Long‐term culture and in vitro maturation of macroencapsulated adult and neonatal porcine islets

Encapsulated porcine islets could be used to treat type I diabetes without necessitating severe immunosuppression. Islet survival and secretory function in the encapsulation device need to be preserved to ensure efficient insulin output in response to surrounding stimuli. In the present study, we evaluated stimulated insulin secretion from adult and neonatal pig islets seeded on an acellular collagen matrix and encapsulated in alginate during long‐term culture. Pig islets survived longer and secreted more insulin when cultured on acellular porcine dermis compared to human fascia. Islets from neonatal pigs could survive up to 33 weeks in vitro, and their insulin secretion increased during the first 5 weeks of culture in a beta‐cell maturation medium. In fact, by the 4th week of culture, insulin secretion from neonatal islets attained the same level as adult islets and even surpassed it by the 18th week. Our results show that in vitro maturation of encapsulated neonatal porcine islets is possible and can actually compensate the initial low insulin secretion from these islets while allowing enough time to perform complete functional and biosafety characterization of islets before transplantation.     

Advantages of combined fetal and adult porcine islets as donor tissue for transplantation

Abstract: The molecular similarity between human and porcine insulin is the primary reason for selecting the pig as a donor for islet xenotransplantation in order to treat type I diabetes. Porcine islets can be prepared from adult, newborn, or fetal pigs. However, each of these islet types possesses advantages and disadvantages due to their anatomical, developmental, and physiological characteristics. We have evaluated both adult and fetal porcine islets for their ability to reverse diabetes using an allograft model. Based on our accumulated data, we recommend transplantation of combined adult and fetal porcine islets as a prospective approach for pig to man islet xenotransplantation. The advantages of combined adult and fetal islet transplantation are as follows: 1) Adult porcine islets can reverse hyperglycemia soon after transplantation; 2) well controlled blood glucose at the critical developmental stage of fetal islets will provide the time and environment necessary to achieve the optimal proliferation, growth and maturation of fetal β cells; 3) after 2 to 3 months, fetal islets will functionally mature and begin to share the insulin-producing load previously carried by adult islets, while new islets will continuously emerge and proliferate from stem cells; and 4) islets from fetal pigs will subsequently play a primary role in controlling hyperglycemia, and provide long-lasting insulin independence.     

Neonatal pig pancreata as a source of insulin-producing tissue for xenotransplantation

Abstract: Islet transplantation has been proven as a viable option for treating type 1 diabetics; however, widespread clinical application of this technique will necessitate an alternative source of insulin-producing tissue. Porcine pancreata may be a potential source of islets, but adult porcine islets are difficult to isolate, are fragile, and have a tendency to fall apart during culture. In this study, we investigated the feasibility of preparing functionally viable islet cells in large quantities from the neonatal pig pancreas. Following collagenase digestion and 7 days of tissue culture, a mean of 48,000 neonatal islet cell (NIC) aggregates were recovered per pancreas. These NICs were responsive to glucose in vitro and were capable of correcting hyperglycemia in alloxan-induced diabetic nude mice. These results indicate that the neonatal porcine pancreas can be used to produce large numbers of functionally viable islet cells.     

Real‐time assessment of encapsulated neonatal porcine islets prior to clinical xenotransplantation

