Natural Cryoprotective and Cytoprotective Agents in Cryopreservation: A Focus on Melatonin.

Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the cryopreservation process by examining its drawbacks and the role of cytoprotective agents in protecting cell physiology. Natural cryoprotective and cytoprotective agents, such as antifreeze proteins, sugars and natural deep eutectic systems, have been compared with synthetic ones, addressing their mechanisms of action and efficacy of protection. The final part of this article focuses melatonin, a hormonal substance with antioxidant properties, and its emerging role as a cytoprotective agent for somatic cells and gametes, including ovarian tissue, spermatozoa and spermatogonial stem cells.

Co-microencapsulation of human umbilical cord-derived mesenchymal stem and pancreatic islet-derived insulin producing cells in experimental type 1 diabetes.

INTRODUCTION: Post-partum umbilical cord Wharton Jelly-derived adult mesenchymal stem cells (hUCMS) hold anti-inflammatory and immunosuppressive properties. Human pancreatic islet-derived progenitor cells (hIDC) may de-differentiate, and subsequently re-differentiate into insulin producing cells. The two cell types share common molecules that facilitate their synergistic interaction and possibly crosstalk, likely useful for the cell therapy of type 1 diabetes (T1D). MATERIALS AND METHODS: Upon microencapsulation in sodium alginate (AG), hUCMS and hIDC were able to form cell co-aggregates that looked well integrated and viable. We then grafted microencapsulated hUCMS/hIDC co-aggregates into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, and observed an acquired ability of cells to produce and store hormones. Finally, we transplanted these biohybrid constructs into NOD mice with recent onset, spontaneous overt diabetes, observing a decline of blood glucose levels. RESULTS: In vitro, we have shown that hUCMS inhibited proliferation of allogeneic polymorphonuclear blood cells from patients with T1D, while promoting expansion of FoxP3+ Tregs. Reversal of hyperglycemia in diabetic NODs seems to suggest that hUCMS and hIDC, upon co-microencapsulation, anatomically and functionally synergized to accomplish two goals: maintain tracer insulin output by hIDC, while exploting the immunoregulatory properties of hUCMS. CONCLUSION: We have gathered preliminary evidence that the two adult stem cell types within AG microcapsules, may synergistically promote tracer insulin production, while "freezing" the autoimmune disease process, and help reversal of the recent onset hyperglycemia in a spontaneous, autoimmune rodent model of diabetes, the NOD mouse, with no need for pharmacologic immunosuppression.

Remission of hyperglycemia in spontaneously diabetic NOD mice upon transplant of microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS).

BACKGROUND: Our previous in vitro demonstration of the immunoregulatory effects of microencapsulated hUCMS on human peripheral blood mononuclear cells (PBMCs) extracted from patients with recent onset, type 1 diabetes mellitus (DM), prompted us to test our product for xenograft (TX) in non obese diabetic (NOD) mice with spontaneous DM. METHODS: We transplanted microencapsulated hUCMS into the peritoneal cavity of NOD mice with either severe or mild DM. Blood glucose (BG) levels were monitored following TX, in either basal or upon glucose stimulation. RESULTS: Only the NODs with mild DM showed full and sustained remission of hyperglycemia throughout 216 days post-TX, unlike recipients with severe DM, where no remission of hyperglycemia was attained, as reflected by erratic BG levels at all times. CONCLUSIONS: These data suggest that the stage of DM disease process in NOD mice, reflecting steady decline of residual b-cell mass, plays a pivotal role in determining the success of this cell therapy approach for treatment of DM.

A Comparison of Lysosomal Enzymes Expression Levels in Peripheral Blood of Mild- and Severe-Alzheimer's Disease and MCI Patients: Implications for Regenerative Medicine Approaches.

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer's Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (ß-Hexosaminidase, ß-Galctosidase, ß-Galactosylcerebrosidase, ß-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of ß-Galctosidase (Gal), ß-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of Aß-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD.

Restoration of t cell substes of patients with type 1 diabetes mellitus by microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells: An in vitro study.

Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) might apply to treating chronic autoimmune disorders, as already shown for Sjögren's syndrome, including type 1 diabetes mellitus (T1D). Since naked hUCMS grafts encountered restraints, we enveloped hUCMS, within immunoisolatory microcapsules (CpS-hUCMS), made of our endotoxin-free, clinical grade alginate. We then examined the vitro effects of interferon (IFN)-γ-pretreated CpS-hUCMS on Th17 and Treg of T1D patients (n=15) and healthy controls (n=10). Peripheral blood mononuclear cells (PBMCs) were co-cultured with PBMC/CpS-hUCMS: lymphocyte proliferation was assessed by carboxyfluorescein succinimidyl esther (CFSE) dilution assay, and phenotypic analysis of regulatory and effector Tc was also performed. Cytokine expression was performed by bead array and qPCR on IFN-γ-pretreated hUCMS before PBMCs co-culture. CpS-hUCMS restored a correct Treg/Th17 ratio, relevant to the T1D disease process. In summary, we have preliminarily developed a new biohybrid system, associated with immunoregulatory properties, that is ready for in vivo application.

Xenograft of microencapsulated Sertoli cells restores glucose homeostasis in db/db mice with spontaneous diabetes mellitus.

BACKGROUND: Increased abdominal fat and chronic inflammation in the expanded adipose tissue of obesity contribute to the development of insulin resistance and type 2 diabetes mellitus (T2D). The emerging immunoregulatory and anti-inflammatory properties of Sertoli cells have prompted their application to experimental models of autoimmune/inflammatory disorders, including diabetes. The main goal of this work was to verify whether transplantation of microencapsulated prepubertal porcine Sertoli cells (MC-SC) in the subcutaneous abdominal fat depot of spontaneously diabetic and obese db/db mice (homozygous for the diabetes spontaneous mutation [Leprdb ]) would: (i) improve glucose homeostasis and (ii) modulate local and systemic immune response and adipokines profiles. METHODS: Porcine prepubertal Sertoli cells were isolated, according to previously established methods and enveloped in Barium alginate microcapsules by a mono air-jet device. MC-SC were then injected in the subcutaneous abdominal fat depot of db/db mice. RESULTS: We have preliminarily shown that graft of MC-SC restored glucose homeostasis, with normalization of glycated hemoglobin values with improvement of the intraperitoneal glucose tolerance test in 60% of the treated animals. These results were associated with consistent increase, in the adipose tissue, of uncoupling protein 1 expression, regulatory B cells, anti-inflammatory macrophages and a concomitant decrease of proinflammatory macrophages. Furthermore, the treated animals showed a reduction in inducible NOS and proinflammatory molecules and a significant increase in an anti-inflammatory cytokine such as IL-10 along with concomitant rise of circulating adiponectin levels. The anti-hyperglycemic graft effects also emerged from an increased expression of GLUT-4, in conjunction with downregulation of GLUT-2, in skeletal muscle and liver, respectively. CONCLUSIONS: Preliminarily, xenograft of MC-SC holds promises for an effective cell therapy approach for treatment of experimental T2D.

In vitro immunomodulatory effects of microencapsulated umbilical cord Wharton jelly-derived mesenchymal stem cells in primary Sjögren's syndrome.

OBJECTIVE: Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) are easy to retrieve in bulk. They may interact with immune cells by either cell contact or soluble factors. Little evidence is currently available on potential therapeutic application of hUCMS to systemic autoimmune disorders such as primary SS (pSS). We have recently developed an endotoxin-free alginate gel that can be used to microencapsulate different cell types for graft into non-immunosuppressed hosts. We aimed to assess the in vitro effects of IFN-γ-pretreated microencapsulated (CpS)-hUCMS on T cells of pSS. METHODS: Ten pSS patients and 10 healthy donors were selected. Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood to establish co-cultures with CpS-hUCMS. Lymphocyte proliferation and phenotypic analysis was performed by flow cytometry and real-time PCR on IFN-γ-pretreated hUCMS was performed before PBMCs co-culture. RESULTS: We found that CpS-hUCMS suppress pSS T cell proliferation and restore the Treg/Th17 ratio, thereby possibly positively impacting the pSS disease process. CONCLUSION: We have developed a new biohybrid drug delivery system that now waits for clinical application in autoimmune diseases, including pSS.

Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy.

BACKGROUND: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGß-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation.

Stem cells for pancreatic ß-cell replacement in diabetes mellitus: actual perspectives.

