Flexible ZnO-mAb nanoplatforms for selective peripheral blood mononuclear cell immobilization.

Cancer is the second cause of death worldwide. This devastating disease requires specific, fast, and affordable solutions to mitigate and reverse this trend. A step towards cancer-fighting lies in the isolation of natural killer (NK) cells, a set of innate immune cells, that can either be used as biomarkers of tumorigenesis or, after autologous transplantation, to fight aggressive metastatic cells. In order to specifically isolate NK cells (which express the surface NKp30 receptor) from peripheral blood mononuclear cells, a ZnO immunoaffinity-based platform was developed by electrodeposition of the metal oxide on a flexible indium tin oxide (ITO)-coated polyethylene terephthalate (PET) substrate. The resulting crystalline and well-aligned ZnO nanorods (NRs) proved their efficiency in immobilizing monoclonal anti-human NKp30 antibodies (mAb), obviating the need for additional procedures for mAb immobilization. The presence of NK cells on the peripheral blood mononuclear cell (PBMCs) fraction was evaluated by the response to their natural ligand (B7-H6) using an acridine orange (AO)-based assay. The successful selection of NK cells from PBMCs by our nanoplatform was assessed by the photoluminescent properties of AO. This easy and straightforward ZnO-mAb nanoplatform paves the way for the design of biosensors for clinic diagnosis, and, due to its inherent biocompatibility, for the initial selection of NK cells for autotransplantation immunotherapies.

NK-Cell-Encapsulated Porous Microspheres via Microfluidic Electrospray for Tumor Immunotherapy.

Immunotherapy has recently garnered significant research interest in the field of clinical cancer management. The potential of tumor immunotherapy has been demonstrated for targeting a variety of tumors, both in vivo and in vitro, yielding some remarkable therapeutic effects. Herein, inspired by the stem cell niche, we developed a scale-up approach to generating porous microspheres with encapsulated natural killer (NK) cells via microfluidic electrospray for in situ tumor immunotherapy. The generated microspheres contained porous microstructures with tunable morphologies because of versatile but precise fluid control in the microfluidic electrospray system. NK-92MI cells encapsulated in porous microspheres were protected from the outer complex environment, allowing for improved proliferation and functionality. As observed, perforin and granzymes were sustainably secreted from the encapsulated NK-92MI cells, which exhibited robust killing effects on tumors both in vitro and in vivo. With continual proliferation, NK-92MI cells budded from the surface of porous microspheres and migrated into the surrounding residual tumor tissues, further destroying tumor cells. More importantly, no side effects owing to the native host immune system were observed by injecting the NK-92MI cell-encapsulated microspheres into tumors in vivo. Therefore, the NK-cell-encapsulated porous microspheres show great potential for tumor immunotherapy, offering a robust and attractive treatment option for cancer patient management.

Immunoisolation of pancreatic islets via thin-layer surface modification.

Islet transplantation is an alternative method of replacing exogenous insulin to treat type 1 diabetes. However, transplantation of allo- or xenograft islets causes the activation of host's immune reaction, which leads to the failure of the transplanted grafts. Immunosuppressive-sparing strategies have been introduced to avoid adverse effects associated with a long-term use of the immunosuppressive drugs. In this regard, macro/microencapsulation, surface camouflage, and surface modification with immune-privileged cells have been performed to protect the transplanted islets against instant blood-mediated inflammatory reactions or immune reactions. However, the increased size of the encapsulated islets after transplantation leads to insufficient oxygen and nutrients for the islets, causing most of them to undergo apoptosis. Therefore, recent studies have aimed at reducing the capsule thickness while maintaining immunoprotective ability of encapsulated islets. In this review, we discuss several techniques of thin-layer surface coating of pancreatic islets using a variety of polymers, therapeutic agents (TA), TA-loaded nano or microparticles, and living cells.

3D-3 Tumor Models in Drug Discovery for Analysis of Immune Cell Infiltration.

