Complete protection of islets against allorejection and autoimmunity by a simple barium-alginate membrane

We describe a new technique for microencapsulation with high-mannuronic acid (high-M) alginate crosslinked with BaCl(2) without a traditional permselective component, which allows the production of biocompatible capsules that allow prolonged survival of syngeneic and allogeneic transplanted islets in diabetic BALB/c and NOD mice for >350 days. The normalization of the glycemia in the transplanted mice was associated with normal glucose profiles in response to intravenous glucose tolerance tests. After explantation of the capsules, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated islets. The retrieved capsules were free of cellular overgrowth and islets responded to glucose stimulation with a 5- to 10-fold increase of insulin secretion. Transfer of splenocytes isolated from transplanted NOD mice to NOD/SCID mice adoptively transferred diabetes, indicating that NOD recipients maintained islet-specific autoimmunity. In conclusion, we have developed a simple technique for microencapsulation that prolongs islet survival without immunosuppression, providing complete protection against allorejection and the recurrence of autoimmune diabetes.     

Evaluation of different types of alginate microcapsules as bioreactors for producing endostatin

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.     

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept

BACKGROUND: Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates. METHODS: Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation. RESULTS: Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response. CONCLUSIONS: Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.     

Co-encapsulation of Sertoli enriched testicular cell fractions further prolongs fish-to-mouse islet xenograft survival

BACKGROUND: We previously demonstrated that alginate microencapsulation can prolong fish (tilapia) islet xenograft survival in diabetic animals. However, at present, microencapsulation does not provide complete immune protection to discordant islet xenografts, and long-term graft survival requires supplemental low-dose systemic immunosuppression. In the present study, fish islets were co-encapsulated with Sertoli enriched testicular cell fractions to find out whether this would further prolong fish islet graft survival in diabetic mice. METHODS: Sertoli enriched testicular cell fractions were enzymatically harvested from adult Balb/c or Wistar-Furth rats. They were cultured and co-encapsulated with fragmented tilapia islets in alginate microcapsules. Encapsulated islets alone or islets co-encapsulated with Sertoli cells were then intraperitoneally transplanted into streptozotocin-diabetic Balb/c mice, and graft survival times were compared. Encapsulated and co-encapsulated islet function was also confirmed in streptozotocin-diabetic athymic nude mice. RESULTS: Co-encapsulation with Sertoli enriched testicular cell fractions further prolonged mean fish islet graft survival time from 21+/-6.7 days (encapsulated islet cells alone) to >46+/-6.3 days (co-encapsulated with syngeneic murine Sertoli cells), without additional systemic immunosuppression. Testicular cells harvested from xenogeneic Wistar-Furth rats produced similar protective results (>46+/-10.9 days). CONCLUSIONS: Our results support the theory that Sertoli cells produce local immunosuppressive factors. These factors supplement the immune protective feature of alginate microcapsules in our model. Testicular cell fractions may be an important naturally occurring facilitator in the development of new microencapsulation systems for islet xenotransplantation.     

Alginate polylysine microcapsules as immune barrier: Permeability of cytokines and immunoglobulins over the capsule membrane

Transplantation of pancreatic islets in alginate polylysine microcapsules is a potential useful method for treating type I diabetes. In this study, the permeability for alginate-polylysine microcapsules to cytokines an immunoglobulines has been investigated by a newly developed method. Magnetic monodisperse polymer particles (Dynabeads) coated with antibodies against selected proteins were encapsulated in 0.7 mm alginate polylysine microcapsules. The capsule membrane permeability to IgG (150 kDa), Transferrin (81 kDa), Tumor necrosis factor (TNF, 51 kDa), Interleukin-1β (IL-1β, 17.5 kDa), and insulin (5.8 kDa) was estimated by measuring the binding of ¹²⁵I-labeled proteins to the encapsulated antibody coated Dynabeads. Capsules with an inhomogeneous solid gel core were made of alginates with high guluronic or high mannuronic acid content and poly-l (PLL)- or poly-d-lysine (PDL) of concentrations varied from 0.05–0.2%. The various capsules examined were all impermeable to IgG. The capsules made with a PLL-, but not PDL-membranes were permeable for transferrin. IL-1β was found to penetrate all of the different capsule types. The high-G capsules, however, could be made impermeable to TNF and still allowed transferrin to pass. The permeability of these capsules to IL-1β, but not to TNF was confirmed in an assay where mouse islets of Langerhans were incubated with TNF and IL-1β, and comparing the IL-6 for encapsulated and nonencapsulated islets.     

