Inspired by the relatively simple morphological blueprint provided by batoid fish such as stingrays and skates, we created a biohybrid system that enables an artificial animal—a tissue-engineered ...ray—to swim and phototactically follow a light cue. By patterning dissociated rat cardiomyocytes on an elastomeric body enclosing a microfabricated gold skeleton, we replicated fish morphology at 1/10 scale and captured basic fin deflection patterns of batoid fish. Optogenetics allows for phototactic guidance, steering, and turning maneuvers. Optical stimulation induced sequential muscle activation via serpentine-patterned muscle circuits, leading to coordinated undulatory swimming. The speed and direction of the ray was controlled by modulating light frequency and by independently eliciting right and left fins, allowing the biohybrid machine to maneuver through an obstacle course.
Biomedical research has relied on animal studies and conventional cell cultures for decades. Recently, microphysiological systems (MPS), also known as organs-on-chips, that recapitulate the structure ...and function of native tissues in vitro, have emerged as a promising alternative. However, current MPS typically lack integrated sensors and their fabrication requires multi-step lithographic processes. Here, we introduce a facile route for fabricating a new class of instrumented cardiac microphysiological devices via multimaterial three-dimensional (3D) printing. Specifically, we designed six functional inks, based on piezo-resistive, high-conductance, and biocompatible soft materials that enable integration of soft strain gauge sensors within micro-architectures that guide the self-assembly of physio-mimetic laminar cardiac tissues. We validated that these embedded sensors provide non-invasive, electronic readouts of tissue contractile stresses inside cell incubator environments. We further applied these devices to study drug responses, as well as the contractile development of human stem cell-derived laminar cardiac tissues over four weeks.
Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it ...reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease.
Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate ...the complex genetic profile found in patients are urgently needed.
We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines.
We show that multiplex gene editing of mouse lung organoids using the CRISPR-Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell-mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti-PD-1 therapy.
We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage-inducing therapies in the treatment of LSCC.
Wounds in the fetus can heal without scarring. Consequently, biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have emerged as promising ...pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional (∼1.2 ×105 s−1) and shear (∼3 ×105 s−1) strain rates in rotary jet spinning (RJS) can drive high throughput Fn fibrillogenesis (∼10 mL/min), thus producing nanofiber scaffolds that are used to effectively enhance wound healing. When tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also significantly improved tissue restoration, recovering dermal and epidermal structures as well as skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.
Historically, soy protein and extracts have been used extensively in foods due to their high protein and mineral content. More recently, soy protein has received attention for a variety of its ...potential health benefits, including enhanced skin regeneration. It has been reported that soy protein possesses bioactive molecules similar to extracellular matrix (ECM) proteins and estrogen. In wound healing, oral and topical soy has been heralded as a safe and cost‐effective alternative to animal protein and endogenous estrogen. However, engineering soy protein‐based fibrous dressings, while recapitulating ECM microenvironment and maintaining a moist environment, remains a challenge. Here, the development of an entirely plant‐based nanofibrous dressing comprised of cellulose acetate (CA) and soy protein hydrolysate (SPH) using rotary jet spinning is described. The spun nanofibers successfully mimic physicochemical properties of the native skin ECM and exhibit a high water retaining capability. In vitro, CA/SPH nanofibers promote fibroblast proliferation, migration, infiltration, and integrin β1 expression. In vivo, CA/SPH scaffolds accelerate re‐epithelialization and epidermal thinning as well as reduce scar formation and collagen anisotropy in a similar fashion to other fibrous scaffolds, but without the use of animal proteins or synthetic polymers. These results affirm the potential of CA/SPH nanofibers as a novel wound dressing.
A plant‐based biomimetic cellulose/soy protein nanofibrous wound dressing is fabricated using rotary jet spinning. Its physicochemical properties mimic native extracellular matrix in skin and exhibit a high water‐retaining capability for enhanced wound healing. The plant hybrid wound dressing accelerates in vitro dermal fibroblast proliferation, spreading, and migration. Furthermore, it promotes re‐epithelialization and reduces epidermal thickness and scar area in mouse model.
