Long-Term Graft Function of Adult Rat and Human Islets Encapsulated in Novel Alginate-Based Microcapsules After Transplantation in Immunocom
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糖尿病学杂志 2005年第3期
1 Division of Endocrinology and Metabolism, Medical Department I, University of Mainz, Germany
2 Department of Biotechnology, Biozentrum, University of Wezburg, Germany
3 Department of Cryobiophysics and Cryotechnology, Fraunhofer Institute for Biomedical Technique, St. Ingbert, Germany
ABSTRACT
We describe the results of the first study to show that adult rat and human islets can be protected against xenogenic rejection in immunocompetent diabetic mice by encapsulating them in a novel alginate-based microcapsule system with no additional permselective membrane. Nonencapsulated islets lost function within 4eC8 days after being transplanted into diabetic Balb/c mice, whereas transplanted encapsulated adult rat or human islets resulted in normoglycemia for >7 months. When rat islet grafts were removed 10 and 36 weeks after transplantation, the mice became immediately hyperglycemic, thus demonstrating the efficacy of the encapsulated islets. The explanted capsules showed only a mild cellular reaction on their surface and a viability of >85%, and responded to a glucose stimulus with a 10-fold increase in insulin secretion. Furthermore, transplanted mice showed a slight decrease in the glucose clearance rate in response to intraperitoneal glucose tolerance tests 3eC16 weeks after transplantation; after 16 weeks, the rate remained stable. Similar results were obtained for encapsulated human islets. Thus we provide the first evidence of successful transplantation of microencapsulated human islets. In conclusion, we have developed a novel microcapsule system that enables survival and function of adult rat and human islets in immunocompetent mice without immunosuppression for >7 months.
As has recently been shown (1), long-term normoglycemia can be achieved in type 1 diabetic patients by allogenic human islet transplantation. However, this technique requires the administration of immunosuppressives, which are well known to be associated with serious side effects. The severe shortage of available human islets is another drawback of this promising therapeutic approach. A bioartificial pancreas consisting of islets immunoprotected by an alginate microcapsule provides an alternative approach to overcoming the rejection problem (2eC4). Such immunoprotection is also appealing because it provides improved prospects of obtaining an abundant supply of human islets from ductal tissue (5) and the possibility of using xenogenic islets (e.g., porcine islets) (6). In most studies, islets have been encased in Ca2+eCcross-linked alginate-polylysine-alginate microcapsules following the protocol of Lim and Sun (7). However, the results obtained by various groups using this microcapsule formulation in the last two decades have been inconsistent. Most importantly, graft survival is random and generally too limited for therapeutic application of these microcapsules (8eC10). The reasons for this are presumably the cytotoxicity of the polyamino acid and the mechanical instability of the microcapsules (2,11,12). Microcapsules made of alginate cross-linked with Ba2+do not share these disadvantages (13). Duvivier-Kali et al. (14) have reported that these microcapsules protect islets against allorejection and autoimmunity. Furthermore, Omer et al. (15) found that Ba2+eCcross-linked alginate microcapsules protect neonatal pancreatic cell clusters (NPCCs) when transplanted in the streptozotocin-induced diabetic Balb/c mouse model for 20 weeks. We have recently shown that using highly purified and ultraeChigh-iscosity alginates when cross-linked internally and externally with Ba (2) results in microcapsules of extremely high stability, particularly when proteins (e.g., human serum albumin [HSA]) are simultaneously incorporated (16,17). A recent study has shown that rat islets encapsulated in these novel microcapsules exhibit a well-preserved insulin secretion in tissue culture over 3 weeks (16).
Here we demonstrate that adult rat as well as adult human islets encapsulated in these novel microcapsules and transplanted into diabetic mice are protected against xenogenic rejection for extremely long periods of time without the need for immunosuppression.
RESEARCH DESIGN AND METHODS
Isolation of rat islets.
Isolation of rat islets was performed according to the protocol previously described (17,18). Briefly, SD rats (Central Animal Facility, University of Mainz) age 8 weeks and weighing 280eC330 g were used as islet donors. Rats were anesthetized by pentobarbital administration (60 mg/kg, i.p.). A midline abdominal incision was made and the pancreas was exposed and injected via the pancreatic duct with Hank’s balanced salt solution (HBSS; Gibco BRL, Long Island, NY) containing 1.7 mg/ml collagenase (Serva PanPlus, Heidelberg, Germany). After the animal was killed, the pancreatic tissue was surgically removed and incubated for 10 min at 37°C in the collagenase solution. Mechanical disruption of the digested pancreatic tissue was achieved by incubating the tissue in the collagenase solution at 37°C for 10 min, with the incubation being interrupted every 2 min by shaking for 30 s. Digestion was stopped by adding cold (4°C) HBSS plus 10% FCS (Greiner, Frickenhausen, Germany). Islet purification was achieved using a discontinuous three-phase Ficoll density gradient (densities: 1.090, 1.077, and 1.040).
Isolation of human islets.
Human pancreases were obtained from brain-dead cadaveric multiorgan donors with the informed consent of their relatives. Tissue procurements were approved by the Human Ethics Committee of the Landesrztekammer Rheinland-Pfalz. Human islets were isolated from two consecutively processed human pancreases after a vascular flush with University of Wisconsin (UW) solution. Briefly, pancreases were intraductally distended with 250 ml HBSS containing Liberase HI (Roche, Mannheim, Germany). Distended pancreases were digested in a continuous digestion-filtration device at 37°C. After a subsequent 60-min cold storage of the digest in UW solution, human islets were purified using a continuous hyperosmolar Hank’s-Ficoll gradient (Biochrom KG, Berlin, Germany) on a Cobe 2991.