The cross talk between tumor cells and other cells present in the tumor microenvironment such as stromal and immune cells highly influences the behavior and progression of disease. Understanding the underlying mechanisms of interaction is a prerequisite to develop new treatment strategies and to prevent or at least reduce therapy failure in the future. Specific reactivation of the patient's immune system is one of the major goals today. However, standard two-dimensional (2D) cell culture techniques lack the necessary complexity to address related questions. Novel three-dimensional (3D) in vitro models-embedded in a matrix or encapsulated in alginate-recapitulate the in vivo situation much better. Cross talk between different cell types can be studied starting from co-cultures. As cancer immune modulation is becoming a major research topic, 3D in vitro models represent an important tool to address immune regulatory/modulatory questions for T, NK, and other cells of the immune system. The 3D systems consisting of tumor cells, fibroblasts, and immune cells (3D-3) already proved as a reliable tool for us. For instance, we made use of those models to study the molecular mechanisms of the cross talk of non-small cell lung cancer (NSCLC) and fibroblasts, to unveil macrophage plasticity in the tumor microenvironment and to mirror drug responses in vivo. Generation of those 3D models and how to use them to study immune cell infiltration and activation will be described in the present book chapter.

Designing Magnetically Responsive Biohybrids Composed of Cord Blood-Derived Natural Killer Cells and Iron Oxide Nanoparticles.

We report the generation of magnetically responsive, cord blood-derived natural killer (NK) cells using iron oxide nanoparticles (IONPs). NK cells are a promising immune cell population for cancer cell therapy as they can target and lyse target tumor cells without prior education. However, NK cells cannot home to disease sites based on antigen recognition, instead relying primarily on external stimuli and chemotactic gradients for transport. Hence, we hypothesized that conjugating IONPs onto the surface of NK cells provides an added feature of magnetic homing to the NK cells, improving their therapeutic function. We describe a robust design for conjugating the IONPs onto the surface of NK cells, which maintains their intrinsic phenotype and function. The conferred magnetic-responsiveness is utilized to improve the cytolytic function of the NK cells for target cells in 2D and 3D models. These findings demonstrate the feasibility of improving NK cell homing and therapeutic efficacy with our NK:IONP "biohybrid".

Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. In this study, we examined the differences in early innate immune response to human PSC or unpurified stroma (stromal vascular fraction [SVF]) during the in vivo process of bone formation. Briefly, SVF or PSC from the same patient sample were implanted intramuscularly in the hindlimb of severe combined immunodeficient (SCID) mice using an osteoinductive demineralized bone matrix carrier. Histological examination of early inflammatory infiltrates was examined by hematoxylin and eosin and immunohistochemical staining (Ly-6G, F4/80). Results showed significantly greater neutrophilic and macrophage infiltrates within and around SVF in comparison to PSC-laden implants. Differences in early postoperative inflammation among SVF-laden implants were associated with reduced osteogenic differentiation and bone formation. Similar findings were recapitulated with PSC implantation in immunocompetent mice. Exaggerated postoperative inflammation was associated with increased IL-1α, IL-1ß, IFN-γ, and TNF-α gene expression among SVF samples, and conversely increased IL-6 and IL-10 expression among PSC samples. These data document a robust immunomodulatory effect of implanted PSC, and an inverse correlation between host inflammatory cell infiltration and stromal progenitor cell-mediated ossification.

Nonoptical Detection of Allergic Response with a Cell-Coupled Gate Field-Effect Transistor.