Survival of microencapsulated islets at 400 days posttransplantation in the omental pouch of NOD mice

The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, beta-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.     

Xenografts of rat islets into diabetic mice. An evaluation of new smaller capsules.

Healthy rat islets were encapsulated in alginate-polylysine-alginate capsules measuring 0.25-0.35 mm in diameter using a modified encapsulation technique. The encapsulated islets were transplanted intraperitoneally in nonimmunosuppressed streptozotocin-induced diabetic BALB/c mice. The diabetic condition of the experimental animals was reversed within two days following the transplantation and the animals remained normoglycemic for up to 308 days, with a mean xenograft survival of 219.8 +/- 46.2 days. Four and six months posttransplant the capsules were removed from two recipients. This resulted in regression to a hyperglycemic state. After a second transplant of encapsulated islets, the animals returned to normoglycemia. In control mice that received free unencapsulated islets, the xenografts remained functional for no more than 12 days. Our study clearly demonstrates that the encapsulation of islets in the new smaller capsules can effectively prolong xenograft survival without immunosuppression.     

Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with type 1 diabetes mellitus

To comply with regulatory restrictions, with regard to graft of human islets immunoprotected within artificial microcapsules, into patients with type 1 diabetes mellitus (T1DM) with no recipient immunosuppression, we have prepared standard protocols on: (1) sodium alginate purification (clinical grade) for microcapsule fabrication; (2) preparation of biocompatible and permselective microcapsules containing human islets; and (3) minimally invasive techniques for grafting of the encapsulated human islets into the recipients' peritoneal cavity. As to no. 1, starting from pharmaceutical grade, raw sodium alginate powder, we prepared a pyrogen- and endotoxin-free 1.6% alginate solution by means of dialysis, multiple filtrations, and dilution/osmolality adjustments. As to no. 2, we have selected human islet preparations associated with >80% purity/viability, which underwent careful functional quality control testing prior to encapsulation; namely, most capsules contained one islet. As for no. 3, we have devised a simple intraperitoneal injection method under abdominal echography guidance with only local anesthesia to deposit the encapsulated islets in saline within the peritoneal leaflets. These technical protocols were officially approved by the Italian Ministry of Health which has released permission to conduct a phase I, closed human trial in 10 patients using encapsulated human islet grafts into nonimmunosuppressed patients with T1DM.     

Xenotransplantation of cells using biodegradable microcapsules

BACKGROUND: The use of immunoisolation to protect transplanted cells from the immune system of the host has broad application to the treatment of major diseases such as diabetes and a wide range of other disorders resulting from functional defects of native cell systems. In most cases, limitations in functional cell longevity will necessitate periodic replenishment of the cells. We describe a hydrogel-based microcapsule that breaks down at a rate that can be adjusted to correspond to the functional longevity of the encapsulated cells. These injectable capsules can be engineered to degrade over several weeks to months for short-term drug delivery, or to remain intact and immunoprotective for more extended periods. When the supply of cells needs to be replenished, no surgery will be required to localize and remove the old capsules. METHODS: Porcine and bovine islets were immobilized in "composite" microcapsules fabricated from alginate and low-relative molecular mass (Mr) poly (L-lysine[PLL]) (Mr exclusion <120 Kd) and implanted into the peritoneum of normal and streptozotocin-induced diabetic rats. In addition to demonstrating long-term islet viability and function, a series of in vitro studies were carried out to determine the permeability and biodegradability of the microcapsules used in the present system. RESULTS: Xenogeneic islets implanted in nonimmunosuppressed rats remained in excellent condition indefinitely (>40 weeks)(viability was comparable to that of preimplant control specimens). In contrast, no islets survived in uncoated alginate spheres after 2 weeks postimplantation. By changing the concentration of the alginate, it was possible to vary the rate of capsule breakdown in rats from mechanically unstable (outer matrix <0.5-0.75% alginate) to stable for >1 year (> or =1.5% alginate). In addition to in vivo breakdown studies, the biodegradability of the capsular components was verified in vitro using a mixture of tritosomes (enzymes isolated from animal cells). CONCLUSIONS: We have designed a microcapsule system with controllable biodegradability which allows breakdown and absorption of implants when the cells die or become functionally inactive. These results may have application to other alginate-PLL encapsulation systems. The ability to cross species lines using these biodegradable microcapsules has the potential to expand dramatically the number of patients and the scope of diseases that can be successfully treated with cellular therapy.     

