Using bioengineered cardiac tissue to model neurological disorders and ataxia with cardiac dysfunction

Abstract

The present disclosure provides a system for screening therapeutic cardiac compounds in cells, tissues and organoids of patients with diseases such as neurological diseases or conditions with serious cardiac complications. The system comprises: a medical device apparatus suitable for single-layer screening of cardiac active compounds, which comprises a human ventricular cardiac anisotropic sheet (hvCAS) and a human ventricular cardiac tissue strip (hvCTS); a two-layer system further comprising a human ventricular cardiac organoid chamber (hvCOC); or a three-layer system further comprising a medical device with multiple organoids, which include the same or different types of tissues or organoids (e.g., heart, liver, pancreas, kidney). Another aspect of the present disclosure is a method suitable for use with the system, which comprises screening for compounds that have cardiac effects on cells, tissues or organoids of patients with non-cardiac diseases such as neurological diseases that exhibit cardiac effects. The method can also be used to evaluate the toxicity of compounds to various cells, tissues or organoids of such patients.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 649,468, filed on March 28, 2018, which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates generally to the fields of medical health and cardiac physiology, and more particularly to the field of in vitro models and screens for cardiac function in cells from diseased patients. Background Art

[0004] Friedreich's ataxia (FRDA) is an inherited neurodegenerative disorder caused by mutations in the first intron of the frataxin (FXN) gene on chromosome 9. As a result, the trinucleotide guanine-adenine-adenine (GAA) sequence, which normally repeats up to 40 times in healthy individuals, appears hundreds to over a thousand times. Even in FRDA patients who produce a fully functional wild-type FXN protein, the intronic mutation results in a significant reduction in FXN expression, with only 5% to 30% of the protein remaining compared to non-carriers. FXN is a mitochondrial protein involved in the biosynthesis of iron-sulfur clusters, essential for ATP production.

[0005] The heart is the primary site of pathology in patients with FRDA, due to its reliance on mitochondria for energy consumption. FRDA-induced cardiac symptoms are first detected as abnormal electrocardiograms (EKGs) and can progress to compensated hypertrophic cardiomyopathy, dilated cardiomyopathy, and then to cardiomyocyte (CM) death and fibrosis, leading to heart failure and arrhythmias. In fact, the leading cause of death in FRDA patients has been attributed to cardiac problems. However, a correlation between the severity of FRDA-induced cardiac symptoms and FXN expression has not been established.

[0006] Given the high amino acid identity between FXN genes across species, mouse models have been developed to study FRDA pathology. One mouse model is achieved by conditional ablation of the Fxn gene, resulting in complete Fxn knockout in target organs. 11 Although this particular model presents expected FRDA phenotypes, such as cardiomyopathy and mitochondrial defects, its genotype does not reflect that of FRDA patients. Another mouse model that mimics the human genotype has been generated by combining transgenic expression of the human FXN gene with expanded GAA repeats with knockout of the murine Fxn gene; however, the severity of the disease phenotype does not match that observed in FRDA patients. 14,15Therefore, in vitro models using human CMs appear to be more appropriate and relevant models to study the cardiac pathogenesis of FRDA. However, the availability of patient biopsies is limited and insufficient to recapitulate the functional phenotypes of the disease, such as systolic dysfunction.

[0007] With advances in reprogramming, human induced pluripotent stem cells (hiPSCs) can be derived from FRDA patients and differentiated into CMs to study disease progression in human in vitro models. However, to date, there have been no reports or publications examining or suggesting the examination of the contractile or electrophysiological properties of such CMs in multicellular cardiac mimetic models.

[0008] Much effort has been expended to improve our understanding of the human cardiovascular system and human cardiac care, but there remains a need in the art for biological models that can rapidly and accurately reveal cardiac effects in cells from patients with diseases traditionally understood to be non-cardiac in nature. Summary of the Invention

[0009] To improve and prolong the lives of patients with neurological diseases or conditions that present with deleterious cardiac comorbidities, therapeutics targeting cardiac symptoms, often a leading cause of death, would be of greatest value. Testing candidate therapeutics that could potentially target and treat these cardiac effects requires the creation of an effective in vitro platform that adequately recapitulates the disease phenotype—an appropriate disease model. Over the past few decades, animal models have been extensively used, but they are not very predictive of patient outcomes due to significant differences between the human and non-human heart. Unfortunately, the supply of patient cardiac biopsies is very limited, and such isolated tissues do not survive long-term in vitro. Recent advances in pluripotent stem cell technology have enabled the mass production of human cardiac cells (cardiomyocytes), providing an attractive platform for disease modeling. However, the power of cultured cardiomyocytes for disease modeling is limited because single-cell properties are difficult to extrapolate to tissue-level phenotypes, and traditional monolayer cultures fail to recapitulate the anisotropic three-dimensional structure and function of cardiac tissue. Engineered human cardiac tissue could address this issue, as it can be functionally assessed and its design can be customized to allow for readouts that are more readily correlated with clinical symptoms. Thus, a comprehensive suite of engineered cardiac tissue assays offers advantages over any single system for comprehensively evaluating therapeutic agents for cardiac diseases with a neurological basis. To illustrate the benefits of the present disclosure in evaluating candidate therapeutics and identifying treatments for cardiac effects associated with neurological diseases, the following description focuses on addressing cardiac defects associated with Friedreich's ataxia. This description is exemplary of the benefits of the present disclosure and can be readily applied by those skilled in the art to model other neurological diseases with cardiac effects.

[0010] The studies disclosed herein are the first to show the effects of reduced FXN levels on cardiac electrophysiology and contractile properties using multiple engineered tissue constructs composed of FXN-deficient human ventricular CMs (hvCMs) derived from human embryonic stem cells (hESCs) or hiPSCs (collectively referred to as human pluripotent stem cells or hPSCs), in which FXN levels were reduced by short hairpin RNA (shRNA)-mediated knockdown of FXN (in hESCs and healthy hiPSCs) or naturally reduced due to a mutation in the FXN gene in the donor patient (in FRDA hiPSCs). The tissue platform utilized in the studies disclosed herein provides human ventricular cardiac anisotropic slices (hvCAS) for modeling cardiac electrophysiology and arrhythmias. 19-22 Human ventricular cardiac tissue strips (hvCTS) for simulating cardiac force generation and contractility defects 23-24 and / or human ventricular cardiac organoid chambers (hvCOCs) designed to mimic more physiological conditions through electrically and mechanically coupled CMs. These tissue platforms are superior models for studying the pathogenesis of FRDA, enabling readouts such as conduction velocity, force generation, and cardiac output that are not possible with single cells or many other engineered tissue models.

[0011] In general, the present disclosure provides a system and related methods to facilitate drug discovery / screening using engineered hvCAS, hvCTS, and hvCOC that mimic neurological diseases with at least one deleterious cardiac effect. These engineered constructs can be assembled using (a) healthy hPSC-derived CMs (or hPSC-CMs), which are engineered to reproduce the effects of pathogenic mutations in patient CMs, for example, by knockdown of one or more specific genes mediated by lentiviral shRNA, or (b) intrinsically diseased hiPSC-derived CMs (or hiPSC-CMs) from patients carrying one or more pathogenic mutations. The biological fidelity of the model is verified for clinically observed patient symptoms (e.g., changes in electrocardiogram patterns, contractile dysfunction) and compared to relevant healthy controls (e.g., healthy hPSC-CMs transduced with non-targeting shRNA lentiviral controls, or hiPSC-CMs derived from healthy subjects) to ensure model specificity. The validated model can be used to screen for therapeutics with beneficial or toxic effects on cardiac tissue. These engineered constructs can be used to evaluate the electrophysiological and contractile effects of candidate treatments on the heart in terms of toxicity and efficacy, with the goal of identifying treatments that can improve, rescue, or eliminate disease symptoms without causing harmful side effects. For example, for FRDA, which exhibits both electrophysiological and contractile symptoms, both electrophysiological (hvCAS) and contractile (hvCTS / hvCOC) models can be used for efficacy and safety screening. Single or combined treatments that can induce improvement in both symptoms are expected to be promising candidate therapeutic agents.

[0012] The hvCAS disease model is a biohybrid material in the form of (1) human ventricular cardiomyocytes (hvCMs) and (2) a microfabricated substrate that provides attachment points for the growth and orderly development of hvCMs, as fundamental components that have been strategically aligned by design. In use, the hvCMs form a monolayer of cells (referred to as human ventricular cardiac anisotropic sheets or hvCASs) overlying the microfabricated substrate, which provides an environment conducive to the development of anisotropic properties unique to human cardiac cells in vivo. The disease modeling hvCMs can be (a) healthy hPSC-derived CMs (or hPSC-CMs) that are engineered to recapitulate the effects of a pathogenic mutation in a patient's CMs, for example, by lentiviral shRNA-mediated knockdown of one or more specific genes, or (b) intrinsically diseased hiPSC-derived CMs (or hiPSC-CMs) derived from a patient carrying one or more pathogenic mutations. In some embodiments, the microfabricated substrate comprises grooves oriented along a single axis of the substrate. In some embodiments, the grooves have similar dimensions. In some embodiments, the grooves are 1-30 μm wide, including embodiments in which the grooves are 5-15 μm wide, such as embodiments in which the grooves are 15 μm wide. In some embodiments, the grooves are about 5 μm deep. In some embodiments, the spacing between the grooves is about 5 μm.

[0013] One aspect of the present disclosure provides a method for modeling the electrophysiological phenotype of a disease, comprising: (a) fabricating an hvCAS using a disease-modeling CM engineered from healthy hPSCs or derived from patient hiPSCs; (b) stimulating an anisotropic layer of cells at one or more points; (c) detecting the propagation of electrical signals in the anisotropic layer of cells; and (d) evaluating the electrophysiological properties of the hvCAS. In various embodiments, the electrophysiological properties of the hvCAS evaluated are action potential duration, transverse conduction velocity, longitudinal conduction velocity, anisotropy ratio, automaticity (presence of spontaneous action potentials), maximum capture frequency, maximum rise velocity, maximum decay velocity, rise time, conduction pattern, occurrence of arrhythmic events in the form of spiral electrical propagation waves, and / or any other physiological parameter of cardiomyocytes that can be monitored using an anisotropic sheet of cardiomyocytes. In some embodiments, the anisotropic layer of cells comprises ventricular cardiomyocytes derived from human heart. Embodiments of this aspect of the present disclosure exist wherein the microfabricated substrate is polystyrene. In some embodiments, the cardiac anisotropic layer of cells is stimulated at a single point, for example, where the stimulation is 5-30 volts with a pulse duration of 5-30 milliseconds. In one exemplary embodiment, the stimulation is 10 volts with a pulse duration of 10 milliseconds. In some embodiments of this aspect of the present disclosure, the electrical stimulation induces helical electrical propagation waves in the hvCAS disease model, thereby mimicking the arrhythmias that are clinically present in the disease. In some embodiments, the hvCAS disease model exhibits altered electrophysiological parameters, such as action potential duration and conduction velocity.

[0014] More specifically, one aspect of the present disclosure relates to a system for screening compounds for electrophysiological effects on cardiomyocytes or engineered cardiomyocytes of a diseased organism, the system comprising: (1) human ventricular cardiomyocytes (hvCMs), and (2) a microfabricated substrate that provides attachment points for the growth and orderly development of hvCMs, as a building block that has been strategically aligned by design. In use, the hvCMs form a monolayer of cells (referred to as a human ventricular cardiac anisotropic sheet or hvCAS) overlying the microfabricated substrate, which provides an environment conducive to the development of anisotropic properties unique to human cardiac cells in vivo. The method is suitable for screening candidate compounds to identify compounds that have electrophysiological effects on cardiomyocytes of a diseased organism, and for screening therapeutic agents known to be useful in treatment to identify those that have electrophysiological effects on cardiomyocytes. Disease modeling hvCMs can be (a) healthy hPSC-derived CMs (or hPSC-CMs) engineered to recapitulate the effects of a pathogenic mutation in a patient's CMs, for example, by lentiviral shRNA-mediated knockdown of one or more specific genes, or (b) intrinsically diseased hiPSC-derived CMs (or hiPSC-CMs) derived from a patient carrying one or more pathogenic mutations. In some embodiments, the grooves have similar dimensions. In some embodiments, the grooves have a width of 1-30 μm, including embodiments in which the grooves have a width of 5-15 μm, such as embodiments in which the grooves have a width of 15 μm. In some embodiments, the grooves have a depth of about 5 μm. In some embodiments, the spacing between the grooves is about 5 μm. One aspect of the present disclosure provides a method for screening for the electrophysiological effects of a treatment, comprising: (a) contacting an hvCAS disease model with a treatment; (b) stimulating the hvCAS disease model at one or more sites; (c) detecting electrical signal propagation in the hvCAS disease model; and (d) determining whether the compound exhibits efficacy (e.g., by eliminating arrhythmias, as indicated by the absence or reduction of helical electrical propagation waves) or toxicity (e.g., by inducing or exacerbating helical electrical propagation waves). Embodiments of this aspect of the present disclosure exist wherein the micromachined substrate is polystyrene. In some embodiments, the hvCAS is stimulated at a single site, e.g., wherein the stimulation is 5-30 volts with a pulse duration of 5-30 milliseconds. In one exemplary embodiment, the stimulation is 10 volts with a pulse duration of 10 milliseconds. In some embodiments of this aspect of the present disclosure, the electrical stimulation induces helical electrical propagation waves in the hvCAS disease model, thereby mimicking arrhythmias induced by the treatment. In some embodiments, the treatment induces changes in electrophysiological parameters, such as action potential duration and conduction velocity, in the hvCAS disease model.