Culture conditions.
Islets were cultured in RPMI 1640 medium (Biochrom KG) supplemented with 100 mg/dl glucose, 25 mmol/l HEPES (Greiner), 0.2 g/l Glutamax (Gibco BRL, Paisley, Scotland), 10% FCS (Greiner), and antibiotics (100 units/ml penicillin, 100 e/ml streptomycin [Gibco BRL, Paisley, Scotland], and 10 e/ml ciprofloxacin [Ciprobay; Aventis, Frankfurt, Germany]). Incubation lasted 12 h at 37°C. Islets were then transferred into a slightly modified culture medium containing 3% HSA (Pharmacia, Erlangen, Germany) instead of FCS and an elevated concentration of glucose (300 mg/dl). After being cultured for 12 h at 37°C, the islets were used for encapsulation or transplantation.
Islet encapsulation.
Microencapsulation was performed using ultra-higheCviscosity alginate of clinical grade (viscosity of a 0.1% wt/vol solution in distilled water was 20eC30 mPa · s). The extraction and purification protocol has been described elsewhere (19,20). For microcapsule formation, the alginate was dissolved in sterile, endotoxin-free 0.9% NaCl solution at a concentration of 0.7% (wt/vol). Then 3% HSA (Pharmacia) was added to the alginate solution before it was cross-linked. For microcapsule formation, a two-channel air jet droplet generator was used. The islet-containing microcapsules were dropped into a 20-mmol/l BaCl2 solution adjusted to 290 mOsm with appropriate amounts of NaCl and buffered to pH 7.0 with 5 mmol/l histidine. After 20 min, the islet-containing microcapsules were washed three times with 0.9% NaCl solution and treated with 6 mmol/l Na2SO4 saline solution for 30 min at 37°C to precipitate excess Ba2+ ions (20). The microcapsules obtained by this encapsulation procedure had a diameter of 700eC800 e and on average contained one islet or, in rare cases, two or three.
Animals.
Male Balb/c mice (Central Animal Facility) age 6eC8 weeks were used as recipients for nonencapsulated (controls) or encapsulated islets. Mice were rendered diabetic with intraperitoneal injections of streptozotocin (250 mg/kg body wt; Sigma, St. Louis, MO) freshly dissolved in citrate buffer 2 weeks before islet transplantation. Only animals exhibiting blood glucose concentrations >350 mg/dl in four consecutive measurements were used as recipients.
Transplantation of microencapsulated islets.
Transplants were made in a Class 100 biological safety cabinet under sterile conditions. Encapsulated rat (n = 8) or human (n = 3) islets were injected into the peritoneal cavity with a 20-gauge needle (Vasofix; Braun, Melsungen, Germany). Before treatment, the Balb/c mice were anesthetized with ketamine (65 mg/kg i.p.; Pfizer, Karlsruhe, Germany) and xylazine hydrochloride (13 mg/kg i.p.; Bayer, Leverkusen, Germany). Each mouse received 1,800 microencapsulated islets. Control animals (n = 4) received the same number of nonencapsulated rat or human islets, respectively.
Intraperitoneal glucose tolerance test.
After islet transplantation, animals’ body weight was determined once a week and blood glucose levels of islet recipients were monitored biweekly. Blood samples were taken from the tail vein under nonfasting conditions (8:00 A.M.; standard laboratory diet ad libitum overnight) and determined using a glucometer (Accutrend sensor, Roche Diagnostics, Germany). The mice were considered to be normoglycemic when blood glucose levels were <200 mg/dl. An intraperitoneal glucose tolerance test (IPGTT) was performed in mice transplanted with encapsulated rat islets 3 (n = 8), 9 (n = 7), 16 (n = 5), and 36 (n = 2) weeks after transplantation, using a 5% glucose solution (1 g/kg body wt). In mice transplanted with encapsulated human islets, an IPGTT was carried out at the same times (n = 2 at 3, 9, and 16 weeks; n = 1 at 36 weeks). IPGTTs were also performed in all diabetic animals 3 days before islet transplantation and in body weighteCmatched nondiabetic Balb/c mice. Blood glucose levels were measured at 0, 10, 20, 30, 60, and 120 min.
Microcapsule explantation.
Grafts were retrieved from two animals that had received encapsulated adult rat islets 10 (n = 1) and 36 weeks (n = 1) after islet transplantation. Under anesthesia, a 2-cm incision was made in the abdomen and capsules were explanted by repeated flushes with warm 0.9% NaCl solution. The abdomen was sutured and the animals were checked daily for blood glucose concentrations. Explanted microcapsules were evaluated for fibrotic overgrowth under a Wilowert S microscope (Hund GMBH, Wetzlar, Germany), as previously described (21). Images were captured with a digital camera and stored as jpg files. The viability of the encapsulated islets was assessed using the fluorescein diacetate method, as described elsewhere (16,22).
Assessment of insulin secretion by a static incubation assay.
The static incubation assay was used to assess the insulin secretion response of nonencapsulated and encapsulated rat or human islets after a glucose challenge both before transplantation and after retrieval, as previously described (16,17). For each animal, 10 samples of 10 nonencapsulated or encapsulated islets of equal size and shape were hand picked and transferred into a culture insert (membrane pore diameter 12 e; Millicell PCF, Millipore, France). The insert was put into a well of a 24-well culture-plate (Falcon Multiwell; Becton, Dickinson). Basal insulin secretion was measured after a 1.5-h incubation at 37°C in RPMI 1640 + 1 g/l D-glucose + 3% HSA. The inserts with islets were transferred into hyperglycemic culture medium (RPMI 1640 + 3 g/l D-glucose + 3% HSA) for an additional 1.5 h. Aliquots of the medium were stored at eC20°C after inserts were removed. Rat insulin was measured with the rat-insulin enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden) and human insulin was determined with a chemiluminiscent immunoassay (ADVIA Centaur insulin assay; Bayer). Results are expressed as nanograms per islet per 1.5 h for rat islets and as microunits per islet per 1.5 h for human islets.