PURPOSE OF REVIEW: Type 1 and type 2 diabetes mellitus represent a widespread metabolic disorder, related to autoimmune ß-cell destruction and insulin resistance, leading to ß-cell dysfunction, respectively, that are associated with severe chronic complications with irreversible multiorgan morphological and functional damage. Conventional treatment, based on exogenous insulin or oral agents may control and delay but not prevent the disease complications, which has lead, so far, to a steady increase in mortality and morbidity. ß-Cell substitution cell therapy, initially pursued by whole pancreatic and isolated islet transplantation, with scarce and limited efficiency, now is looking at the new technologies for cell and molecular therapy for diabetes, based on stem cells. RECENT FINDINGS: Pancreatic endocrine cells regeneration might replenish the destroyed ß-cell pool, with neogenerated ß-cell derived from pancreatic and extrapancreatic stem cell sources. Additionally, embryonic or adult stem cells derived from different cell lineages, and able to differentiate into ß-like cell elements, may not only restore the original insulin secretory patterns but also exert the immunomodulatory effects aimed at interrupting the ß-cell-directed autoimmune destruction vicious cycle. SUMMARY: These new strategies may, one day, provide for the final cure of diabetes mellitus.

Biomaterials for extrusion-based bioprinting and biomedical applications.

Amongst the range of bioprinting technologies currently available, bioprinting by material extrusion is gaining increasing popularity due to accessibility, low cost, and the absence of energy sources, such as lasers, which may significantly damage the cells. New applications of extrusion-based bioprinting are systematically emerging in the biomedical field in relation to tissue and organ fabrication. Extrusion-based bioprinting presents a series of specific challenges in relation to achievable resolutions, accuracy and speed. Resolution and accuracy in particular are of paramount importance for the realization of microstructures (for example, vascularization) within tissues and organs. Another major theme of research is cell survival and functional preservation, as extruded bioinks have cells subjected to considerable shear stresses as they travel through the extrusion apparatus. Here, an overview of the main available extrusion-based printing technologies and related families of bioprinting materials (bioinks) is provided. The main challenges related to achieving resolution and accuracy whilst assuring cell viability and function are discussed in relation to specific application contexts in the field of tissue and organ fabrication.

GLP-1 RAs and SGLT2i: two antidiabetic agents associated with immune and inflammation modulatory properties through the common AMPK pathway.

Immune cells and other cells respond to nutrient deprivation by the classic catabolic pathway of AMPK (Adenosine monophosphate kinase). This kinase is a pivotal regulator of glucose and fatty acids metabolism, although current evidence highlights its role in immune regulation. Indeed AMPK, through activation of Foxo1 (Forkhead box O1) and Foxo3 (Forkhead box O3), can regulate FOXP3, the key gene for differentiation and homeostasis of Tregs (T regulators lymphocytes). The relevance of Tregs in the onset of T1D (Type 1 diabetes) is well-known, while their role in the pathogenesis of T2D (Type 2 diabetes) is not fully understood yet. However, several studies seem to indicate that Tregs may oppose the progression of diabetic complications by mitigating insulin resistance, atherosclerosis, and damage to target organs (as in kidney disease). Hence, AMPK and AMPK-activating agents may play a role in the regulation of the immune system. The connection between metformin and AMPK is historically known; however, this link and the possible related immune effects are less studied about SGLT2i (Sodium-glucose co-transport 2 inhibitors) and GLP1-RAs (Glucagon-like peptide-1 receptor agonists). Actual evidence shows that the negative caloric balance, induced by SGLT2i, can activate AMPK. Conversely and surprisingly, an anabolizing agent like GLP-1RAs can also upregulate this kinase through cAMP (Cyclic adenosine monophosphate) accumulation. Therefore, both these drugs can likely lead to the activation of the AMPK pathway and consequential proliferation of Tregs. These observations seem to confirm not only the metabolic but also the immunoregulatory effects of these new antidiabetic agents.

Human induced pluripotent stem cells (hiPSC), enveloped in elastin-like recombinamers for cell therapy of type 1 diabetes mellitus (T1D): preliminary data.

Introduction: Therapeutic application and study of type 1 diabetes disease could benefit from the use of functional ß islet-like cells derived from human induced pluripotent stem cells (hiPSCs). Considerable efforts have been made to develop increasingly effective hiPSC differentiation protocols, although critical issues related to cost, the percentage of differentiated cells that are obtained, and reproducibility remain open. In addition, transplantation of hiPSC would require immunoprotection within encapsulation devices, to make the construct invisible to the host's immune system and consequently avoid the recipient's general pharmacologic immunosuppression. Methods: For this work, a microencapsulation system based on the use of "human elastin-like recombinamers" (ELRs) was tested to envelop hiPSC. Special attention was devoted to in vitro and in vivo characterization of the hiPSCs upon coating with ERLs. Results and Discussion: We observed that ELRs coating did not interfere with viability and function and other biological properties of differentiated hiPSCs, while in vivo, ELRs seemed to afford immunoprotection to the cell grafts in preliminary in vivo study. The construct ability to correct hyperglycemia in vivo is in actual progress.