[0015] The hvCTS disease model or the hvCAS disease model can be paired with the hvCOC disease model in a two-tier screening, where the first tier screening uses hvCTS to assess contractile effects on the heart or hvCAS to assess one or more physiological parameters of cardiomyocytes, and then a second tier screening is performed on the higher-order biological structure of the hvCOC system to confirm the results of the first tier screening and reveal any organoid or organ-level effects that were not obvious from the cells used in the first tier screening. The present disclosure also provides the possibility of a third tier screening, which can be considered as a third tier screening that is optimally combined with the first and second tier screening. In the third tier screening, multiple organoids are screened using a multi-organoid system. The data generated by the third tier screening is even more reliable than the data obtained from the two tier screening. In addition, the multi-organoid format allows the use of multiple organoids of the same type, such as cardiac organoids, in the screening, and / or the use of different interconnected organoids simultaneously in the third tier screening, using the universal multi-organoid system disclosed herein. (As used herein, "organoid" generally refers to organ-like biomaterials, but the term can also refer to engineered tissues that can be considered organ-like biomaterials. The meaning of the terms used herein will be apparent from the context in which they are used.) In some embodiments, human ventricular cardiac tissue comprises hPSC-derived ventricular cardiomyocytes (vCMs). Our group has characterized the single-cell properties of such cells, such as electrophysiology (action potential, Ca 2+ extensively characterized the transcriptome, proteome, etc. 30-48 As disclosed herein, various cells, such as human ventricular cardiomyocytes, can be used to develop organoids for the second-tier hvCOC system and for developing organoids for the third-tier multi-organoid system.

[0016] The multi-organoid system used in the second and third layers of the multi-layer system and method of the present invention is a platform that simultaneously characterizes multiple in vitro tissue-engineered tissues or organoids, including a mirror arrangement and a single detection device. Equipped with a fluid exchange network, organoids are interconnected on this platform to build a "mini human body" system that simulates the systemic drug response of human patients and can be used to replace animal testing as a default in vivo model. The semi-automated platform includes multiple features to help investigate the functional response to delivered drugs, such as environmental control (such as temperature and CO2), high-speed cameras, synchronized pressure-volume recordings, interconnected fluid exchange systems, drug perfusion, pressure control in the organoid, mechanical stimulation and electrical stimulation. These features are designed to improve culture operations, allow inspection of long-term drug exposure of tissues or organoids, and allow simultaneous multi-tissue and / or multi-organ drug reactions. Current in vitro therapeutic agent screening typically only assesses the acute response of a single tissue or organoid, which expands the challenge and is costly. By using a single camera with a mirror arrangement to perform multi-organoid imaging, this system is more scalable than currently available designs.

[0017] To develop the next generation of in vitro human models, the bioreactor platform includes a modular organoid box system and a fluid exchange network that enables flexible systems biology approaches. The "plug-and-play" organoid box speeds up the process of imaging various tissues and / or organoid combinations of interest within the bioreactor. In addition, the circulation and exchange of culture medium between tissues or organoids can reproduce the human circulatory system. Signaling factors and metabolites can be freely exchanged between tissues or organoids and can affect the drug response of one or more tissues or organoids. This "body-in-a-jar" technology promotes drug discovery and precision or personalized medical research, and is superior to organ chip technology, which often cannot fully reproduce organ function due to the lack of three-dimensional tissue.

[0018] New molecular entities are characterized using clinically relevant endpoints (e.g., ejection fraction in cardiac tissue, permeability in lung tissue) and then classified using automated computer algorithms (e.g., machine learning) trained to detect biological activity and toxicity patterns. The multi-organoid imaging platform disclosed herein increases throughput and can be used for screening of higher levels. By increasing throughput, the system becomes easier for preclinical drug screening. The platform is also used to detect basic biology in tissue-engineered human constructs derived from patients with neurological diseases or conditions.

[0019] In addition to a layer of hvCTS systems and methods, the present disclosure provides a universal bioreactor platform for developing engineered organoid tissues that more closely mimic the in vivo structure and function of corresponding human organs. Utilizing a combination of high-speed cameras and pressure sensors, the present disclosure provides a method for combining the spatiotemporal motion of displaced tissues or organoids (e.g., contracting heart organoids) with pressure recordings to measure pressure-volume relationships. In addition, in contrast to some systems that use simple hydrostatic pressure systems, the present disclosure provides a complex system for performing fluid exchange within a bioreactor platform by collaboratively using fluid pumps, three-way valves, and fluid boxes. The fluid exchange system also provides for connecting any number of organoids within a bioreactor. The fluid exchange system of the present disclosure provides the additional functionality of controlling culture medium delivery for feeding, aspirating culture medium, mixing bioactive components (e.g., therapeutic agents), and injecting bioactive agents on an acute schedule (e.g., bolus) or chronic schedule (e.g., perfusion). Bioactive components may include, but are not limited to, drug compounds, viral vectors, conditioned culture media, progenitor cells, and extracellular vesicles. The fluid exchange system also provides for cleaning, rinsing, or flushing of fluid lines. In addition, the present disclosure provides mechanical stimulation to developing tissues or organoids by applying a method for mechanical stretching to tissues or organoids with chambers. By applying mechanical stretching, the present disclosure provides a means for manipulating tissues or organoids, as long as the mechanical stretching can act as a mechanical transduction signal. In contrast to electrical pacing of human cardiac organoids via field stimulation, the present disclosure provides point stimulation of such organoids, resulting in more precise and refined stimulation of organoid tissue. Using point stimulation, conductivity measurements within tissues or organoids (such as hearts) can be completed, for example, by using optical mapping technology. In addition, according to the present disclosure, the application of machine learning principles in tissue or organoid behavior analysis is expected to improve the assessment of treatment response results by comprehensively analyzing and understanding high-dimensional parameter spaces. The present disclosure provides all benefits in an integrated package, which represents a major advancement in the field of therapeutic agent screening, including new methods, i.e., experimental assays, which are expected to improve the prevention, treatment and / or improvement of cardiac symptoms associated with various neurological diseases, disorders and conditions.

[0020] More specifically, one aspect of the present disclosure relates to a system for screening compounds for cardiac effects (e.g., contractile effects) on cardiomyocytes or engineered cardiomyocytes of a diseased organism, the system comprising: (a) a screening device comprising: (i) a biocompatible gel containing a plurality of cardiomyocytes or engineered cardiomyocytes of a diseased organism; (ii) a biocompatible support device for suspending the biocompatible gel, wherein the biocompatible gel and the biocompatible support device form a cardiac tissue strip containing cardiomyocytes or engineered cardiomyocytes of a diseased organism; (iii) a detection device for detecting movement of the biocompatible gel; and (iv) a power source for applying electrical pacing stimulation to the biocompatible gel. The compound can be a therapeutic agent known to be useful for cardiac treatment due to its contractile effect on cardiomyocytes, or the compound can be a candidate therapeutic agent. In some embodiments, the system for screening compounds further comprises: (a) a secondary screening apparatus comprising: (i) at least one organoid module comprising at least one organoid cassette, wherein the organoid cassette comprises a culture medium inlet, a culture medium outlet, and at least one wall compatible with an external detection device, wherein each organoid cassette comprises cardiomyocytes or engineered cardiomyocytes of a diseased organism, and wherein at least one organoid cassette comprises cardiomyocytes or engineered cardiomyocytes of a diseased organism; and (ii) a detection device for observing the monitored biological development of the cardiomyocytes or engineered cardiomyocytes of the diseased organism in each organoid cassette. In some embodiments, the secondary screening apparatus disclosed herein further comprises a mirror arrangement for simultaneously monitoring any biological development of the cardiomyocytes or engineered cardiomyocytes of the diseased organism in each organoid cassette.

[0021] Another aspect of the present disclosure is a system for screening compounds for cardiac effects on cardiomyocytes or engineered cardiomyocytes of a diseased organism, the system comprising: (a) a screening device comprising: (i) an anisotropic layer of cardiomyocytes on a micromachined substrate; (ii) a power source for stimulating the anisotropic layer of cells at one or more points; and (iii) a detection device for detecting the propagation of electrical signals in the anisotropic layer of cells. In some embodiments, the micromachined substrate comprises grooves oriented along a single axis of the substrate. In some embodiments, the grooves have a width of 1-30 μm, a depth of about 5 μm, and a spacing between the grooves of about 5 μm, or a combination thereof. In some embodiments, the power source is at least one electrode, such as a single electrode or an array of electrodes, or at least one charged portion of the substrate. The partially or fully charged substrate is capable of establishing an electric field, which can be uniform or can exhibit a gradient. In some embodiments, a system for screening compounds for cardiac effects comprises a screening apparatus further comprising: (a) a secondary screening device comprising: (i) at least one organoid module comprising at least one organoid cassette, wherein the organoid cassette comprises a culture medium inlet, a culture medium outlet, and at least one wall compatible with an external detection device, wherein each organoid cassette comprises cardiomyocytes or engineered cardiomyocytes from a diseased organism, and wherein at least one organoid cassette comprises cardiomyocytes or engineered cardiomyocytes from a diseased organism; and (ii) a detection device for observing the biological development of the cardiomyocytes or engineered cardiomyocytes from the diseased organism in each organoid cassette monitored.

[0022] In some embodiments of the system according to the present disclosure, the cardiomyocytes are human cardiomyocytes, such as human ventricular cardiomyocytes, for example, wherein the cardiomyocytes are at least 10 6 The concentration of cells / ml was present.

[0023] In some embodiments of the system, the cardiomyocytes are engineered cardiomyocytes or are generated from hiPSCs derived from a patient with a neurological disease, including but not limited to Friedreich's ataxia (FRDA), Kearns-Sayre syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers). In some embodiments, the cardiomyocytes are engineered to have low FXN expression or are derived from hiPSCs from a patient with, for example, Friedreich's ataxia (FRDA).

[0024] In some embodiments of the system, the cardiomyocytes are genotypically normal and are derived from healthy hPSCs, including established hESCs or hiPSCs from healthy volunteers, and are engineered to recapitulate a mutant disease phenotype typical of a neurological disease, including but not limited to Friedreich's ataxia (FRDA), Kaplan-Meier syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers), for example, wherein the cardiomyocytes are engineered to express reduced levels of frataxin (FXN) protein, as typically observed in FRDA patients.

[0025] In some embodiments, the biocompatible gel comprises matrigel, e.g., a gel wherein the matrigel is present at a concentration of at least 0.5 mg / ml. In some embodiments, the biocompatible gel further comprises collagen, e.g., human type I collagen. In some embodiments, the collagen is present at a concentration of at least 1 mg / ml.

[0026] The support device for the biocompatible gel can be at least two vertical support members. In some embodiments, the vertical support members are made of polydimethylsiloxane. In specific embodiments, there are two vertical support members, each of which has a substantially circular cross-section and a diameter of approximately 0.5 mm. In some embodiments, the cardiac tissue strip is approximately 26.5 mm long, approximately 16 mm wide, and approximately 6 mm high.

[0027] The systems disclosed herein contemplate embodiments in which the detection device is a high-speed camera. In some embodiments, the mirror arrangement of the second-stage screening apparatus comprises at least one conic mirror. Some embodiments of the screening system comprise a second-stage screening apparatus further comprising electrodes in adjustable relationship to the tissue or organoid in at least one organoid cassette. In some embodiments, the second-stage screening apparatus further comprises a temperature control element, a light source, a module access port, or any combination thereof. In some embodiments, the system comprising the second-stage screening apparatus further comprises a data processor in electronic communication with the detection device, the temperature control element, the light source, the module access port, or any combination thereof. In some embodiments, the detection device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor.