Recent reports suggest the utility of extracellular matrix (ECM) molecules as raw components in scaffolding of engineered materials. However, rapid and tunable manufacturing of ECM molecules into ...fibrous structures remains poorly developed. Here we report on an immersion rotary jet-spinning (iRJS) method to show high-throughput manufacturing (up to ∼1 g/min) of hyaluronic acid (HA) and other ECM fiber scaffolds using different spinning conditions and postprocessing modifications. This system allowed control over a variety of scaffold material properties, which enabled the fabrication of highly porous (70–95%) and water-absorbent (swelling ratio ∼2000–6000%) HA scaffolds with soft-tissue mimetic mechanical properties (∼0.5–1.5 kPa). Tuning these scaffolds’ properties enabled the identification of porosity (∼95%) as a key facilitator for rapid and in-depth cellular ingress in vitro. We then demonstrated that porous HA scaffolds accelerated granulation tissue formation, neovascularization, and reepithelialization in vivo, altogether potentiating faster wound closure and tissue repair. Collectively, this scalable and versatile manufacturing approach enabled the fabrication of tunable ECM-mimetic nanofiber scaffolds that may provide an ideal first building block for the design of all-in-one healing materials.
BACKGROUND:Modeling of human arrhythmias with induced pluripotent stem cell–derived cardiomyocytes has focused on single-cell phenotypes. However, arrhythmias are the emergent properties of cells ...assembled into tissues, and the impact of inherited arrhythmia mutations on tissue-level properties of human heart tissue has not been reported.
METHODS:Here, we report an optogenetically based, human engineered tissue model of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia caused by mutation of the cardiac ryanodine channel and triggered by exercise. We developed a human induced pluripotent stem cell–derived cardiomyocyte–based platform to study the tissue-level properties of engineered human myocardium. We investigated pathogenic mechanisms in CPVT by combining this novel platform with genome editing.
RESULTS:In our model, CPVT tissues were vulnerable to developing reentrant rhythms when stimulated by rapid pacing and catecholamine, recapitulating hallmark features of the disease. These conditions elevated diastolic Ca levels and increased temporal and spatial dispersion of Ca wave speed, creating a vulnerable arrhythmia substrate. Using Cas9 genome editing, we pinpointed a single catecholamine-driven phosphorylation event, ryanodine receptor–serine 2814 phosphorylation by Ca/calmodulin-dependent protein kinase II, that is required to unmask the arrhythmic potential of CPVT tissues.
CONCLUSIONS:Our study illuminates the molecular and cellular pathogenesis of CPVT and reveals a critical role of calmodulin-dependent protein kinase II–dependent reentry in the tissue-scale mechanism of this disease. We anticipate that this approach will be useful for modeling other inherited and acquired cardiac arrhythmias.
Microphysiological systems and organs-on-chips promise to accelerate biomedical and pharmaceutical research by providing accurate in vitro replicas of human tissue. Aside from addressing the ...physiological accuracy of the model tissues, there is a pressing need for improving the throughput of these platforms. To do so, scalable data acquisition strategies must be introduced. To this end, we here present an instrumented 24-well plate platform for higher-throughput studies of engineered human stem cell-derived cardiac muscle tissues that recapitulate the laminar structure of the native ventricle. In each well of the platform, an embedded flexible strain gauge provides continuous and non-invasive readout of the contractile stress and beat rate of an engineered cardiac tissue. The sensors are based on micro-cracked titanium-gold thin films, which ensure that the sensors are highly compliant and robust. We demonstrate the value of the platform for toxicology and drug-testing purposes by performing 12 complete dose-response studies of cardiac and cardiotoxic drugs. Additionally, we showcase the ability to couple the cardiac tissues with endothelial barriers. In these studies, which mimic the passage of drugs through the blood vessels to the musculature of the heart, we regulate the temporal onset of cardiac drug responses by modulating endothelial barrier permeability in vitro.
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