Metallomic mapping of gut and brain in heavy metal exposed earthworms: A novel paradigm in ecotoxicology.

This study explored the uptake of lead in the epigeic earthworm Dendrobaena veneta exposed to 0, 1000, and 2500 µg Pb/g soil. The soil metal content was extracted using strong acid digestion and water leaching, and analysed by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to estimate absolute and bioavailable concentrations of metals in the soil. The guts and heads of lead-exposed earthworms were processed into formalin-fixed and paraffin embedded sections for high-resolution multi-element metallomic imaging via Laser Ablation ICP-MS (LA-ICP-MS). Metallomic maps of phosphorus, zinc, and lead were produced at 15-µm resolution in the head and gut of D. veneta. Additional 4-µm resolution metallomic maps of the earthworm brains were taken, revealing the detailed localisation of metals in the brain. The Pb bioaccumulated in the chloragogenous tissues of the earthworm in a dose-dependent manner, making it possible to track the extent of soil contamination. The bioaccumulation of P and Zn in earthworm tissues was independent of Pb exposure concentration. This approach demonstrates the utility of LA-ICP-MS as a powerful approach for ecotoxicology and environmental risk assessments.

Bio-electrospraying 3-D Organotypic Human Skin Cultures.

Organotypic 3D tissue models have greatly contributed to understand a wide range of molecular and cellular characteristics within a functional or diseased tissue. Human skin reconstructs which act as models are most useful for a wide range of investigations, ranging from tissue engineering and regenerative medicine, drug development, screening, and discovery to name a few. There are many approaches for reconstructing 3D skin tissue models, however, to date there have been very few that are able to generate organotypic 3D constructs with a single technology having minimal processing steps to finally scalability. The many manifestations of 3D bioprinting have contributed to this endeavor, having said that, the technology's limitations have tempered those reconstructed models, as they are known to contain low cell numbers/concentrations to those having damaged/dead molecules/cells within the reconstructed tissue, which are not desirable, for exploring as tissues models. Contrary to 3D bioprinting approaches, bio-electrosprays have been demonstrated to possess the ability to handle large concentrations of cells and molecules to whole fertilized embryos without damaging them from a molecular level upwards. Consequently, this article demonstrates, for the first time, bio-electrospray's capacity to reconstruct skin-like structures in vitro and its potential in reconstructing full-thickness 3D organotypic human skin tissues.

General Computational Methodology for Modeling Electrohydrodynamic Flows: Prediction and Optimization Capability for the Generation of Bubbles and Fibers.

The application of an electric field on a fluid in motion gives rise to unique features and flow manipulation capabilities. Technologies ranging from bubble formation, droplet generation, fiber spinning, and many others are predicated on this type of flows, often referred to as Electrohydrodynamics (EHD). In this paper, we present a numerical methodology that allows for the modeling of such processes in a generalized way. The method can account for the premixing of various liquid species, the injection of gases in the mixture and the interaction of such complex multiphase flow with an electric field, static or AC. The domain in which these processes take place can be of arbitrary geometric complexity, allowing for design and optimization of complex EHD devices. Our study looks at the critical phases of some of these processes and emphasizes the strong coupling of fluid mechanics and electric fields and the influence of the electric field on fluid flow and vice versa. The conservation of mass and momentum, with appropriate additional force terms coming from the presence of the electric field, and the electrostatic equations are coupled together and solved using the Finite Volume method. The Volume of Fluid (VoF) technique is used to track free surfaces dynamically. The solution procedure iteratively computes electric body and surface forces and then includes those into the Navier-Stokes equation to predict the velocity field and other fluid parameters. No initial shape is assumed for the fluid(s) and charge distributions. The methodology presented handles two-dimensional, axisymmetric. and full three-dimensional cases of arbitrary geometric complexity, allowing for mixing and microfluidic configurations of high levels of realism. We highlight the capability of the method by demonstrating cases like the formation of a Taylor cone, microfluidic bubble generation, jet evolution, and droplet breakup. Results agree well with both existing experimental and computational reports.

The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction.

Controlled Generation of Microspheres Incorporating Extracellular Matrix Fibrils for Three-Dimensional Cell Culture.