Emerging of a new CD3+CD31HCD184+ tang cell phenothype in Sjögren's syndrome induced by microencapsulated human umbilical cord matrix-derived multipotent stromal cells.

Background: Sjögren's syndrome (SS) is an autoimmune disease hallmarked by infiltration and destruction of exocrine glands. Currently, there is no therapy that warrants full recovery of the affected tissues. Umbilical cord-derived multipotent stromal cells, microincapsulated in an endotoxin-free alginate gel (CpS-hUCMS), were shown to modulate the inflammatory activity of PBMCs in SS patients in vitro, through release of soluble factors (TGFß1, IDO1, IL6, PGE2, VEGF). These observations led us to set up the present study, aimed at defining the in vitro effects of CpS-hUCMS on pro- and anti-inflammatory lymphocyte subsets involved in the pathogenesis of SS. Methods and results: Peripheral blood mononuclear cells (PBMCs) upon collection from SS patients and matched healthy donors, were placed in co-culture with CpS-hUCMS for five days. Cellular proliferation and T- (Tang, Treg) and B- (Breg, CD19+) lymphocyte subsets were studied by flow cytometry, while Multiplex, Real-Time PCR, and Western Blotting techniques were employed for the analysis of transcriptome and secretome. IFNγ pre-treated hUCMS were assessed with a viability assay and Western Blotting analysis before co-culture. After five days co-culture, CpS-hUCMS induced multiple effects on PBMCs, with special regard to decrease of lymphocyte proliferation, increase of regulatory B cells and induction of an angiogenic T cell population with high expression of the surface marker CD31, that had never been described before in the literature. Conclusion: We preliminarily showed that CpS-hUCMS can influence multiple pro- and anti-inflammatory pathways that are deranged in SS. In particular, Breg raised and a new Tang phenothype CD3+CD31HCD184+ emerged. These results may considerably expand our knowledge on multipotent stromal cell properties and may open new therapeutic avenues for the management of this disease, by designing ad hoc clinical studies.

The role behind the scenes of Tregs and Th17s in Hashimoto's thyroiditis: Toward a pivotal role of FOXP3 and BACH2.

In Hashimoto's thyroiditis (HT), the genetic bases play a central role in determining development of the disease. In particular, the most frequent genes involved in the onset of HT are the Human Leukocyte Antigen (HLA). However, there are other genes and transcription factors in the autoimmune background of HT, both isolated and as part of autoimmune polyendocrine syndromes (APS). Recently more interest is being fueled toward BACH2 (BTB Domain and CNC Homolog 2), that promotes Tregs (T regulators lymphocytes) differentiation and enhances Treg-mediated immunity. The synergistic interaction between environmental agents and the aforementioned genes leads to the onset of autoimmunity and ultimately to damage of the thyroid gland. In this scenario, the role of Th17 (T helper-17 lymphocytes) and Treg cells is still less defined as compared to action of Th1 cells (T helper-1 lymphocytes) and cytotoxic lymphocytes (CD8 + T lymphocytes). Evidences show that an imbalance of Th17/Treg ratio represents a prognostic factor with respect to the gland damage. Moreover, the deficient ability of Treg to inhibit the proliferation of T cells against the self can break the immune balance. In light of these considerations, the use of genetic panels and the progress of immunotherapy could allow for better targeting treatment and preventive interventions in subjects with potential or early stage of HT.

Immunoisolation of pancreatic islet grafts with no recipient's immunosuppression: actual and future perspectives.

In spite of steady and remarkable progress, islet transplantation in patients with type 1 diabetes mellitus (T1DM) continues to face two major bottlenecks: inadequate availability of human pancreatic donors and necessity to totally immunosuppress the graft recipients lifelong. Microencapsulation of the islet grafts within highly biocompatible and selective permeable biomembranes could obviate use of the immunosuppressants, while potentially offering the opportunity to use a wide array of insulin-producing cells, in active development, including xenogeneic pig islets. Although macrodevices and microcapsules, which essentially differ by size/configuration, and both serve for immunoisolation devices, have been used for many years with initial human applications, new products on development in both areas might open new perspectives for more focused use in patients with T1DM. Physical-chemical properties and material engineering of these devices are critically reviewed to assess where we actually stand and where the future expansion of these technologies may go.

