6). Variables of isolated islets were evaluated in term of Islet Equivalents (IE) per kilogram of recipient weight, purity, isolated islet volume, viability, morphology, functionally and sterility. Islet yields and purity were assessed after dithizone staining of islets under microscope (1, 6, 10). Islet volume was measured as tissue volume (1). Islet viability was assessed after purification using acridine orange (10 μmol/L) and propidium iodide (15 μmol/L) (AO/PI) staining to visualize living and dead islet cells simultaneously (6, 10, 11). Islet morphology was qualitatively assessed by two independent investigators scoring the islets for shape, border, integrity, uniformity of stain and diameter (6, 10). Islet function was assessed by monitoring the insulin secretory response of the purified islets during glucose stimulation according to a procedure described by Shapiro and colleagues (1). Sterility was assessed by gram stain and endotoxin measurement (1, 6). These procedures were performed without xenogeneic serum (1). All values are described as means±SE.

This study was approved by the Ethics Committee of the Kyoto University Graduate School and Faculty of Medicine.

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RESULTS

Donor characteristics are shown in Table 1. We procured eight human pancreata together with sixteen kidneys by mean of ISRC-PK modified from its exclusive use for nephrectomies on NHBDs for kidney transplantation. Whereas all donors had abnormal creatinine and blood urine nitrogen (BUN) levels, seven of the eight cases of kidneys were transplanted. Four of the eight cases showed high amylase levels (normal range: 39–129 IU/L) from 164 to 1818 IU/L with a mean±SE level of 550.0±243.5 IU/L. Lipase levels were not measured. Six cases had high blood glucose levels with a mean level of 285.0±68.9 mg/dl. These facts indicated that the donors used for this study were marginal. Pancreata characteristics shown in Table 2 indicate that one donor had hemorrhagic pancreatitis and two donors had chronic pancreatitis. Pancreatitis was diagnosed by high amylase level and histological findings (Fig. 2). We speculate that the patient of hemorrhagic pancreatitis was due to several hypotensive episodes during his long hospital stay.

TABLE 2

TABLE 2

FIGURE 2.

FIGURE 2.

The case of anoxia was the case of suicide. The period of cardiorespiratory arrest was estimated at about 20 minutes.

By using ISRC-PK, the average warm ischemic time could be limited to 3.0±0.8 min. All the pancreata were exposed to minimal warm ischemic period because they were almost immediately perfused with cold lactate ringer solutions. There is no correlation between warm ischemic time and islet yield before (R2=0.1811, P=0.2931) or after (R2=0.0047, P=0.8719) purification.

The operations for procurement of the kidneys and pancreata lasted 45.6±3.6 min and 10.6±1.8 min, respectively. Total storage period was between 105 and 305 min.

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Pancreatic Islet Variables and Transplantation

Islet characteristics are shown in Table 3. Islet yield per isolation was 444,426±35,172 islet equivalent, purity was 51.9±5.0%, and viability was 95.5±1.4%, whereas previous studies reported a successful islet isolation rate of only 55 to 61% (2, 12). All of our cases (100%) met the criteria for islet transplantation based on the Edmonton protocol, and seven of the eight islet preparations were transplanted into four patients with type 1 diabetes. The remaining case was our first human pancreas isolation, but this islet preparation was not transplanted because at the time of the isolation there was not yet a procedure in Japan for the use of fresh islets. Therefore, that islet preparation was cryopreserved. At that time, we did not have any agreement of fresh islet transplantation in Japan. After this case, the Japan committee of pancreas and islet transplantation fixed the protocol for the fresh islet transplantation. We transplanted the first islets into a type 1 diabetic patient for the first time in Japan on April 7, 2004. The patient was sedated and a percutaneous transhepatic approach was used for the access to the portal vein. The Kumpe catheter was placed within the main portal vein. Islets were infused by gravity using bag technique (13). This patient received a second transplantation three months after the initial transplantation, and became insulin independent 19 days after secondary transplantation (7). Furthermore, all patients were showed after PIT that insulin requirements were improved from 0.6±0.1 units/kg to 0.3±0.1 units/kg and also hemoglobin A1c levels changed from 8.0±0.4% to 5.1±0.2%, respectively.

TABLE 3

TABLE 3

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DISCUSSION

The first successful PIT in Japan was performed by our team. The favorable outcome may spread islet transplantation as an alternative to whole organ pancreas transplantation for the type 1 diabetes patients. Theoretically, PIT has several advantages over whole organ transplantation. It is less invasive because the procedure can be performed percutaneously and recipients do not require prolonged hospitalization.