Statistics.
Data are given as means ± SE. Statistical significance of differences was calculated with an unpaired Student’s t test (two-sided).
RESULTS
Graft survival.
Before encapsulated islets were transplanted, their insulin secretion capacity after being cultured for 1 day was determined. As shown in Table 1, encapsulated rat or human islets responded well to a high-glucose stimulus. The insulin increase after a glucose challenge was comparable with that of the corresponding nonencapsulated islets, even though the absolute amount of insulin was lower, presumably due to diffusion restrictions within the cross-linked alginate matrix (16). Transplantation of 1,800 nonencapsulated rat islets into the peritoneal cavity of diabetic Balb/c mice resulted in normoglycemia for 4eC8 days. Similar results were obtained for nonencapsulated human islets (Table 2). In contrast, when encapsulated rat and human islets were transplanted under the same conditions, normoglycemia was achieved for >270 days (Fig. 1). As indicated in Table 2, part of the transplantation experiments are still going on. Loss of graft function was observed in only one case. This happened 69 days after transplantation of encapsulated rat islets and was due to a severe infection of the tail as a result of repeated blood collection. Normoglycemic blood glucose levels were generally reached within 24 h of transplantation; normoglycemia was not established in only one animal that had received encapsulated human islets. This was presumably due to a heavy infection at the abdominal wall induced by the transplantation procedure. Graft explantation from two animals 10 and 36 weeks after transplantation resulted in the immediate reappearance of hyperglycemia associated with blood glucose concentrations > 500 mg/dl. In addition, transplantation of microencapsulated rat or human islets led to an increase in body weight, as depicted in Fig. 2.
Intraperitoneal glucose tolerance test.
Figure 3 illustrates the blood glucose profiles of Balb/c mice transplanted with encapsulated rat islets during IPGTTs at different times (3, 9, 16, and 36 weeks) after transplantation. During the IPGTTs, the blood glucose concentration profiles were always significantly lower compared with those of diabetic controls (Figs. 3AeCD). As indicated in Fig. 3A, 3 weeks after receiving transplanted encapsulated rat islets, mice showed the same blood glucose concentration after overnight fasting (t = 0) as the nondiabetic controls. Interestingly, the subsequent glucose clearance kinetics were much faster and the final glucose value reached after 120 min was considerably lower than in nondiabetic mice (Fig. 3A). In contrast, 9 weeks after transplantation, blood glucose concentration and kinetics in rat isleteCtransplanted mice were similar to those observed of nondiabetic control mice (Fig. 3B). The fasting and peak glucose levels of the rat isleteCtransplanted mice during IPGTT 16 and 36 weeks after transplantation were similar to those of the controls, but their subsequent glucose clearance rate was significantly delayed and, in turn, their blood glucose levels remained higher than those of the nondiabetic controls at the end of the IPGTT (Figs. 3C and D).
Because of the limited number of mice transplanted with encapsulated human islets, the blood glucose kinetics during the IPGTT could not be determined as thoroughly as for encapsulated rat islets. However, as shown in Fig. 4, the blood glucose kinetics of these mice were apparently not significantly different from those of the nondiabetic control mice 3, 9, and 16 weeks after transplantation. Consistent with the finding for mice with encapsulated rat islets, 36 weeks after transplantation, glucose clearance in human isleteCtransplanted mice was delayed, but the blood glucose concentration at t = 0 (after fasting) and the glucose peak value were comparable with the corresponding values measured in nondiabetic controls.
Retrieved microcapsules.
The majority of rat isleteCcontaining microcapsules retrieved 10 or 36 weeks after transplantation exhibited only a mild cellular reaction on their external surface (Figs. 5C and F). As assessed by fluorescein diacetate staining, encapsulated rat islets retrieved 10 as well as 36 weeks after transplantation exhibited good viability (>85%) (Fig. 5A, B, D, and E). Consistent with the results of the IPGTTs, the retrieved encapsulated rat islets responded well to a glucose challenge. Their insulin secretion capacity, however, was slightly reduced compared with their initial insulin secretion capacity before transplantation (Table 1). The reduction in insulin secretion apparently occurred during the first two months of transplantation, as the insulin secretion capacity of encapsulated rat islets retrieved after 36 weeks was not significantly different from that of microcapsules retrieved after 10 weeks.
DISCUSSION
Over the past two decades, many different hydrogels and synthetic polymers have been proposed for immunoisolation and encapsulation of cells (23). Among these, alginate is one of the most promising natural polymers, as demonstrated by animal studies and small-scale clinical trials (24,25). One of the main requirements for long-term alginate-based transplantation is the stability of the microcapsules. This is achieved by cross-linking ultra-higheCviscosity alginate of clinical grade (i.e., free of mitogenic and cytotoxic ingredients as well as of gram-negative and gram-positive bacterial spores) (19,20) with Ba2+ and by simultaneously incorporating stabilizing agents such as proteins into the microcapsules (17). As shown elsewhere (16), HSA can preserve insulin secretion for up to 3 weeks in tissue culture when this microcapsule design is used for encapsulation of rat islets. In contrast to FCS, HSA meets the safety criteria of the U.S. Food and Drug Administration and the American Society for Testing and Materials (26). The animal studies reported here confirm the enormous clinical potential of HSA-stabilized alginate-based microcapsules. The encapsulated islets of adult rats or humans maintained insulin secretion for >7 months, implying the absence of xenogenic rejection and the stability of the microcapsules. This finding was also confirmed by studies of insulin secretion of transplants explanted after 10 and 36 weeks. Failure of one graft was obviously due to severe infection of the tail induced by the blood collection procedure. Similarly, the finding that normoglycemia in one mouse was not established after transplantation of encapsulated human islets can most likely be traced back to a heavy infection of the abdominal wall.
