Yasunaru Sakuma

Shipment of Human Islets for Transplantation

The use of regional human islet cell processing centers (ICPC) supporting distant clinical islet transplantation programs (CITP) has proven successful in recent clinical trials. Standardization of islet shipping protocols is needed to preserve cell product identity, quantity, quality and sterility, and to meet criteria for transplantation. We evaluated the use of gas‐permeable bags for human islet preparation shipment from a single ICPC to two remote CITPs. Product release tests (counts, purity, viability, sterility and potency) were performed at both centers using identical protocols to determine adequacy for transplantation. Thirty‐five islet preparations were shipped either immediately after isolation (n = 20) or following culture (n = 15). Islet recovery rate after shipment was higher in cultured preparations, when compared to those not cultured (91.2 ± 4.9% vs. 72.9 ± 4.7%, respectively; p < 0.05), though the overall recovery rate based on isolation and pre‐transplant counts was comparable (72.9 ± 4.7% vs. 70.4 ± 3.5%, respectively; p = N.S.). All preparations met product release criteria for transplantation. Additional experiments showed that gas‐permeable bags led to improved recovery and potency, when compared to 50‐mL conical tubes or to non‐gas‐permeable bags for shipment. Collectively, our data demonstrate that the use of gas‐permeable bags is efficient for clinical‐grade and should be preferred also for the shipment of research‐grade islet preparations.

Factors That Affect Human Islet Isolation

More than 10,000 IEQ/kg recipient weight of islets is often necessary to achieve insulin independence in patients with type 1 diabetes mellitus. Several studies have identified high donor body mass index (BMI) and pancreas size as important factors for the success of human islet isolation. However, the donor shortage underscores the need to improve isolation outcomes from lower BMI pancreas donors and/or small pancreata. The aim of this study was to identify the critical factors that affect isolation outcome. We analyzed the data from 207 isolations performed from 2002 to 2006 with respect to donor characteristics, pancreas condition, and processing variables. More than 3000 IEQ/g pancreas weight was considered to be an acceptable isolation outcome. This goal was obtained from donors with a BMI >30 kg/m2 (P = .002). The pancreatic surface integrity was also a significant factor (P = .02). Moreover, longer digestion times (P = .04) and a greater proportion of trapped islets negatively affected success rates (P = .004). As previously reported, pancreata from high BMI donors were suitable for islet isolation and transplantation, as they yielded higher total islet particle numbers and higher IEQ/g. Although BMI and pancreas size are not controllable due to the organ donor shortage, factors such as pancreatic surface integrity, shorter digestion time, and lower proportions of trapped islets were found to be significant to obtain higher success rates. The development of better protocols and systematic training of processing/procurement teams will be of assistance to increase the number of successful human islet isolations.

Deterioration and variability of highly purified collagenase blends used in clinical islet isolation.

Background. The quality and stability of enzyme blends used in islet cell processing are critical for successful human islet isolation. A wide variability in enzymatic activity among lots of Liberase HI has been reported. This study examines the interlot and intralot variability of Liberase HI and the over-time deterioration of enzyme quality based on the analysis of islet isolation outcomes. Methods. The data of 169 human isolations processed for clinical islet transplantation, using five different lots of Liberase HI, were retrospectively analyzed. Inter- and intralot variables in the islet isolation were assessed over a 15-month period. Results. The analysis revealed significant interlot differences in the digestion time, prepurification islet counts, percent recovery, viability, and glucose stimulation insulin index. Moreover, a significant decrease in the pre- and postpurification islet yield per pancreas weight (IEQ/g) in isolations processed by two different enzyme lots used over a 15-month period was observed, suggesting a progressive deterioration of enzyme quality. Conclusions. Our data demonstrate a significant lot-to-lot related variability in islet isolation outcomes. In addition, the over-time decline in isolation outcomes processed using a single enzyme lot was observed even when the enzyme blends were used within the expiration dating specified by the manufacturer.