Microencapsulation of cells and molecular therapy of type 1 diabetes mellitus: The actual state and future perspectives between promise and progress.

The history of microencapsulation of live cells started with an idea of Thomas MS Chang in 1964, thereafter applied to isolated pancreatic islets by Anthony M Sun in 1980. The original aim was to provide isolated cells with an immune-protective shield, to prevent physical contact between the transplanted cells and the host's immune system, with retention of the microcapsules' biocompatibility and physical-chemical properties over time. In particular, this revolutionary approach essentially applied to islet grafts, in diabetic recipients who are not immunosuppressed, at a preclinical (rodents) and, subsequently, clinical level. Among the different chemistries potentially suitable for microencapsulation of live cells, alginic acid-based polymers, originally proposed by Sun, proved to be superior to all others in the following decades. In fact, only alginic acid-based microcapsules, containing allogeneic islets, ultimately entered pilot human clinical trials in patients with type 1 diabetes mellitus, as immuno-selectiveness and biocompatibility of alginic acid-hydrogels were never matched by other biopolymers. With problems related to human islet procurement coming into a sharper focus, in conjunction with technical limits of the encapsulated islet grafting procedures, new challenges are actually being pursued, with special regard to developing both new cellular systems - able to release immunomodulatory molecules and insulin itself - and new microencapsulation methods, with the use of novel polymeric formulations, under actual scrutiny. The use of embryonic and adult stem cells, within microcapsules, should address the restricted availability of cadaveric human donor-derived islets, whereas a new generation of newly-engineered microcapsules could better fulfill issues with graft site and long-term retention of biopolymer properties.

Microencapsulated Wharton Jelly-derived adult mesenchymal stem cells as a potential new therapeutic tool for patients with COVID-19 disease: an in vitro study.

BACKGROUND: The recent newly appeared Coronavirus disease (COVID-19), caused by an enveloped RNA virus named "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", is associated with severe respiratory morbidity and mortality. Recent studies have shown that lymphopenia and a cytokine mass release represent important pathogenic features, with clinical evidence of dyspnea and hypoxemia, often leading to acute respiratory distress syndrome (ARDS), in severely ill patients, with a high death toll. Currently, stem cells are actively being investigated for their potential use in many "untreatable" diseases. In this regard and in particular, Mesenchymal Stem Cells (MSC), due to their intrinsic features, including either ability to impact on regulation of the immune system, or association with both anti-viral and anti-inflammatory properties, or potential for differentiation into several cell lineages, have become a promising tool for cell and molecular-based therapies. On this background, we wished to explore whether human umbilical cord-derived mesenchymal stem cells (hUCMS) would represent a potential viable therapeutic approach for the management of critically ill COVID19 patients. METHODS: We tested the hUCMS effects on peripheral blood mononuclear cell (PBMCs) retrieved from patients with COVID19 (Ethical Committee CEAS Umbria, Italy CER N°3658/20 7, May, 2020), both as free cell monolayers and after envelopment in sodium alginate microcapsules. Both cell systems, after priming with IFN-γ, proved able to produce several immunomodulatory molecules such as IDO1 and HLAG5, although only the microencapsulated hUCMS were associated with massive and dose-dependent production of these factors. RESULTS: The microencapsulated hUCMS improved allo-suppression in mixed lymphocytes reactions (MLRs), while also blunting T helper 1 and T helper 17 responses, that are involved with the cytokine storm and greatly contribute to the patient death. Moreover, we observed that both free and microencapsulated hUCMS permitted 5 days survival of in vitro culture maintained PBMCs extracted from very ill patients. CONCLUSION: We have provided evidence that microencapsulated hUCMS in vitro, seem to represent a powerful tool to impact on several immune pathways, clearly deranged in COVID19 patients. Further study is necessary to begin in vivo assessment of this experimental system, upon determining both, the most appropriate time of the disease onset for intervention, and cell dosage/patient of our experimental product.

Bioactive long-term release from biodegradable microspheres preserves implanted ALG-PLO-ALG microcapsules from in vivo response to purified alginate.

PURPOSE: To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release. METHODS: Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry. RESULTS: Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group. CONCLUSIONS: Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.

Microencapsulated G3C Hybridoma Cell Graft Delays the Onset of Spontaneous Diabetes in NOD Mice by an Expansion of Gitr+ Treg Cells.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet ß-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr-/- mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25-/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.