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Long-Term Allogeneic Islet Graft Survival in Prevascularized Subcutaneous Sites Without Immunosuppressive Treatment

2014; Elsevier BV; Volume: 14; Issue: 7 Linguagem: Inglês

10.1111/ajt.12739

1600-6143

Nguyen Minh Luan, Hiroo Iwata,

Diabetes Management and Research

American Journal of TransplantationVolume 14, Issue 7 p. 1533-1542 Original ArticleFree Access Long-Term Allogeneic Islet Graft Survival in Prevascularized Subcutaneous Sites Without Immunosuppressive Treatment N. M. Luan, N. M. Luan Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanSearch for more papers by this authorH. Iwata, Corresponding Author H. Iwata Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanCorresponding author: Hiroo Iwata, iwata@frontier.kyoto-u.ac.jpSearch for more papers by this author N. M. Luan, N. M. Luan Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanSearch for more papers by this authorH. Iwata, Corresponding Author H. Iwata Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanCorresponding author: Hiroo Iwata, iwata@frontier.kyoto-u.ac.jpSearch for more papers by this author First published: 06 June 2014 https://doi.org/10.1111/ajt.12739Citations: 49AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Establishment of noninvasive and efficient islet transplantation site together with the avoidance of immunosuppressive drugs for islet engraftment is currently the two major tasks for islet transplantation approach to treat patients with type 1 diabetes. Here, we proposed a method to achieve long-term allogeneic islet graft function without immunosuppression after transplantation in subcutaneous sites. Two agarose rods with basic fibroblast growth factor and heparin were implanted for 1 week in dorsal subcutaneous sites in diabetic rats. After rod removal, 1500 islets were transplanted into the prevascularized pockets. Islets transplanted in prevascularized but not nontreated subcutaneous sites rapidly reverted hyperglycemia in all streptozotocin-induced diabetic rats. In contrast to transient normalization of blood glucose when allogeneic islets were transplanted into liver, allogeneic islets transplanted into this prevascularized subcutaneous site demonstrated long-term graft survival and function in all three rat strain combinations (Fisher 344 to ACI, Lewis to ACI and Fisher 344 to Wistar), evidenced by nonfasting blood glucose level, plasma insulin concentration, intraperitoneal glucose tolerance test and immunohistochemistry. These results indicated that a subcutaneous site prevascularized by this method is potentially a suitable site for successful allogeneic islet transplantation without immunosuppression. Abbreviations bFGF basic fibroblast growth factor ELISA enzyme-linked immunosorbent assay FITC fluorescein isothiocyanate H&E hematoxylin and eosin IDDM insulin-dependent diabetes mellitus IPGTT intraperitoneal glucose tolerance test islets islets of Langerhans PBS phosphate buffered saline STZ streptozotocin Introduction Transplantation of islets of Langerhans (islets) has been used to treat patients with insulin-dependent diabetes mellitus (IDDM) 1-3. However, the number of patients treated by islet transplantation is limited due to a shortage of pancreas donors. In recent years, insulin-releasing cells and quasi-islets have been derived from embryonic stem/induced pluripotent stem cells and other adult stem cells, and long-term normoglycemia has been achieved in animal models of diabetes by transplantation of these cells 4-7. These promising results indicate that the shortage of pancreas donors can be overcome. However, there are obstacles to cell transplantation becoming a prevailing method for treating IDDM patients. Cells should be transplanted into a patient using a minimally invasive procedure and the grafted cells should be maintained without the administration of immunosuppressive drugs. Different sites, including the peritoneal cavity, portal vein of the liver, subcapsular space of the kidney and subcutaneous sites, have been examined 8, 9. Subcutaneous sites are the most attractive transplant sites due to easy transplantation of islets