[0028] The present disclosure further contemplates a system further comprising a monitor. In some embodiments, the system comprising the secondary screening device comprises a plurality of organoid modules. Some embodiments of the system further comprise an interconnected fluid exchange network, wherein the network comprises a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank. Some embodiments further comprise a port for introducing a compound. In some embodiments, the interconnected fluid exchange network comprises fluid communication between at least two organoid cassettes. In some embodiments, the fluid is culture medium. Some embodiments further comprise an air pressure controller, such as an embodiment in which the air pressure controller controls the concentration of at least one of O2 and CO2 in at least one module or one or more organoid cassettes. Some embodiments further comprise a drug perfusion device for delivering compounds to cells, tissues, or organoids. Some embodiments further comprise a culture medium mixer.

[0029] Another aspect of the present disclosure is a method for screening a compound having a cardiac effect, comprising: (a) pacing a cardiac tissue strip disclosed herein with electrical stimulation at a pacing frequency of 0.5 Hz, 1.0 Hz, 1.5 Hz, or 2.0 Hz in the presence or absence of a candidate cardiac compound; (b) detecting any movement of the paced cardiac tissue strip in the presence or absence of the candidate cardiac compound; (c) comparing the movement of the cardiac tissue strip paced in the presence of the candidate cardiac compound with the movement of the cardiac tissue strip paced in the absence of the candidate cardiac compound; and (d) determining that the candidate cardiac compound is a cardiac compound when the movement of the cardiac tissue strip paced in the presence of the compound is different from the movement of the cardiac tissue strip paced in the absence of the compound. In some embodiments of the method, the pacing frequency is 1.0 Hz. In some embodiments, the compound is a drug, a viral vector, a conditioned medium, an extracellular vesicle, other cells, or any combination thereof. In some embodiments, the method further comprises an assay for measuring the toxicity of the compound.

[0030] In some embodiments of the method, the cardiomyocytes are engineered cardiomyocytes or cardiomyocytes generated from hiPSCs derived from a patient with a neurological disease, including but not limited to Friedreich's ataxia (FRDA), Kaspersky syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers). In some embodiments, the cardiomyocytes are engineered to have low FXN expression or are derived from a patient with Friedreich's ataxia (FRDA). In some embodiments, the engineered cardiomyocytes are genotypically normal and are derived from healthy hPSCs, including established embryonic stem cell lines or hiPSCs from healthy volunteers, and are engineered to recapitulate a mutant disease phenotype typical of a neurological disease, including but not limited to Friedreich's ataxia (FRDA), Kaspersky syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers), for example, wherein the cardiomyocytes are engineered to express reduced levels of frataxin (FXN) protein, as typically observed in FRDA patients. In some embodiments of the compound screening method involving monitoring motion of a paced cardiac tissue strip, the method further comprises: (a) contacting an anisotropic layer of cardiac cells on a microfabricated substrate with a compound; (b) stimulating the anisotropic layer of cells at one or more points using an electrical source; (c) detecting propagation of electrical signals in the anisotropic layer of cells; and (d) determining whether the cardiac cells exhibit a cardiac effect in the presence of the compound compared to in the absence of the compound.

[0031] Another aspect of the present disclosure is a tissue monitoring system comprising (a) at least one organoid module comprising a plurality of organoid cassettes, wherein each organoid cassette comprises a culture medium inlet, a culture medium outlet, and at least one wall compatible with an external detection device, wherein each of the plurality of organoid cassettes comprises cardiomyocytes or engineered cardiomyocytes of a diseased organism; (b) a mirror arrangement for simultaneously monitoring any biological development of the cardiomyocytes or engineered cardiomyocytes of the diseased organism in each of the at least two organoid cassettes; and (c) a detection device for observing the biological development of the cardiomyocytes or engineered cardiomyocytes of the diseased organism in each of the at least two organoid cassettes being monitored. In some embodiments, the cardiomyocytes are engineered cardiomyocytes or cardiomyocytes derived from a patient with a neurological disease, including but not limited to Friedreich's ataxia (FRDA), Kaspersky syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers). In some embodiments, the cardiomyocytes are engineered to have low FXN expression or are derived from a patient with Friedreich's ataxia (FRDA). In some embodiments of the system, the cardiomyocytes are engineered cardiomyocytes that are genotypically normal and derived from healthy hPSCs, including established embryonic stem cell lines or hiPSCs from healthy volunteers, and are engineered to recapitulate a mutant disease phenotype typical of a neurological disease, including but not limited to Friedreich's ataxia (FRDA), Kaplan-Meier syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers), for example, wherein the cardiomyocytes are engineered to express reduced levels of frataxin (FXN) protein, as typically observed in FRDA patients.

[0032] In some embodiments, the mirror arrangement comprises at least one conic mirror. Some embodiments further comprise an electrode in adjustable relationship with the cells, tissue, or organoid in at least one organoid cartridge. In some embodiments, the detection device is a recording device. Some embodiments further comprise a temperature control element, a light source, a module access port, or any combination thereof. Some embodiments further comprise a data processor in electronic communication with the detection device, the temperature control element, the light source, the module access port, or any combination thereof. In some embodiments, the recording device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor. Some embodiments further comprise a tissue comprising at least one human cell. Some embodiments of the system further comprise a monitor.

[0033] In some embodiments, the system comprises a plurality of organoid modules. Some embodiments further comprise an interconnected fluid exchange network, wherein the network comprises a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank. Some embodiments further comprise a port for introducing a compound. In some embodiments, the fluid is culture medium. Some embodiments further comprise an air pressure controller, such as an embodiment in which the air pressure controller controls the concentration of at least one of O2 and CO2 in at least one module or one or more organoid boxes. Some embodiments further comprise a plurality of module access ports. Some embodiments further comprise a drug perfusion device for delivering a therapeutic agent to the cells, tissues, or organoids.

[0034] Other features and advantages of the disclosed subject matter will become apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 In vitro modeling of FRDA using engineered cardiac tissue constructs from FXN-deficient hPSCs. A) Experimental timeline for generating cardiac tissue models, human ventricular cardiac anisotropic sheets (hvCAS), and human ventricular cardiac tissue strips (hvCTS), for electrophysiological and contractile assessments, respectively. B) FXN transcript and protein expression in hESCs (n=9), hiPSCs (n=9), and FRDA-hiPSC lines 68 (n=10) and 03665 (n=3), normalized to GAPDH expression. Data are shown as mean ± SEM. *Indicates statistical significance, p<0.05.

[0036] Figure 2hESC-derived isogenic FRDA cardiac model. A) FXN transcript and protein expression in hESC-hvCMs transduced with Lv-shFXN1 (n=4) and Lv-shFXN2 (n=4) (normalized to GAPDH expression) relative to controls transduced with Lv-shNT (n=8). Data are shown as mean + SEM. B) Representative twitch force traces of hES2-hvCTS transduced with Lv-shFXN1 and Lv-shFXN2 at day 12, with Lv-shNT as a control. C) Twitch force generation by 1 Hz paced hESC-hvCTS transduced with Lv-shFXN1 (n=14) and Lv-shFXN2 (n=7) at days 7, 9, and 12, compared to controls transduced with Lv-shNT (n=17), shown as medians with interquartile ranges. D) Kinetic analysis of hESC-hvCTS force production at day 12. All force production data are shown as median with interquartile range. * indicates statistical significance, p < 0.05.

[0037] Figure 3 .FRDA cardiac model derived from FRDA-hiPSC. A) FXN transcript and protein expression (normalized to GAPDH expression) of healthy control hiPSC-hvCM (n=5), FRDA(68)-hiPSC-hvCM (n=4), and FRDA(03665)-hiPSC-hvCM (n=4). B) Correlation of force generated in hESC- and hiPSC-hvCTS with FXN expression (at day 12, pacing at 1 Hz). Data are shown as mean + SEM. *Indicates statistical significance, p < 0.05.

[0038] Figure 4 Electrophysiological measurements of FXN-deficient hvCAS derived from hESCs and hiPSCs relative to their respective healthy controls. A) Representative action potentials and isochrone plots from control and Lv-shFXN-transduced hESC-hvCAS, as well as healthy control and FRDA patient-derived hiPSC-hvCAS. B) Maximum capture frequency (MCF) of control (n=32) vs. shFXN (n=33) hESC-hvCAS and healthy control (n=25) vs. FRDA (n=35) hiPSC-hvCAS. C) Action potential duration at 50% repolarization (APD50) and 90% repolarization (APD90) derived from optical mapping of control vs. Lv-shFXN-transduced hESC-hvCAS and healthy control vs. FRDA hiPSC-hvCAS. Data are shown as median with interquartile range. *Indicates statistical significance, p<0.05.

[0039] Figure 5FXN expression rescue in the FRDA hvCTS model. A) FXN protein expression and representative Western blot images of FRDA(03665)-hiPSC-hvCMs transduced with Lv-RFP control (n=3) relative to the rescue group transduced with Lv-FXN (n=3). B) Twitch force generated at 1 Hz pacing in FFRDA(03665)-hiPSC-hvCTS transduced with Lv-FXN (n=10) relative to the Lv-RFP control (n=9). FXN expression data are shown as mean + SEM, and force generation data are shown as median with interquartile range. * and ** indicate statistical significance at p<0.05 and p<0.01, respectively. C) Isometric force measurements of hESC-derived hvCTS double-transduced with Lv-shFXN and Lv-FXN relative to controls transduced with Lv-shFXN and Lv-RFP at 1 Hz (Lv-shFXN + Lv-FXN: n = 17; Lv-shFXN + Lv-RFP: n = 14). FXN expression data are shown as mean + SEM, and force generation data are shown as median with interquartile range. * indicates statistical significance, p < 0.05.

[0040] Figure 6 FRDA hvCOC disease model. FRDA models of hvCOC created using hESC-hvCMs transduced with Lv-shFXN or patient-derived FRDAhiPSC-hvCMs are physically intact and can pump fluid and generate pressure. These models can be cross-compared with healthy hESC-hvCMs on functional properties including stroke work, stroke volume, ejection fraction, developed pressure, and cardiac output.

[0041] Figure 7 A) Schematic diagram of a bioreactor system comprising an organoid module 10, a computer-controlled detection / recording device 2 (e.g., a camera) for simultaneously imaging (and optionally storing images of) up to four organoid cassettes 20 via a retroreflector 13, each containing an organoid 1 (at least one of which is cardiac, while the others can be any organoid, such as heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gallbladder, lung, small intestine, colon, bladder, prostate, uterus, blood, blood vessel, tumor, eye, or skin). An organoid module 10 can contain multiple organoids 1 of the same type or multiple organoid types. Organoid cassettes 20 can be used interchangeably with organoid chambers 20. B) Schematic diagram of an imaging bioreactor platform comprising a computer or data processor 5 that controls the array of organoid modules 10.

[0042] Figure 8A three-dimensional rendering of an organoid module 10 with four organoid cassettes 20. An isometric view and a side view are shown. Also shown are the organoids 1, a detection / recording device 2 connected to a lens 3, a light 12 (e.g., an LED light), a cuboctahedron 13, the organoid cassettes 20, a temperature control element 4 (e.g., a heater), and a mixer 19, such as a stirring bar.

[0043] Figure 9 A) Schematic diagram of the fluid exchange system for an organoid cartridge, including fluid lines, pumps, valves, pressure sensors, and a fluid tank. Specific configurations of valves and pumps are used depending on the function, e.g., B) aspiration or C) addition of fresh medium to a medium reservoir. A detailed description of an illustrated embodiment of the bioreactor system is provided in Example 6.

[0044] Figure 10 A) Diagrammatic representation of the fluid exchange system, which consists of fluid lines, pumps, and valves that direct culture medium between multiple organoid cassettes within a module. Multiple organoid types can be connected to simulate a "body in a jar." B) A cardiac organoid with sufficient pumping capacity can be used as the sole biological pump to form a self-powered "body in a jar." Example 6 provides additional descriptions of these embodiments of the bioreactor system.

[0045] Figure 11 A) An example of a bioreactor system is shown, showing the inlet and outlet paths for medium exchange through an organoid 1 controlled by valves (left pane: cardiac organoid; right pane: liver organoid). B) Schematic diagram of a mechanical stimulation system, where a reversible fluid pump is connected to the organoid 1 to inflate and deflate it. Based on the pressure changes delivered by the stimulation system and the flexibility of the organoid 1, the organoid 1 is stretched.