Kitzmann JP, Law L, Shome A, Muzina M, Elliott RB, Mueller KR, Schuurman H‐J, Papas KK. Real‐time assessment of encapsulated neonatal porcine islets prior to clinical xenotransplantation. Xenotransplantation 2012; 19: 333–336. © 2012 John Wiley & Sons A/S. Abstract: Background: Porcine islet transplantation is emerging as an attractive option for the treatment of patients with type 1 diabetes, with the possibility of providing islets of higher and more consistent quality and in larger volumes than available from human pancreata. The use of encapsulated neonatal porcine islets (ENPI) is appealing because it can address islet supply limitations while reducing the need for anti‐rejection therapy. Pre‐transplant characterization of ENPI viability and potency is an essential component of the production process. We applied the validated assay for oxygen consumption rate normalized for DNA content (OCR/DNA) to characterize ENPI viability. Methods: ENPI of low viscosity and high m alginate were prepared according to standard methods and characterized at various culture time points up to 5 weeks. Results: The OCR/DNA (nmol/min·mgDNA ± SEM) of ENPI (235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14, n = 13). After encapsulation, NPI OCR/DNA was sustained over a culture period of up to 5 weeks. The average OCR/DNA of ENPI cultured longer than 9 days was higher than that of freshly encapsulated NPI. Conclusion: This is the first characterization of ENPI by a validated and more sensitive method for product viability. The NPI encapsulation process does not compromise viability as measured by OCR/DNA, and ENPI can be cultured for up to 5 weeks with maintenance of viability. ENPI meet or exceed current adult porcine islet product release criteria (established at the University of Minnesota) for preclinical xenotransplantation in terms of OCR/DNA.     

Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets

There is considerable interest in the viral modification of insulin-producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre-transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post-transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue-specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1-alpha promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H-2K(b) and the pancreatic-specific promoters insulin and human pdx-1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans.     

Adult porcine islets produce MCP-1 and recruit human monocytes in vitro

Abstract:  Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4⁺ T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1β and TNF-α. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of α- and β-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1β nor TNF-α were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1β and TNF-α were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.     

Sertoli cell-induced effects on functional and structural characteristics of isolated neonatal porcine islets

A lack of a sufficient number of human donor pancreases has stimulated interest in isolation and cryopreservation techniques for islets from the porcine pancreas. But because of a poorly developed outer membrane porcine islets are particularly susceptible to damage during cryopreservation. The aims of this study were two-fold: 1) to develop a method for isolation and storage of islets from neonatal porcine pancreas and, 2) to examine effects of Sertoli cells on islet yield and function in Sertoli cell-islet cell cocultures. A total of 170 neonatal porcine pancreases were processed by means of a short period of digestion with collagenase and culture of the tissues at 32°C for periods up to 7 days following isolation. Results were: The mean ± SEM, number of viable islets, and percentage loss of cells following 7 days of culture were 29, 442 ± 1,119 and 22.2 ± 1.2, respectively. Cryopreservation had a marked impact on recovery of viable islets: In absence of Sertoli cells an average of only 64% of islets remained viable; by contrast, when cryopreserved islets were cocultured with Sertoli cells, a mean of 82% was recovered. Glucose at 16.7 mmol/L had the capacity to elicit insulin release from 3-day-old cultured islets. The concentration in absence of Sertoli cells was 57.3 ± 3.8, uU/mL/10 islets; in the presence of Sertoli cells the level increased to a mean ± SEM of 112.8 ± 17.7, uU/mL/10 islets. Similar results were obtained following cryopreservation: glucose at 16.7 mmol/L stimulated a mean ± SEM of 27.9 ± 6.6, uU/mL/10 islets, of insulin in absence of, and 44.9 ± 9.9, uU/mL/10 islets, in presence of, Sertoli cells. Our results show that isolation and cryopreservation of neonatal porcine islets can be successfully accomplished. In addition, coculture with Sertoli cells significantly improves both the yield and functional capacity of islets following cryopreservation.     

Heterogenous expression of endocrine and progenitor cells within the neonatal porcine pancreatic lobes–Implications for neonatal porcine islet xenotransplantation