Survival of Microencapsulated Adult Pig Islets in Mice In Spite of an Antibody Response

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.     

Xenotransplantation of microencapsulated fetal rat islets

Fetal pancreatic islets were isolated from 21-day pregnant Wistar rats and enclosed in semipermeable alginate-polylysine-alginate capsules. Encapsulated islets that had been previously cultured for eight days in vitro were shown to secrete insulin in response to glucose challenge: low-glucose, high-glucose, and high-glucose + 3-isobutyl-1-methyl-xanthine (IBMX). Transplants of 800-1000 encapsulated cultured fetal islets into the peritoneal cavities of BALB/c mice with streptozotocin-induced diabetes restored normoglycemia for up to 171 days without immunosuppression. When the capsules were removed from 2 of the recipients they both quickly regressed to a diabetic state. Control groups of diabetic mice received unencapsulated, uncultured islets or empty capsules. The mortality rate among these animals was high and none experienced relief from hyperglycemia for longer than 6 days. These results demonstrate that cultured microencapsulated fetal rat islets of Langerhans can release insulin in response to an in vitro glucose challenge, and that transplants of these islets into diabetic mice can restore normoglycemia without the need for immunosuppressive therapy.     

Synthetic poly(ethylene glycol)‐based microfluidic islet encapsulation reduces graft volume for delivery to highly vascularized and retrievable transplant site

Transplant of hydrogel‐encapsulated allogeneic islets has been explored to reduce or eliminate the need for chronic systemic immunosuppression by creating a physical barrier that prevents direct antigen presentation. Although successful in rodents, translation of alginate microencapsulation to large animals and humans has been hindered by large capsule sizes (≥500 μm diameter) that result in suboptimal nutrient diffusion in the intraperitoneal space. We developed a microfluidic encapsulation system that generates synthetic poly(ethylene glycol)‐based microgels with smaller diameters (310 ± 14 μm) that improve encapsulated islet insulin responsiveness over alginate capsules and allow transplant within vascularized tissue spaces, thereby reducing islet mass requirements and graft volumes. By delivering poly(ethylene glycol)‐encapsulated islets to an isolated, retrievable, and highly vascularized site via a vasculogenic delivery vehicle, we demonstrate that a single pancreatic donor syngeneic islet mass exhibits improved long‐term function over conventional alginate capsules and close integration with transplant site vasculature. In vivo tracking of bioluminescent allogeneic encapsulated islets in an autoimmune type 1 diabetes murine model showed enhanced cell survival over unencapsulated islets in the absence of chronic systemic immunosuppression. This method demonstrates a translatable alternative to intraperitoneal encapsulated islet transplant.     

Histological and immunopathological analysis of recovered encapsulated allogeneic islets from transplanted diabetic BB/W rats.

Allogeneic islets encapsulated in an alginate/poly-L-lysine membrane and transplanted into diabetic BB/W rats resulted in graft failure within 2 weeks of transplantation. Graft failure was associated with a dense pericapsular infiltrate (PCI) that resulted in necrosis of the encapsulated islets. The PCI could be inhibited by immunosuppressive agents, including cyclosporine and dexamethasone, and this resulted in a significant increase in graft survival. Immunopathological characterization of the PCI indicated that there was a predominance of macrophages. T helper cells also appeared to be present in this PCI. Empty capsules were also found to induce a similar PCI that was identical in composition to that found around encapsulated islets. Thus alginate/poly-L-lysine capsules do not appear to be biocompatible and may account for the variable results in islet graft survival found with these capsules.     