[0046] Figure 12 Schematic diagram of the LabVIEW front panel used to operate a bioreactor platform or system. A) Acquisition preview window for multiple organoids. B) Environmental control panel. C) Electrical stimulation parameters. The user can control voltage and power, change frequency, select the chamber to stimulate, and decide whether to deliver continuous stimulation or single pulses. D) Real-time pressure and volume data for four different organoids. Pressure is represented by a gray line, while organoid volume is represented by a black line. E) Recording parameters.

[0047] Figure 13 MATLAB analysis to generate average PV loops from the acquisition. A) Calculation of the average volume contraction curve for the tissue. Each volume contraction of the beat is plotted as a scatter plot, with the maximum contraction time set at t = 0 seconds. The average curve is represented by a solid red line. B) Plot of the average PV loop summarizing multiple contractions. Red circles indicate the values ​​at the sampling time points.

[0048] Figure 14. Flowchart of LabVIEW software for monitoring cells, tissues, and organoids in the systems and devices disclosed herein. The flowchart schematically illustrates software-based control of environmental variables, such as temperature and CO2 levels, and software-based control of system and device features, such as lens control, lighting control, and electrical stimulation of cells, tissues, and / or organoids contained in the system or device. DETAILED DESCRIPTION

[0049] The present disclosure provides a system and related methods for screening compounds for beneficial or toxic cardiac effects derived from cardiomyocytes derived from patients with a neurological disease, disorder, or condition that may have at least one cardiac effect. A typical configuration of the system includes an efficacy screen followed by a toxicity screen, depending on the phenotype of the disease to be modeled. The initial efficacy screen is used to identify treatments that can improve, rescue, or eliminate disease symptoms, followed by a toxicity screen to eliminate treatments with deleterious side effects. To model cardiac electrophysiology and arrhythmias, a cardiac anisotropic sheet, such as a human ventricular cardiac anisotropic sheet (hvCAS), comprising a monolayer of human ventricular cardiomyocytes coated on a microfabricated substrate, is provided that provides an environment conducive to the development of anisotropic properties unique to human cardiac cells in vivo. Together with well-defined inclusion / exclusion quality control criteria and algorithms, CAS platforms (e.g., hvCAS) can reproduce key electrophysiological characteristics of the native human heart while minimizing the variability commonly seen in conventional hPSC-CM electrophysiological assays for systematic assessment of disease and drug-induced arrhythmogenicity. A significant advantage of biohybrid materials is that in vitro assays can be performed that more accurately reflect in vivo physiological effects. To assess contractility, a multi-layer system, such as a two-layer system and related methods, is employed, wherein the first layer is designed to provide accurate yet rapid, versatile and cost-effective preliminary screening of compounds for beneficial cardiac effects. The first-layer system comprises a cardiac tissue strip (CTS), such as a human ventricular cardiac tissue strip (hvCTS) supported in a manner that allows significant flexibility in gel movement, and an associated detection (e.g., recording) device that captures gel movement in the presence or absence of a test compound. The CTS is easy to prepare and is used in a simple manner to screen compounds for their ability to induce gel movement in embedded cells. The first-layer system and method are suitable for high-throughput formats as well as conventional formats.

[0050] The second layer of screening systems and methods involves tissues and organoids that are developed and maintained in an organoid box or cavity, typically located in an organoid module, as described herein. The second layer of screening involves exposing the tissue or organoid to a candidate compound in a box or cavity placed in an environment where a detection (e.g., recording) device can monitor the behavior of the organoid. The environment also typically provides for the delivery and removal of fluids, such as culture media and fluids containing compounds, under controlled conditions, requiring various controls to maintain an environment compatible with the viability of the tissue or organoid. The two-layer system is used in a two-layer method that reveals compounds that have beneficial cardiac effects at the cellular, tissue, and / or organoid or organ level, thereby improving the accuracy and reliability of the results obtained when screening for compounds with such effects. In addition, the present disclosure provides a three-layer system and method that relates to the above-mentioned two-layer system and method and supplements a third-layer system and method involving a multi-organoid (or multi-tissue or mixed tissue and organoid) module system and related methods. In this third layer, multiple tissues and / or organoids are developed and maintained in different organoid boxes or cavities that can be conveniently located in a single organoid module (it should be understood that the organoid boxes and organoid modules can contain tissues or organoids), which can be of the same type (e.g., heart) or different types. This typical arrangement conveniently allows a single mirror system (e.g., a conic mirror system) to be used in conjunction with a single detection (e.g., recording) device. As the compound is subjected to the three-layer system and method, information is obtained about the beneficial cardiac effects of the compound on the cells and the effects of the compound on one or more homologous tissues, organoids or organs or on multiple different tissues, organoids or organs. The three-layer screening system and method further enhances the data obtained in terms of accuracy, reliability, and repeatability, and adds a manageable cost in terms of money and time.

[0051] The following terms are defined herein.

[0052] "APD50" means action potential duration at 50% repolarization.

[0053] "APD90" means action potential duration at 90% repolarization.

[0054] "FRDA" stands for Friedreich's ataxia.

[0055] "FXN" means the protein frataxin, and "FXN" means the polynucleotide encoding FXN.

[0056] "Engineered cardiomyocytes" are cardiomyocytes that have been recombinantly engineered to exhibit a specific genotype and phenotype. As used herein, typical engineered cardiomyocytes include cardiomyocytes into which exogenous nucleic acids, such as short hairpin RNAs directed against FXN, have been introduced using any known vector, such as the lentiviral vectors disclosed herein.

[0057] “hESC” means human embryonic stem cell, and “hESCs” means human embryonic stem cells.

[0058] “hiPSC” means human induced pluripotent stem cells, and “hiPSCs” means a plurality of human induced pluripotent stem cells.

[0059] “hPSC” means human pluripotent stem cell, and “hPSCs” means human pluripotent stem cells.

[0060] “hvCAS” means human ventricular cardiac anisotropic sheet, and “hvCASs” means a plurality of human ventricular cardiac anisotropic sheets.

[0061] “hvCM” means human ventricular cardiomyocytes, and “hvCMs” means a plurality of human ventricular cardiomyocytes.

[0062] “hvCOC” means human ventricular cardiac organoid chamber, and “hvCOCs” means multiple human ventricular cardiac organoid chambers.

[0063] "hvCTS" means human ventricular cardiac tissue strip, and "hvCTSs" means a plurality of human ventricular cardiac tissue strips.

[0064] In general, the present disclosure encompasses neurological diseases or conditions and ataxias with cardiac dysfunction, such as those that disrupt the neurocardiac axis. Inherited neurological diseases and conditions may have direct or indirect cardiovascular effects, including effects on cardiac physiology. Exemplary neurological diseases and conditions with the potential for such effects include, but are not limited to, Friedreich's ataxia (FRDA), as described herein; Kaspersky syndrome, which is a mitochondrial myopathy with cardiac conduction abnormalities and cardiomyopathy; carbohydrate deficiency glycoprotein type Ia syndrome, which is a neurological disease with malformations and cardiac manifestations (mean onset of cardiac damage is 5 months, 20% die within the first year of life, usually due to severe cardiac complications); spinocerebellar ataxias, which have cardiovascular abnormalities, particularly abnormal heart rate variability; Wilson's disease, which is a The following are some of the most common diseases: a copper metabolism disorder with concentric remodeling and supraventricular tachycardia; arrhythmias; Dandy-Walker syndrome, which is characterized by cardiac malformations; dilated cardiomyopathy with ataxia, which presents with dilated cardiomyopathy and long QT, with 70% of patients progressing to heart failure or sudden cardiac death; Leigh disease, a neurological disorder that may be associated with hypertrophic cardiomyopathy; MEFAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), a mitochondrial disease with FV hypertrophy; and MERRF (myoclonic epilepsy with ragged red fibers), a neuromuscular disorder with cardiomyopathy.

[0065] To maintain the energy expenditure required for constant cardiac contraction, cardiomyocytes (the single working unit of the heart) are endowed with the highest mitochondrial density of all cells. 26 FRDA, an exemplary non-cardiac disease with deleterious effects on the heart, is caused by a genetic mutation that reduces FXN production and, consequently, the biosynthesis of numerous iron-sulfur proteins essential for mitochondrial oxidative metabolism. Therefore, the disease is expected to adversely affect cardiomyocytes, which are primarily dependent on mitochondrial ATP production. Not surprisingly, heart failure and arrhythmias are the leading causes of death in FRDA patients. 3-6 , indicating contractile and electrophysiological dysfunction at the cellular level. In the experimental studies disclosed herein, the effects of FXN expression on contractile and electrophysiological function in hvCM models generated from hESCs or hiPSCs, including cell lines reprogrammed from FRDA patient cells, were tested. Indeed, FRDA-hiPSC lines from two different patients both showed lower levels of FXN transcripts and protein than healthy hESCs and hiPSCs, indicating that hvCMs derived from FRDA-hiPSCs provide an in vitro model for studying FRDA.

[0066] To eliminate possible differential responses in contraction and electrophysiological function due to variations in the genetic background of different hPSC lines, an isogenic FRDA model was generated by knocking down FXN expression in hESCs using lentiviral-delivered Lv-shFXN to mimic low FXN expression as reported in FRDA patients, as disclosed in the examples below. This strategy has been shown to be effective as demonstrated by the reduction of FXN expression at both transcript and protein levels ( Figure 2 A, Example 3). More importantly, contractile dysfunction was observed for the first time in cardiac tissue hvCTS engineered from FXN-deficient hvCMs. Unlike healthy hESC-hvCTS, which showed a gradual increase in developmental force over time, contractile force decreased and remained low in FXN-deficient hESC-hvCTS ( Figure 2 B and 2C, Example 3). Furthermore, the rise and decay of force development were slower in hESC-hvCTS cells lacking FXN. These observations suggest that FXN deficiency impacts the ability of hvCMs to generate force, a phenomenon that becomes more pronounced as hvCTS cells mature over time.

[0067] The effects of FXN deficiency on cardiac contractile dysfunction were further validated in the FRDA-hiPSC-derived hvCM model. Similar to the isogenic FXN-deficient hESC-hvCM model, FRDA-hiPSC-derived hvCMs also demonstrated reduced FXN synthesis relative to healthy hiPSC-hvCM controls by qPCR and western blotting, despite the different genetic backgrounds between the cell lines. The relationship between developmental force and FXN expression was validated in six different hESC- and hiPSC-hvCTS models of FRDA (including isogenic FXN knockdown models in healthy hESC- and hiPSC-hvCTS, their respective healthy hESC- and hiPSC-hvCTS controls, and FRDA patient-derived hiPSC-hvCTS from two patients). It is important to note that there was a strong positive correlation between the magnitude of active force and FXN expression ( Figure 3 B, Example 4), as shown by a Pearson's coefficient of 0.84, where values ​​>0.5 indicate a strong positive correlation. Thus, the data disclosed herein establish for the first time the FRDA contractile phenotype in two in vitro models: an isogenic model derived from FXN knockdown and another model directly from a FRDA patient with an intrinsic mutation in the FXN gene.

[0068] The impaired contractility of FXN-deficient hvCTS could be attributed to reduced contractility of individual hvCMs or a reduction in the number of contractile cells. Since live-dead staining of hvCTS did not reveal differences in the number of dead cells between FXN-deficient and healthy hvCTS, we can rule out the possibility that a loss of contractile hvCMs over time is responsible for the reduced contractility of FXN-deficient hvCTS. Reduced contractility of individual hvCMs is reasonable to expect, as contraction is associated with high energy expenditure, and FXN is required for iron-sulfur proteins such as aconitase and succinate dehydrogenase, which are involved in oxidative metabolism, the primary method of ATP production in mature cardiomyocytes. Detrimental effects on ATP production would be expected to lead to impaired force generation.

[0069] FXN deficiency induces electrophysiological changes in hvCMs

[0070] Given that one of the main causes of death in FRDA patients is cardiac arrhythmia 3-6Therefore, extensive studies have been conducted to determine the effects of FXN on electrophysiological properties in hvCAS (monolayers of aligned hvCMs). This tissue structure allows measurement not only of action potential parameters of individual cells, but also of the electrical conductivity of these cells as syncytia, which is the only way to truly test arrhythmogenicity. Arrhythmogenicity is assessed by the incidence of automaticity and the incidence of reentrant arrhythmias represented by spiral waves. Although no statistically significant increase in arrhythmias was detected in FXN-deficient hvCAS compared to controls, it is important to note that hvCAS are composed of ventricular CMs, and supraventricular arrhythmias are a common type of arrhythmia in patients with FRDA.