A growing body of evidence suggests that studying cell biology in classical two-dimensional formats, such as cell culture plasticware, results in misleading, non-physiological findings. For example, some aspects of cancer biology cannot be observed in 2D, but require 3D culture methods to recapitulate observations in vivo. Therefore, we developed a microsphere-based model to permit 3D cell culture incorporating physiological extracellular matrix components. Bio-electrospraying was chosen as it is the most advanced method to produce microspheres, with THP-1 cells as a model cell line. Bio-electrospraying parameters, such as nozzle size, polymer flow rate, and voltage, were systematically optimized to allow stable production of size controlled microspheres containing extracellular matrix material and human cells. We investigated the effect of bio-electrospraying parameters, alginate type and cell concentration on cell viability using trypan blue and propidium iodide staining. Bio-electrospraying had no effect on cell viability nor the ability of cells to proliferate. Cell viability was similarly minimally affected by encapsulation in all types of alginate tested (MVM, MVG, chemical- and food-grade). Cell density of 5 × 106 cells ml-1 within microspheres was the optimum for cell survival and proliferation. The stable generation of microspheres incorporating cells and extracellular matrix for use in a 3D cell culture will benefit study of many diverse diseases and permit investigation of cellular biology within a 3D matrix.

Cell electrospinning: an in vitro and in vivo study.

Cell electrospinning and aerodynamically assisted bio-threading are novel bioplatforms for directly forming large quantities of cell-laden scaffolds for creating living sheets and vessels in three-dimensions. The functional biological architectures generated will be useful in both the laboratory and the clinic.

Cell electrospinning cardiac patches for tissue engineering the heart.

Cell electrospinning has tremendous applicability to a wide range of uses within both the laboratory and clinic. This has directly resulted from the technology's unique ability to immobilize multiple cell types with a wide range of molecules simultaneously within a fiber during the scaffold generation process. The technology has been shown to generate many cell laden complex architectures from true three-dimensional sheets to those multi-core vessels. Although those studies have demonstrated the versatility of this platform biotechnology, we show here for the first time the ability to immobilize primary cardiac myocytes within these fibers in our quest to develop this technology for creating three-dimensional cardiac patches which could be used for repairing, replacing and rejuvenating damaged, diseased and/or ageing cardiac tissues. These advances are unrivalled by any other technology currently available in the regenerative medicine toolbox, and have many interesting ramifications for repairing a damaged heart.

Cell electrospinning: a novel tool for functionalising fibres, scaffolds and membranes with living cells and other advanced materials for regenerative biology and medicine.

Recent years have seen interest in approaches for directly generating fibers and scaffolds following a rising trend for their exploration in the health sciences. In this review the author wishes to briefly highlight the many approaches explored to date for generating such structures, while underlining their advantages and disadvantages, and their contribution in particular to the biomedical sciences. Such structures have been demonstrated as having implications in both the laboratory and the clinic, as they mimic the native extra cellular matrix. Interestingly the only materials investigated until very recently for generating fibrous architectures employed either natural or synthetic polymers with or without the addition of functional molecule(s). Arguably although such constructs have been demonstrated to have many applications, they lack the one unit most important for carrying out the ability to directly reconstruct a three-dimensional functional tissue, namely living cells. Therefore recent findings have demonstrated the ability to directly form cell-laden fibers and scaffolds in useful quantities from which functional three-dimensional living tissues can be conceived. These recent developments have far-reaching ramifications to many areas of research and development, a few of which range from tissue engineering and regenerative medicine, a novel approach to analyzing cell behavior and function in real time in three-dimensions, to the advanced controlled and targeted delivery of experimental and/or medical cells and/or genes for localized treatment. At present these developments have passed all in vitro and in vivo mouse model based challenge trials and are now spearheading their journey towards initiating human clinical trials.

Cell Electrospinning: Revolutionising Cell Scaffolding for Healthcare.

Electrospinning is a century-old technology, which has recently found its vast applicability to many areas of research and development and its utility in industry. In the context of the life and health sciences, electrospinning for many years has been explored as a unique approach to scaffolding, on which cells are manually or through automated means seeded with cells. Unfortunately, this approach has seen little being achieved, as the voids generated between fibers within a scaffold negate cell infiltration throughout the entire scaffold. This limitation is a bottleneck for electrospinning in its true applicability to the healthcare and medical sciences.

In vitro and in vivo interrogation of bio-sprayed cells.

Bio-sprays can directly form pre-organized cell-bearing structures for applications ranging from engineering functional tissues to the forming of cultures, most useful for modeling disease, to the discovery and development of drugs. Bio-electrosprays and aerodynamically assisted bio-jets, are leading approaches that have been demonstrated as having far-reaching ramifications for regenerative biology and medicine.

Aerodynamically assisted bio-jetting of hematopoietic stem cells.