Prolactin Supplementation to Culture Medium Improves β-cell Survival

Objectives. Recent studies demonstrated that prolactin (PRL) has beneficial effects on &bgr; cells for islet transplantation. We examined the effect of human recombinant PRL (rhPRL) supplementation to the culture media to determine its potential use in the context of clinical islet transplantation. Materials and Methods. Each human islet isolated from 14 deceased multiorgan donors was cultured in Miami modified media-1 supplemented with or without rhPRL (500 &mgr;g/L) for 48 hr. &bgr;-Cell survival and proliferation (BrdU and Ki-67) were determined by laser scanning cytometry. The cytoprotective effects of rhPRL against noxious stimuli were assessed by flow cytometry (tetramethylrhodamine ethyl ester). Cytokine/chemokine and tissue factor productions were measured in vitro, and islet potency was assessed in vivo in diabetic immunodeficient mice. Results. &bgr;-Cell survival during culture was 37% higher in the rhPRL group than in control (P=0.029). rhPRL protected &bgr; cells in vitro from cytokines, Nitric oxide donor, and H2O2. The exposure to rhPRL did not affect human &bgr;-cell proliferation with our protocol. rhPRL treatment did not alter cytokine/chemokine and tissue factor production in vitro or affected human islet functionality in vivo: recipient mice achieved normoglycemia with a comparable tempo, whereas loss of graft function was observed in two of the seven mice in the control group and in none of the rhPRL group (p=n.s.). Conclusion. rhPRL supplementation to islet culture media improved human &bgr;-cell-specific survival without altering islet quality. Addition of rhPRL to cultured islets may grant a more viable &bgr;-cell mass in culture. The development of &bgr;-cell cytoprotective strategies will be of assistance in improving islet transplantation outcomes.

Diagnosis and treatment of pediatric patients with late-onset portal vein stenosis after living donor liver transplantation.

Portal vein stenosis (PVS) after living donor liver transplantation (LDLT) is a serious complication that can lead to graft failure. Few studies of the diagnosis and treatment of late‐onset (≥3 months after liver transplantation) PVS have been reported. One hundred thirty‐three pediatric (median age 7.6 years, range 1.3–26.8 years) LDLT recipients were studied. The patients were followed by Doppler ultrasound (every 3 months) and multidetector helical computed tomography (once a year). Twelve patients were diagnosed with late‐onset PVS 0.5–6.9 years after LDLT. All cases were successfully treated with balloon dilatation. Five cases required multiple treatments. Early diagnosis of late‐onset PVS and interventional radiology therapy treatment may prevent graft loss.

Generation of donor hematolymphoid cells after rat-limb composite grafting.

Background. Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. Methods. Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP+ donor onto the GFP− recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. Results. The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP+ skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP+ lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. Conclusions. The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.

Double-balloon enteroscopy for bilioenteric anastomotic stricture after pediatric living donor liver transplantation.

Bilioenteric anastomotic stricture after liver transplantation is still frequent and early detection and treatment is important. We established the management using double‐balloon enteroscopy (DBE) and evaluated the intractability for bilioenteric anastomotic stricture after pediatric living donor liver transplantation (LDLT). We underwent DBE at Jichi Medical University from May 2003 to July 2009 for 25 patients who developed bilioenteric anastomotic stricture after pediatric LDLT. The patients were divided into two types according to the degree of dilatation of the anastomotic sites before and after interventional radiology (IVR) using DBE. Type I is an anastomotic site macroscopically dilated to five times or more, and Type II is an anastomotic site dilated to less than five times. The rate of DBE reaching the bilioenteric anastomotic sites was 68.0% (17/25), and the success rate of IVR was 88.2% (15/17). There were three cases of Type I and 12 cases of Type II. Type II had a significantly longer cold ischemic time and higher recurrence rate than Type I (P = 0.005 and P = 0.006). In conclusion, DBE is a less invasive and safe treatment method that is capable of reaching the bilioenteric anastomotic site after pediatric LDLT and enables IVR to be performed on strictures, and its treatment outcomes are improving. Type II and long cold ischemic time are risk factors for intractable bilioenteric anastomotic stricture.

