Functional validation of the simplified in vitro 3D Co-culture based BBB model.

Various methods of generating 2D and 3D in vitro blood-brain barrier (BBB) models have previously been published with the objective of developing therapeutics for brain diseases. In general, published methods including our published method demonstrate that in vivo-like semi-permeable barrier can be generated. To further verify that an in vitro BBB model closely represents BBB, functional validation is required. Here, we functionally validate our in vitro 3D BBB model using rituximab as a representative therapeutic antibody and previously published anti-TfR (transferrin receptor) antibodies as representative BBB-penetrating antibodies. We demonstrate that our BBB model can efficiently block rituximab while allowing receptor-mediated transcytosis (RMT) of anti-TfR antibodies. In addition, we showed that RMT efficacy of anti-TfR antibodies with different binding affinity can be displayed using our BBB model. In conclusion, this demonstrates that our BBB model functionally mimics the BBB as well as having BBB-like physical properties, further establishing our BBB model as a screening tool for discovery and development of therapeutics for brain diseases.

Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell.

The blood-brain barrier (BBB) is a major hurdle for treatment of brain diseases. To overcome this, precise and reproducible BBB model is one of the key factors for successful evaluation of BBB-penetrating efficacy of developmental drugs. Thus, in vitro BBB model recapitulating the physiological structure of the BBB is a valuable tool for drug discovery and development for brain diseases. Here, we develop a simplified 3D co-culture-based BBB model using immortalized human brain endothelial cells and immortalized human astrocytes mixed with Matrigel allowing model preparation within 30 min. We directly compare our 3D BBB model to a 2D BBB model comprised solely of immortalized brain endothelial cells, to demonstrate that our 3D BBB model blocks penetration of Dextran molecules with various molecular weights, remain durable and impermeable even in a BBB-degrading condition, and rapidly form tight junctions while the 2D BBB model do not. In conclusion, this establishes our simplified 3D BBB model as a valuable tool for high throughput screening of drug candidates for brain diseases.

Rapamycin and abundant TCR stimulation are required for the generation of stable human induced regulatory T cells.

OBJECTIVES: Regulatory T cells (Tregs) are a vital sub-population of CD4+ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4+ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF-ß, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α-CD3/CD28 bead ratios on human iTreg stability. METHODS: We assess iTregs generated with various concentrations of rapamycin and differing ratios of α-CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg-specific demethylation region (TSDR) status. RESULTS: iTregs generated in the presence of TGF-ß, ATRA, rapamycin and a higher ratio of α-CD3/CD28 beads were highly suppressive and stable upon in vitro re-stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation. CONCLUSION: This work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re-stimulation. Further validation in pre-clinical models will be needed to ensure its suitability for applications in adoptive transfer.

Bioprinting an Artificial Pancreas for Type 1 Diabetes.

PURPOSE OF REVIEW: Pancreatic islet cell transplantation is currently the only curative cell therapy for type 1 diabetes mellitus. However, its potential to treat many more patients is limited by several challenges. The emergence of 3D bioprinting technology from recent advances in 3D printing, biomaterials, and cell biology has provided the means to overcome these challenges. RECENT FINDINGS: 3D bioprinting allows for the precise fabrication of complex 3D architectures containing spatially distributed cells, biomaterials (bioink), and bioactive factors. Different strategies to capitalize on this ability have been investigated for the 3D bioprinting of pancreatic islets. In particular, with co-axial bioprinting technology, the co-printability of islets with supporting cells such as endothelial progenitor cells and regulatory T cells, which have been shown to accelerate revascularization of islets and improve the outcome of various transplantations, respectively, has been achieved. 3D bioprinting of islets for generation of an artificial pancreas is a newly emerging field of study with a vast potential to improve islet transplantation.

Development of a Coaxial 3D Printing Platform for Biofabrication of Implantable Islet-Containing Constructs.

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes.

Murine and Non-Human Primate Dendritic Cell Targeting Nanoparticles for in Vivo Generation of Regulatory T-Cells.

Porous silicon nanoparticles (pSiNP), modified to target dendritic cells (DC), provide an alternate strategy for the delivery of immunosuppressive drugs. Here, we aimed to develop a DC-targeting pSiNP displaying c-type lectin, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), and CD11c monoclonal antibodies. The in vivo tracking of these fluorescent DC-targeting nanoparticles was assessed in both C57BL/6 mice and common marmosets ( Callithrix jacchus) by intravenous injection (20 mg/kg). Rapamycin and ovalbumin (OVA)323-339 peptide loaded pSiNP were employed to evaluate their ability to generate murine CD4+CD25+FoxP3+ regulatory T-cells in vivo within OVA sensitized mice. In vivo, pSiNP migrated to the liver, kidneys, lungs, and spleen in both mice and marmosets. Flow cytometry confirmed pSiNP uptake by splenic and peripheral blood DC when functionalized with targeting antibodies. C57BL/6 OVA sensitized mice injected with CD11c-pSiNP loaded with rapamycin + OVA323-339 produced a 5-fold higher number of splenic regulatory T-cells compared to control mice, at 40 days post-pSiNP injection. These results demonstrate the importance of the immobilized targeting antibodies to enhance cellular uptake and enable the in vivo generation of splenic regulatory T-cells.