In this study, we report the label-free and reliable detection of allergic response using a cell-coupled gate field-effect transistor (cell-based FET). Rat basophilic leukemia (RBL-2H3) cells were cultured as a signal transduction interface to induce allergic reaction on the gate oxide surface of the FET, because IgE antibodies, which bind to Fcε receptors at the RBL-2H3 cell membrane, are specifically cross-linked by allergens, resulting in the allergic response of RBL-2H3 cells. In fact, the surface potential at the FET gate decreased owing to secretions such as histamine from the IgE-bound RBL-2H3 cells, which reacted with the allergen. This is because histamine, as one of the candidate secretions, shows basicity, resulting in a change in pH around the cell/gate interface. That is, the RBL-2H3-cell-based FET used in this study was originally from an ion-sensitive FET (ISFET), whose oxide surface (Ta2O5) with hydroxyl groups is fully responsive to pH on the basis of the equilibrium reaction. The allergic response of RBL-2H3 cells on the gate was also confirmed by estimating the amount of ß-hexosaminidase released together with histamine and was analyzed using the electrical properties based on an inflammatory response of secreted histamine with the vascular endothelial cell-based FET. Thus, the allergic responses were monitored in a nonoptical and real-time manner using the cell-based FETs with the cellular layers on the gate, which reproduced the in vivo system and were useful for the reliable detection of the allergic reaction.

Co-encapsulation and co-transplantation of mesenchymal stem cells reduces pericapsular fibrosis and improves encapsulated islet survival and function when allografted.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated islets. A strategy to combat PFO is the use of mesenchymal stem cells (MSC). MSC have anti-inflammatory properties and their potential can be enhanced by stimulation with proinflammatory cytokines. This study investigated whether co-encapsulation or co-transplantation of MSC with encapsulated islets would reduce PFO and improve graft survival. Stimulating MSC with a cytokine cocktail of IFN-γ and TNF-α enhanced their immunosuppressive potential by increasing nitric oxide production and secreting higher levels of immunomodulatory cytokines. In vitro, co-encapsulation with MSC did not affect islet viability but significantly enhanced glucose-induced insulin secretion. In vivo, normoglycemia was achieved in 100% mice receiving islets co-encapsulated with stimulated MSC as opposed to 71.4% receiving unstimulated MSC and only 9.1% receiving encapsulated islets alone. Microcapsules retrieved from both unstimulated and stimulated MSC groups had significantly less PFO with improved islet viability and function compared to encapsulated islets alone. Levels of peritoneal immunomodulatory cytokines IL-4, IL-6, IL-10 and G-CSF were significantly higher in MSC co-encapsulated groups. Similar results were obtained when encapsulated islets and MSC were co-transplanted. In summary, co-encapsulation or co-transplantation of MSC with encapsulated islets reduced PFO and improved the functional outcome of allotransplants.

RareCyte® CTC Analysis Step 2: Detection of Circulating Tumor Cells by CyteFinder® Automated Scanning and Semiautomated Image Analysis.

The RareCyte CyteFinder instrument is an automated scanner that allows rapid identification of circulating tumor cells (CTCs) on microscope slides prepared by the AccuCyte process (see Chapter 13 ) and stained by immunofluorescence. Here, we present the workflow for CyteFinder scanning, analysis, and CyteMapper scan review which includes CTC confirmation and report generation.

RareCyte® CTC Analysis Step 3: Using the CytePicker® Module for Individual Cell Retrieval and Subsequent Whole Genome Amplification of Circulating Tumor Cells for Genomic Analysis.

The CytePicker module built into the RareCyte CyteFinder instrument allows researchers to easily retrieve individual cells from microscope slides for genomic analyses, including array CGH, targeted sequencing, and next-generation sequencing. Here, we describe the semiautomated retrieval of CTCs from the blood processed by AccuCyte (see Chapter 13) and amplification of genomic DNA so that molecular analysis can be performed.

Minocycline modulates NFκB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds. METHODS: Modulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague-Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student's t test was used to determine p values for all assays with multiple or two conditions, respectively. RESULTS: Minocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization. CONCLUSIONS: Minocycline modulates the NFκB pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.

Historical Perspectives and Current Challenges in Cell Microencapsulation.