and easy removal of the grafts using minimally invasive procedures under local anesthesia. Various islet modifications, such as encapsulation of islets in a semipermeable membrane (bioartificial pancreas) or culturing under low temperature and ultraviolet irradiation, have been performed 10-14. The modifications have innate disadvantages: the large volume of the graft for bioartificial pancreas and islet deterioration for the latter two treatments. In this study, we propose a method to achieve long-term allogeneic islet graft survival in prevascularized subcutaneous sites of rats with streptozotocin (STZ)-induced diabetes without immunosuppressive treatment. Materials and Methods Animals Eight-week-old male Fisher 344 (F344, RT-1lv1) and Lewis rats (RT-1l) were used as donors. Eight-week-old male ACI/NSIc rats (RT-1a) and mongrel Wistar rats were used as recipients. All animals were obtained from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). All animal experiments were carried out according to the guidelines of The Kyoto University Animal Care Committee. Preparation of basic fibroblast growth factor device A rod-shaped agarose scaffold (4 mm diameter, 2.5 cm length) incorporating basic fibroblast growth factor (bFGF) and heparin was prepared as follows. A 4.5% agarose solution was prepared by mixing 450 mg agarose (Seakem GTG agarose; Cambrex Bio Science Rockland, Inc., Rockland, ME) in 10-mL double-distilled water and autoclaved to dissolve and sterilize the solution. The agarose solution was collected in a tube with an inner diameter of 4 mm and kept on ice to induce gelation. The agarose gel was then cut into rods 2.5 cm in length, frozen at −30°C overnight and freeze-dried for 24 h under reduced pressure. The bFGF solution (20–200 µL of 500 µg/mL solution; Kaken Pharmaceutical Co., Tokyo, Japan) was uniformly dropped onto the freeze-dried agarose rod and allowed to absorb for 2 min. Then, 100 µL of heparin solution (250 µg/mL) was added drop-wise. The agarose gel rod with bFGF and heparin was stored at 4°C overnight before use. Neovascularization of subcutaneous tissue An agarose rod with bFGF–heparin was implanted into each of the two dorsal subcutaneous sites in the diabetic rat to induce neovascularization. In nondiabetic rats, agarose rods with bFGF–heparin (5 and 25 µg/rod, respectively) effectively induce blood capillaries in subcutaneous sites (data not shown). However, in this study, recipients were in severe diabetic state and this decreased the effect of bFGF to induce neovascularization in subcutaneous sites. Rods with 10 or 50 µg bFGF and 25 µg heparin per rod were examined to induce similar degree of vascularity in subcutaneous sites. The rods were removed 1 week after implantation. The granular tissue contacting the device was dissected for histological analysis. To estimate the level of neovascularization, the amount of hemoglobin in the subcutaneous tissue was determined as described previously 15. Briefly, the dissected tissue was weighed, minced and incubated in 10 mL of Gay's solution (0.75% NH4Cl in 17 mM Tris–HCl buffer solution, pH 7.6) for 48 h at 4°C with periodic, gentle agitation to extract the hemoglobin. Hemoglobin concentrations were determined using the hemoglobin assay kit (Wako Pure Chemicals Co., Kyoto, Japan). Islet isolation Islets were isolated from donor rats using the collagenase digestion method as described previously 16-18. Islets were purified from digested tissues by centrifugation using a discontinuous density gradient of Ficoll/Conray solutions. Islets were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin for 2 days before transplantation. Islet transplantation Recipient ACI rats and Wistar rats were rendered diabetic by a single intraperitoneal injection of STZ (Nacalai Tesque, Kyoto, Japan) at a dose of 60 mg/kg body weight in citrate buffer (pH 4.5). Rats with blood glucose levels exceeding 400 mg/dL for 2 consecutive days were used as diabetic recipients. STZ-ACI rats were divided into four groups. In the prevascularization groups, agarose rods with bFGF–heparin (50 and 25 µg/rod, respectively) were implanted into the left and right dorsal subcutaneous