Although the number of experiments with human islet cells were limited due to the restricted availability of intact human pancreatic tissue, our human islet cell data are in good accordance with the results obtained with rat islet cells. As depicted in Figs. 3 and 4, the pattern of glucose clearance differed from that of the nondiabetic control mice and changed with time after transplantation. Glucose clearance was faster in rat isleteCtransplanted mice than in controls 3 weeks after transplantation. This was likely due to the different secretory properties of rat islets. It is well known that rat islets have a lower set point than mouse islets (27,28), as evidenced by the lower fed and fasted blood glucose levels measured in normal rats. In contrast, a decrease in glucose clearance was observed 3eC16 weeks after transplantation, as indicated by the considerably higher glucose values recorded 120 min after intraperitoneal glucose injection. It was interesting to observe that a further decrease in the glucose clearance rate with ongoing transplantation did not occur. A possible explanation for this finding is the restricted diffusion of nutrients and oxygen by an intense fibrotic overgrowth of the microcapsules during the first weeks of transplantation. However, although microcapsules retrieved 10 and 36 weeks after transplantation exhibited a reduced insulin secretion after glucose challenge, a strong fibrosis was not observed. The retrieved microcapsules showed only a weak cellular reaction close to the surface. -Cell injury by complement-dependent components can also be excluded as a possible cause for the reduction in the glucose clearance rate, as the microcapsules used here were protected against these cytotoxic components, as shown recently by in vitro studies (16). The most likely explanation for these findings is that the microcapsules float freely in the peritoneal cavity without any vascularization. The supply of the encapsulated islets with nutrients and oxygen is, therefore, rather limited as evidenced by 19F nuclear magnetic resonance imaging (29). Therefore, compromised nutrient and oxygen supply to the islets might lead to mass loss (8) and, in turn, a reduction in insulin secretion. This could also explain the fact that rather high numbers of encapsulated rat and human islets (1,800 islets) were needed to achieve a stable graft function in this study as compared with the lower numbers of nonencapsulated rat (400) and human (1,200) islets reported elsewhere (30,31). However, the decrease in islet mass was obviously stopped a few weeks after transplantation when the encapsulated islets equilibrated to their unfavorable environment.
The data presented here demonstrate that similar to rat islets, encapsulated human islets can reverse diabetes for a long period of time without the need for immunosuppression. This is a very promising finding, because long-term graft function of human islets has not been reported in the literature. Andersson et al. (32) found that the function of human islets entrapped in a chamber device and transplanted in nondiabetic nude mice was limited to 8 weeks. Even significantly shorter survival times of transplants were reported by Scharp et al. (33). These authors showed that 150eC200 allogeneic adult human islets encapsulated in permselective hollow fibers provided protection against the immune response of diabetic and nondiabetic patients for up to 2 weeks. Treatment of a type 1 diabetic patient with Ca2+-alginate/polylysine-based, intraperitoneally transplanted allogeneic adult islets resulted in insulin independence for only 30 days, despite administration of cyclosporin (34). Even though the literature is extremely limited, the data reported here suggest that microcapsules made of ultra-higheCviscosity, clinical-grade alginate cross-linked with Ba2+ are superior to macrocapsule systems as well as alginate/polylysine microcapsules cross-linked with Ca2+. Our data extend the results obtained by Omer et al. (15), who established normoglycemia in streptozotocin-induced diabetic mice for up to 20 weeks using NPCCs immunoisolated by Ba2+eCcross-linked alginate. However, in this study, diabetes could be reversed only by transplantation of 10,000 islet equivalents, and normoglycemia was observed relatively late, as NPCCs contain large amounts of precursor cells and immature islets that limit initial insulin production. In clinical practice, however, fetal human islets are not available in sufficient amounts; for clinical application of xenotransplantation, the use of adult xenogenic islets has the advantage of rapid onset of normoglycemia and reduction of the transplant volume (6).
In conclusion, our study demonstrated that microcapsules made up of ultra-higheCviscosity alginate cross-linked with Ba2+ and stabilized with HSA protect adult rat and human islets against xenorejection for long periods after transplantation. These data are promising as they provide the first evidence that human islets can be protected by alginate-based microcapsules without an additional permselective membrane.
ACKNOWLEDGMENTS
This work was supported by research grants from Novo Nordisk Pharma, Germany (M.M.W. and S.S.); the Stiftung Rheinland-Pfalz fe Innovation (386261/525 to S.S. and M.M.W.); the Bundesministerium fe Forschung und Bildung (16 SV 1329 to U.Z. and 16 SV 1366/0 and 03N8707 to H.Z.); the Forum Programm Ruhr-Universitt Bochum (Forum AZ437-2004 to S.S.); and the Deutsche Forschungsgemeinschaft (Zi 99/16-1 to U.Z.).