The research reported in this communication demonstrates the emerging direct cell handling technology now widely referred to as aerodynamically assisted bio-jetting. This is a non-electric field driven approach which directly competes with bio-electrosprays. The technology in these investigations has been explored for the direct handling of live murine primary hematopoietic stem cells. The viability studies demonstrate the complete inertness of this technology for handling such cells for a wide range of applications in both basic biology and clinical medicine. Interestingly these studies pave the way for this technology to undergo development as a flow cell for utility as a sheathless cell most useful in flow cytometry.

Bio-electrosprays: from bio-analytics to a generic tool for the health sciences.

Electrosprays or electrospraying is a process by which an aerosol is generated between two charged electrodes. This aerosol generation methodology has been known for well over a century, and has undergone exploration in aerosol and materials sciences, to many other areas of research and development. In one such exploration, electrosprays were partnered with mass spectrometry for the accurate characterisation of molecules. This technology now widely referred to as electrospray ionisation mass spectrometry (ESI MS) significantly contributes to molecular analysis and cancer biology to name a few. In fact these findings were recognised by the Chemistry Nobel Committee in 2002, and have catapulted electrosprays to many areas of research and development. In this review, the author wishes to introduce and discuss another such recent discovery, where electrosprays have been investigated for directly handling living cells and whole organisms. Over the past few years these electrosprays now referred to as "bio-electrosprays" have undergone rigorous developmental studies both in terms of understanding all the associate physical, chemical and biological sciences for completely assessing their effects, if any on the direct handling of living biological materials. Therefore, the review will bring together all the work that has contributed to fully understanding that bio-electrosprays are an inert technology for directly handling living biological materials, while elucidating some unique features they possess over competing technologies. Hence, demonstrating this approach as a flexible methodology for a wide range of applications spanning bio-analytics, diagnostics to the possible creation of synthetic tissues, for repairing and replacing damaged/ageing tissues, to the targeted and controlled delivery of personalised medicine through experimental and/or medical cells and/or genes. Therefore, elucidating the far reaching ramifications bio-electrosprays have to our health sciences and well-being.

Biosprayed spleen cells integrate and function in mouse models.

Bio-electrospraying (BES) and aerodynamically assisted bio-jetting (AABJ), two non-contact direct cell handling approaches, have recently undergone rigorous scientific testing to assess whether cells retain chemical, physical and more importantly biological functions similarly to their unmanipulated counterparts. Previous in vitro validation of these two approaches has shown that they are inert for the direct handling and distributing of cells with great accuracy. In the present investigation we aim to validate, in vivo, that the spray techniques do not functionally or phenotypically alter splenic cells. By taking advantage of an adoptive transfer mouse model we demonstrated that the in vivo behaviour of treated cells is indistinguishable from unmanipulated cells following adoptive transfer into C57/BL6 mice. Indeed, sprayed cells survived and proliferated in response to antigen activation to similar levels observed in unmanipulated cells. In addition, in vivo sprayed cells displayed identical migratory characteristics to those observed in unmanipulated cells. Thus, demonstrating the inertness of these biosprays. Hence these biotechniques hold great potential for use in the development of three-dimensional cultures, tracking and monitoring cell-interactions and in vitro modelling of disease-states and therapeutics.

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

Molecular characterisation of post-bio-electrosprayed human brain astrocytoma cells.

Bio-electrospraying (BES) is a method for directly jetting living cells under conditions that allow their distribution in the x, y, and z axes. Previous work has been focused on achieving jetting in stable cone-jet mode, which is required for precision placement, and these studies have demonstrated that there are no significant effects of bio-electrospraying on cell morphology or viability. In this work, we examine the biological properties of bio-electrosprayed cells using assays of cellular function that range from the molecular level through to integrated cellular systems, and include proteomics, signal transduction, cell growth and proliferation, and the characterisation of apoptotic blebs. From these molecular methods, we have determined that bio-electrospraying, under the electric field conditions used to achieve stable cone-jet mode, causes no alterations to the biological properties and function of the cells being jetted. Bio-electrosprayed and control cells had similar viability, proliferation properties and virtually indistinguishable cell cycle profiles. The biophysical properties of large conducting (BK) potassium channels were unchanged, as were the pharmacological responses of the endogenous muscarinic and exogenous P2Y(11) receptors, both of which are cell surface receptors of the 7TM superfamily. Proteomic analyses revealed that although three proteins had subtle differences in expression level between bio-electrosprayed and control cells, none of these fold differences was above the 1.5-fold cut-off threshold required for further analyses. These findings support the further development of bio-electrosprays as a viable technology for a wide diversity of tissue engineering, regenerative biology, advanced cellular therapeutics and medicinal applications, having significance in the clinic.

