Riccardo Calafiore

Cell encapsulation: Promise and progress

In cell encapsulation, transplanted cells are protected from immune rejection by an artificial, semipermeable membrane, potentially allowing transplantation (allo- or xenotransplantation) without the need for immunosuppression. Yet, despite some promising results in animal studies, the field has not lived up to expectations, and clinical products based on encapsulated cell technology continue to elude the scientific community. This commentary discusses the reasons for this, summarizes recent progress in the field and outlines what is needed to bring this technology closer to clinical application.

Long-Term Metabolic and Immunological Follow-Up of Nonimmunosuppressed Patients With Type 1 Diabetes Treated With Microencapsulated Islet Allografts Four cases

OBJECTIVE To assess long-term metabolic and immunological follow-up of microencapsulated human islet allografts in nonimmunosuppressed patients with type 1 diabetes (T1DM). RESEARCH DESIGN AND METHODS Four nonimmunosuppressed patients, with long-standing T1DM, received intraperitoneal transplant (TX) of microencapsulated human islets. Anti-major histocompatibility complex (MHC) class I–II, GAD65, and islet cell antibodies were measured before and long term after TX. RESULTS All patients turned positive for serum C-peptide response, both in basal and after stimulation, throughout 3 years of posttransplant follow-up. Daily mean blood glucose, as well as HbA1c levels, significantly improved after TX, with daily exogenous insulin consumption declining in all cases and being discontinued, just transiently, only in patient 4. Anti-MHC class I–II and GAD65 antibodies all tested negative at 3 years after TX. CONCLUSIONS The grafts did not elicit any immune response, even in the cases where more than one preparation was transplanted, as a unique finding, compatible with encapsulation-driven “bioinvisibility” of the grafted islets. This result had never been achieved with the recipient’s general immunosuppression.

Transplantation of Pancreatic Islets Contained in Minimal Volume Microcapsules in Diabetic High Mammalians

ABSTRACT: To minimize technical problems relating to excessive size (600–800μ in diameter) of standard alginate microcapsules (CSM) for pancreatic islet graft immunoisolation, we have developed two novel minimal volume, chemically identical, capsule prototypes (MVC): 1) coherent microcapsules (CM), and 2) medium‐size microcapsules (300–400μ, MSM). CM, which envelop each individual islet within a thin alginate hydrogel cast, are prepared by emulsification, whereas MSM are made by atomizing the islet‐alginate suspension through a special microdroplet generator. Upon graft into diabetic rodents, CM have shown to immunoprotect both allo‐ and xenogeneic nondiscordant islets, and restored normoglycemia. In higher mammals, at sub‐therapeutic doses, CM fully immunoprotected islet allografts (pig→pig), but only temporarily xenografts (dog→pig). We then used MSM to immunoisolate canine islet allografts in the peritoneal cavity of dogs with spontaneous insulin‐dependent diabetes. Of three grafted dogs, two showed full remission of hyperglycemia with insulin withdrawal. MSM could represent an intermediate solution between CSM and CM for peritoneal immunoisolated islet transplants.

Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine

BACKGROUND & AIMS Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. METHODS MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed. RESULTS Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-β, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. CONCLUSIONS BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.

Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone

Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective treatment of the disease might require rescuing β cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional β cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-β–dependent emergence of autoantigen-specific regulatory T cells and recovery of β cell function in the diabetic recipients.

A method for the massive separation of highly purified, adult porcine islets of langerhans

A method for the massive and reproducible isolation of highly purified, adult porcine islets of Langerhans is described. The successful combination of donor animal-strain selection with original procedures for pancreas retrieval and enzymatic digestion permitted us to separate uniquely massive concentrations of pure porcine islets with no need for mechanical disruption of the pancreatic tissue. Following our procedure, porcine islets, which fully retain viability and function, can be harvested easily and rapidly. Xenotransplantation of such islets, immunoprotected within algin/polyaminoacidic microcapsules, was associated with complete reversal of hyperglycemia in rodents with either spontaneous or streptozotocin-induced diabetes mellitus.

Alginate microcapsules for pancreatic islet cell graft immunoprotection: struggle and progress towards the final cure for type 1 diabetes mellitus

Lights and shadows have been associated with the use of alginate/polyaminoacidic microcapsules for transplantation of pancreatic islet cells for the therapy of diabetes mellitus, with no recipient pharmacological immunosuppression. In fact, preliminary success in rodents has generally not matched the results achieved in diabetic higher mammals. The restricted availability of cadaveric human donor organs/tissue, coupled to regulatory hurdles in the use of microcapsules in patients, has significantly delayed the progress of microencapsulated islet grafts into pilot clinical trials. While the basic formulation of microcapsules from the author’s laboratory, originally comprised of an alginate gel (AG) core, a double poly-L-ornithine (PLO) coat and an outer AG coat, has virtually remained unchanged, highly purified ‘clinical grade’ AG has been introduced in order to try to surmount regulatory restrictions. In parallel, novel insulin-producing cell types have been employed to fill the capsules, with particular regard to non-human tissue, such as adult and, more recently, neonatal porcine islets. In particular, using neonatal porcine islets enveloped in AG-PLO microcapsules, hyperglycaemia has been corrected in several diabetic animal models. Should standardisation and optimisation problems associated with both AG procurement and other membrane physical–chemical fabrication parameters be surmounted, microcapsules containing either human or, possibly, pig islets, could be close to approval for Phase I human clinical trials.

Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non-immunosuppressed animals.

Pancreatic‐islet‐cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated‐islet‐transplant outcome in several animal models of diabetes are reported.

Production and Characterization of Alginate Microcapsules Produced by a Vibrational Encapsulation Device

The optimization, through a Design of Experiments (DoE) approach, of a microencapsulation procedure for isolated neonatal porcine islets (NPI) is described. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An alginate/polyornithine encapsulation procedure, developed and validated in our laboratory for almost a decade, was used to embody pancreatic islets. We analyzed different experimental parameters including frequency of vibration, amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. We produced calcium—alginate gel microbeads with excellent morphological characteristics as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology, viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

Immunoprotection of pancreatic islet grafts within artificial microcapsules.

To circumvent pancreatic islet graft-directed immune destruction we enveloped porcine islets within highly biocompatible and selectively permeable algin/polyaminoacid microcapsules. These special microspheres were deposited between the inner (permeable) and the outer (impermeable) layers of an artificial, coaxial vascular prosthesis, directly anastomized to blood vessels. Five dogs with spontaneous, insulin-dependent diabetes received microencapsulated porcine islets in arterio-vein iliac prosthesis by-passes. One showed complete and the remainder partial, sustained reversal of hyperglycemia. Microencapsulation may be a potential solution to immunological problems related to islet transplantation.