Neonatal porcine islets (NPIs) are a source of islets for xenotransplantation. In the pig, the pancreatic lobes remain separate, thus, when optimizing NPI isolation, the pancreatic lobes included in the pancreatic digest should be specified. These lobes are the duodenal (DL), splenic (SL) and connecting (CL) lobe that correspond to the head, body-tail, and uncinate process of the human pancreas. In this study we are the first to evaluate all three neonatal porcine pancreatic lobes and NPIs isolated from these lobes. We report, a significant difference in endocrine and progenitor cell composition between lobes, and observed pancreatic duct glands (PDG) within the mesenchyme surrounding exocrine ducts in the DL and CL. Following in vitro differentiation, NPIs isolated from each lobe differed significantly in the percent increase of endocrine cells and final cell composition. Compared to other recipients, diabetic immunodeficient mice transplanted with NPIs isolated from the SL demonstrated euglycemic control as early as 4 weeks (p < 0.05) and achieved normoglycemia by 6 weeks post-transplant (p < 0.01). For the first time we report significant differences between the neonatal porcine pancreatic lobes and demonstrate that NPIs from these lobes differ in xenograft function.     

Long-term survival of pig-to-mouse Islet xenografts

Abstract: Several laboratories are currently able to prepare large amounts of purified porcine islets of proven in vitro viability. The long-term in vivo function of pig islet xenografts has been evaluated in both "nonimmunocompetent" animals (i.e., the nude mouse) and "immunocompetent" animals. In the nude mouse, documentation has been provided for pig islet function for up to 4 months, even though the issue of how quickly porcine islet xenografts restore normal blood glucose in this animal model is still controversial. Interestingly, pig islet xenografts drive glucose metabolism to maintain plasma glucose concentrations at the donor species levels. Porcine islets have been also transplanted into varying "immunocompetent" animals species. Long-term pig islet xenograft survival in rats and larger animals has been achieved by transplanting islets immunoisolated by either macro- or microencapsulation techniques. In the pig-to-mouse experimental model, freshly prepared, nonimmunoisolated islets survived long-term (for up to 50–60 days) when anti-CD4 antibody treatment was given temporarily posttransplant. Neither the addition of either mouse and/or pig anti-lymphocyte serum, nor the use of 1 week, low-temperature cultured, or cryopreserved islets did further prolong the survival. When 2 to 3 week cultured islets were transplanted into anti-CD4 antibody treated mice, function of the xenografts was observed at 100 days posttransplant in 75% of the animals. Thus, long-term survival of pig-to-mouse islet xenografts in both nonimmunocompetent and immunocompetent animals is achievable. Although further studies are needed to fully understand the hormonal and metabolic effects of the islet xenografts, as well as to extend some of the results obtained in mice to larger animal models, the in vivo data available so far support the use of pig islets for potential use in human xenotransplantation studies.     

Isolation of porcine pancreatic islets for xenotransplantation studies: Effects of low collagenase digestion temperatures

Abstract: Islets of Langerhans were isolated from porcine pancreata by a modification of our previously described method. The modification involved the use of a low temperature of collagenase digestion (30°C) during the process of islet isolation. The resulting islets were then evaluated in vitro and in vivo and compared to islets isolated at the regular 37°C temperature.
The islets produced at the low temperature were more compact compared to the control islets. In the dextran density gradient these islets were deposited at the interface of the 1.060 and 1.068 g/ml density bands as compared to 1.050 and 1.060 g/ml for the control islets. In addition, the experimental islets contained a higher proportion of compact, unfragmented islets (68%) compared to the regular islets (55%), and their uptake of the dithizone stain was considerably slower than with the control islets. All ten batches of freshly isolated microencapsulated islets produced at both temperatures responded to the glucose stimulation. After 4 weeks of in vitro culture the islets of both groups microencapsulated in alginate-polylysine-alginate (APA) microcapsules still retained glucose responsiveness, with the experimental islets demonstrating significantly higher responsiveness to the high glucose (16.7 mM) and 0.1 mM IB MX stimulation. The morphology of unencapsulated islets in the experimental group following 4 weeks of in vitro culture indicates much firmer islet structure compared to the control islets. In addition, the unencapsulated experimental islets following the 4 week culture were still found to have secreted insulin when exposed to glucose. In transplantation studies both the experimental and the control islets normalized diabetic hyperglycemia in diabetic mice in a comparable fashion. In general, the low temperature digestion results in superior islets in terms of their morphology, viability, and physiological function.     

