The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that ...may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)—the “β-Air”—that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immunoprotective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.
Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the ...long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression. A patient with type-1 diabetes received a transplanted chamber and was followed for 10 mo. Persistent graft function in this chamber system was demonstrated, with regulated insulin secretion and preservation of islet morphology and function without any immunosuppressive therapy. This approach may allow for future widespread application of cell-based therapies.
Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein ...islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO
Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO
to a supraphysiological level that maintains all islets viable and functional. These levels were determined from measurements of pO
profiles in islet-alginate slabs. Encapsulated islets implanted with surface density as high as 4,800 islet equivalents/cm
in diabetic rats maintained normoglycemia for more than 7 months and provided near-normal intravenous glucose tolerance tests. Nearly 90% of the original viable tissue was recovered after device explantation. Damaged islets failed after progressively shorter times. The required values of gas chamber pO
were predictable from a mathematical model of oxygen consumption and diffusion in the device. These results demonstrate feasibility of developing retrievable macroencapsulated devices small enough for clinical use and provide a firm basis for design of devices for testing in large animals and humans.
Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or ...organs. To reduce the xenotransplantation-associated risk of infection of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of microorganisms was examined in Göttingen Minipigs by PCR methods. Since zoonotic transmission of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly established. In addition, sera were examined by Western blot analysis using two recombinant capsid proteins of HEV as antigens. HEV RNA was not detected in pigs older than one year including gilts, but it was detected in the sera of three of ten animals younger than 1 year. Furthermore, HEV was also detected in the sera of three sows six days after delivery and their offspring, indicating vertical transmission of the virus. PCR amplicons were cloned, sequenced and the viruses were found to belong to the HEV genotype (gt) 3/4. Anti-HEV immunoglobulins G were detected in one sow and maternal antibodies in her six day old piglet. Since Göttingen minipigs were negative for many xenotransplantation-relevant microorganisms, they can now be classified as safe. HEV may be eliminated from the Ellegaard herd by selection of negative animals and/or by treatment of the animals.
Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of ...complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing β-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.
Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects ...especially due to the failure to restore normal function of the hypothalamic–pituitary–adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, D-Trp6LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21–25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell–cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis.
Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, ...blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.
Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for ...transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.
Background
To establish the safety of xenotransplantation when cells, tissues, or organs of pigs are used, an effective screening for potential zoonotic microorganisms has to be performed. In doing ...so, special attendance has to be paid to porcine endogenous retroviruses (PERVs) that are widely distributed as proviruses in the genome of pigs. PERV‐A and PERV‐B are present in all pigs, they infect human cells in vitro and therefore represent a direct risk. PERV‐C infects only pig cells; however, recombinant PERV‐A/C infecting human cells and replicating at a higher rate were found in pigs indicating an indirect risk. To prevent the transmission of PERV, it was suggested to use animals characterized by a low expression of PERV‐A and PERV‐B that are free of PERV‐C and cannot generate recombinants. Göttingen minipigs are used for numerous biomedical investigations and they are well characterized; however, the prevalence and the expression of PERV in these animals were not yet investigated.
Methods
The presence and expression of all PERVs including a new variant (nv) of PERV‐C and PERV‐A/C were analyzed using PCR and real‐time PCR methods. Altogether, 15 animals belonging to different families were analyzed. To make a low expression better measurable, peripheral blood mononuclear cells (PBMCs) of the animals were stimulated with phytohaemagglutinin generally increasing the expression of PERV and allowing a better classification into animals with high and low expression. As a major end point, the release of virus particles able to infect susceptible human 293 cells was investigated.
Results
PERV‐A, PERV‐B, PERV‐C, and PERV‐Cnv were found in the genome of all investigated Göttingen minipigs, but recombinant PERV‐A/Cs were not found. When the expression of PERV was compared with that in previously analyzed pig strains, it was higher than in German landrace and some other pigs, but lower than in Yucatan miniature pigs. Virus particles able to infected human 293 cells were not detected even after mitogen treatment of the PBMCs.
Conclusion
The Göttingen minipigs are well defined concerning their physiologic parameters, their health status, and their genetics, and therefore, they may be considered as donor animals for at least cell xenotransplantation. When the prevalence and the expression of PERVs were analyzed in these animals, it was demonstrated that although PERV‐A, ‐B, and ‐C proviruses were found in all animals, their expression was low. Additional investigations are required to assess the suitability of Göttingen minipigs and other animals for xenotransplantation in terms of microbiological safety.
Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem ...cell–derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The βAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the βAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1‐2 βAir devices, each containing 155 000‐180 000 islet equivalents (ie, 1800‐4600 islet equivalents per kg body weight), and monitored for 3‐6 months, followed by the recovery of devices. Implantation of the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C‐peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose‐stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the βAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).
An investigator‐initiated phase 1 study of the safety and efficacy of implanting the macroencapsulation device βAir with an incorporated refillable oxygen tank containing allogeneic human pancreatic islets into 4 patients with type 1 diabetes shows that the device is safe and can support survival of allogeneic islets for several months, though the function of the transplanted cells is limited.
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