HBSS, Hank’s balanced salt solution; HSA, human serum albumin; IPGTT, intraperitoneal glucose tolerance test; NPCC, neonatal pancreatic cell cluster; UW, University of Wisconsin
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2 Department of Biotechnology, Biozentrum, University of Wezburg, Germany
3 Department of Cryobiophysics and Cryotechnology, Fraunhofer Institute for Biomedical Technique, St. Ingbert, Germany
ABSTRACT
We describe the results of the first study to show that adult rat and human islets can be protected against xenogenic rejection in immunocompetent diabetic mice by encapsulating them in a novel alginate-based microcapsule system with no additional permselective membrane. Nonencapsulated islets lost function within 4eC8 days after being transplanted into diabetic Balb/c mice, whereas transplanted encapsulated adult rat or human islets resulted in normoglycemia for >7 months. When rat islet grafts were removed 10 and 36 weeks after transplantation, the mice became immediately hyperglycemic, thus demonstrating the efficacy of the encapsulated islets. The explanted capsules showed only a mild cellular reaction on their surface and a viability of >85%, and responded to a glucose stimulus with a 10-fold increase in insulin secretion. Furthermore, transplanted mice showed a slight decrease in the glucose clearance rate in response to intraperitoneal glucose tolerance tests 3eC16 weeks after transplantation; after 16 weeks, the rate remained stable. Similar results were obtained for encapsulated human islets. Thus we provide the first evidence of successful transplantation of microencapsulated human islets. In conclusion, we have developed a novel microcapsule system that enables survival and function of adult rat and human islets in immunocompetent mice without immunosuppression for >7 months.
As has recently been shown (1), long-term normoglycemia can be achieved in type 1 diabetic patients by allogenic human islet transplantation. However, this technique requires the administration of immunosuppressives, which are well known to be associated with serious side effects. The severe shortage of available human islets is another drawback of this promising therapeutic approach. A bioartificial pancreas consisting of islets immunoprotected by an alginate microcapsule provides an alternative approach to overcoming the rejection problem (2eC4). Such immunoprotection is also appealing because it provides improved prospects of obtaining an abundant supply of human islets from ductal tissue (5) and the possibility of using xenogenic islets (e.g., porcine islets) (6). In most studies, islets have been encased in Ca2+eCcross-linked alginate-polylysine-alginate microcapsules following the protocol of Lim and Sun (7). However, the results obtained by various groups using this microcapsule formulation in the last two decades have been inconsistent. Most importantly, graft survival is random and generally too limited for therapeutic application of these microcapsules (8eC10). The reasons for this are presumably the cytotoxicity of the polyamino acid and the mechanical instability of the microcapsules (2,11,12). Microcapsules made of alginate cross-linked with Ba2+do not share these disadvantages (13). Duvivier-Kali et al. (14) have reported that these microcapsules protect islets against allorejection and autoimmunity. Furthermore, Omer et al. (15) found that Ba2+eCcross-linked alginate microcapsules protect neonatal pancreatic cell clusters (NPCCs) when transplanted in the streptozotocin-induced diabetic Balb/c mouse model for 20 weeks. We have recently shown that using highly purified and ultraeChigh-iscosity alginates when cross-linked internally and externally with Ba (2) results in microcapsules of extremely high stability, particularly when proteins (e.g., human serum albumin [HSA]) are simultaneously incorporated (16,17). A recent study has shown that rat islets encapsulated in these novel microcapsules exhibit a well-preserved insulin secretion in tissue culture over 3 weeks (16).
Here we demonstrate that adult rat as well as adult human islets encapsulated in these novel microcapsules and transplanted into diabetic mice are protected against xenogenic rejection for extremely long periods of time without the need for immunosuppression.
RESEARCH DESIGN AND METHODS
Isolation of rat islets.
Isolation of rat islets was performed according to the protocol previously described (17,18). Briefly, SD rats (Central Animal Facility, University of Mainz) age 8 weeks and weighing 280eC330 g were used as islet donors. Rats were anesthetized by pentobarbital administration (60 mg/kg, i.p.). A midline abdominal incision was made and the pancreas was exposed and injected via the pancreatic duct with Hank’s balanced salt solution (HBSS; Gibco BRL, Long Island, NY) containing 1.7 mg/ml collagenase (Serva PanPlus, Heidelberg, Germany). After the animal was killed, the pancreatic tissue was surgically removed and incubated for 10 min at 37°C in the collagenase solution. Mechanical disruption of the digested pancreatic tissue was achieved by incubating the tissue in the collagenase solution at 37°C for 10 min, with the incubation being interrupted every 2 min by shaking for 30 s. Digestion was stopped by adding cold (4°C) HBSS plus 10% FCS (Greiner, Frickenhausen, Germany). Islet purification was achieved using a discontinuous three-phase Ficoll density gradient (densities: 1.090, 1.077, and 1.040).
Isolation of human islets.
Human pancreases were obtained from brain-dead cadaveric multiorgan donors with the informed consent of their relatives. Tissue procurements were approved by the Human Ethics Committee of the Landesrztekammer Rheinland-Pfalz. Human islets were isolated from two consecutively processed human pancreases after a vascular flush with University of Wisconsin (UW) solution. Briefly, pancreases were intraductally distended with 250 ml HBSS containing Liberase HI (Roche, Mannheim, Germany). Distended pancreases were digested in a continuous digestion-filtration device at 37°C. After a subsequent 60-min cold storage of the digest in UW solution, human islets were purified using a continuous hyperosmolar Hank’s-Ficoll gradient (Biochrom KG, Berlin, Germany) on a Cobe 2991.
Culture conditions.