微囊化新生猪甲状旁腺细胞异种移植的实验研究

目的 探讨微囊化新生猪甲状旁腺细胞异种移植治疗大鼠甲状旁腺功能低下症的效果。方法 应用微囊化技术，制备微囊化（海藻酸钠－聚赖氨酸－海藻酸钠生物微胶囊）新生猪甲状旁腺细胞，32只去甲状旁腺的Wistar大鼠随机分成微囊组、非微囊组、空囊组和对照组，分别移植微囊化新生猪甲状旁腺细胞、甲状旁腺细胞、空微囊及生理盐水。移植后监测血钙及甲状旁腺素水平40周，40周后回收移植物，透射电镜检查。结果 移植后，微囊组大鼠血钙及甲状旁腺素水平恢复到正常范围内，直至观察结束时（40周），透射电镜检查显示移植物存活良好；非微囊组、空囊组和对照组大鼠的血钙及甲状旁腺素水平无改善。结论 微囊化新生猪甲状旁腺细胞异种移植在不用免疫抑制剂情况下，可以在大鼠体内存活，且有功能；海藻酸钠－聚赖氨酸－海藻酸钠生物微胶囊对免疫活性细胞及抗体具有屏蔽作用。     

Transplantation of alginate microcapsules: generation of antibodies against alginates and encapsulated porcine islet-like cell clusters

BACKGROUND: Microencapsulation of islets of Langherhans in alginate poly-L-lysine capsules provides an effective protection against cell-mediated immune destruction, and ideally should allow the transplantation of islets in the absence of immunosuppression. It has previously been suggested that alginate rich in mannuronic acid (high M) is more immunogenic than alginate rich in guluronic acid (high G). The ability of these alginates to induce an antibody response in the recipient or act as an adjuvant to antibody responses against antigens leaked from the capsule was investigated in the present study. METHODS: Empty capsules made from these different types of alginate were transplanted intraperitoneally to Wistar rats or Balb/c mice. In addition, some animals were also injected with bovine serum albumin to assess the ability of the alginates to act as an adjuvant to this antigen. Antibody responses to intraperitoneally transplanted free and microencapsulated fetal porcine islet like cell clusters (ICC) were also evaluated, in animals treated with or without cyclosporine. RESULTS: Antibodies against high M-alginate capsules were detected in the sera of mice transplanted with this capsule type. However, this response was not seen after the transplantation of high G capsules. When Wistar rats were used as recipients, no antibody responses were detected against any type of alginate capsules. Neither type of capsule acted as an adjuvant. Antibodies against ICC were present, in rats transplanted with both nonencapsulated and encapsulated ICCs. Administration of cyclosporine could abolish this production of antibodies against ICC. CONCLUSIONS: High G-alginate capsules are less immunogenic than high M capsules. Because encapsulation did not protect against the generation of antibodies against ICC, it can be assumed that antigen leakage from the capsules occurs, as no evidence was found for capsules breaking in vivo.     

The effects of microencapsulation on pancreatic islet osmotically induced volumetric response

Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 microm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22 degrees C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37 degrees C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 +/- 1.04% (mean +/- SEM) and 70.11 +/- 1.05%, and in 2 M EG to 72.89 +/- 1.93% and 69.33 +/- 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 +/- 0.91% and 67.67 +/- 1.91% for canine and 62.22 +/- 0.66.% or 65.89 +/- 1.30% for human islets. Minimum volumes were reached within 30-40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 +/- 1.47% of their original volume in 2 M and 80.33 +/- 0.89% in 3 M DMSO, and 87.33 +/- 1.86% in 2 M and 82.80 +/- 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 +/- 2.13% of their original volume in 2 M and 78.22 +/- 0.95% in 3 M DMSO, and 85.44 +/- 1.92% in 2 M and 78.11 +/- 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30-50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.     

Biodritin microencapsulated human islets of Langerhans and their potential for type 1 diabetes mellitus therapy

BACKGROUND: Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. METHODS: Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. RESULTS: The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1beta expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. CONCLUSION: This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.     

