3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks.

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.

3D Bioprinting of Collagen-based Microfluidics for Engineering Fully-biologic Tissue Systems.

Microfluidic and organ-on-a-chip devices have improved the physiologic and translational relevance of in vitro systems in applications ranging from disease modeling to drug discovery and pharmacology. However, current manufacturing approaches have limitations in terms of materials used, non-native mechanical properties, patterning of extracellular matrix (ECM) and cells in 3D, and remodeling by cells into more complex tissues. We present a method to 3D bioprint ECM and cells into microfluidic collagen-based high-resolution internally perfusable scaffolds (CHIPS) that address these limitations, expand design complexity, and simplify fabrication. Additionally, CHIPS enable size-dependent diffusion of molecules out of perfusable channels into the surrounding device to support cell migration and remodeling, formation of capillary-like networks, and integration of secretory cell types to form a glucose-responsive, insulin-secreting pancreatic-like microphysiological system.

Embedded 3D Printing of Thermally-Cured Thermoset Elastomers and the Interdependence of Rheology and Machine Pathing.

Thermoset elastomers are widely used high-performance materials due to their thermal stability, chemical resistance, and mechanical properties. However, established casting and molding techniques limit the overall 3D complexity of parts that can be fabricated. Advanced manufacturing methods such as 3D printing have improved design flexibility and reduced development time but have proved challenging using thermally-cured thermosets due to their viscosity, slow gelation kinetics and high surface tension. To address this, freeform reversible embedding (FRE) 3D printing extrudes thermosets such as polydimethylsiloxane (PDMS) elastomer within a carbomer support bath, but due to the liquid-like state of the prepolymer during extrusion has been limited to hollow structures. Here, we have significantly improved FRE printing through rheological modification of PDMS with a thixotropic additive (1.0-10.0 wt%) that imparts a yield stress (30-120 Pa) to help control filament morphology. Further, to minimize the interaction of the nozzle with previously printed PDMS we implemented print process controls consisting of region-specific slicing, filament retraction, and non-print travel moves outside of the print. The combined result is the FRE printing of PDMS in complex 3D parts with high fidelity, establishing a 3D printing methodology that can be used broadly with thermally-cured thermoset elastomers and related polymers.

Development of a high-performance open-source 3D bioprinter.

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.

Long-Fiber Embedded Hydrogel 3D Printing for Structural Reinforcement.

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.

FRESH 3D bioprinting a contractile heart tube using human stem cell-derived cardiomyocytes.

Here we report the 3D bioprinting of a simplified model of the heart, similar to that observed in embryonic development, where the heart is a linear tube that pumps blood and nutrients to the growing embryo. To this end, we engineered a bioinspired model of the human heart tube using freeform reversible of embedding of suspended hydrogels 3D bioprinting. The 3D bioprinted heart tubes were cellularized using human stem cell-derived cardiomyocytes and cardiac fibroblasts and formed patent, perfusable constructs. Synchronous contractions were achieved ∼3-4 days after fabrication and were maintained for up to a month. Immunofluorescent staining confirmed large, interconnected networks of sarcomeric alpha actinin-positive cardiomyocytes. Electrophysiology was assessed using calcium imaging and demonstrated anisotropic calcium wave propagation along the heart tube with a conduction velocity of ∼5 cm s-1. Contractility and function was demonstrated by tracking the movement of fluorescent beads within the lumen to estimate fluid displacement and bead velocity. These results establish the feasibility of creating a 3D bioprinted human heart tube and serve as an initial step towards engineering more complex heart muscle structures.

In situvolumetric imaging and analysis of FRESH 3D bioprinted constructs using optical coherence tomography.

As 3D bioprinting has grown as a fabrication technology, so too has the need for improved analytical methods to characterize engineered constructs. This is especially challenging for engineered tissues composed of hydrogels and cells, as these materials readily deform when trying to assess print fidelity and other properties non-destructively. Establishing that the 3D architecture of the bioprinted construct matches its intended anatomic design is critical given the importance of structure-function relationships in most tissue types. Here we report development of a multimaterial bioprinting platform with integrated optical coherence tomography forin situvolumetric imaging, error detection, and 3D reconstruction. We also report improvements to the freeform reversible embedding of suspended hydrogels bioprinting process through new collagen bioink compositions, gelatin microparticle support bath optical clearing, and optimized machine pathing. This enables quantitative 3D volumetric imaging with micron resolution over centimeter length scales, the ability to detect a range of print defect types within a 3D volume, and real-time imaging of the printing process at each print layer. These advances provide a comprehensive methodology for print quality assessment, paving the way toward the production and process control required for achieving regulatory approval and ultimately clinical translation of engineered tissues.

Endothelial superoxide dismutase 2 is decreased in sickle cell disease and regulates fibronectin processing.