[0071] Although no increase in arrhythmogenicity was detected in FXN-deficient hvCAS transduced with Lv-shFXN or carrying a genetic defect in FXN, the mean MCF of cardiac tissue constructs generated by either method was consistently lower compared to their respective healthy controls ( Figure 4 B, Example 5). MCF is an indicator of cell connectivity - lower MCF in FXN-deficient hvCAS indicates weaker electrical coupling between these cells. Indeed, disruption of the organization of connexin 43, which forms gap junctions at intercalated discs, has been observed in sections of cardiac tissue samples from FRDA patients. 27

[0072] Interestingly, APD50 and APD90 of isogenic FXN-deficient hESC-hvCAS and FRDA-hiPSC-hvCAS were consistently prolonged compared with corresponding controls ( Figure 4 C, Example 5). This result suggests that the disease phenotype is robust and unaffected by background genetic differences. Interestingly, the first signs of FRDA-related pathological symptoms due to metabolic stress in the heart can be detected by abnormal electrophysiology, particularly the presence of T-wave inversions in the EKG, which reflect abnormal cellular repolarization. 6 This observation is consistent with the delayed repolarization observed in FXN-deficient hvCAS. The delayed repolarization may be due to the intracellular Ca 2+ The increase in Ca 2+ The sarcoplasmic reticulum / endoplasmic reticulum Ca transported back to the sarcoplasmic reticulum (SR) 2 + The pumping activity of ATPase (SERCA) is reduced, which affects the Na2+ transport across the sarcolemma. + -Ca 2+ The consistency of APD prolongation across models suggests that the APD parameter can be used as a reliable readout for screening pharmacological treatments using in-dish disease models.

[0073] Restoration of FXN expression rescues impaired contractile function

[0074] By inducing FXN expression in both hiPSCs derived from FRDA patients and hESCs with FXN knockdown, force generation in hvCTS was significantly improved in both models, demonstrating for the first time that restoring FXN expression rescues impaired contractile function in a three-dimensional tissue model of human FRDA. Figure 5 , Example 6). This is consistent with the inducible and reversible murine FRDA model, which showed that restoration of FXN expression can reverse the pathological effects. 16 These findings suggest that restoration of FXN expression is an effective strategy for treating patients with FRDA by preventing and reversing the pathological cardiac symptoms that are a major contributor to FRDA lethality. Importantly, the studies disclosed herein demonstrate that human pluripotent stem cell-derived three-dimensional tissues with appropriately selected sensitive readouts, as demonstrated using an isometric force measurement system, can serve as sensitive and accurate disease models for therapeutic testing and drug screening.

[0075] FXN-deficient hvCOC model

[0076] In addition to hvCTS, fluid-jet human ventricular cardiac organoid chambers (hvCOCs) provide advanced engineered cardiac tissue for modeling FRDA disease phenotypes. The hvCOC model can recapitulate physiologically complex behaviors such as pressure-volume relationships, stroke work, and cardiac output, and also provides a pro-maturation environment to enhance cardiac mimic properties, especially those related to contractility. The hvCOC FRDA model created with Lv-shFXN-transduced hESC-hvCMs or patient-derived FRDA hiPSC-hvCMs is physically intact and can pump fluid and generate pressure ( Figure 6 When compared to control hESC-hvCOCs, these models displayed impaired functional properties including stroke work, stroke volume, ejection fraction, developed pressure, and cardiac output ( Figure 6 ). They add a higher level of assay for treatments producing positive outcomes in hvCTS disease models, providing more physiological and mature tissue constructs with increased sensitivity to confirm positive effects on contractility.

[0077] Current and future status of the FRDA in vitro heart model

[0078] For the experiments disclosed in the following examples, two human cardiac FRDA models were developed using hESCs with FXN knockdown and hiPSCs reprogrammed from FRDA patient cells. Each model has its own unique advantages over the other. Although generating a FRDA model by FXN knockdown in hESCs mimics FXN deficiency and creates isogenic diseased cells to compare with healthy cells, thereby reducing readout variability by eliminating genetic background differences, the FRDA model generated from FRDA patient-derived hiPSCs is expected to be a more physiologically accurate model of heart disease in a dish, in which FXN expression is suppressed by pathological GAA expansion in the FXN gene. This latter model is expected to be suitable for in vitro mechanistic studies of FRDA pathogenesis. By utilizing both isogenic and patient-specific FRDA models, we were able to determine robust contractility (developed force at 1 Hz pacing) and electrophysiological (MCF and APD) readouts, as indicated by the consistent phenotypes exhibited in both FRDA models tested.

[0079] The experiments disclosed below demonstrate contractile and electrophysiological dysfunction of cardiac tissue in FXN-deficient hPSC-derived ventricular CM models, engineered from either shRNA knockdown of healthy hESCs and hiPSCs or FRDA patient-specific hiPSCs, compared to corresponding controls. Specifically, reduced force generation was detected in FXN-deficient hvCTS, and electrical coupling dysfunction and action potential prolongation were measured in FXN-deficient hvCAS, reflecting clinical symptoms of cardiomyopathy and T-wave inversion, respectively. Assessment of these parameters is expected to provide reliable pharmacological screening readouts for in vitro engineered cardiac tissue models using FRDA or other non-cardiac diseases that can exhibit deleterious cardiac effects. For diseases with electrophysiological and contractile symptoms similar to FRDA, both electrophysiological (hvCAS) and contractile (hvCTS / hvCOC) models can be used for efficacy and safety screening.

[0080] Importantly, we have demonstrated in two human FRDA in vitro models that the pathological effects on cardiac contractile function associated with FXN deficiency can be rescued by restoring FXN expression. This suggests that treatments that stimulate FXN expression in patient hearts could rescue the contractile symptoms of FRDA, thus revealing a potential therapeutic strategy.

[0081] The following examples illustrate embodiments of the present invention. Example 1 discloses the materials and methods used in the experiments disclosed herein. Example 2 shows that hiPSCs reprogrammed from FRDA patient fibroblasts exhibit reduced FXN expression levels. Example 3 determines that hESC-hvCMs with isogenic FXN knockdown exhibit reduced FXN expression and reduced contractile function. Example 4 shows that FXN-deficient hvCTS have a reduced ability to generate contractile force. Example 5 reveals that FXN-deficient hvCAS exhibit altered electrophysiological properties. Example 6 shows that rescuing FXN deficiency can restore contractile force in hvCTS. Example 7 shows FXN-deficient hvCOCs with contractile dysfunction under baseline conditions and when treated with 0.1 μM isoproterenol, as indicated by impaired stroke volume, stroke volume, developed pressure, and cardiac output.

[0082] Example 1

[0083] This Example describes the materials and methods used in the experiments disclosed in the Examples herein.

[0084] hPSC culture and differentiation into CMs

[0085] Healthy hESCs (HES2; ESI, NIH code ESO2) and FRDA patient-specific hiPSCs (FRDA (68) and FRDA (03665)) were cultured on hESC-defined Matrigel (Corning) with mTeSR1 medium (Stem Cell Technologies) at 37°C with 5% CO2. hiPSCs (PB02) reprogrammed from peripheral blood mononuclear cells by episomal nucleofection of transcription factors OCT3 / 4, SOX2, KLF4, L-MYC, and LIN28 plus p53 interference shRNA were cultured on Essential 8 medium (Gibco) on hESC-defined Geltrex (Gibco). To differentiate hPSCs into cardiomyocytes, dissociated hPSCs were allowed to form cell clusters overnight in ultra-low attachment plates in mTeSR1 with 1 ng / ml bone morphogenetic protein 4 (BMP4) under hypoxia. From day 1 to day 4, cell clusters were treated with 50 μg / ml ascorbic acid (Sigma-Aldrich), 10 ng / ml activin A, and 10 ng / ml BMP4 in StemPro-34 medium supplemented with GlutaMAX (Thermo Fisher Scientific) under hypoxia. Next, cell clusters were treated with 50 μg / ml ascorbic acid and 5 mM IWR-1 in StemPro-34 medium under hypoxia until day 8. After day 8, cell clusters were maintained under normoxic conditions with StemPro-34 medium containing 50 μg / ml ascorbic acid. Using this differentiation protocol 25 , a ventricular subtype yield exceeding 70% of hPSC-derived CMs can be achieved. These differentiated cells are termed human ventricular (hv) CMs and were used in all experiments disclosed herein.

[0086] Knockdown and overexpression of FXN in hvCMs

[0087] To mimic FXN deficiency in FRDA in hESC- and hiPSC-derived vCMs, we generated FXN-deficient cells in both types of hvCMs by following Figure 1FXN was knocked down using the schedule described in A using lentiviral shRNA transduction (Lv-shFXN1: TRCN0000006137 or Lv-shFXN2: TRCN0000010996 inserted into the pLKO.1 vector backbone) at a multiplicity of infection (MOI) of 5. Respective control hvCMs were transduced with a mammalian non-targeting shRNA (Lv-shNT). To restore FXN expression, FXN-deficient hvCMs were transduced with lentivirus overexpressing FXN (Lv-FXN; GE Dharmacon OHS5835-EG2395), while a lentivirus delivering red fluorescent protein (Lv-RFP) served as a control. To assess and compare electrophysiological and contractile function between healthy, FXN-deficient, and / or FXN-overexpressing hvCMs, two tissue construct platforms were constructed from these cells, each specifically designed to enable functional assessments, as described below.

[0088] Contractility Assessment of Human Ventricular Cardiac Tissue Strips (hvCTS)

[0089] To assess contractile function by measuring force generation, hvCM were assessed as hvCTS as described previously. 23-24 Briefly, cardiac clusters at day 15 of hPSC cardiac differentiation were dissociated into single cells and allowed to recover in an incubator for 3 days before being constructed into hvCTS. Each hvCTS consisted of 1.3 × 10 6 cardiomyocytes and 1.3×10 5 Human foreskin fibroblasts were cultured in a mixture of 40% 5 mg / ml collagen I (Thermo Fisher Scientific), 10% 9.3 mg / ml Matrigel, 6% 10× PBS, 2% 1 M NaOH, 8% 10× Minimum Essential Medium (Sigma-Aldrich), 8% 0.2 M HEPES, 10% hvCTS maintenance medium (Dulbecco's Minimum Essential Medium with 10% newborn calf serum), and sterile water to a final volume of 100 μl. The cell-collagen mixture was added to a polydimethylsiloxane (PDMS) mold with force-sensing cantilever posts at each end of the strip to form the hvCTS. The hvCTS was maintained in Dulbecco's Membrane Oil (DMEM) supplemented with 10% newborn calf serum (Gibco).

[0090] The force generated by the hvCTS was measured by recording the displacement of the cantilever post using a custom-designed force measurement system in DMEM medium with HEPES without phenol red at 37°C. The hvCTS was paced with electric field stimulation to measure the force-frequency response. An overview of the experimental schedule is available in Figure 1 Shown in A.

[0091] Electrophysiological evaluation of human ventricular myocardial anisotropy slices (hvCAS)

[0092] To evaluate the electrophysiological properties of hvCMs, which are aligned and electrically coupled syncytia with anisotropic conduction properties similar to in vivo ventricular CMs, cardiac clusters at day 20 of hPSC cardiac differentiation were dissociated into single cells and plated at 4.5 × 10 cells per cm². 5 The cells were plated as a monolayer on a Matrigel-coated microgrooved substrate made of polystyrene shrink film (Shrinky Dinks 'CrystalClear', K&B Innovations) with a groove width of 15 μm, a groove depth of 5 μm, and a groove spacing of 5 μm to form hvCAS, as described previously. 20 After allowing 8 days to recover, action potentials of hvCAS were optically imaged using the MiCAM ULTIMA imaging system (SciMedia) using the voltage-sensitive fluorophores Di-8-ANEPPS and Pluronic F-127 (Thermo Fisher Scientific) in Tyrode's solution (Sigma-Aldrich) containing blebbistatin. Automaticity, threshold voltage, and maximum capture frequency (MCF) of each hvCAS were first determined, and then reentrant arrhythmias were tested by programmed electrical stimulation. An overview of the experimental schedule is available in Figure 1 Shown in A.

