a-micropatterning-assay-for-measuring-cell-chirality

A Micropatterning Assay for Measuring Cell Chirality

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique ...

Columbia University

Columbia College

Organs-on-a-chip (OOCs) are miniature tissues and organs grown in vitro that enable modeling of human physiology and disease. The technology has emerged from converging advances in tissue engineering, semiconductor fabrication, and human cell sourcing. Encompassing innovations in human stem cell technology, OOCs offer a promising approach to emulate human patho/physiology in vitro, and address limitations of current cell and animal models. Here, we review the design considerations for single and multi-organ OOCs, discuss remaining challenges, and highlight the potential impact of OOCs as a fast-track opportunity for tissue engineering to advance drug development and precision medicine.

Organs-on-a-Chip: A Fast Track for Engineered Human Tissues in Drug Development.

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease. However, the predictive power of these models is presently limited by the immature state of the cells. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype.

Traditional drug screening models are often unable to faithfully recapitulate human physiology in health and disease, motivating the development of microfluidic organs-on-a-chip (OOC) platforms that can mimic many aspects of human physiology and in the process alleviate many of the discrepancies between preclinical studies and clinical trials outcomes. Linsitinib, a novel anti-cancer drug, showed promising results in pre-clinical models of Ewing Sarcoma (ES), where it suppressed tumor growth. However, a Phase II clinical trial in several European centers with patients showed relapsed and/or refractory ES. We report an integrated, open setting, imaging and sampling accessible, polysulfone-based platform, featuring minimal hydrophobic compound binding. Two bioengineered human tissues - bone ES tumor and heart muscle - were cultured either in isolation or in the integrated platform and subjected to a clinically used linsitinib dosage. The measured anti-tumor efficacy and cardiotoxicity were compared with the results observed in the clinical trial. Only the engineered tumor tissues, and not monolayers, recapitulated the bone microenvironment pathways targeted by linsitinib, and the clinically-relevant differences in drug responses between non-metastatic and metastatic ES tumors. The responses of non-metastatic ES tumor tissues and heart muscle to linsitinib were much closer to those observed in the clinical trial for tissues cultured in an integrated setting than for tissues cultured in isolation. Drug treatment of isolated tissues resulted in significant decreases in tumor viability and cardiac function. Meanwhile, drug treatment in an integrated setting showed poor tumor response and less cardiotoxicity, which matched the results of the clinical trial. Overall, the integration of engineered human tumor and cardiac tissues in the integrated platform improved the predictive accuracy for both the direct and off-target effects of linsitinib. The proposed approach could be readily extended to other drugs and tissue systems.

Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety.

Most cancer deaths are due to tumor metastasis rather than the primary tumor. Metastasis is a highly complex and dynamic process that requires orchestration of signaling between the tumor, its local environment, distant tissue sites, and immune system. Animal models of cancer metastasis provide the necessary systemic environment but lack control over factors that regulate cancer progression and often do not recapitulate the properties of human cancers. Bioengineered "organs-on-a-chip" that incorporate the primary tumor, metastatic tissue targets, and microfluidic perfusion are now emerging as quantitative human models of tumor metastasis. The ability of these systems to model tumor metastasis in individualized, patient-specific settings makes them uniquely suitable for studies of cancer biology and developmental testing of new treatments. In this review, we focus on human multi-organ platforms that incorporate circulating and tissue-resident immune cells in studies of tumor metastasis.

Engineered models of tumor metastasis with immune cell contributions.

Cardiomyocytes derived from human induced pluripotent stem (iPS) cells enable the study of cardiac physiology and the developmental testing of new therapeutic drugs in a human setting. In parallel, machine learning methods are being applied to biomedical science in unprecedented ways. Machine learning has been used to distinguish healthy from diseased cardiomyocytes using calcium (Ca) transient signals. Most Ca transient signals are obtained via terminal assays that do not permit longitudinal studies, although some recently developed options can circumvent these concerns. Here, we describe the use of machine learning to identify healthy and diseased cardiomyocytes according to their contractility profiles, which are derived from brightfield videos. This noncontact, label-free approach allows for the continued cultivation of cells after they have been evaluated for use in other assays and can be readily extended to organs-on-chip. To demonstrate utility, we assessed contractility profiles of cardiomyocytes obtained from patients with Timothy Syndrome (TS), a long QT disease which can lead to fatal arrhythmias, and from healthy individuals. The videos were processed and classified using machine learning methods and their performance was evaluated according to several parameters. The trained algorithms were able to distinguish the TS cardiomyocytes from healthy controls and classify two different healthy controls. The proposed computational machine learning evaluation of human iPS cell-derived cardiomyocytes' contractility profiles has the potential to identify other genetic proarrhythmic events, screen therapeutic agents for inducing or suppressing long QT events, and predict drug-target interactions. The same approach could be readily extended to the evaluation of engineered cardiac tissues within single-tissue and multi-tissue organs-on-chip.

Machine Learning Techniques to Classify Healthy and Diseased Cardiomyocytes by Contractility Profile.

We explore the utility of bioengineered human tissues-individually or connected into physiological units-for biological research. While much smaller and simpler than their native counterparts, these tissues are complex enough to approximate distinct tissue phenotypes: molecular, structural, and functional. Unlike organoids, which form spontaneously and recapitulate development, "organs-on-a-chip" are engineered to display some specific functions of whole organs. Looking back, we discuss the key developments of this emerging technology. Thinking forward, we focus on the challenges faced to fully establish, validate, and utilize the fidelity of these models for biological research.

Article	Year
Organs-on-a-Chip: A Fast Track for Engineered Human Tissues in Drug Development. Cell stem cell\| PubMed ID: 29499151	2018
Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature\| PubMed ID: 29618819	2018
A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling. Cell\| PubMed ID: 30686581	2019
Author Correction: Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature\| PubMed ID: 31363231	2019
Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype. Nature protocols\| PubMed ID: 31492957	2019
Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety. Lab on a chip\| PubMed ID: 32955072	2020
Engineered models of tumor metastasis with immune cell contributions. iScience\| PubMed ID: 33718831	2021
Machine Learning Techniques to Classify Healthy and Diseased Cardiomyocytes by Contractility Profile. ACS biomaterials science & engineering\| PubMed ID: 34152732	2021
Organs-on-a-chip models for biological research. Cell\| PubMed ID: 34478657	2021

Kacey Ronaldson-Bouchard

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