The differentiation and engraftment potential of mouse hematopoietic stem cells is maintained after bio-electrospray.

The bio-electrospray technique has been recently pioneered to manipulate living, immortalised and primary cells, including a wide range of stem cells. Studies have demonstrated that the creation of viable, fully functional in vitro microenvironments is possible using this technique. By modifying the bio-electrospray procedure (referred to as cell electrospinning), a variety of microenvironment morphologies have been fabricated. Because bio-electrospraying of biological material is a relatively new technique, it is important to determine if there are any unwanted consequences to the manipulated cells as a result of the procedure. Here, we establish the validity of the process using a heterogeneous, living population of hematopoietic stem/progenitor cells, using a functional in vitro assay and in vivo mouse model to investigate for side-effects that previous in vitro assays may not have detected. Our studies demonstrate that these bio-protocols have no obvious negative effects, thus indicating significant promise for utility in biological sciences and for a plethora of healthcare applications.

Reimagining Flow Cytometric Cell Sorting.

In this review, a brief history of this unrivaled technology, flow cytometry, is provided, highlighting its past and present advances, with particular focus on "flow cell" technologies. Flow cytometry has truly revolutionized high-throughput single cell analysis, which has tremendous implications, from laboratory to the clinic. This technology embodies what is truly referred to as cross fertile research, merging the physical with the life sciences. This review introduces the recent notable advancements in flow cell technology. This advancement sees the complete removal of liquid sheath flow, which has advanced the technology with the possibility of both the reduction in its foot print, while also simplifying the flow cells explored in cytometry. Interestingly, the novel sheathless flow cell technology demonstrated herein has the flexibility for handling both heterogeneous cell populations and whole organisms, thus demonstrating a versatile flow cell technology for both flow cytometry and fluorescent-activated cell sorting.

Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential.

Background: Bio-electrospray (BES) is a jet-based delivery system driven by an electric field that has the ability to form micro to nano-sized droplets. It holds great potential as a tissue engineering tool as it can be used to place cells into specific patterns. As the human central nervous system (CNS) cannot be studied in vivo at the cellular and molecular level, in vitro CNS models are needed. Human neural stem cells (hNSCs) are the CNS building block as they can generate both neurones and glial cells. Methods: Here we assessed for the first time how hNSCs respond to BES. To this purpose, different hNSC lines were sprayed at 10 kV and their ability to survive, grow and differentiate was assessed at different time points. Results: BES induced only a small and transient decrease in hNSC metabolic activity, from which the cells recovered by day 6, and no significant increase in cell death was observed, as assessed by flow cytometry. Furthermore, bio-electrosprayed hNSCs differentiated as efficiently as controls into neurones, astrocytes and oligodendrocytes, as shown by morphological, protein and gene expression analysis. Conclusions: This study highlights the robustness of hNSCs and identifies BES as a suitable technology that could be developed for the direct deposition of these cells in specific locations and configurations.

Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-α.

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-α is responsible for accelerated Mtb growth, and TNF-α neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-α immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-α concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-α secretion.

Bio-electrospraying living Xenopus tropicalis embryos: investigating the structural, functional and biological integrity of a model organism.

Bio-electrosprays, a recently pioneered direct cell engineering approach, have been demonstrated to handle living cells including stem cells for the development of active specialized and unspecialized microenvironments. This electric field driven technique is currently undergoing vigorous development where the technique is racing towards possible clinical utility. Although this direct cell engineering approach has been elucidated to have no significant effects on the processed cells from a molecular level upwards, the technique needs to demonstrate its potential for use with whole organisms (multi-cellular systems). We believe this is mandatory for whole organisms such as model embryos; developing multi-cellular biological structures are sensitive systems and could possibly be prone to a wide range of embryological disruptions during their dynamic development, post-treatment. Therefore our studies presented herein have investigated the effects on embryos in terms of their structure, function and biological integrity post-bio-electrospraying in comparison to several controls. Our investigations demonstrate the absence of any detectable gross effects on the embryos from a genetic level upwards on post-treated embryos. In fact, these studies clearly elucidate no significant disruptions on the dynamic development of these treated embryos in comparison to those respective controls, thus validating the utility of bio-electrosprays for the careful handling of dynamically developing multi-cellular organisms.