Islet autotransplantation studies in pigs to assess viability and implantation site as a prelude to xenotransplantation

Abstract: Before pig-to-human xenotransplantation trials can begin, it must be demonstrated that porcine islets can engraft and function in pig recipients. Collagenase dispersion of the pancreas followed by density gradient centrifugation is effective in preparing viable discrete islets from species such as rodents and dogs, but these methods frequently results in marked fragmentation of porcine islets. Thus, we hypothesized that by limiting the digestion period and eliminating the purification step to minimize islet fragmentation, a viable preparation suitable for transplantation would be obtained. Purification is known to be unnecessary in dogs and humans as long as a site able to accommodate impure tissue is used. In this study, we autotransplanted impure islets prepared by two different methods (collagenase/semi-automated or mechanical chopping) without further purification into either the peritoneal cavity (N=46), spleen (N=5), or liver (N=5) of 56 pancreatectomized pigs. The semi-automated method (N=45) yielded a mean of 426,000 ± 41,696 islets, and the mechanical chopping method (N=11) yielded 165,438 ± 19,611 islets (P<0.05). Intraportal transplantation of impure islets was uniformly lethal due to portal hypertension. Intraperitoneal transplants were well tolerated with no pigs dying periooperatively. None of the mechanically prepared islet grafts transplanted in the peritoneal cavity functioned (N=11). Of the grafts prepared by the collagenase/semi-automated method, none transplanted into the spleen functioned; however, 13 of the 35 (37%) transplanted into the peritoneal cavity did function (30 day insulin-independent survival). Of these, four recipients were normoglycemic (blood glucose <200 mg/dl), and glucose disposal during an intravenous glucose tolerance test was similar to normal pigs. Although islets prepared by the semi-automated/collagenase digestion function in less than half of recipients, the intraperitoneal site generally is less receptive than other sites of engraftment. Unlike pigs, humans can tolerate the transplantation of impure islets intraportally. Thus, the proportion that functioned in this study likely under estimates the potential function rate using the intraportal site in humans. This study demonstrates by in vivo testing the viability of an impure porcine islet preparation and supports its use in preclinical xenograft models and human xenograft trials.     

Studies of the isolation, viability, and preservation of purified islets after surgical pancreatectomy in large pigs

Abstract: Previous studies have shown separation of mass quantities of islets from slaughterhouse pig pancreases. In these studies, we examined the isolation, viability, and preservation of pig islets obtained by surgical pancreatectomy. Pigs aged 6 to 30 months weighing 100 to 200 kg were subjected to laparotomy under general anesthesia. The splenic lobe of pancreas was mobilized without warm ischemia. Islets were immediately isolated by collagenase perfusion through the duct, automated dissociation, and Ficoll purification. Yields of islets (150 μm size) were 8500 to 9300/g for two different collagenases before purification. Purification yielded 90% pure islets, but yields decreased to 400 to 600/g. Perifusate insulin release was biphasic after glucose/theophylline with 3 to 5-fold stimulation. Following culture/cryopreservation marked islet losses occurred but viability was preserved. Quantities of 1,500 islet equivalents resulted in euglycemia in nude mice. These data show that mass quantities of viable islets can be isolated after pancreatectomy, but there is marked islet loss during purification and subsequent preservation due to inherent islet fragility.     

