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Hydrogels are candidate building blocks in a wide range of biomaterial applications including soft and biohybrid robotics, microfluidics, and tissue engineering. Recent advances in embedded 3D printing have broadened the design space accessible with hydrogel additive manufacturing. Specifically, the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) technique has enabled the fabrication of complex 3D structures using extremely soft hydrogels, e.g., alginate and collagen, by assembling hydrogels within a fugitive support bath. However, the low structural rigidity of FRESH printed hydrogels limits their applications, especially those that require operation in nonaqueous environments. In this study, we demonstrated long-fiber embedded hydrogel 3D printing using a multihead printing platform consisting of a custom-built fiber extruder and an open-source FRESH bioprinter with high embedding fidelity. Using this process, fibers were embedded in 3D printed hydrogel components to achieve significant structural reinforcement (e.g., tensile modulus improved from 56.78 ± 8.76 to 382.55 ± 25.29 kPa and tensile strength improved from 9.44 ± 2.28 to 45.05 ± 5.53 kPa). In addition, we demonstrated the versatility of this technique by using fibers of a wide range of sizes and material types and implementing different 2D and 3D embedding patterns, such as embedding a conical helix using electrochemically aligned collagen fiber via nonplanar printing. Moreover, the technique was implemented using low-cost material and is compatible with open-source software and hardware, which facilitates its adoption and modification for new research applications.

Mechanical forces are integral to cellular migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution with minimal perturbation in living sytems. Here, we fabricate, calibrate, and test a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to the surface of cells and tissues to measure the magnitude, direction, and strain dynamics from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultra-thin FN lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2–100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrates 2D and 3D surface strain tracking during mechanical deformation of known materials and is validated with finite element modeling. Analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles highlights the NMBS’s ability to dynamically track microscopic tensile and compressive strains across diverse biological systems where forces guide structure and function., The ability to measure strain in cells and tissues in vitro with minimal perturbation and at high spatial resolution has proven challenging. Here the authors develop a fluorescently-labelled fibronectin square lattice mesh that can be applied to the surface of cells and tissues to enable direct quantification and mapping of strain over time.

If I only had a heart 3D bioprinting is still a fairly new technique that has been limited in terms of resolution and by the materials that can be printed. Lee et al. describe a 3D printing technique to build complex collagen scaffolds for engineering biological tissues (see the Perspective by Dasgupta and Black). Collagen gelation was controlled by modulation of pH and could provide up to 10-micrometer resolution on printing. Cells could be embedded in the collagen or pores could be introduced into the scaffold via embedding of gelatin spheres. The authors demonstrated successful 3D printing of five components of the human heart spanning capillary to full-organ scale, which they validated for tissue and organ function. Science , this issue p. 482 ; see also p. 446

Recent advances in embedded three-dimensional (3D) bioprinting have expanded the design space for fabricating geometrically complex tissue scaffolds using hydrogels with mechanical properties comparable to native tissues and organs in the human body. The advantage of approaches such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) printing is the ability to embed soft biomaterials in a thermoreversible support bath at sizes ranging from a few millimeters to centimeters. In this study, we were able to expand this printable size range by FRESH bioprinting a full-size model of an adult human heart from patient-derived magnetic resonance imaging (MRI) data sets. We used alginate as the printing biomaterial to mimic the elastic modulus of cardiac tissue. In addition to achieving high print fidelity on a low-cost printer platform, FRESH-printed alginate proved to create mechanically tunable and suturable models. This demonstrates that large-scale 3D bioprinting of soft hydrogels is possible using FRESH and that cardiac tissue constructs can be produced with potential future applications in surgical training and planning.

This protocol details the step-by-step fabrication process to create an extracellular matrix (ECM) protein-based nanomechanical biosensor (NMBS) via an adaptation of the surface-initiated assembly (SIA) procedure1. The NMBS is a fluorescently labeled, ultra-thin fibronectin lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2-100 µm. By applying the NMBS to the surface of cells and tissues one can directly measure deformation from subcellular to tissue length-scales. The procedure outlined here covers all aspects of NMBS construction from pattern design, photolithography for mold creation, casting of polydimethylsiloxane (PDMS) silicone stamps, conjugation of fluorescent dye to fibronectin, microcontact printing of fibronectin, creation of gelatin carriers2, transfer of the NMBS to gelatin carriers, and the use of gelatin carriers for application of the NMBS onto cells and tissue. The protocol can be broken down into three phases: material preparation (2 days), NMBS patterning (4 hours), and transfer to cell or tissue (1 hour). Material preparation and NMBS patterning can be performed ahead of time and the patterned NMBS can be stored for up to 1 year prior to use.

Mechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

Recent advances in embedded 3D bioprinting have significantly improved the resolution of individual filaments to below 100 μm; however, printing with such small filaments requires accurate extrusion of nanoliter volumes of bioink. Commercially available bioprinters and extruders are expensive and most utilize pneumatic control, which limits the minimum extrusion volume and prevents retraction (pulling bioink back into the reservoir), which is essential to printing high resolution features and complex internal geometry. Here we present a new generation of our open-source syringe pump designed for extrusion-based 3D bioprinting of soft materials: the Replistruder 4. The Replistruder 4 takes advantage of the geometry customizability and ease of 3D plastic printing while improving performance by integrating mass produced high-precision linear motion components. Simultaneously this new syringe pump remains compact and lightweight enough for several to be utilized on a 3D bioprinter for multimaterial bioprinting. To facilitate multiple use cases the Replistruder 4 is compatible with a range of syringes including disposable BD and Hamilton gastight syringes. In addition, we describe the process of designing clamps for other syringes. We demonstrate the performance of a Replistruder 4 with a 2.5 mL Hamilton gastight syringe by printing collagen type I constructs with individual filaments comprising 3.35 nL and patent channels down to 300 μm in width. With smaller volume Hamilton gastight syringes this performance can be further improved. Thus, the Replistruder 4 provides an open-source solution to print soft materials at the resolution limits of current embedded bioprinting platforms.

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, we developed a versatile, bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers. We term this family of materials UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) were each used as backbone polymers to create inks with storage moduli spanning 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 were 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.

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 Ca(2+)-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 sp(2) 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-018-0525-z) contains supplementary material, which is available to authorized users.

A current challenge for proteomics is detecting proteins over the large concentration ranges found in complex biological samples such as whole-cell extracts. Currently, no unbiased, whole-proteome analysis scheme is capable of detecting the full range of cellular proteins. This is due in part to the limited dynamic range of the detectors used to sense proteins or peptides. We present a new technology, structured illumination (SI) gel imager, which detects fluorescently labeled proteins in electrophoretic gels over a 1 000 000-fold concentration range. SI uses computer-generated masks to attenuate the illumination of highly abundant proteins, allowing for long exposures of low-abundance proteins, thus avoiding detector saturation. A series of progressively masked gel images are assembled into a single, very high dynamic range image. We demonstrate that the SI imager can detect proteins over a concentration range of approximately 1 000 000-fold, making it a useful tool for comprehensive, unbiased proteome-wide surveys.