Islets were cultured in RPMI 1640 medium (Biochrom KG) supplemented with 100 mg/dl glucose, 25 mmol/l HEPES (Greiner), 0.2 g/l Glutamax (Gibco BRL, Paisley, Scotland), 10% FCS (Greiner), and antibiotics (100 units/ml penicillin, 100 e/ml streptomycin [Gibco BRL, Paisley, Scotland], and 10 e/ml ciprofloxacin [Ciprobay; Aventis, Frankfurt, Germany]). Incubation lasted 12 h at 37°C. Islets were then transferred into a slightly modified culture medium containing 3% HSA (Pharmacia, Erlangen, Germany) instead of FCS and an elevated concentration of glucose (300 mg/dl). After being cultured for 12 h at 37°C, the islets were used for encapsulation or transplantation.
Islet encapsulation.
Microencapsulation was performed using ultra-higheCviscosity alginate of clinical grade (viscosity of a 0.1% wt/vol solution in distilled water was 20eC30 mPa · s). The extraction and purification protocol has been described elsewhere (19,20). For microcapsule formation, the alginate was dissolved in sterile, endotoxin-free 0.9% NaCl solution at a concentration of 0.7% (wt/vol). Then 3% HSA (Pharmacia) was added to the alginate solution before it was cross-linked. For microcapsule formation, a two-channel air jet droplet generator was used. The islet-containing microcapsules were dropped into a 20-mmol/l BaCl2 solution adjusted to 290 mOsm with appropriate amounts of NaCl and buffered to pH 7.0 with 5 mmol/l histidine. After 20 min, the islet-containing microcapsules were washed three times with 0.9% NaCl solution and treated with 6 mmol/l Na2SO4 saline solution for 30 min at 37°C to precipitate excess Ba2+ ions (20). The microcapsules obtained by this encapsulation procedure had a diameter of 700eC800 e and on average contained one islet or, in rare cases, two or three.
Animals.
Male Balb/c mice (Central Animal Facility) age 6eC8 weeks were used as recipients for nonencapsulated (controls) or encapsulated islets. Mice were rendered diabetic with intraperitoneal injections of streptozotocin (250 mg/kg body wt; Sigma, St. Louis, MO) freshly dissolved in citrate buffer 2 weeks before islet transplantation. Only animals exhibiting blood glucose concentrations >350 mg/dl in four consecutive measurements were used as recipients.
Transplantation of microencapsulated islets.
Transplants were made in a Class 100 biological safety cabinet under sterile conditions. Encapsulated rat (n = 8) or human (n = 3) islets were injected into the peritoneal cavity with a 20-gauge needle (Vasofix; Braun, Melsungen, Germany). Before treatment, the Balb/c mice were anesthetized with ketamine (65 mg/kg i.p.; Pfizer, Karlsruhe, Germany) and xylazine hydrochloride (13 mg/kg i.p.; Bayer, Leverkusen, Germany). Each mouse received 1,800 microencapsulated islets. Control animals (n = 4) received the same number of nonencapsulated rat or human islets, respectively.
Intraperitoneal glucose tolerance test.
After islet transplantation, animals’ body weight was determined once a week and blood glucose levels of islet recipients were monitored biweekly. Blood samples were taken from the tail vein under nonfasting conditions (8:00 A.M.; standard laboratory diet ad libitum overnight) and determined using a glucometer (Accutrend sensor, Roche Diagnostics, Germany). The mice were considered to be normoglycemic when blood glucose levels were <200 mg/dl. An intraperitoneal glucose tolerance test (IPGTT) was performed in mice transplanted with encapsulated rat islets 3 (n = 8), 9 (n = 7), 16 (n = 5), and 36 (n = 2) weeks after transplantation, using a 5% glucose solution (1 g/kg body wt). In mice transplanted with encapsulated human islets, an IPGTT was carried out at the same times (n = 2 at 3, 9, and 16 weeks; n = 1 at 36 weeks). IPGTTs were also performed in all diabetic animals 3 days before islet transplantation and in body weighteCmatched nondiabetic Balb/c mice. Blood glucose levels were measured at 0, 10, 20, 30, 60, and 120 min.
Microcapsule explantation.
Grafts were retrieved from two animals that had received encapsulated adult rat islets 10 (n = 1) and 36 weeks (n = 1) after islet transplantation. Under anesthesia, a 2-cm incision was made in the abdomen and capsules were explanted by repeated flushes with warm 0.9% NaCl solution. The abdomen was sutured and the animals were checked daily for blood glucose concentrations. Explanted microcapsules were evaluated for fibrotic overgrowth under a Wilowert S microscope (Hund GMBH, Wetzlar, Germany), as previously described (21). Images were captured with a digital camera and stored as jpg files. The viability of the encapsulated islets was assessed using the fluorescein diacetate method, as described elsewhere (16,22).
Assessment of insulin secretion by a static incubation assay.
The static incubation assay was used to assess the insulin secretion response of nonencapsulated and encapsulated rat or human islets after a glucose challenge both before transplantation and after retrieval, as previously described (16,17). For each animal, 10 samples of 10 nonencapsulated or encapsulated islets of equal size and shape were hand picked and transferred into a culture insert (membrane pore diameter 12 e; Millicell PCF, Millipore, France). The insert was put into a well of a 24-well culture-plate (Falcon Multiwell; Becton, Dickinson). Basal insulin secretion was measured after a 1.5-h incubation at 37°C in RPMI 1640 + 1 g/l D-glucose + 3% HSA. The inserts with islets were transferred into hyperglycemic culture medium (RPMI 1640 + 3 g/l D-glucose + 3% HSA) for an additional 1.5 h. Aliquots of the medium were stored at eC20°C after inserts were removed. Rat insulin was measured with the rat-insulin enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden) and human insulin was determined with a chemiluminiscent immunoassay (ADVIA Centaur insulin assay; Bayer). Results are expressed as nanograms per islet per 1.5 h for rat islets and as microunits per islet per 1.5 h for human islets.
