Search

The application of 3D printing to biological research has provided the tissue engineering community with a method for organizing cells and biological materials into complex 3D structures. While many commercial bioprinting platforms exist, they are expensive, ranging from $5000 to over $1,000,000. This high cost of entry prevents many labs from incorporating 3D bioprinting into their research. Due to the open-source nature of desktop plastic 3D printers, an alternative option has been to convert low-cost plastic printers into bioprinters. Several open-source modifications have been described, but there remains a need for a user-friendly, step-by-step guide for converting a thermoplastic printer into a bioprinter using components with validated performance. Here we convert a low-cost 3D printer, the FlashForge Finder, into a bioprinter using our Replistruder 4 syringe pump and the Duet3D Duet 2 WiFi for total cost of less than $900. We demonstrate that the accuracy of the bioprinter's travel is better than 35 µm in all three axes and quantify fidelity by printing square lattice collagen scaffolds with average errors less than 2%. We also show high fidelity reproduction of clinical-imaging data by printing a scaffold of a human ear using collagen bioink. Finally, to maximize accessibility and customizability, all components we have designed for the bioprinter conversion are provided as open-source 3D models, along with instructions for further modifying the bioprinter for additional use cases, resulting in a comprehensive guide for the bioprinting field. [ABSTRACT FROM AUTHOR]

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response. [Display omitted] • Endogenous WNK1 forms hyperosmotic stress-induced condensates • The intrinsically disordered C-terminal domain mediates WNK1 phase separation • Phase behavior of the WNK1 CTD is evolutionarily conserved despite sequence divergence • WNK1 is a molecular crowding sensor and mediator of regulatory volume increase WNK1 is a molecular crowding sensor that undergoes cell shrinkage-dependent phase separation to restore cell volume. This study provides a clear link between the intrinsically disordered region-mediated phase separation of a protein and cell physiology. [ABSTRACT FROM AUTHOR]

R6022 --> 730.11 --> The pursuit of an ideal model system to study the pathophysiology of vascular disease has included the use of tissue engineered blood vessels (TEBV), vascularizing engineered volumetric tissue, silicone‐based "organ‐on‐a‐chip" platform, and tissue decellularization; yet we are still far from generating engineered vascular tissue that recapitulate native physiology. Despite advances such as 3D bioprinting that have led to new approaches for creating fluidic channels, it remains challenging to produce continuous networks with vessels ranging from large (>6 mm) to small (<1 mm) using native‐like extracellular matrix (ECM) that recapitulates the mechanical properties, geometric organization, and complexity of the cellular and extracellular microenvironment. An ideal engineering strategy would combine the controlled fluid flow achieved by organ‐on‐a‐chip platforms with the ECM structure, composition, and biomechanics of decellularized tissue. Here, using Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting, we spatially define ECM composition and vascular geometry to create collagen‐based microfluidics and an engineered resistance artery scaffold to study how ECM composition, mechanical properties, and fluid flow contribute to vascular disease. A 3D vascular microfluidic CAD model was designed with a hierarchically branching network containing a single inlet and outlet of 1 mm that branched down to 250 µm channels embedded within a collagen scaffold. This model was FRESH printed with 24 mg/mL collagen‐I and yielded a high‐fidelity scaffold with patent internal vasculature. 3D imaging and gauging analysis of the CAD model compared to the collagen scaffold revealed an average positive deviation of 9.9 µm and an average negative deviation of 56.3 µm. To interface with and perfuse the soft collagen microfluidic scaffold we 3D printed a custom bioreactor chamber for long‐term culture capable of >1‐week perfusion under physiological conditions. In addition to embedded vascular networks, FRESH bioprinting allows for direct fabrication of suspended vessels. We designed an ECM scaffold scaled to match a resistance vessel consisting of a collagen‐I frame and suspended vessel (inner diameter of 300 µm, outer diameter of 500 µm). The collagen frame provides overall scaffold stability and allows interfacing to the custom perfusion bioreactor. Using multimaterial FRESH printing, we varied the ECM composition by patterning fibronectin along the inner lumen and collagen‐I within the outer adventitial wall. Biomechanical evaluation using pressure and wire myography revealed a burst pressure >140 mmHg and an average peak tension of 600 mg, comparable to mouse mesenteric resistance arteries. Together, this work highlights the exciting potential for ECM‐based microfluidic scaffolds and vascular tissue engineering that will set the foundation for future work to replicate a healthy and hypertensive vessel microenvironment. In addition to vascular biology, the collagen‐based microfluidics are amenable to other organ systems, and can be utilized to study cell proliferation, metastasis, and ECM remodeling in a 3D environment. [ABSTRACT FROM AUTHOR]

Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage- independent growth , and loss of TMEM16A resulted in inhibition of tumor growth both and in vitro in vitro in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal--regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation . Together, our findings provide a in vitro mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a genetic red blood cell disorder characterized by increased reactive oxygen species (ROS) and a concordant reduction in antioxidant capacity in the endothelium. Superoxide dismutase 2 (SOD2) is a mitochondrial-localized enzyme that catalyzes the dismutation of superoxide to hydrogen peroxide. Decreased peripheral blood expression of SOD2 is correlated with increased hemolysis and cardiomyopathy in SCD. Here, we report for the first time that endothelial cells exhibit reduced SOD2 protein expression in the pulmonary endothelium of SCD patients. To investigate the impact of decreased SOD2 expression in the endothelium, SOD2 was knocked down in human pulmonary microvascular endothelial cells (hPMVECs). We found that SOD2 deficiency in hPMVECs results in endothelial cell dysfunction, including reduced cellular adhesion, diminished migration, integrin protein dysregulation, and disruption of permeability. Furthermore, we uncover that SOD2 mediates changes in endothelial cell function via processing of fibronectin through its inability to facilitate dimerization. These results demonstrate that endothelial cells are deficient in SOD2 expression in SCD patients and suggest a novel pathway for SOD2 in regulating fibronectin processing. [ABSTRACT FROM AUTHOR]

Introduction: Cell-cell communication plays a pivotal role in biological systems’ coordination and function. Electrical properties have been linked to specification and differentiation of stem cells into targeted progeny, such as neurons and cardiomyocytes. Currently, there is a critical need in developing new ways to complement fluorescent indicators, such as Ca2+-sensitive dyes, for direct electrophysiological measurements of cells and tissue. Here, we report a unique transparent and biocompatible graphene-based electrical platform that enables electrical and optical investigation of human embryonic stem cell-derived cardiomyocytes’ (hESC-CMs) intracellular processes and intercellular communication. Methods: Graphene, a honeycomb sp2 hybridized two-dimensional carbon lattice, was synthesized using low pressure chemical vapor deposition system, and was tested for biocompatibility. Au and graphene microelectrode arrays (MEAs) were fabricated using well-established microfabrication methods. Au and graphene MEAs were interfaced with hESC-CMs to perform both optical and electrical recordings.Results: Optical imaging and Raman spectroscopy confirmed the presence of monolayer graphene. Viability assay showed biocompatibility of graphene. Electrochemical characterization proved graphene’s functional activity. Nitric acid treatment further enhanced the electrochemical properties of graphene. Graphene electrodes’ transparency enabled both optical and electrical recordings from hESC-CMs. Graphene MEA detected changes in beating frequency and field potential duration upon β-adrenergic receptor agonist treatment.Conclusion: The transparent graphene platform enables the investigation of both intracellular and intercellular communication processes and will create new avenues for bidirectional communication (sensing and stimulation) with electrically active tissues and will set the ground for investigations reported diseases such as Alzheimer, Parkinson’s disease and arrhythmias. [ABSTRACT FROM AUTHOR]

Three‐dimensional (3D) bioprinting is a promising technology to produce tissue‐like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, a versatile bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers is developed. This family of materials is termed UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin‐like protein (ELP), and polyethylene glycol (PEG) are each used as backbone polymers to create inks with storage moduli spanning from 200 to 10 000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix‐adherent human corneal mesenchymal stromal cells and non‐matrix‐adherent human induced pluripotent stem cell‐derived neural progenitor spheroids are printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting. [ABSTRACT FROM AUTHOR]

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion. [ABSTRACT FROM AUTHOR]