The principle of immunoisolation of cells is based on encapsulation of cells in immunoprotective but semipermeable membranes that protect cells from hazardous effects of the host immune system but allows ingress of nutrients and outgress of therapeutic molecules. The technology was introduced in 1933 but has only received its deserved attention for its therapeutic application for three decades now.In the past decade important advances have been made in creating capsules that provoke minimal or no inflammatory responses. There are however new emerging challenges. These challenges relate to optimal nutrition and oxygen supply as well as standardization and documentation of capsule properties.It is concluded that the proof of principle of applicability of encapsulated grafts for treatment of human disease has been demonstrated and merits optimism about its clinical potential. Further innovation requires a much more systematic approach in identifying crucial properties of capsules and cellular grafts to allow sound interpretations of the results.

Immunological Challenges Facing Translation of Alginate Encapsulated Porcine Islet Xenotransplantation to Human Clinical Trials.

Transplantation of alginate-encapsulated islets has the potential to treat patients suffering from type I diabetes, a condition characterized by an autoimmune attack against insulin-secreting beta cells. However, there are multiple immunological challenges associated with this procedure, all of which must be adequately addressed prior to translation from trials in small animal and nonhuman primate models to human clinical trials. Principal threats to graft viability include immune-mediated destruction triggered by immunogenic alginate impurities, unfavorable polymer composition and surface characteristics, and release of membrane-permeable antigens, as well as damage associated molecular patterns (DAMPs) by the encapsulated islets themselves. The lack of standardization of significant parameters of bioencapsulation device design and manufacture (i.e., purification protocols, surface-modification grafting techniques, alginate composition modifications) between labs is yet another obstacle that must be overcome before a clinically effective and applicable protocol for encapsulating islets can be implemented. Nonetheless, substantial progress is being made, as is evident from prolonged graft survival times and improved protection from immune-mediated graft destruction reported by various research groups, but also with regard to discoveries of specific pathways involved in explaining observed outcomes. Progress in the latter is essential for a comprehensive understanding of the mechanisms responsible for the varying levels of immunogenicity of certain alginate devices. Successful translation of encapsulated islet transplantation from in vitro and animal model testing to human clinical trials hinges on application of this knowledge of the pathways and interactions which comprise immune-mediated rejection. Thus, this review not only focuses on the different factors contributing to provocation of the immune reaction by encapsulated islets, but also on the defining characteristics of the response itself.

Encapsulation of Adenovirus BMP2-Transduced Cells with PEGDA Hydrogels Allows Bone Formation in the Presence of Immune Response.

Gene therapy approaches have been difficult to implement due to pre-existing immunity against the virus used for delivery. To circumvent this problem, a cell-based approach was developed that avoided the use of free virus within the animal. However, even cells transduced in vitro with E1- to E3-deleted adenovirus encoding bone morphogenetic protein 2 (AdBMP2) resulted in the production of virus-neutralizing antibodies in mice. Furthermore, when mice received an intramuscular injection of nonencoding adenovirus (AdEmpty)-transduced cells, AdBMP2-transduced cells were unable to launch bone formation when an intramuscular injection of these BMP2-producing cells was delivered 1 week later. This phenomenon was not observed in NOD/SCID mice, and could be overcome in C57BL/6 mice by encapsulating the adenovirus-transduced cells in a nondegradable hydrogel poly(ethylene glycol) diacrylate (PEGDA). Data collectively suggest that PEGDA hydrogel encapsulation of AdBMP2-transduced cells prevents pre-existing immunity from suppressing BMP2-induced bone formation.

Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies.

A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies.

Protection Provided by an Encapsulated Live Attenuated ΔabcBA Strain of Brucella ovis against Experimental Challenge in a Murine Model.

This study aimed to evaluate the Brucella ovis ΔabcBA strain as a vaccine candidate in the murine model. BALB/c mice were subcutaneously or intraperitoneally immunized with a single dose or three doses of the B. ovis ΔabcBA strain and then were challenged with wild-type B. ovis. Single or multiple immunizations provided only mild protection, with significantly smaller numbers of wild-type B. ovis CFU in the livers of immunized mice but not in the spleens. Encapsulation of B. ovis ΔabcBA significantly improved protection against experimental challenges in both BALB/c and C57BL/6 mice. Furthermore, immunization with encapsulated B. ovis ΔabcBA markedly prevented lesions in the spleens and livers of experimentally challenged mice. These results demonstrated that the encapsulated B. ovis ΔabcBA strain confers protection to mice; therefore, this strain has potential as a vaccine candidate for rams.