[0093] Disease Modeling with Human Ventricular Cardiac Organoid Chambers (hvCOCs)

[0094] To assess cardiac function in a 3D cardiac mimetic construct, hvCM was assessed in the form of hvCOC as previously described. 49 Briefly, cardiac clusters at day 15 of hPSC cardiac differentiation were dissociated into single cells and allowed to recover in an incubator for 3 days before being constructed into hvCOCs. Each hvCOC consisted of 1 × 10 7 hvCM and 1×10 6Human foreskin fibroblasts were inoculated in a mixture of 40% 5mg / ml collagen I (Thermo Fisher Scientific), 10% 9.3mg / ml Matrigel, 6% 10× PBS, 2% 1M NaOH, 8% 10× Minimum Essential Medium (Sigma-Aldrich), 8% 0.2M HEPES, 10% hvCTS maintenance medium (Dulbecco's Minimum Essential Medium with 10% novice calf serum), and sterile water to a final volume of 1ml. The ice-cold, sterile tissue mixture was transferred to a custom-designed bioreactor. The entire device was incubated at 37°C and 5% CO₂ for 2 hours to initiate gel polymerization, after which it was immersed in hvCTS maintenance medium. The tissue was maintained in this environment at 37°C and 5% CO₂ for 10 days, with half of the medium changed daily. During this time, the tissue compacted around the mammosphere core, forming a hollow human engineered cardiac organoid.

[0095] After 10-12 days of cultivation, the silicone rubber balloon was deflated and carefully removed from the organoid. A high-sensitivity pressure catheter (Millar) was pushed into the lumen of the hvCOC cavity and the catheter entry point was sealed. A high-speed camera (Allied Vision, Exton, PA, USA) was installed outside the bioreactor and allowed to capture images at 100 frames per second for direct tissue monitoring. Chamber pressure and digital video were collected simultaneously using a custom acquisition program built into LABVIEW (National Instruments, Austin, TX, USA), and the chamber cross-sectional area was extracted from the video using a custom script in MATLAB (MathWorks, Natick, MA, USA). The chamber volume was then estimated by assuming an equivalent sphere with the same cross-sectional area. According to the resulting PV loop, stroke work was calculated using the product of development pressure and stroke volume. When needed, electric field stimulation was induced using a pair of carbon electrodes with a spacing of 19 mm at a 10V amplitude with a stimulation pulse width of 50 ms. Prior to data analysis, both pressure and video signals were digitally low-pass filtered with a 13-Hz cutoff frequency in MATLAB. hvCOC contraction was measured during spontaneous beating and 1.0-Hz electric field stimulation.

[0096] Example 2

[0097] hiPSCs reprogrammed from FRDA patient fibroblasts exhibit reduced FXN expression

[0098] FXN expression was assessed in one healthy hESC, one healthy hiPSC, and two FRDA-hiPSC lines reprogrammed from two patients, FRDA (68) and FRDA (03665). The two FRDA-hiPSC lines expressed comparable FXN at the transcript level, with both expressing >50% less FXN than the healthy hESC and hiPSC lines ( Figure 1 B). FXN protein levels were consistent with transcript expression, with FRDA-hiPSCs expressing the lowest levels of FXN relative to healthy hPSC lines. The expression differences were statistically significant for both FRDA lines compared to hESCs and FRDA (68) compared to hiPSCs ( Figure 1 B).

[0099] Example 3

[0100] Isogenic FXN knockdown models generated from hESC-hvCMs exhibit reduced FXN expression and contractile function

[0101] To model and study the progression of FRDA in isogenic human CMs in vitro, hESC-derived hvCMs were transduced with two Lv-shFXN constructs, Lv-shFXN1 and Lv-shFXN2, and non-targeting Lv-shNT transduction was used as a control. Both Lv-shFXNs successfully induced knockdown of FXN at the transcriptional level, as demonstrated by a ~70% reduction in expression levels in hESC-hvCMs relative to the Lv-shNT-transduced control. Figure 2 A). Lv-shFXN1 and Lv-shFXN2 reduced FXN protein in hvCMs by 60% and 80%, respectively. Notably, hvCMs transduced with Lv-shFXN showed reduced FXN expression relative to healthy hESCs, which was comparable to the reduction in FXN expression in FRDA-hiPSCs compared to their corresponding healthy controls ( Figure 1 B).

[0102] Contractile function was assessed as twitch force generation developed during 1 Hz pacing. hvCTS typically reestablished spontaneous contraction within 6-7 days after construction. Twitch force generation ( ) was measured from the hvCTS under 1 Hz electrical pacing on days 7, 9, and 12 after hvCTS construction. Figure 2 Contractile force in control hESC-hvCTS gradually increased from a median of 5 μN on day 7 to 135 μN on day 12. In contrast, hESC-hvCTS in which FXN was knocked down by either Lv-shFXN failed to significantly increase developed force over time. By day 12, the median force of FXN-deficient hvCTS was 75-80% lower than that of controls ( Figure 2 C).

[0103] Example 4

[0104] Reduced contractile force production in FXN-deficient hvCTS

[0105] FXN expression in hvCMs derived from FRDA(68)-hiPSCs and healthy hiPSCs was assessed at both the transcript and protein levels. Compared to healthy controls, hvCMs differentiated from FRDA(68)-hvCMs expressed approximately 50% less FXN at the transcript level and nearly 80% less at the protein level ( Figure 3 A). Force was measured after establishing FRDA-hiPSC-hvCMs into hvCTS and again compared to healthy hiPSC controls. To correlate the magnitude of force generation with FXN expression levels, force generated in hvCTS on day 12 by 1 Hz pacing stimulation was plotted relative to FXN transcript expression ( Figure 3 B). Typically, FXN-deficient hvCTS (due to knockdown in healthy hESC- or hiPSC-derived cardiomyocytes or due to genetic defects in FRDA-hiPSC-derived cardiomyocytes) exhibit impaired force production, developing less than half the force of healthy FXN-expressing controls. Thus, there is a strong direct correlation between developed force levels and FXN expression, as indicated by a Pearson coefficient of 0.81.

[0106] Example 5

[0107] Altered electrophysiological properties of FXN-deficient hvCAS

[0108] The electrophysiological function of hvCMs derived from hPSCs lacking FXN by knockdown (control hESCs vs. Lv-shFXN hESCs) or reprogrammed from FRDA patient-specific somatic cells (control hiPSCs vs. FRDA-hiPSCs) was assessed by optical mapping of hvCAS. Action potentials and isochronal maps of hvCAS were derived from optical recordings ( Figure 4 A). The incidence of spiral conduction waves and automaticity, or spontaneous generation of action potentials, was not statistically different in hESC-hvCAS transduced with Lv-shFXN compared to controls transduced with Lv-shNT. The maximum capture frequency (MCF) of hESC-hvCAS transduced with Lv-shFXN, with a median of 2.0 Hz, was statistically lower than that of controls, with a median of 2.5 Hz ( Figure 4 B). FRDA-hiPSC-derived hvCAS were also more difficult to capture at higher pacing frequencies compared to controls—median MCF of 1.5 Hz for FRDA-hiPSC-hvCAS and 2.5 Hz for healthy hiPSC-hvCAS ( Figure 4 B).

[0109] Action potential and conduction parameters quantified from optical recordings revealed electrophysiological differences between healthy hvCAS and FXN-deficient hvCAS. Figure 4 C). For both hESC and hiPSC groups, the action potential rise and decay speeds and conduction velocities between FXN-deficient hvCAS and controls showed no consistent differences in either longitudinal or transverse directions. However, hESC-hvCAS transduced with Lv-shFXN (APD50: 153ms, APD90: 268ms) significantly prolonged the action potential duration at 50% repolarization (APD50) and 90% repolarization (APD90) relative to controls (APD50: 138ms, APD90: 236ms). Figure 4 C). Notably, compared with healthy controls, FRDA-hiPSC-hvCAS also prolonged APD50 and APD90 ( Figure 4 C).

[0110] Example 6

[0111] Rescue of FXN deficiency restores the contractile function of hvCTS

[0112] As a first step to assess whether restoration of FXN deficiency could rescue the impaired contractile function in the hvCTS FRDA model, forced expression of FXN was achieved by transducing FRDA(03665)-hvCTS with Lv-FXN. FXN transcript and protein expression was >30-fold higher compared to controls transduced with Lv-RFP, indicating that FXN deficiency was corrected in FRDA(03665)-hvCTS transduced with Lv-FXN. Figure 5 A). On day 17 after construction, the contraction kinetics and force generated by Lv-FXN-transduced FRDA(03665)-hvCTS at 1 Hz pacing were significantly higher than those of the control (53 μN), with a median contraction force of 167 μN ( Figure 5 B).

[0113] Next, the functional consequences of restored FXN expression were assessed in a complementary isogenic hESC-hvCTS FRDA model, which included two groups doubly transduced with a combination of FXN knockdown and forced expression—the Lv-shFXN+Lv-FXN group with FXN knockdown and forced expression and the Lv-shFXN+Lv-RFP group with FXN knockdown and RFP control ( Figure 5C). As expected, FXN protein expression was restored in hESC-hvCTS transduced with Lv-shFXN+Lv-FXN, with expression levels significantly higher than those in the Lv-shFXN+Lv-RFP control. More detailed physiological isometric force measurements, performed by manipulating muscle strip length in steps of 0.225 mm (2.5% strain), showed that on day 18 after construction, twitch force developed under 1 Hz pacing increased with increasing muscle strip length in all hvCTS groups, reaching higher twitch forces at Lmax (length at maximum twitch force). Similar to the FXN rescue effect observed in the FRDA-hiPSC model, in the rescued hESC-hvCTS FRDA model with stable FXN expression, twitch force was significantly higher than that of the FXN-deficient control at 50 μN within the physiological range of 17.5-27.5% strain, exhibiting a median force of 109 μN at Lmax. In the hESC-hvCTS FRDA model with restored FXN expression, force dynamics were also significantly accelerated, with a median contraction velocity of 0.89 μN / ms compared to control levels of 0.42 μN / ms at Lmax, and a median relaxation velocity of 0.57 μN / ms compared to a control velocity of 0.24 μN / ms at Lmax.

[0114] Example 7

[0115] Organoid Module

[0116] In some embodiments of the bioreactor disclosed herein, a housing (approximately 25 x 25 x 15 cm) made of sterilizable material with a detection / recording device 2 (e.g., a camera) and a temperature control element 4 (e.g., a heating unit) attached to the top of the organoid module 10, all as shown. Figure 7 . In some embodiments, a temperature control element 4, for example in the form of a heater, is placed within the housing. A vertical camera is focused onto four 45 degree conic mirrors 13, which reflect the side profile of one or more organoids 1 upwards to the camera. An angled LED light 12 evenly illuminates the side profile of each organoid 1. An access door allows a replaceable organoid cartridge 20 to be simply inserted into the organoid module 10 for monitoring and then removed for other experimental analysis (e.g. optical mapping). Following therapeutic administration to the organoid 1, the culture medium 93 is mixed using a mixer 19 in the form of a micro magnetic stirrer (e.g. a ThermoSci Micro Stirrer), which can be turned on and off using software control. See Figure 8. Each organoid module 10 is temperature controlled using a temperature control element 4 (e.g., IncuKitMini) comprising a thermostat, a heater, and a fan. For cell culture buffering, the CO2 level is also individually controlled at 5%. The platform's CO2 control system comprises a single box connected to a pressure regulator, which is first connected to a solenoid valve manifold (e.g., Takasago CTV-2-4MIC) before being connected to each organoid module 10, and finally to a flow meter (Dwyer Mini-Master Flowmeter). Each valve is individually controlled by a multi-channel digital output module (e.g., NI-9472). A CO2 sensor (e.g., SprintIR) within the housing measures the CO2 level and controls the valve to switch between open and closed states. Consider incorporating other sensors (e.g., O2 sensors) to further control the specific partial pressure within the enclosed environment.

[0117] Because microtissues lack key features of larger organs (e.g., diffusion limitations of thicker tissues), they are not ideal for mimicking human organ responses. Bioreactors that allow fluid exchange between multiple macroorganoids recapitulate key physiological and pharmacological features of the human body. The ability to measure multiple functional properties in simplified human biomimetic models provides a new approach to bridge the long-standing gap between traditional cell culture systems, in vivo animal models, and clinical trials. Combined with induced somatic reprogramming of hPSCs, the “human in a jar” system has the potential to become a versatile platform for next-generation drug discovery, cardiotoxicity screening, disease modeling, and other race-, sex-, and patient-specific applications.

