Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical ...investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.
Engineered cardiac patches for treating damaged heart tissues after a heart attack are normally produced by seeding heart cells within three-dimensional porous biomaterial scaffolds. These ...biomaterials, which are usually made of either biological polymers such as alginate or synthetic polymers such as poly(lactic acid) (PLA), help cells organize into functioning tissues, but poor conductivity of these materials limits the ability of the patch to contract strongly as a unit. Here, we show that incorporating gold nanowires within alginate scaffolds can bridge the electrically resistant pore walls of alginate and improve electrical communication between adjacent cardiac cells. Tissues grown on these composite matrices were thicker and better aligned than those grown on pristine alginate and when electrically stimulated, the cells in these tissues contracted synchronously. Furthermore, higher levels of the proteins involved in muscle contraction and electrical coupling are detected in the composite matrices. It is expected that the integration of conducting nanowires within three-dimensional scaffolds may improve the therapeutic value of current cardiac patches.
The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been ...made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.
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.
Abstract In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of ...engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 ± 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 ± 2.1 kPa and generated another 5.1 ± 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.
Smoking represents a major risk factor for chronic obstructive pulmonary disease (COPD), but it is difficult to characterize smoke-induced injury responses under physiological breathing conditions in ...humans due to patient-to-patient variability. Here, we show that a small airway-on-a-chip device lined by living human bronchiolar epithelium from normal or COPD patients can be connected to an instrument that “breathes” whole cigarette smoke in and out of the chips to study smoke-induced pathophysiology in vitro. This technology enables true matched comparisons of biological responses by culturing cells from the same individual with or without smoke exposure. These studies led to identification of ciliary micropathologies, COPD-specific molecular signatures, and epithelial responses to smoke generated by electronic cigarettes. The smoking airway-on-a-chip represents a tool to study normal and disease-specific responses of the human lung to inhaled smoke across molecular, cellular and tissue-level responses in an organ-relevant context.
Display omitted
•Smoking lung airway chip recapitulated clinical oxidative stress molecular profiles•New smoke-induced ciliary micropathologies were identified•This technology supported study of potential toxic effects of electronic cigarettes•COPD-specific responses were reproduced in vitro and novel biomarkers were identified
Benam et al. describe a microengineered in vitro model system that permits analysis of the effects of whole smoke, from both conventional tobacco and electronic cigarettes, delivered under physiologically relevant flow conditions that mimic breathing on the pathophysiology of differentiated human mucociliated bronchiolar epithelium cultured in a microfluidic small airway-on-a-chip.
The spatial and temporal scales of cardiac organogenesis and pathogenesis make engineering of artificial heart tissue a daunting challenge. The temporal scales range from nanosecond conformational ...changes responsible for ion channel opening to fibrillation which occurs over seconds and can lead to death. Spatial scales range from nanometre pore sizes in membrane channels and gap junctions to the metre length scale of the whole cardiovascular system in a living patient. Synchrony over these scales requires a hierarchy of control mechanisms that are governed by a single common principle: integration of structure and function. To ensure that the function of ion channels and contraction of muscle cells lead to changes in heart chamber volume, an elegant choreography of metabolic, electrical and mechanical events are executed by protein networks composed of extracellular matrix, transmembrane integrin receptors and cytoskeleton which are functionally connected across all size scales. These structural control networks are mechanoresponsive, and they process mechanical and chemical signals in a massively parallel fashion, while also serving as a bidirectional circuit for information flow. This review explores how these hierarchical structural networks regulate the form and function of living cells and tissues, as well as how microfabrication techniques can be used to probe this structural control mechanism that maintains metabolic supply, electrical activation and mechanical pumping of heart muscle. Through this process, we delineate various design principles that may be useful for engineering artificial heart tissue in the future.
Analyses of drug pharmacokinetics (PKs) and pharmacodynamics (PDs) performed in animals are often not predictive of drug PKs and PDs in humans, and in vitro PK and PD modelling does not provide ...quantitative PK parameters. Here, we show that physiological PK modelling of first-pass drug absorption, metabolism and excretion in humans-using computationally scaled data from multiple fluidically linked two-channel organ chips-predicts PK parameters for orally administered nicotine (using gut, liver and kidney chips) and for intravenously injected cisplatin (using coupled bone marrow, liver and kidney chips). The chips are linked through sequential robotic liquid transfers of a common blood substitute by their endothelium-lined channels (as reported by Novak et al. in an associated Article) and share an arteriovenous fluid-mixing reservoir. We also show that predictions of cisplatin PDs match previously reported patient data. The quantitative in-vitro-to-in-vivo translation of PK and PD parameters and the prediction of drug absorption, distribution, metabolism, excretion and toxicity through fluidically coupled organ chips may improve the design of drug-administration regimens for phase-I clinical trials.
Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. ...Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quantitative knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiological forces, but their buckling wavelength is reduced significantly because of mechanical coupling to the surrounding elastic cytoskeleton. We quantitatively explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mechanical behavior of the cell than previously thought possible.
PDF