Macrophage embedded fibrin gels: an in vitro platform for assessing inflammation effects on implantable glucose sensors.

The erroneous and unpredictable behavior of percutaneous glucose sensors just days following implantation has limited their clinical utility for diabetes management. Recent research has implicated the presence of adherent inflammatory cells as the key mitigating factor limiting sensor functionality in this period of days post-implantation. Here we present a novel in vitro platform to mimic the cell-embedded provisional matrix that forms adjacent to the sensor immediately after implantation for the focused investigation of the effects of early stage tissue response on sensor function. This biomimetic surrogate is formed by imbibing fibrin-based gels with physiological densities of inflammatory RAW 264.7 macrophages. When surrounding functional sensors, macrophage-embedded fibrin gels contribute to sensor signal declines that are similar in both shape and magnitude to those observed in previous whole blood and small animal studies. Signal decline in the presence of gels is both metabolically-mediated and sensitive to cell type and activation. Computational modeling of the experimental setup is also presented to validate the design by showing that the cellular glucose uptake parameters necessary to achieve such experimental declines align well with literature values. Together, these data suggest this in vitro provisional matrix surrogate may serve as an effective screening tool for testing the biocompatibility of future glucose sensor designs.

Requirement of the expression of 3-phosphoglycerate dehydrogenase for traversing S phase in murine T lymphocytes following immobilized anti-CD3 activation.

Murine resting (G(0)) T lymphocytes contained no detectable mRNA of 3-phosphoglycerate dehydrogenase (PHGDH) catalyzing the first step in the phosphorylated pathway of l-serine biosynthesis. Immobilized anti-CD3 activation of G(0) T cells expressed the PHGDH mRNA in G(1) with a maximum level in S phase. G(0) T cells activated with either immobilized anti-CD3 plus CsA or PBu(2), which failed to drive the activated T cells to enter S phase, did not express the PHGDH mRNA unless exogenous rIL-2 was added. Blocking of IL-2R signaling by adding anti-IL-2 and anti-IL-2Rα resulted in no expression of the PHGDH mRNA during immobilized anti-CD3 activation of G(0) T cells. Deprivation of l-serine from culture medium or addition of antisense PHGDH oligonucleotide significantly reduced [(3)H]TdR incorporation of activated T cells. These results indicate that the PHGDH gene expression, dictated by IL-2R signaling, is a crucial event for DNA synthesis during S phase of activated T cells.

A study of the transport and immobilisation mechanisms of human red blood cells in a paper-based blood typing device using confocal microscopy.

Recent research on the use of bioactive paper for human blood typing has led to the discovery of a new method for identifying the haemagglutination of red blood cells (RBCs). When a blood sample is introduced onto paper treated with the grouping antibodies, RBCs undergo haemagglutination with the corresponding grouping antibodies, forming agglutinated cell aggregates in the paper. A subsequent washing of the paper with saline buffer could not remove these aggregates from the paper; this phenomenon provides a new method for rapid, visual identification of the antibody-specific haemagglutination reactions and thus the determination of the blood type. This study aims to understand the mechanism of RBC immobilization inside the paper which follows haemagglutination reactions. Confocal microscopy is used to observe the morphology of the free and agglutinated RBCs that are labelled with FITC. Chromatographic elution patterns of both agglutinated and non-agglutinated RBCs are studied to gain insight into the transport behaviour of free RBCs and agglutinated aggregates. This work provides new information about RBC haemagglutination inside the fibre network of paper on a microscopic level, which is important for the future design of paper-based blood typing devices with high sensitivity and assaying speed.