The most important issue affecting islet transplantation is related to donor quality (14, 15). In spite of a serious shortage of donors in Japan, it is prohibited by law to use the pancreas from heart-beating brain death donors for PIT. This fact made it necessary to perform PIT by using islets isolated from NHBD. Although some institutions have tried to perform PIT with islets isolated from NHBD, it proved to be difficult to isolate human islets successfully from NHBD (3, 4). In fact, only one case of successful islet transplantation with NHBD was reported (3). To obtain better results for islet isolation from marginal donors, we implemented several procurement strategies.

First, we used ISRC to prevent from warm ischemic injury during organ procurement. Kato et al. developed ISRC for the kidney procurement (5). The only modification we made was the position of the double balloon catheter to ensure pancreas and kidney protection. This ISRC system reduced warm ischemic time to only 3 minutes, whereas the University of Pennsylvania group had 20 minutes of warm ischemic time (3). We used lactate ringer solution instead of University of Wisconsin solution for perfusion. Lactate ringer solution has low potassium concentration and low viscosity compared with University of Wisconsin solution. Low potassium concentration could prevent potassium induced vasospasms and low viscosity help rapid perfusion. Therefore, using lactate ringer for perfusion might be important in ISRC. Our results showed no correlation between islet yield and warm ischemia time. In our speculation, this was caused by remarkable shortening of warm ischemic time in all cases by ISRC (3, 16).

Second, the pancreas procurement team met with the kidney procurement team to discuss details of the procedure and to explain any risk of organ injury during the procurement procedures. This communication enabled us to protect pancreas during kidney retrieval.

Third, after the peritoneal cavity had been opened, the lesser sac was filled with some crushed ice. Lee et al. have previously reported that for their procurement technique involving mobilization of the pancreas, slush was packed onto the posterior and anterior surfaces of the pancreas to maintain a core pancreas temperature of 4°C (12). Our ISRC did not require mobilization of the pancreas. After the ISRC had been performed, the cadaver body—especially “the back”—was chilled down immediately. It was reflected that the core of pancreas was chilled suitably.

Fourth and last, KPT usually do not consider the pancreas, so it is vulnerable to injury. Therefore, the injuries of the pancreas had been avoided by one surgeon from the pancreas procurement team. Of special interest in connection with our procurement procedure was that there was no reduction in the number of renal transplant or function of the kidney grafts. Additionally, our novel technique prevents the possibility of contaminating the kidney since kidney was procured first. If kidney is excised after the pancreas procurement, contamination of the kidney might occur. (Note: Under “Procurement of Human Pancreas,” it says that under Japanese rules pancreas procurement for islet must be performed after kidney procurement.)

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CONCLUSION

We developed a novel procurement technique in cooperation with our kidney procurement team. This collaborative effort has resulted in successful islet isolation in all cases. Our current protocol for the procurement of pancreas and kidney from a NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.

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ACKNOWLEDGMENTS

The authors wish to thank Tetsuo Kanno, Hirotoshi Sano, Motoi Shoda, Yoko Kato, Fumihiro Imai, Takafumi Kaito, Shingo Maeda, Tsunetoshi Araki, Hirotoshi Ishise, Takamitsu Morikawa, Hitomi Sasaki, Mamoru Kusaka, Nobuyuki Oyama, Hironobu Akino, Osamu Yokoyama, Yoshikazu Tsuji, Kenji Mizuno, Masahiro Ito, Shin Ishihara, Yuji Iwase, Akihiko Horiguchi, Makoto Hayakawa, Naotatsu Niwamoto, Yasumitsu Ukai, Chikao Yamazaki, Osamu Kato, Akika Uwano, Tomoko Asai, Yukiko Kobayashi, Yumiko Yonemitsu, Kazuyo Fujii and Miyuki Hara for their assistance and arrangement of organ procurement; Yasunari Kasai, Taira Maekawa, Yusuke Nakai, Michiko Ueda, Yurika Uchida, Yoko Nakagawa, Hiroko Muramatsu, Akemi Ishii, Yumi Yoshida for their technical assistance and maintenance of the GMP facility; and Kazuhito Fukuda, Yuichiro Yamada, Katsushi Tsukiyama as well as all members of the Department of Diabetes and Clinical Nutrition of the Kyoto University Graduate School of Medicine.

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REFERENCES

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Keywords:

Islet transplantation; Nonheart-beating donor; Organ procurement

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