The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats

This study investigates whether capsular overgrowth on alginate-polylysine microencapsulated islets is influenced by (1) the presence of islet tissue, (2) MHC incompatibility between donor and recipient, or (3) the presence of autoimmune diabetes. Encapsulated Albino Oxford (AO, n=6, isografts) and Lewis (n=6, allografts) rat islets, and encapsulated human islets (n=5, xenografts) were implanted intraperitoneally into streptozotocin-diabetic AO rats. Also, encapsulated AO islets were implanted into autoimmune diabetic Bio Breeding/Organon (BB/O) rats (n=5, allografts). Five isografts, five allografts, and three xenografts in AO recipients and five allografts in BB/O recipients resulted in normoglycemia. Two weeks after implantation, islets containing capsules were retrieved by peritoneal lavage, after which all animals that had become normoglycemic after transplantation returned to a state of hyperglycemia. Recovery rates of the capsules of these successful grafts, expressed as percentages of the initially implanted graft volume, varied from 72%±7% to 80%±9%. The associated pericapsular infiltrates (PCI) were similar in all groups and varied from 3.2%±1.4% to 8.3%±2.6%. Similar recovery rates and PCI were also found with empty capsules. However, the recovery rates of recipients with graft failures were lower and showed more PCI. Immunohistological staining of PCI showed no differences in the types of cells in the PCI on capsules with or without islets. We conclude that this early PCI is a capsule-induced foreign body reaction that is not influenced by MHC incompatibility or by the presence of autoimmune diabetes, and it should be avoided by improving the biocompatibility of the capsules.     

Microencapsulation of Crystalline Insulin or Islets of Langerhans: An Insulin Diffusion Study

Microcapsules containing insulin crystals or islets of Langerhans were made by extruding a mixture of insulin crystals or islets and sodium alginate into a calcium chloride solution, and then coating it with poly-l-lysine. When these microcapsules were incubated at 37 degrees C, insulin could be detected readily in the medium, indicating that the microcapsular membrane is permeable to insulin. The efficiency of insulin encapsulation with crystalline insulin declined as the concentration in the sodium alginate mixture increased. Over 90% of the entrapped insulin was released after 3 days of incubation at 37 degrees C, indicating that the rate of insulin release from the microcapsules requires modification if the microcapsules are to be used as a long-term insulin delivery system. The amount of insulin secreted by the encapsulated islets was not significantly different from that of unencapsulated islets, suggesting the islets were not affected by the modified encapsulation process.     

Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats

Omer A, Keegan M, Czismadia E, De Vos P, Van Rooijen N, Bonner-Weir S and Weir GC. Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats. Xenotransplantation 2003; 10: 240–251. © Blackwell Munksgaard 2003
Background: Macrophages can accumulate on the surface of empty and islet-containing alginate capsules, leading to loss of functional tissue. In this study, the effect of peritoneal macrophage depletion on the biocompatibility of alginate macrocapsules and function of macroencapsulated porcine neonatal pancreatic cell clusters (NPCCs) was investigated. Methods: Clodronate liposomes were injected into the peritoneal cavities of normoglycemic Lewis rats 5 and 2 days before the transplantation. Empty or NPCC-containing Ca-alginate poly L -lysine (PLL)-coated macrocapsules were transplanted into the peritoneal cavities of rats injected with either clodronate liposomes or saline. On days 7, 14 and 21, samples were evaluated by immunohistochemistry for cellular immune responses on the surface of the macrocapsules and for macrophage populations in omental tissue. To assess the function of macroencapsulated NPCCs, insulin secretory responses to glucose and theophylline were measured after capsule retrieval. Results: In saline-injected control groups, all of the empty and NPCC-containing macrocapsules were overgrown with macrophages, this being especially severe on NPCC-containing macrocapsules. In the clodronate liposomes-injected group, the majority of the empty macrocapsules were free of macrophage accumulation and the NPCC-containing macrocapsules were less overgrown than in control animals. Higher insulin responses to glucose and theophylline were observed in NPCCs retrieved from rats injected with clodronate liposomes. Conclusion: We conclude that depletion of peritoneal macrophages with clodronate liposomes improve the survival of macroencapsulated NPCCs.