sites of STZ rats. Upon removal of the rods 1 week later, 1500 F344 islets or 1500 Lewis islets were transplanted into the two prevascularized dorsal pockets. The wounds were closed by suturing. In the control groups, 3000 F344 islets were transplanted into the liver through the portal vein or untreated dorsal subcutaneous sites. In the other donor–recipient combination, 1500 F344 islets were transplanted into each of the two prevascularized dorsal pockets of STZ-Wistar rats. No immunosuppressive drugs were used in any recipients during the experiments. Blood samples were collected from the tail veins of the recipients between 11:00 a.m. and 1:00 p.m. every 2–3 days for the initial 2 weeks and once a week thereafter to determine nonfasting blood glucose levels. Glucose levels were determined using a glucose sensor (DIAmeter-a glucocard; Arkray, Kyoto, Japan). Graft rejection was defined as two consecutive blood glucose measurements exceeding 300 mg/dL. Plasma insulin analysis Between 40 and 90 days after islet transplantation, blood was collected from the tail veins of recipients to determine plasma insulin levels. Blood samples from normal and untreated diabetic rats were used as controls. All blood samples were kept on ice, then centrifuged at 600g at 4°C for 15 min. Plasma was collected and stored at −30°C. The plasma insulin concentration was determined by enzyme-linked immunosorbent assay (ELISA) (Shibayagi, Gunma, Japan). Intraperitoneal glucose tolerance test One to three months after islet transplantation, recipient rats were subjected to an intraperitoneal glucose tolerance test (IPGTT) to evaluate islet graft function 19. Normal nondiabetic rats were used as controls. After 14 h of fasting, rats received a glucose solution intraperitoneally (2 g glucose/kg body weight). Blood glucose levels were determined 0, 15, 30, 60, 90 and 120 min after glucose loading using a glucose sensor. Effect of transplantation of donor-specific islets or splenocytes on an accepted islet graft STZ-ACI recipients with long-term functional F344 islet grafts (<150 mg/dL) were subjected to intrahepatic transplantation of 1500 (n = 1) or 3000 F344 islets (n = 3) or intraperitoneal injection of 107 F344 splenocytes (n = 6). The islets and splenocytes were both isolated from donor-specific F344 rats. Before receiving islets or splenocytes, the left dorsal graft was retrieved and subjected to histological examination. The blood glucose levels of all recipients were determined every 2 days after the second islet transplantation or splenocyte injection. The liver or subcutaneous tissues including the islet grafts were recovered for histological examination 4 and 14 days after receiving islets or splenocytes. Histological study Recovered tissues containing islet grafts were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and thin tissue sections (4 µm in thickness) prepared following the conventional procedure. Hematoxylin and eosin (H&E) staining and immunofluorescence staining for insulin, CD4 T cells, CD8 T cells, macrophages and granulocytes were performed 19. For immune cell staining, spleen tissues and samples without primary antibodies were used as positive controls and negative controls, respectively. Antibodies against CD4 T cells (rabbit polyclonal; Novus Biologicals, Littleton, CO), CD8 T cells (Mouse monoclonal, OX-8; Novus Biologicals), macrophages (CD68) (Mouse monoclonal; ED1, Novus Biologicals) and granulocytes (Mouse monoclonal; HIS48, Novus Biologicals) were purchased. Protease-induced antigen recovery was performed using 0.1% trypsin solution in PBS. The thin tissue sections were treated with Blocking One solution (Nacalai Tesque) for 1 h to block nonspecific adsorption of antibodies, followed by incubation of a primary antibodies against CD4 T cells (1:50 in Blocking One solution), against CD8 T cells (1:50 in Blocking One solution), against macrophages (1:50 in Blocking One solution) and granulocytes (1:50 in Blocking One solution) at 4°C overnight. After washing with PBS containing 0.05% Tween-20, slides were treated by Alexa Fluor 594 anti-rabbit IgG antibodies or Alexa Fluor 488 anti-mouse IgG antibodies (1:200 in Blocking One solution; Invitrogen, Life Technologies, Carlsbad, CA) for 1 h at room temperature and washed with PBS containing 0.05% Tween-20. Nucleus was counterstained with Hoechst 33258 (Dojindo, Kumamoto, Japan). Intravital perfusion of fluorescein isothiocyanate-lectin for examination of functional vasculature ACI rats prevascularized for 1 week or ACI recipients of F344 islets at Day 30 after translantation were intravenously injected with 1 mL of saline solution containing 200 µg fluorescein isothiocyanate (FITC)-lectin (Vector Labs, Burlingame, CA) and 1000 IU heparin. After 15 min circulation, subcutaneous tissue and pancreas were rapidly dissected and briefly washed with saline solution twice, fixed and snap-frozen in liquid nitrogen. Thin tissue sections (4 µm in thickness) were prepared and immunofluorescence stainings for insulin were performed following the conventional procedure. Statistical analysis Two groups were compared using the Student's t-test. p < 0.05 was considered significant. All statistical calculations were performed using JMP ver.5.1.1 (http://www.jmp.com/). Results Induction of blood capillaries in subcutaneous tissue by agarose rods with bFGF–heparin To induce blood capillaries in subcutaneous tissue, we used freeze-dried agarose rods as a carrier to deliver bFGF and heparin. The agarose rods with bFGF–heparin were implanted in the dorsal subcutaneous sites of diabetic rats for 1 week. Untreated subcutaneous tissues were used as controls. The subcutaneous tissue of a normal rat is very poorly vascularized, as indicated by the macroscopic and microscopic images in Figure 1A-1, B-1 and B-2. When rats received agarose rods without bFGF–heparin, no apparent improvement in vascularization was observed (Figure 1A-2). In contrast, when rats received agarose with bFGF–heparin (50 and 25 µg/rod, respectively) for 1 week, the subcutaneous tissue was granulated and formed a thick vascularized pocket surrounding the agarose rod (Figure 1A-3, C-1 and C-2). The agarose rod was removed easily without tissue adhesion. Numerous blood vessels were observed in the vicinity of the implanted bFGF device (Figure 1C-2). We also performed intravital perfusion of FITC-lectin to examine the functional vasculature in prevascularized subcutaneous sites as shown in Figure 1D. Numerous lectin-positive vessels could be seen in prevascularized subcutaneous sites, suggesting the formation of dense functional vascularity in this site, which is expected to supply sufficient nutrients and oxygen for islet survival. Figure 1Open in figure viewerPowerPoint Induction of prevascularization in subcutaneous tissue by agarose rods with basic fibroblast growth factor (bFGF)–heparin. (A) Macroscopic images of subcutaneous tissue from untreated diabetic ACI rats (A-1) or 1 week after implantation with bare agarose rods (A-2) or agarose rods incorporated with 50 µg/rod bFGF and 25 µg/rod heparin (A-3). The illustration on the right depicts the position of the agarose rod in the subcutaneous space and the opening method. (B, C) Hematoxylin and eosin staining of untreated subcutaneous tissue (B-1, B-2) and tissue prevascularized with agarose rods with bFGF–heparin for 1 week (C-1, C-2). Scale bar: (B-1, C-1) 500 µm, (B-2, C-2) 50 µm. The dotted lines indicate the interface between the agarose rods and subcutaneous tissue. (D) Prevascularized subcutaneous site after intravital perfusion of fluorescein isothiocyanate-lectin. Functional vasculature is identified as green fluorescent-lectin-positive vessels. Scale bar 100 µm. (E) Hemoglobin content indicates the degree of vascularization in subcutaneous tissue from streptozotocin-ACI rats before and 1 week after the implantation of agarose rods incorporated with different amounts of bFGF (0–50 µg) and 25 µg heparin. Six replicates were examined for each group. The asterisk (*) indicates statistically significant difference (p < 0.01). Because blood vessels contain hemoglobin, the hemoglobin content of the subcutaneous tissue was used to semi-quantitatively compare the induction of blood capillaries 15. As shown in Figure 1E, the hemoglobin content of untreated subcutaneous