Statistics.
Data are given as means ± SE. Statistical significance of differences was calculated with an unpaired Student’s t test (two-sided).
RESULTS
Graft survival.
Before encapsulated islets were transplanted, their insulin secretion capacity after being cultured for 1 day was determined. As shown in Table 1, encapsulated rat or human islets responded well to a high-glucose stimulus. The insulin increase after a glucose challenge was comparable with that of the corresponding nonencapsulated islets, even though the absolute amount of insulin was lower, presumably due to diffusion restrictions within the cross-linked alginate matrix (16). Transplantation of 1,800 nonencapsulated rat islets into the peritoneal cavity of diabetic Balb/c mice resulted in normoglycemia for 4eC8 days. Similar results were obtained for nonencapsulated human islets (Table 2). In contrast, when encapsulated rat and human islets were transplanted under the same conditions, normoglycemia was achieved for >270 days (Fig. 1). As indicated in Table 2, part of the transplantation experiments are still going on. Loss of graft function was observed in only one case. This happened 69 days after transplantation of encapsulated rat islets and was due to a severe infection of the tail as a result of repeated blood collection. Normoglycemic blood glucose levels were generally reached within 24 h of transplantation; normoglycemia was not established in only one animal that had received encapsulated human islets. This was presumably due to a heavy infection at the abdominal wall induced by the transplantation procedure. Graft explantation from two animals 10 and 36 weeks after transplantation resulted in the immediate reappearance of hyperglycemia associated with blood glucose concentrations > 500 mg/dl. In addition, transplantation of microencapsulated rat or human islets led to an increase in body weight, as depicted in Fig. 2.
Intraperitoneal glucose tolerance test.
Figure 3 illustrates the blood glucose profiles of Balb/c mice transplanted with encapsulated rat islets during IPGTTs at different times (3, 9, 16, and 36 weeks) after transplantation. During the IPGTTs, the blood glucose concentration profiles were always significantly lower compared with those of diabetic controls (Figs. 3AeCD). As indicated in Fig. 3A, 3 weeks after receiving transplanted encapsulated rat islets, mice showed the same blood glucose concentration after overnight fasting (t = 0) as the nondiabetic controls. Interestingly, the subsequent glucose clearance kinetics were much faster and the final glucose value reached after 120 min was considerably lower than in nondiabetic mice (Fig. 3A). In contrast, 9 weeks after transplantation, blood glucose concentration and kinetics in rat isleteCtransplanted mice were similar to those observed of nondiabetic control mice (Fig. 3B). The fasting and peak glucose levels of the rat isleteCtransplanted mice during IPGTT 16 and 36 weeks after transplantation were similar to those of the controls, but their subsequent glucose clearance rate was significantly delayed and, in turn, their blood glucose levels remained higher than those of the nondiabetic controls at the end of the IPGTT (Figs. 3C and D).
Because of the limited number of mice transplanted with encapsulated human islets, the blood glucose kinetics during the IPGTT could not be determined as thoroughly as for encapsulated rat islets. However, as shown in Fig. 4, the blood glucose kinetics of these mice were apparently not significantly different from those of the nondiabetic control mice 3, 9, and 16 weeks after transplantation. Consistent with the finding for mice with encapsulated rat islets, 36 weeks after transplantation, glucose clearance in human isleteCtransplanted mice was delayed, but the blood glucose concentration at t = 0 (after fasting) and the glucose peak value were comparable with the corresponding values measured in nondiabetic controls.
Retrieved microcapsules.
The majority of rat isleteCcontaining microcapsules retrieved 10 or 36 weeks after transplantation exhibited only a mild cellular reaction on their external surface (Figs. 5C and F). As assessed by fluorescein diacetate staining, encapsulated rat islets retrieved 10 as well as 36 weeks after transplantation exhibited good viability (>85%) (Fig. 5A, B, D, and E). Consistent with the results of the IPGTTs, the retrieved encapsulated rat islets responded well to a glucose challenge. Their insulin secretion capacity, however, was slightly reduced compared with their initial insulin secretion capacity before transplantation (Table 1). The reduction in insulin secretion apparently occurred during the first two months of transplantation, as the insulin secretion capacity of encapsulated rat islets retrieved after 36 weeks was not significantly different from that of microcapsules retrieved after 10 weeks.
DISCUSSION
Over the past two decades, many different hydrogels and synthetic polymers have been proposed for immunoisolation and encapsulation of cells (23). Among these, alginate is one of the most promising natural polymers, as demonstrated by animal studies and small-scale clinical trials (24,25). One of the main requirements for long-term alginate-based transplantation is the stability of the microcapsules. This is achieved by cross-linking ultra-higheCviscosity alginate of clinical grade (i.e., free of mitogenic and cytotoxic ingredients as well as of gram-negative and gram-positive bacterial spores) (19,20) with Ba2+ and by simultaneously incorporating stabilizing agents such as proteins into the microcapsules (17). As shown elsewhere (16), HSA can preserve insulin secretion for up to 3 weeks in tissue culture when this microcapsule design is used for encapsulation of rat islets. In contrast to FCS, HSA meets the safety criteria of the U.S. Food and Drug Administration and the American Society for Testing and Materials (26). The animal studies reported here confirm the enormous clinical potential of HSA-stabilized alginate-based microcapsules. The encapsulated islets of adult rats or humans maintained insulin secretion for >7 months, implying the absence of xenogenic rejection and the stability of the microcapsules. This finding was also confirmed by studies of insulin secretion of transplants explanted after 10 and 36 weeks. Failure of one graft was obviously due to severe infection of the tail induced by the blood collection procedure. Similarly, the finding that normoglycemia in one mouse was not established after transplantation of encapsulated human islets can most likely be traced back to a heavy infection of the abdominal wall.