Living Donor Liver Transplantation for Neonates Using Segment 2 Monosubsegment Graft

The prognosis of liver transplantation for neonates with fulminant hepatic failure (FHF) continues to be extremely poor, especially in patients whose body weight is less than 3 kg. To address this problem, we have developed a safe living donor liver transplantation (LDLT) modality for neonates. We performed LDLTs with segment 2 monosubsegment (S2) grafts for three neonatal FHF. The recipient age and body weight at LDLT were 13–27 days, 2.59–2.84 kg, respectively. S2 or reduced S2 grafts (93–98 g) obtained from their fathers were implanted using temporary portacaval shunt. The recipient portal vein was reconstructed at a more distal site, such as the umbilical portion, to have the graft liver move freely during hepatic artery (HA) reconstruction. The recipient operation time and bleeding were 11 h 58 min–15 h 27 min and 200–395 mL, respectively. The graft‐to‐recipient weight ratio was 3.3–3.8% and primary abdominal wall closure was possible in all cases. Although hepatic artery thrombosis occurred in one case, all cases survived with normal growth. Emergency LDLT with S2 grafts weighing less than 100 g can save neonates with FHF whose body weight is less than 3 kg. This LDLT modality using S2 grafts could become a new option for neonates and very small infants requiring LT.

Living-Donor Liver Transplantation in 126 Patients with Biliary Atresia: Single-Center Experience

OBJECTIVES To describe our experience with 126 consecutive living-donor liver transplantation (LDLT) procedures performed because of biliary atresia and to evaluate the optimal timing of the operation. PATIENTS AND METHODS Between May 2001 and January 2010,126 patients with biliary atresia underwent 130 LDLT procedures. Mean (SD) patient age was 3.3 (4.2) years, and body weight was 13.8 (10.7) kg. Donors included 64 fathers, 63 mothers, and 3 other individuals. The left lateral segment was the most commonly used graft (75%). Patients were divided into 3 groups according to body weight: group 1, less than 8 kg (n = 40); group 2,8 to 20 kg (n = 63); and group 3, more than 20 kg (n = 23). Medical records were reviewed retrospectively. Follow up was 4.5 (2.7) years. RESULTS All group 3 donors underwent left lobectomy, and all group 1 donors underwent left lateral segmentectomy. No donors required a second operation or died. Comparison of the 3 groups demonstrated that recipient Pediatric End-Stage Liver Disease score in group 1 was highest, operative blood loss in group 2 was lowest (78 mL/kg), and operative time in group 3 was longest (1201 minutes). Hepatic artery complications occurred more frequently in group 1 (17.9%), and biliary stenosis (43.5%) and gastrointestinal perforation (8.7%) occurred more frequently in group 3. The overall patient survival rates at 1, 5, and 9 years was 98%, 97%, and 97%, respectively. Five-year patient survival rate in groups 1,2, and 3 were 92.5%, 100%, and 95.7%, respectively. Gastrointestinal perforation (n = 2) was the primary cause of death. CONCLUSIONS Living-donor liver transplantation is an effective treatment of biliary atresia, with good long-term outcome. It seems that the most suitable time to perform LDLT to treat biliary atresia is when the patient weighs 8 to 20 kg.

Living donor liver transplantation for ornithine transcarbamylase deficiency

Wakiya T, Sanada Y, Mizuta K, Umehara M, Urahasi T, Egami S, Hishikawa S, Fujiwara T, Sakuma Y, Hyodo M, Murayama K, Hakamada K, Yasuda Y, Kawarasaki H. Living donor liver transplantation for ornithine transcarbamylase deficiency.
Pediatr Transplantation 2011: 15: 390–395.