tissue was 0.35 ± 0.55 mg/g tissue. The hemoglobin content of tissue is dependent on the amount of bFGF incorporated in the agarose rod. When 10 µg of bFGF was added to a rod, the hemoglobin content of the tissue was 3.30 ± 1.35 mg/g, which is not significantly different from the tissue not treated with bFGF. When bFGF increased to 50 µg/rod, the hemoglobin content of the tissue significantly increased to 9.40 ± 2.84 mg/g. Further increases in the amount of bFGF applied to a rod did not significantly increase the hemoglobin content (data not shown). Therefore, in all following experiments, agarose rods with bFGF–heparin (50 and 25 µg/rod, respectively) were used to induce blood capillaries in subcutaneous sites. Outcome of allogeneic islet transplantation in different experimental groups Normoglycemic periods in the recipients of islet grafts are summarized in Tables 1 and S1. No immune-suppressive therapy was given to any of the recipients. As a fully MHC-incompatible combination, F344 rats (RT-1lv1) were used as islet donors and ACI rats (RT-1a) were rendered diabetic by intraperitoneal injection of 60 mg/kg STZ. In prevascularization group, subcutaneous spaces with blood capillaries were prepared on both dorsal sides of diabetic rats by implanting agarose rods containing bFGF and heparin for 1 week. A total of 1500 islets were allogeneically transplanted into each space after removing the rods, resulting in 3000 islets transplanted in each recipient. Nine of ten STZ-ACI rats demonstrated stable normoglycemia for more than 100 days before additional treatments were performed, such as intraperitoneal splenocytes and intraportal islet transplantation (Table 1; Figure 2C). The rats' body weight continuously increased from 175.6 ± 13.2 to 297.2 ± 10.4 (Figure S2C). In contrast, STZ-ACI rats receiving 3000 F344 islets in the liver through the portal vein demonstrated 7–10 days of transient normalization of blood glucose levels (Figure 2A). The grafts were rejected as reported previously 20. Body weights of the recipient rats increased during the initial several days, but subsequently remained almost constant (Figure S2A). STZ-ACI rats that received islets in subcutaneous sites without prevascularization did not demonstrate normalized blood glucose levels (Figure 2B). The rats' body weight decreased during initial observations (Figure S2B). The graft might deteriorate due to insufficient oxygen or nutrient supply because subcutaneous tissue is poorly vascularized. Table 1 also includes the normoglycemic periods of other donor–recipient combinations, such as Lewis (RT-1l)-ACI (RT-1a) rats and F344-mongrel Wistar rats. Table 1. Normoglycemic periods in different experimental groups Donor–recipient Transplantation site Prevascularization Normoglycemic periods F344–ACI Subcutaneous With Subcutaneous Without Liver – Lewis–ACI Subcutaneous With >60†† Days for graft removal., >102†† Days for graft removal., >152†† Days for graft removal., >200†† Days for graft removal. F344–Wistar Subcutaneous With >130 (2)†† Days for graft removal. *Normoglycemic days before transplantation of F344 islets into liver. **Normoglycemic days before intraperitoneal injection of F344 splenocytes. ***Graft rejection days after intraportal F344 islet transplantation to recipients with functional grafts. † Days for graft removal. ‡Statistical significance (p < 0.01). Figure 2Open in figure viewerPowerPoint Outcome of allogeneic islet transplantation in diabetic rats in different experimental groups. Blood glucose levels of streptozotocin (STZ)-ACI rats transplanted with 3000 F344 islets into livers (A), nontreated subcutaneous (B) and prevascularized subcutaneous spaces (C). Double-slash (//) indicates recipients subjected to additional experiments to give some insight into the tolerance state (see Figure 4). (D) Blood glucose levels of STZ-ACI rats transplanted with 3000 Lewis islets in prevascularized subcutaneous tissue. Arrows indicate total graft recovery. (E) Intraperitoneal glucose tolerance test on normal ACI rats (triangle, n = 3) and STZ-ACI rats transplanted with 3000 F344 islets (square, n = 3) or 3000 Lewis islets (circle, n = 3). In all