Advances in 3D Bioprinted Cardiac Tissue Using Stem Cell-Derived Cardiomyocytes.

The ultimate goal of cardiac tissue engineering is to generate new muscle to repair or replace the damaged heart. This requires advances in stem cell technologies to differentiate billions of cardiomyocytes, together with advanced biofabrication approaches such as 3D bioprinting to achieve the requisite structure and contractile function. In this concise review, we cover recent progress in 3D bioprinting of cardiac tissue using pluripotent stem cell-derived cardiomyocytes, key design criteria for engineering aligned cardiac tissues, and ongoing challenges in the field that must be addressed to realize this goal.

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.

Electrocatalytic on-site oxygenation for transplanted cell-based-therapies.

Implantable cell therapies and tissue transplants require sufficient oxygen supply to function and are limited by a delay or lack of vascularization from the transplant host. Previous exogenous oxygenation strategies have been bulky and had limited oxygen production or regulation. Here, we show an electrocatalytic approach that enables bioelectronic control of oxygen generation in complex cellular environments to sustain engineered cell viability and therapy under hypoxic stress and at high cell densities. We find that nanostructured sputtered iridium oxide serves as an ideal catalyst for oxygen evolution reaction at neutral pH. We demonstrate that this approach exhibits a lower oxygenation onset and selective oxygen production without evolution of toxic byproducts. We show that this electrocatalytic on site oxygenator can sustain high cell loadings (>60k cells/mm3) in hypoxic conditions in vitro and in vivo. Our results showcase that exogenous oxygen production devices can be readily integrated into bioelectronic platforms, enabling high cell loadings in smaller devices with broad applicability.

Nonplanar 3D Printing of Epoxy Using Freeform Reversible Embedding.

Thermally cured thermoset polymers such as epoxies are widely used in industry and manufacturing due to their thermal, chemical, and electrical resistance, and mechanical strength and toughness. However, it can be challenging to 3D print thermally cured thermosets without rheological modification because they tend to flow and not hold their shape when extruded due to cure times of minutes to hours. 3D printing inside a support bath addresses this by allowing the liquid polymer to be held in place until the thermoset is fully cured and expands the structures that can be printed as extrusion is not limited to layer-by-layer. Here we report the use of Freeform Reversible Embedding (FRE) to 3D print off-the-shelf thermoset epoxy into lattice structures using non-planar extrusion. To do this we investigate how extrusion direction in 3D space impacts epoxy filament morphology and fusion at filament intersections. Further, we show the advantages of this approach by using non-planar printing to produce lattice geometries that show ~4 times greater specific modulus compared to lattice structures printed using other materials and printing techniques.

Rapid model-guided design of organ-scale synthetic vasculature for biomanufacturing.

Our ability to produce human-scale bio-manufactured organs is critically limited by the need for vascularization and perfusion. For tissues of variable size and shape, including arbitrarily complex geometries, designing and printing vasculature capable of adequate perfusion has posed a major hurdle. Here, we introduce a model-driven design pipeline combining accelerated optimization methods for fast synthetic vascular tree generation and computational hemodynamics models. We demonstrate rapid generation, simulation, and 3D printing of synthetic vasculature in complex geometries, from small tissue constructs to organ scale networks. We introduce key algorithmic advances that all together accelerate synthetic vascular generation by more than 230 -fold compared to standard methods and enable their use in arbitrarily complex shapes through localized implicit functions. Furthermore, we provide techniques for joining vascular trees into watertight networks suitable for hemodynamic CFD and 3D fabrication. We demonstrate that organ-scale vascular network models can be generated in silico within minutes and can be used to perfuse engineered and anatomic models including a bioreactor, annulus, bi-ventricular heart, and gyrus. We further show that this flexible pipeline can be applied to two common modes of bioprinting with free-form reversible embedding of suspended hydrogels and writing into soft matter. Our synthetic vascular tree generation pipeline enables rapid, scalable vascular model generation and fluid analysis for bio-manufactured tissues necessary for future scale up and production.

Evaluation of Senescence and Its Prevention in Doxorubicin-Induced Cardiotoxicity Using Dynamic Engineered Heart Tissues.

Background: Doxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence. Objectives: The aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target. Methods: Biopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell-derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol. Results: Senescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function. Conclusions: Senescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.

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.

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.

3D-bioprinted human tissue and the path toward clinical translation.

Three-dimensional (3D) bioprinting is a transformative technology for engineering tissues for disease modeling and drug screening and building tissues and organs for repair, regeneration, and replacement. In this Viewpoint, we discuss technological advances in 3D bioprinting, key remaining challenges, and essential milestones toward clinical translation.

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.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Modeling neuron growth using isogeometric collocation based phase field method.

We present a new computational framework of neuron growth based on the phase field method and develop an open-source software package called "NeuronGrowth_IGAcollocation". Neurons consist of a cell body, dendrites, and axons. Axons and dendrites are long processes extending from the cell body and enabling information transfer to and from other neurons. There is high variation in neuron morphology based on their location and function, thus increasing the complexity in mathematical modeling of neuron growth. In this paper, we propose a novel phase field model with isogeometric collocation to simulate different stages of neuron growth by considering the effect of tubulin. The stages modeled include lamellipodia formation, initial neurite outgrowth, axon differentiation, and dendrite formation considering the effect of intracellular transport of tubulin on neurite outgrowth. Through comparison with experimental observations, we can demonstrate qualitatively and quantitatively similar reproduction of neuron morphologies at different stages of growth and allow extension towards the formation of neurite networks.