[0118] Data from the hvCOC FRDA disease model also revealed physiological differences between healthy cardiomyocytes and cardiomyocytes derived effectively from FRDA patients ( Figure 6 hvCOC FRDA models created with Lv-shFXN-transduced hESC-hvCMs or patient-derived FRDAhiPSC-hvCMs are physically intact and can pump fluids and generate pressure ( Figure 6 When cross-compared with healthy hESC-hvCMs (transduced with a lentiviral non-targeting shRNA control), these models showed impaired functional properties including stroke work, stroke volume, ejection fraction, developed pressure, and cardiac output both under baseline conditions and upon treatment with 0.1 μM isoproterenol (iso). Figure 6 ).

[0119] Example 8

[0120] Screening equipment or bioreactors

[0121] The present disclosure provides a custom bioreactor for culturing numerous, and in some cases, multiple, tissue-engineered human organoids. The device is designed to allow interconnection and simultaneous measurement of multiple organoids and has features that enhance the reproducibility and efficiency of organoid functional testing by enabling subsequent characterization within the same bioreactor with minimal operator manipulation or intervention.

[0122] Figure 6 A high-level schematic diagram is provided illustrating the versatility of the disclosed bioreactor system. Figure 6 A shows an organoid module 10 containing at least one organoid cassette 20. The organoid cassette 20 contains a single organoid 1 of any type (e.g., heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gallbladder, lung, small intestine, colon, bladder, prostate, uterus, blood, blood vessels, tumor, eye, or skin, etc.), preferably a heart. The organoid module 10 can contain multiple organoid cassettes 20 and can therefore contain multiple organoids 1 of a single type or multiple organoids 1. The organoid module 10 is oriented so that a detection / recording device 2 (e.g., a camera) can detect and record the contents of the organoid module 10, for example by making the surface of the organoid module 10 closest to the detection / recording device 2 and preferably perpendicular to the device substantially or completely transparent to at least one wavelength of the electromagnetic spectrum detected by the detection / recording device 2. Figure 6 Figure B shows a data processor 5, such as a computer, connected to at least one organoid module 10. Organoid module 10 generally corresponds 1:1 with detection / recording device 2, and detection / recording device 2 is in electronic communication with data processor 5 via communication path 7, such as conventional wires or wireless communication. A video monitor 6 may also be connected to data processor 5 via communication path 7.

[0123] Figure 7A perspective view of an organoid module 10 is shown. At least one organoid cassette 20 is located within the organoid module 10. Disposed within or outside the organoid module 10 (not shown) and within or outside the organoid cassettes 20 (not shown) is a mixer 19, such as a movable platform (e.g., a vibrator or rotating platform) on which the organoid module 10 is placed, or a magnetic stirring device (e.g., a stirring bar) located within or outside the organoid cassettes 20. In some embodiments, at least one light source 12 is located within the organoid module 10 for illuminating the organoids 1. Also located within the organoid module 10 is at least one mirror 13 for directing electromagnetic radiation from the organoids 1 to a detection / recording device 2 in the form of direct and / or reflected light images. In some embodiments, the mirror 13 is a cuboidal mirror 13 for directing images from multiple organoid cassettes 20 to a single detection / recording device 2. The cuboidal mirror 13 can merge the images of multiple organoid cassettes 20 into a single, focused viewpoint to maximize image resolution while allowing the individual organoid cassettes 20 to be physically separated from each other.

[0124] Figure 9 Elements of one embodiment of a bioreactor system are shown that relate to fluid movement, such as culture medium flow, and particularly to fluid movement related to adding or supplying fresh culture medium and removing or aspirating spent or waste culture medium. Figure 9 A shows the entire fluid exchange system for a single organoid cassette 20 in an organoid module 10, Figure 9 B provides a combination of activation valve and pump for suction, while Figure 9 C provides a combination for supplying fresh culture medium. The components involved in the movement of fluid (e.g., culture medium) in the system can be located inside or outside the organoid module 10. Figure 9 While illustrating one embodiment of a bioreactor system for providing fluid movement, attention will be focused on the pumping mechanism for culture medium. Figure 9 B and for supplying culture medium to the cells, tissues and organoids of the present disclosure Figure 9 C. It will be appreciated that the combined description of pumping and supply will provide a description of the complete fluid communication in one embodiment of a bioreactor system, such as Figure 9 As shown in A. Figure 9 In the remainder of the description, an accessory should be understood as providing fluid communication between the attached components.

[0125] Turning now to one embodiment of a bioreactor system involving a device for aspirating culture medium Figure 9As shown in FIG. 1B , culture medium 93 comes into contact with the cartridge medium-connector D tubing 86, which is attached to connector D valve 67. Organoid-connector D tubing 85 is also attached to connector D valve 67. Furthermore, connector D valve 67 is attached to connector D-pump C tubing 87, which is attached to pump C 72. Pump C 72 is attached to pump C-connector C tubing 88, which is attached to connector C valve 66. Connector C valve 66 is attached to connector C-mixing / recirculation tank tubing 89, which is in turn attached to mixing / recirculation tank 73. In some embodiments, culture medium 93 is recirculated and directed to mixing / recirculation tank 73. Connector C valve 66 is also attached to connector C-waste tubing 90, which leads from connector C valve 66 to waste.

[0126] In operation, the supply of cells in organoids involves Figure 9 The components highlighted in C include fresh media tank 60, which is attached to fresh media-connector B tubing 78, which is in turn attached to connector B valve 65. Connector B valve 65 is attached to connector B-pump B tubing 79, which is attached to pump B 71. Pump B 71 is in turn attached to pump B-connector E tubing 80, which is attached to connector E valve 68. Connector E valve 68 is also attached to connector E-connector F tubing 82, which is attached to connector F valve 69. Cassette medium-connector F tubing 83 is also attached to connector F valve 69, which also contacts cassette medium 93.

[0127] Additional attachment components are described that provide fluid connectivity within the system and allow for additional functionality, including, but not limited to, dilution of therapeutic additives, perfusion of therapeutic agents, therapeutic washing of organoids, flushing of fluid lines, and the like. Fresh medium tank 60 is attached to and in fluid communication with fresh medium-connector A tubing 74, which is in turn attached to and in fluid communication with connector A valve 64, such as a three-way fluid controller or valve. An additive container 62 (e.g., a therapeutic agent container) is used to deliver at least one therapeutic agent to additive tank 63, which is attached to additive tank-connector A tubing 75, which is in turn attached to connector A valve 64. Connector A valve 64 is also attached to connector A-pump A tubing 76, which is attached to pump A 70. Pump A 70 is attached to pump A-mixing / recirculation tank tubing 77, which is in turn attached to mixing / recirculation tank 73. In some embodiments, medium from fresh medium tank 60 is used to dilute the therapeutic agent from additive tank 63 within mixing / recirculation tank 73. The mixing / recirculation tank 73 is also attached to the mixing / recirculation tank-connector B tubing 92, which is in turn attached to the connector B valve 65. The connector E valve 68 is attached to the connector E-organoid cartridge tubing 81. In some embodiments, culture medium can be delivered through the connector E-organoid cartridge tubing 81 to increase the pressure within the organoid 1. The pressure probe, i.e., pressure sensor 95, detects the pressure and pressure changes within the organoid 1 and converts the pressure into an analog electrical signal, which is typically transmitted to a data processor, allowing the pressure to be monitored and adjusted by the system. In addition, the device provides for cleaning or flushing of the fluid lines. In particular, the connector F valve 69 is attached to the connector F-waste tubing 84, which in turn leads from the connector F valve 69 to waste. In some embodiments, fluid can be removed from the fluid exchange system without contacting the organoid cartridge 20 by draining to waste via the connector F-waste tubing 84.

[0128] Figure 10 A high-level schematic diagram of fluid exchange within the organoid module 10 is shown to illustrate the formation of a "body in a jar." Figure 10 A shows a fluid exchange system that transfers culture medium between at least two organoid cassettes 20 within an organoid module 10. Fluid is directed through the system by a series of valves and pumps. Figure 10 B shows a fluid exchange system where fluids are directed by valves and pumped only by biological pumps (e.g., heart organoid 1), providing a self-powered "body in a jar."

[0129] Figure 11 Methods for flowing fluid into and out of organoid 1 are shown. Figure 11Figure A shows an organoid 1 (left: heart organoid; right: liver organoid) connected to a medium inlet tube 26 and a medium outlet tube 28, allowing fluid to be introduced into the interstices of the organoid 1 and out to a waste path through the medium outlet tube 28. The direction of fluid flow through the organoid 1 is controlled by an inlet valve 27 and an outlet valve 29. Figure 11 B shows a method for applying mechanical pressure to an organoid 1 (e.g., lung organoid 1). A fluid pump controls the flow of fluid (e.g., gas or liquid) to the organoid 1 and adjusts the pressure in the organoid cavity to control the size of the organoid 1. The absolute pressure value depends on the material properties of the organoid 1 and the membrane size required for a given application. The applied relative pressure is adjusted to achieve a maximum mechanical strain of 25%.

[0130] As will be apparent to one skilled in the art, some features of the bioreactor are optional, and most features are present in various embodiments. In some embodiments, cells can be derived from any mammalian species or engineered into organoids from cells and / or extracellular matrix. Any organ tissue type is suitable for use with the disclosed systems, compositions, and methods; cardiac cells, tissues, and organoids are preferred. For example, tissues can serve as replacements for any organ, including but not limited to the heart, brain, nerves, liver, kidney, adrenal glands, stomach, pancreas, gall bladder, lungs, small intestine, colon, bladder, prostate, uterus, blood, blood vessels, tumors, eyes, and skin.

[0131] The organoid cassette 20 containing the organoid 1 is typically a cube made of a transparent solid, which can be disposable or sterilizable, and has at least two access ports, such as doors. Suitable transparent solids include glass and transparent plastics, such as polystyrene, acrylic, and polycarbonate. The organoid cassette 20 can also be of any polygonal shape, as long as the detection / recording device 2 can detect and record the behavior of the cells in the organoid 1 within the cassette 20. Given that the structure of the organoid cassette 20 is constrained by the need to allow the detection / recording device 2 to detect cell behavior, it is clear that a variety of transparent and translucent materials can be used in constructing the organoid cassette 20. In embodiments where the detection / recording device 2 does not detect the transmission of visible light from the organoid 1, even opaque materials are contemplated. The organoid cassette 20 is also constructed to be fluid-tight, allowing it to accommodate the cassette culture medium 93 for supplying the cells of the organoid 1. Furthermore, the cassette lid can provide a hole for the passage of at least one electrode or pressure probe (i.e., pressure sensor 95).

[0132] At least one organoid cassette 20 is contained within the organoid module 10, which is formed from a material similar to that used for the organoid cassettes 20. The organoid module 10 is typically square or rectangular in plan view and has a top in addition to a bottom. The organoid module 10 is sized to accommodate at least one, two, three, four, five, six, eight, ten, or more organoid cassettes 20. The walls, top, and bottom of the organoid module 10 are typically formed from a transparent solid, such as glass or a transparent plastic (e.g., acrylic or polycarbonate), but may also be made from translucent or opaque materials, as long as the detection / recording device 2 can detect and record cell behavior. The organoid module 10 also typically contains one or more light sources 12 and one or more mirrors 13, such as a truncated mirror 13. In some embodiments, at least one light source 12 and at least one surface of a mirror 13 are present for each organoid cassette 20 contained within the organoid module 10.

[0133] The remaining components of this system include boxes such as fresh culture medium box 60, mixing / recirculation box 73 and additive box 63, which are containers for holding the fluid used in the bioreactor. Such boxes can be any various sizes and can be made of various materials, as long as the box constructed can be used in an environment (such as sterile environment) designed to minimize biological contamination, and as long as the material used is compatible with any fluid that it can hold and is compatible with forming one or more fluid movement ports. The embodiment of the bioreactor can also relate to one or more pumps, such as pump A 70, pump B 71 and pump C 72, which can be identical or different and can operate according to any known principle that fluid (such as air and / or culture medium by pipe) is provided to move. Exemplary pumps include peristaltic pumps, siphon pumps compatible with sterile environment, positive displacement pumps such as piston-driven pumps and non-positive displacement pumps such as centrifugal pumps. In certain embodiments, gravity is used to move fluid, and no pump is used to move, such as culture medium.