Although the number of experiments with human islet cells were limited due to the restricted availability of intact human pancreatic tissue, our human islet cell data are in good accordance with the results obtained with rat islet cells. As depicted in Figs. 3 and 4, the pattern of glucose clearance differed from that of the nondiabetic control mice and changed with time after transplantation. Glucose clearance was faster in rat isleteCtransplanted mice than in controls 3 weeks after transplantation. This was likely due to the different secretory properties of rat islets. It is well known that rat islets have a lower set point than mouse islets (27,28), as evidenced by the lower fed and fasted blood glucose levels measured in normal rats. In contrast, a decrease in glucose clearance was observed 3eC16 weeks after transplantation, as indicated by the considerably higher glucose values recorded 120 min after intraperitoneal glucose injection. It was interesting to observe that a further decrease in the glucose clearance rate with ongoing transplantation did not occur. A possible explanation for this finding is the restricted diffusion of nutrients and oxygen by an intense fibrotic overgrowth of the microcapsules during the first weeks of transplantation. However, although microcapsules retrieved 10 and 36 weeks after transplantation exhibited a reduced insulin secretion after glucose challenge, a strong fibrosis was not observed. The retrieved microcapsules showed only a weak cellular reaction close to the surface. -Cell injury by complement-dependent components can also be excluded as a possible cause for the reduction in the glucose clearance rate, as the microcapsules used here were protected against these cytotoxic components, as shown recently by in vitro studies (16). The most likely explanation for these findings is that the microcapsules float freely in the peritoneal cavity without any vascularization. The supply of the encapsulated islets with nutrients and oxygen is, therefore, rather limited as evidenced by 19F nuclear magnetic resonance imaging (29). Therefore, compromised nutrient and oxygen supply to the islets might lead to mass loss (8) and, in turn, a reduction in insulin secretion. This could also explain the fact that rather high numbers of encapsulated rat and human islets (1,800 islets) were needed to achieve a stable graft function in this study as compared with the lower numbers of nonencapsulated rat (400) and human (1,200) islets reported elsewhere (30,31). However, the decrease in islet mass was obviously stopped a few weeks after transplantation when the encapsulated islets equilibrated to their unfavorable environment.
The data presented here demonstrate that similar to rat islets, encapsulated human islets can reverse diabetes for a long period of time without the need for immunosuppression. This is a very promising finding, because long-term graft function of human islets has not been reported in the literature. Andersson et al. (32) found that the function of human islets entrapped in a chamber device and transplanted in nondiabetic nude mice was limited to 8 weeks. Even significantly shorter survival times of transplants were reported by Scharp et al. (33). These authors showed that 150eC200 allogeneic adult human islets encapsulated in permselective hollow fibers provided protection against the immune response of diabetic and nondiabetic patients for up to 2 weeks. Treatment of a type 1 diabetic patient with Ca2+-alginate/polylysine-based, intraperitoneally transplanted allogeneic adult islets resulted in insulin independence for only 30 days, despite administration of cyclosporin (34). Even though the literature is extremely limited, the data reported here suggest that microcapsules made of ultra-higheCviscosity, clinical-grade alginate cross-linked with Ba2+ are superior to macrocapsule systems as well as alginate/polylysine microcapsules cross-linked with Ca2+. Our data extend the results obtained by Omer et al. (15), who established normoglycemia in streptozotocin-induced diabetic mice for up to 20 weeks using NPCCs immunoisolated by Ba2+eCcross-linked alginate. However, in this study, diabetes could be reversed only by transplantation of 10,000 islet equivalents, and normoglycemia was observed relatively late, as NPCCs contain large amounts of precursor cells and immature islets that limit initial insulin production. In clinical practice, however, fetal human islets are not available in sufficient amounts; for clinical application of xenotransplantation, the use of adult xenogenic islets has the advantage of rapid onset of normoglycemia and reduction of the transplant volume (6).
In conclusion, our study demonstrated that microcapsules made up of ultra-higheCviscosity alginate cross-linked with Ba2+ and stabilized with HSA protect adult rat and human islets against xenorejection for long periods after transplantation. These data are promising as they provide the first evidence that human islets can be protected by alginate-based microcapsules without an additional permselective membrane.
ACKNOWLEDGMENTS
This work was supported by research grants from Novo Nordisk Pharma, Germany (M.M.W. and S.S.); the Stiftung Rheinland-Pfalz fe Innovation (386261/525 to S.S. and M.M.W.); the Bundesministerium fe Forschung und Bildung (16 SV 1329 to U.Z. and 16 SV 1366/0 and 03N8707 to H.Z.); the Forum Programm Ruhr-Universitt Bochum (Forum AZ437-2004 to S.S.); and the Deutsche Forschungsgemeinschaft (Zi 99/16-1 to U.Z.).
HBSS, Hank’s balanced salt solution; HSA, human serum albumin; IPGTT, intraperitoneal glucose tolerance test; NPCC, neonatal pancreatic cell cluster; UW, University of Wisconsin
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