recipients, intraperitoneal glucose tolerance test was done between 1 and 3 months after transplantation. (F) Plasma insulin levels in nondiabetic ACI rats (I), diabetic ACI rats (II) and STZ-ACI rats transplanted with 3000 Lewis islets (III) or 3000 F344 islets (IV) into prevascularized subcutaneous spaces. Three recipients of allogeneic islets in each group were examined between 1 and 3 months after transplantation. The transplantation of 3000 Lewis islets into the prevascularized subcutaneous sites of STZ-ACI rats resulted in normalized blood glucose levels within 1–3 days (Figure 2D). When the islet grafts at both dorsal sites were removed 60, 102, 152 and 200 days after transplantation, the blood glucose levels promptly reverted to the high preoperative levels. The body weights of the recipients concomitantly decreased with increasing blood glucose levels (Figure S2D). Figure S1B shows the blood glucose levels of two STZ-Wistar rats carrying 3000 F344 islets in two prevascularized subcutaneous pockets. F344 islets transplanted into prevascularized subcutaneous sites of STZ-Wistar rats normalized blood glucose levels within 3 days. Normoglycemia in both rats were stably maintained for more than 130 days follow-up. These results indicate that the allogeneic islet grafts were accepted in prevascularized subcutaneous and normalized blood glucose levels for long periods without any immunosuppressive treatment in the three donor–recipient combinations we studied. Assessment for graft function IPGTTs were performed for normal ACI rats and STZ-ACI rats with F344 islets or Lewis islets 1–3 months after transplantation to evaluate islet graft function. The results are provided in Figure 2E. Areas under the curve were 19 867 ± 1897, 22 835 ± 2023 and 20 180 ± 1286 mg min/dL for normal ACT rats, STZ-ACI rats with 3000 F344 islets and STZ-ACI rats with 3000 Lewis islets, respectively. Plasma insulin levels of STZ-ACI rats with F344 islets or Lewis islets in prevascularized subcutaneous sites were determined between 40 and 90 days after islet transplantation (1.56 ± 0.23 and 1.733 ± 0.140 ng/mL, respectively) (Figure 2F). The plasma insulin levels of normal and diabetic ACI rats were 1.39 ± 0.32 and 0.31 ± 0.06 ng/mL, respectively. The plasma insulin levels of the recipients were well maintained by the islet grafts. Histological analysis of long-term subcutaneous islet allografts All recipients in prevascularization group carried two grafts, one each on the left and right dorsal sides. The left subcutaneous islet graft of some recipients was retrieved at 91–130 days (Table S1) for histological examination (Figure 3). Normoglycemia was maintained, even after the removal of one graft. Tissue sections were stained with H&E and Alexa488-labeled anti-insulin antibodies. In all three donor–recipient combination group, islets were clearly seen in the H&E slices, and insulin-positive cells were clearly observed in the immunofluorescently stained slices. Many small blood vessels were observed in the islet grafts, but few lymphocytes had infiltrated (Figure S3). We also demonstrated functional assessment of vessels in subcutaneous islet grafts by intravital perfusion approach using FITC-lectin. As shown in Figure 3D, functional vessels could be clearly detected within islet grafts, as indicated by FITC-lectin stained cells surrounded by insulin cells. Figure 3E includes the staining of normal pancreas tissue. Islet morphology and vascularity in these two figures are similar to each other. These data suggested that allo-islets transplanted in prevascularized subcutaneous sites could establish networks of functional vessels in the islet grafts connected to the host vasculature. Figure 3Open in figure viewerPowerPoint Engraftment and revascularization of allogeneic islets transplanted in prevascularized subcutaneous sites. Hematoxylin and eosin (H&E) staining and immunofluorescent staining for insulin and nucleus of a (A) F344-to-ACI islet graft, (B) Lewis-to-ACI islet graft and (C) F344-to-Wistar islet graft recovered at 94, 102 and 130 days, respectively, after allotransplantation into prevascularized subcutaneous spaces. Scale bar: 100 