Xenogeneic transplantation of porcine islets: an overview

The extreme demand for human organs or tissues for transplantation has driven the search for viable alternatives. Pigs are considered a possible source of tissue for a number of reasons including shared physiology, plentiful supply, short gestation, and, more recently, the generation of transgenic animals. Porcine islets show promise as a source of islets for the treatment of type 1 diabetes mellitus. Porcine islets regulate glucose levels in the same physiologic range as humans, and porcine insulin has been used for years as an exogenous source of insulin for glucose control. In this review, we discuss the advantages and disadvantages of the use of adult or neonatal porcine islets, the immunologic challenges facing transplantation of xenogeneic islets, and the concerns regarding transmission of infectious agents between species. Porcine islets isolated from both adult and neonatal pigs are capable of restoring euglycemia in experimental animal models of diabetes. Adult islets are more difficult to isolate, whereas neonatal islets have great proliferation potential but require several weeks to function posttransplantation. Xenogeneic islets are susceptible to complement-mediated lysis after the binding of preformed natural antibodies and cellular immunity involving both macrophages and CD4+ T cells. In addition, the potential for transmission of porcine endogenous retroviruses, porcine cytomegalovirus, and porcine lymphotropic herpesvirus type 1 are all concerns that must be addressed. Despite the challenges facing xenotransplantation, the extreme need for donor organs and tissues continues to drive progress toward overcoming the unique issues associated with transplantation between species.     

Isolation of pig pancreatic islets by a new method with hydraulic shaking: preliminary report

The limited availability of human pancreas represents a serious problem in islet transplantation. In the past few years many efforts have been made to isolate pancreatic islets from large mammals in order to achieve valid and reproducible isolation methods [1–4]. For several reasons swine may be considered an ideal source of islet tissue because of the similarity between human and porcine insulin and because of the easy availability of pig pancreata. Some papers have been published recently on this topic with good results [1, 5–7]. However, some problems, such as islet dissociation into single cells after collagenase digestion, are not completely solved. In this article, an automated method involving a hydraulic shaking system is described for islet isolation from the pig pancreas, developed in our laboratory and derived from Ricordi's model.     

Examining the potential for porcine‐derived islet cells to harbour viral pathogens

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal‐derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture.     

Pig peripheral blood mononuclear cells are directly associated with the thrombotic microangiopathy that complicates the induction of chimerism in pig-to-baboon xenotransplantation

The infusion of large numbers of porcine cells into primates in order to induce specific immunologic tolerance by mixed hematopoietic chimerism, results in thrombotic microangiopathy that can be fatal. For this reason, it is important to study in vitro the interaction of primate endothelial cells with pig cells. We show that pig peripheral blood mononuclear cells (p-PBMC) activate human endothelial cells (hECs) through direct contact. Thus, when endothelial cells are cultured in the presence of p-PBMC, overexpression of VCAM-1 and E-selectin adhesion molecules occurs within 3 h of culture and continues for at least 9 h. The co-culture of p-PBMC and hECs also results in an important adhesion of human platelets to both types of cell. Thus, viewed with the microscope, platelets aggregate above the endothelial cells and also around the pig cells. We present data that suggest that the presence of p-PBMC may be more important with regard to the increase of platelet adhesion to the endothelial cells than the activation alone of the cells. Our results also show that p-PBMC, and not the activated endothelia or the culture supernatant of activated hECs, are able to activate the coagulation cascade because they are able to generate thrombin when added to defibrinated human plasma. Overall, these findings suggest that p-PBMC are of primary importance for the development of the thrombotic disorders that occur in primates transplanted with pig progenitor cells.     

Engraftment of Adult Porcine Islet Xenografts in Diabetic Nonhuman Primates Through Targeting of Costimulation Pathways

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.     

Culture of the islets of Langerhans for transplantation

The isolated islets of Langerhans are the most available donors for transplantation. As the preservation of the isolated islets is difficult, we attempted to keep these tissues viable by use of an organ culture. Islets of Langerhans from adult Wistar rats were isolated by a collagenase technique and cultured in air-CO₂ (95-5%) incubator at 37°C. Insulin contents of the culture media which was changed every 3 days ranged from 1097 to 1434 μU/ml during the 80 days' culture period. Transplantation of these islets into the portal vein of streptozotocin-induced diabetic rats resulted in a good recovery from the diabetic state. These studies indicate that cultured islets do preserve their original biological abilities.     

Accelerated functional maturation of isolated neonatal porcine cell clusters: in vitro and in vivo results in NOD mice

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.