FRESH 3D Bioprinting a Ventricle-like Cardiac Construct Using Human Stem Cell-Derived Cardiomyocytes.

Here we describe a method to engineer a contractile ventricle-like chamber composed of human stem cell-derived cardiomyocytes using freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting. To do this, we print a support structure using a collagen type I ink and a cellular component using a high-density cell ink supplemented with fibrinogen. The gelation of the collagen and the fibrinogen into fibrin is initiated by pH change and enzymatic crosslinking, respectively. Fabrication of the ventricle-like chamber is completed in three distinct phases: (i) materials preparation, (ii) bioprinting, and (iii) tissue maturation. In this protocol, we describe the method to print the construct from a high-density cell ink composed of human stem cell-derived cardiomyocytes and primary fibroblasts (~300 × 106 cells/mL) using our open-source dual-extruder bioprinter. Additional details are provided on FRESH support preparation, bioink preparation, dual-extruder needle alignment, print parameter selection, and post-processing. This protocol can also be adapted by altering the 3D model design, cell concentration, or cell type to FRESH 3D bioprint other cardiac tissue constructs.

Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells.

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (µTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage µT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Organs begin developing during the first few months of pregnancy, while the baby is still an embryo. These early stages of development are known as embryogenesis ­ a tightly organized process, during which the embryo forms different layers of stem cells. These cells can be activated to turn into a particular type of cell, such as a heart or a lung cell. The heart and lungs develop from different layers within the embryo, which must communicate with each other for the organs to form correctly. For example, chemical signals can be released from and travel between layers of the embryo, activating processes inside cells located in the different areas. In mouse models, chemical signals and cells travel between developing heart and lung, which helps both organs to form into the correct structure. But it is unclear how well the observations from mouse models translate to heart and lung development in humans. To find out more, Ng et al. developed a human model of heart and lung co-development during embryogenesis using human pluripotent stem cells. The laboratory-grown stem cells were treated with chemical signals, causing them to form different layers that developed into early forms of heart and lung cells. The cells were then transferred into a specific growing condition, where they arranged into three-dimensional structures termed microtissues. Ng et al. found that lung cells developed faster when grown in microtissues with accompanying developing heart cells compared to microtissues containing only developing lung cells. In addition, Ng et al. revealed that the co-developing heart and lung tissues automatically separate from each other during later stage, without the need for chemical signals. This human cell-based model of early forms of co-developing heart and lung cells may help provide researchers with new strategies to probe the underlying mechanisms of human heart and lung interaction during embryogenesis.

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.

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.

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.

Gain-of-function mutation in ubiquitin-ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues.

The start codon c.1A>G mutation in KLHL24, encoding ubiquitin-ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation, ultimately causing epidermolysis bullosa simplex (EBS). The majority of these EBS-patients are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homologue of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from two patients and three (non)familial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients' explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, that can be prevented by restoring desmin protein levels.

Engineering aligned human cardiac muscle using developmentally inspired fibronectin micropatterns.

Cardiac two-dimensional tissues were engineered using biomimetic micropatterns based on the fibronectin-rich extracellular matrix (ECM) of the embryonic heart. The goal of this developmentally-inspired, in vitro approach was to identify cell-cell and cell-ECM interactions in the microenvironment of the early 4-chambered vertebrate heart that drive cardiomyocyte organization and alignment. To test this, biomimetic micropatterns based on confocal imaging of fibronectin in embryonic chick myocardium were created and compared to control micropatterns designed with 2 or 20 µm wide fibronectin lines. Results show that embryonic chick cardiomyocytes have a unique density-dependent alignment on the biomimetic micropattern that is mediated in part by N-cadherin, suggesting that both cell-cell and cell-ECM interactions play an important role in the formation of aligned myocardium. Human induced pluripotent stem cell-derived cardiomyocytes also showed density-dependent alignment on the biomimetic micropattern but were overall less well organized. Interestingly, the addition of human adult cardiac fibroblasts and conditioning with T3 hormone were both shown to increase human cardiomyocyte alignment. In total, these results show that cardiomyocyte maturation state, cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblast interactions, and cardiomyocyte-ECM interactions can all play a role when engineering anisotropic cardiac tissues in vitro and provides insight as to how these factors may influence cardiogenesis in vivo.

Recent Advances in Cellular and Molecular Bioengineering for Building and Translation of Biological Systems.

In January of 2020, the Biomedical Engineering Society (BMES)- Cellular and Molecular Bioengineering (CMBE) conference was held in Puerto Rico and themed "Vision 2020: Emerging Technologies to Elucidate the Rule of Life." The annual BME-CMBE conference gathered worldwide leaders and discussed successes and challenges in engineering biological systems and their translation. The goal of this report is to present the research frontiers in this field and provide perspectives on successful engineering and translation towards the clinic. We hope that this report serves as a constructive guide in shaping the future of research and translation of engineered biological systems.