µm (left and right columns), 50 µm (middle column). Intravital perfusion of fluorescein isothiocyanate-lectin for examination of functional vasculature in (D) islet grafts in subcutaneous sites at Day 30 after allotransplantation and (E) native islets in normal ACI pancreas. Scale bar: 50 µm. Transplantation of donor-specific islets or splenocytes causes rejection of long-term functioning islet graft To give some insight into the tolerance state, 3000 F344 islets were infused into the liver of STZ-ACI recipients with functional F344 islet grafts after more than 100 days of normoglycemia. Blood glucose levels increased over 350 mg/dL from 7 to 10 days after islet infusion (n = 2, Figure 4A). The islet grafts in both the liver and the subcutaneous site lost their function. Many lymphocytes infiltrated the surrounding islets in the liver and subcutaneous tissues retrieved 14 days after the islet infusion, and the islets severely deteriorated (Figure 4C-1 and C-2). To confirm this finding, the ACI recipients with functional F344 islet grafts were systematically sensitized by intraperitoneal injection of donor-specific F344 splenocytes. Blood glucose levels increased over 350 mg/dL, 4–7 days after F344 splenocyte injection (n = 4, Figure 4B). Many lymphocytes infiltrated the surrounding islets and the number of insulin-positive cells decreased in the tissue retrieved 4 days after splenocyte injection (Figure 4D-1 and D-2). These results indicate that immune reactions caused by donor-specific cells played a vital role in the destruction of accepted islet grafts. Figure 4Open in figure viewerPowerPoint Rejection of established subcutaneous islet allogeneic islet grafts with the transplantation of donor-specific cells. All recipients carried two grafts on the left and right dorsal sides. The left subcutaneous islet graft of recipients (n = 7) was retrieved at 91–109 days. Blood glucose levels indicated the maintenance of normoglycemia, even after one graft was removed. (A) Blood glucose levels of diabetic ACI rats with long-term functional subcutaneous F344 islet grafts after transplantation of 3000 F344 islets into the liver (n = 2). Two arrows indicate the day of F344 islet transplantation into livers. (B) Blood glucose levels of diabetic ACI rats with long-term functional subcutaneous F344 islet grafts after transplantation of 107 F344 splenocytes into the intraperitoneal cavity (n = 4). Recipients carried one-side graft (square and diamond) and carried both-side grafts (triangle and cross). Graft rejection was defined as two consecutive blood glucose measurements exceeding 300 mg/dL. Arrows indicate the day of F344 splenocyte transplantation. (C) Hematoxylin and eosin (H&E) staining of subcutaneous tissue (C-1) and liver (C-2) containing allogeneic islet grafts recovered 14 days after intraportal transplantation of F344 islets. Scale bar: (C-1) 100 µm, (C-2) 50 µm. (D) H&E staining (D-1) and immunofluorescent staining (D-2) for insulin and nucleus of subcutaneous site containing islet allograft recovered 4 days after intraperitoneal injection of F344 splenocytes. Scale bar: 50 µm. Discussion Subcutaneous islet transplantation has been considered an attractive islet transplantation method. Islets could be transplanted into a patient using a minimally invasive procedure, and the islet grafts would be easy to remove in the event of complications. However, there are two major obstacles to be overcome. The first is that the subcutaneous site is too poorly vascularized to provide sufficient oxygen and nutrients for islet survival and function 21-23. The second is the possible rejection of the islet graft by the host immune system. Many trials have involved subcutaneous islet transplantation. We found 152 articles in PubMed using the keywords islet, transplantation and subcutaneous. Several studies proposed methods to solve the former obstacle. Neovascularization of the subcutaneous site might enhance oxygen supply, and thus is expected to improve islet engraftment and function 24-31. For example, in our previous work, we preconditioned subcutaneous sites with bFGF incorporated in polyethylene terephthalate mesh bag device c