[0134] The organoid module 10 can also interface with various tubing to move gases (e.g., air) for providing pressure, such as to inflate an organoid, which can be a balloon (e.g., a 6-Fr silicone Foley catheter balloon) or for moving fluids. The pressure variation is sufficient to control the inflation of the balloon or to move fluids through the system, and this is achieved at pressures that are compatible with the use of a variety of tubing types, not just tubing certified to handle high pressures. For example, clear plastic flexible tubing, such as Tubing. In addition, as described above, various tubes can be combined into a single-pass tubing, and such combined tubing is particularly suitable for use with peristaltic pumps. In addition, the tubing used in a given embodiment can vary in composition, inner diameter, and outer diameter. Another feature of the system is the connector. The connector typically connects or attaches to two or three tubes, which may vary in diameter and composition, as described above. These connectors can be simply conduits, or more typically, valves that can direct a fluid, such as culture medium, from any attached tube to any other one or two other attached tubes. Additional features and variations thereof will become apparent in light of the full disclosure provided herein.

[0135] In some embodiments, the organoid model has inflow and outflow fluid pathways ( Figure 10 A). Valves (e.g., check valves, solenoid valves) control the direction of fluid movement in and out of the lumen-containing organoid. In some embodiments, a single shaft or tube for inflow and outflow is contemplated. Figure 10 B). A fluid pump controls the flow rate of fluid into and out of the organoid. In some embodiments, unequal inflow and outflow rates are used to control the amount of fluid within the organoid. Regulating the volume within the organoid cavity can result in mechanical stretching of the flexible organoid. In some embodiments, stretching is applied in the form of a step function (passive stretch) or a sigmoid function (cyclic stretch). In many organoid types, mechanical stretch is considered a mechanotransduction signal. In some embodiments, a combination of mechanical and electrical stimulation provides a more robust response for therapeutic agent screening.

[0136] The fluid exchange system automates routine media changes, regulates intraluminal pressure, perfuses candidate therapeutics during screening, and exchanges media between organoids. Figure 9 and 10). The fluid system consists of a series of microfluidic pumps, a three-way valve controlled by a digital output board, and a culture medium reservoir. Changing the valve configuration changes the direction of travel of the culture medium. In some embodiments, fluid can be added to or removed from a hollow vertically mounted shaft to which an organoid is connected, thereby regulating the hydrostatic pressure. A pressure sensor 95 and a signal conditioner (e.g., OPP-M and LifeSens) sense the average pressure within the organoid and communicate with the pump via LabVIEW to regulate the required intracavitary pressure. In addition, a fluid exchange system is used to mix and perfuse compounds into the organoid. The solution is pumped out from the additive tank and mixed with the circulating culture medium. The compound is then perfused into the organoid box 20 and passed through the organoid 1, similar to drug delivery via human bloodstream. In some embodiments, the fluid system of pumps and valves connects at least two organoid boxes 20 in the organoid module 10 to allow exchange of culture medium and / or therapeutic agent between or in the middle of the organoid 1. In addition, in some embodiments, the fluid exchange system between the organoid boxes 20 is powered by a biological pump (e.g., cardiac organoid 1) in the form of an organoid 1.

[0137] Example 9

[0138] Bioreactor controls.

[0139] Custom LabVIEW code automates a large portion of the process, including both hardware and software. Each organoid module 10 is discretely controlled by a computer (i.e., data processor 5) driven by LabVIEW. See Figure 14 for an exemplary software flow chart. Thus, multiple organoids 1 and multiple organoid modules 10 ( Figure 12). The LabVIEW code controls associated hardware, such as data acquisition devices, multi-channel digital output sources, valves, pumps, and camera capture cards. Thus, the code electronically controls multiple functions of the bioreactor platform or system, such as automated drug perfusion and mixing, intraluminal pressure control, electrical stimulation, CO2 and temperature control, and pressure conduction with synchronized image capture capabilities. The computer is equipped with sufficient memory and storage space to continuously capture data (e.g., sufficient for at least 24 hours of continuous data collection). Image acquisition is synchronized with other acquisition modes of the bioreactor (e.g., intra-organoid pressure measurement) to achieve clinically relevant endpoint measurements (e.g., pressure-volume loops). Several analysis functions in the LabVIEW code can enhance and simplify user functionality of the bioreactor. Particle analysis of thresholded digital images, for example, quantifies the real-time volume of multiple discrete organoids 1 via a conic mirror 13, which can be used to calculate contractile characteristics of the relevant organoids 1 in real time. These functions are or can be combined with the control of the electrical stimulator for automated maximum capture frequency analysis and related electrophysiological testing protocols. For example, for cardiac organoid 1, the LabVIEW code begins by sending 0.5Hz biphasic electrical stimulation pulses to cardiac organoid 1 and monitors whether organoid 1 has captured the current frequency. The code automatically increases the rate of electrical stimulation until 1:1 capture is lost, at which point the beating frequency of cardiac organoid 1 no longer matches the stimulation rate. The recording date, duration of drug intervention, electrical pacing protocol, and other information about each detected organoid 1 are saved as metadata for archival and quality control purposes.

[0140] Example 10

[0141] Data capture.

[0142] Pressure and volume data from the bioreactor were recorded simultaneously to generate pressure-volume curves in the associated contracting organoids. A high-speed digital camera (Allied Vision) acquired images at up to 100 frames per second. Organoid volumes were estimated by assuming equivalent spheres with the same cross-sectional area. A single acquisition from, for example, a cardiac organoid 1 typically contains multiple contractions. To characterize the average contraction profile of the organoid, the MATLAB code first separated the curve into discrete contractions. The data for each contraction were then aligned and averaged ( Figure 13 A). The mean pressure curve and mean volume curve can then be plotted as the mean PV loop ( Figure 13 B).

[0143] Recorded high-speed brightfield video (e.g., optical flow) is analyzed to characterize the motion patterns of the contracting organoid 1. Changes in the contraction profile are analyzed to confirm the therapeutic effect on the contractile properties of the organoid 1. To handle the large amount of multi-dimensional data acquisition, machine learning algorithms determine key parameters associated with treatment response and ultimately classify unknown therapeutic agents into categories of interest. In addition, machine learning can be performed simultaneously with long-term data acquisition to identify rare abnormal events and minimize data storage. For example, long-term data acquisition can be divided into a series of continuous acquisitions. As further acquisitions continue, completed acquisitions are sent to a buffer for analysis. Machine learning (e.g., binary support vector machines) evaluates any anomalies in function, such as rare abnormal events, based on the data in the buffer. If an anomaly is detected, the relevant data is permanently stored, and the normal functional data is discarded.

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[0194] Each reference cited herein is incorporated herein by reference in its entirety or, in relevant part, as is apparent from the context of the citation.

[0195] It is to be understood that while the claimed subject matter has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not to limit the scope of the claimed subject matter, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A system for screening a compound for cardiac effects on cardiomyocytes or engineered cardiomyocytes of a diseased organism, comprising: (a) A screening apparatus comprising: (i) a biocompatible gel comprising a plurality of cardiomyocytes from a diseased organism or a plurality of engineered cardiomyocytes, wherein the engineered cardiomyocytes are engineered to have low FXN expression; (ii) a biocompatible support device for suspending the biocompatible gel, wherein the biocompatible support device comprises at least two vertical support members, wherein the biocompatible gel and the biocompatible support device form a cardiac tissue strip comprising cardiomyocytes of a diseased organism or engineered cardiomyocytes; (iii) a detection device for detecting the movement of the biocompatible gel; and (iv) a power source for applying electrical pacing stimulation to the biocompatible gel; The system further comprises: (a) A second-stage screening device comprising: (i) a plurality of organoid modules, said modules comprising at least one organoid cartridge, wherein said organoid cartridge comprises a culture medium inlet, a culture medium outlet, and at least one wall compatible with an external detection device, wherein each organoid cartridge comprises an organoid of a diseased organism or an engineered organoid, and wherein at least one organoid cassette comprises a cardiac organoid or engineered cardiac organoid of a diseased organism, wherein the cardiac organoid or engineered cardiac organoid has pumping capacity; and (ii) a detection device for observing the biological development of the cardiomyocytes of the diseased organism or the engineered cardiomyocytes in each monitored organoid cartridge; wherein the detection device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor.

2. The system of claim 1, wherein the secondary screening device further comprises a mirror arrangement for simultaneously monitoring any biological development of the organoids of the diseased organism or the engineered organoids in each organoid cassette.

3. A system for screening a compound for cardiac effects on cardiomyocytes or engineered cardiomyocytes of a diseased organism, comprising: (a) a screening device comprising: (i) an anisotropic layer of cardiomyocytes on a micromachined substrate; the micromachined substrate comprising grooves oriented along a single axis of the substrate; (ii) a power source for stimulating the anisotropic layer of cells at one or more points; as well as (iii) a detection device for detecting the propagation of electrical signals in the anisotropic layer of cells; The system further comprises: (a) A second-stage screening device comprising: (i) a plurality of organoid modules comprising at least one organoid cartridge, wherein the organoid cartridge comprises a culture medium inlet, a culture medium outlet, and at least one wall compatible with an external detection device, wherein each organoid cartridge comprises an organoid or engineered organoid from a diseased organism, and wherein at least one organoid cartridge comprises a cardiac organoid or engineered cardiac organoid from a diseased organism, wherein the cardiac organoid or engineered cardiac organoid has a pumping capability; as well as (ii) a detection device for observing the biological development of the cardiomyocytes of the diseased organism or the engineered cardiomyocytes in each monitored organoid cartridge; wherein the detection device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor. 4 . The system of claim 3 , wherein the grooves have a width of 1-30 μm, the grooves have a depth of 5 μm, the spacing between grooves is 5 μm, or a combination thereof.

5. The system of claim 3, wherein the power source is at least one electrode or at least one charged portion of the substrate. The system of claim 1 , wherein the cardiomyocytes are human cardiomyocytes.

7. The system of claim 6, wherein the human cardiomyocytes are human ventricular cardiomyocytes.

8. The system of claim 1 , wherein the cardiomyocytes are derived from a patient suffering from Friedreich's ataxia (FRDA), Kaspersky syndrome, carbohydrate-deficient glycoprotein type Ia syndrome, spinocerebellar ataxia, Wilson's disease, Dandy-Walker syndrome, dilated cardiomyopathy with ataxia, Leigh's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or MERRF (myoclonic epilepsy with ragged red fibers).

9. The system of claim 8, wherein the cardiomyocytes are derived from a patient with Friedreich's ataxia (FRDA).

10. The system of claim 1, wherein the biocompatible gel comprises Matrigel.

11. The system of claim 10, wherein the matrigel is present at a concentration of at least 0.5 mg / ml. Degree of existence.

12. The system of claim 10, wherein the biocompatible gel further comprises collagen.

13. The system of claim 12, wherein the collagen is type I human collagen.

14. The system of claim 12, wherein the collagen is present at a concentration of at least 1 mg / ml.

15. The system of claim 1, wherein the vertical support members are made of polydimethylsiloxane.

16. The system of claim 1, wherein there are two vertical support members, each vertical support member having a substantially circular cross-section and a diameter of 0.5 mm.

17. The system of claim 1, wherein the cardiac tissue strip is 26.5 mm in length x 16 mm in width x 6 mm in height.

18. The system of claim 1, wherein the detection device of the screening device or the secondary screening device is a high-speed camera.

19. The system of claim 2, wherein the mirror arrangement of the second stage screening device comprises at least one cube corner mirror.

20. The system of claim 1, wherein the screening device further comprises an electrode in adjustable relationship to the tissue or organoid in at least one organoid cartridge.

21. The system of claim 1, wherein the secondary screening device further comprises a temperature control element, a light source, a module access port, or any combination thereof.

22. The system of claim 21, wherein the secondary screening apparatus further comprises a data processor in electronic communication with the detection device, temperature control element, light source, module access port, or any combination thereof.

23. The system of claim 1, further comprising a monitor.

24. The system of claim 1, wherein the plurality of organoid modules comprises a plurality of organoids of the same type.

25. The system of claim 1, wherein the plurality of organoid modules comprises interconnected organoids of different types.

26. The system of claim 1 further comprising an interconnected fluid exchange network, wherein the The network includes a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank.

27. The system of claim 26, further comprising a port for introducing a compound.

28. The system of claim 26, wherein the interconnected fluid exchange network comprises fluid communication between at least two organoid cartridges.

29. The system of claim 26, wherein the fluid is culture medium.

30. The system of claim 1, further comprising an air pressure controller.

31. The system of claim 30, wherein the gas pressure controller controls the concentration of at least one of O2 and CO2 in at least one module or in one or more organoid cassettes.

32. The system of claim 1, further comprising a drug perfusion device for delivering a compound to the cells, cardiac tissue strips, or organoids.

33. The system of claim 1, further comprising a media mixer.

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