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.

3D Bioprinted Patient-Specific Extracellular Matrix Scaffolds for Soft Tissue Defects.

Soft tissue injuries such as volumetric muscle loss (VML) are often too large to heal normally on their own, resulting in scar formation and functional deficits. Decellularized extracellular matrix (dECM) scaffolds placed into these wounds have shown the ability to modulate the immune response and drive constructive healing. This provides a potential solution for functional tissue regeneration, however, these acellular dECM scaffolds are challenging to fabricate into complex geometries. 3D bioprinting is uniquely positioned to address this, being able to create patient-specific scaffolds based on clinical 3D imaging data. Here, a process to use freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting and computed tomography (CT) imaging to build large volume, patient-specific dECM patches (≈12 × 8 × 2 cm) for implantation into canine VML wound models is developed. Quantitative analysis shows that these dECM patches are dimensionally accurate and conformally adapt to the surface of complex wounds. Finally, this approach is extended to a human VML injury to demonstrate the fabrication of clinically relevant dECM scaffolds with precise control over fiber alignment and micro-architecture. Together these advancements represent a step towards an improved, clinically translatable, patient-specific treatment for soft tissue defects from trauma, tumor resection, and other surgical procedures.

A high performance open-source syringe extruder optimized for extrusion and retraction during FRESH 3D bioprinting.

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.

Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication.

In tissue engineering, an unresolved challenge is how to build complex 3D scaffolds in order to recreate the structure and function of human tissues and organs. Additive manufacturing techniques, such as 3D bioprinting, have the potential to build biological material with unprecedented spatial control; however, printing soft biological materials in air often results in poor fidelity. Freeform Reversible Embedding of Suspended Hydrogels (FRESH) is an embedded printing approach that solves this problem by extruding bioinks within a yield-stress support bath that holds the bioinks in place until cured. In this Perspective, we discuss the challenges of 3D printing soft and liquid-like bioinks and the emergence for FRESH and related embedded printing techniques as a solution. This includes the development of FRESH and embedded 3D printing within the bioprinting field and the rapid growth in adoption, as well as the advantages of FRESH printing for biofabrication and the new research results this has enabled. Specific focus is on the customizability of the FRESH printing technique where the chemical composition of the yield-stress support bath and aqueous phase crosslinker can all be tailored for printing a wide range of bioinks in complex 3D structures. Finally, we look ahead at the future of FRESH printing, discussing both the challenges and the opportunities that we see as the biofabrication field develops.

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.

FRESH 3D Bioprinting a Full-Size Model of the Human Heart.

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.

3D printed biaxial stretcher compatible with live fluorescence microscopy.

Mechanical characterization and tensile testing of biological samples is important when determining the material properties of a tissue; however, performing tensile testing and tissue stretching while monitoring cellular changes via fluorescence microscopy is often challenging. Additionally, commercially available cell/tissue stretchers are often expensive, hard to customize, and limited in their fluorescence imaging compatibility. We have developed a 3D printed Open source Biaxial Stretcher (OBS) to be a low-cost stage top mountable biaxial stretching system for use with live cell fluorescence microscopy in both upright and inverted microscope configurations. Our OBS takes advantage of readily available open source desktop 3D printer hardware and software to deliver a fully motorized high precision (10 ± 0.5 µm movement accuracy) low cost biaxial stretching device capable of 4.5 cm of XY travel with a touch screen control panel, and an integrated heated platform with sample bath to maintain cell and tissue viability. Further, we designed a series of tissue mounts and clamps to accommodate varying samples from synthetic materials to biological tissue. By creating a low-profile design, we can directly mount the stretcher onto a microscope stage, and through coordinated biaxial stretching we maintain a constant field of view facilitating real-time sample tracking and time-lapse fluorescence imaging.

Fibronectin-based nanomechanical biosensors to map 3D surface strains in live cells and tissue.

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.

Organ-on-e-chip: Three-dimensional self-rolled biosensor array for electrical interrogations of human electrogenic spheroids.

Cell-cell communication plays a pivotal role in coordination and function of biological systems. Three-dimensional (3D) spheroids provide venues to explore cellular communication for tissue development and drug discovery, as their 3D architecture mimics native in vivo microenvironments. Cellular electrophysiology is a prevalent signaling paradigm for studying electroactive cells. Currently, electrophysiological studies do not provide direct, multisite, simultaneous investigation of tissues in 3D. In this study, 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field-effect transistors or passive microelectrodes were implemented to interface human cardiac spheroids in 3D. The arrays provided continuous and stable multiplexed recordings of field potentials with high sensitivity and spatiotemporal resolution, supported with simultaneous calcium imaging. Our approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment, such as arrhythmias.