Cardiac Organoids and Anti-Electric Mitochondrial Dysynchronization Therapy

Abstract

The present invention provides a method for treating a disease or disorder characterized by electrical mitochondrial desynchronization in a subject in need of such treatment, the method comprising: determining that the disease or disorder is characterized by electrical mitochondrial desynchronization in the subject; and administering an agent that regulates mitochondrial calcium concentration and / or increases mitochondrial calcium channel activity in the tissue of the disease or disorder in the subject. A multi-chamber cardiac organoid is provided, comprising cardiomyocytes and endothelial cells, and at least two chambers that beat synchronously. In addition, a method of using the multi-chamber cardiac organoid and a method of manufacturing the cardiac organoid are provided. A system for performing measurements in cell aggregates or tissues, and its use for testing therapeutic agents are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 242,091, filed September 9, 2021, and U.S. Provisional Patent Application No. 63 / 329,448, filed April 10, 2022, the contents of which are all incorporated herein by reference in their entireties.

[0002] The present invention relates to cardiac organoids, including methods of using the cardiac organoids. [Background technology]

[0003] Cardiovascular disease is the leading cause of death worldwide, affecting nearly half of the adult population in the United States. Recent studies have led to an increased appreciation for the contribution of cardiomyocyte metabolism to disease progression, demonstrating the metabolic changes that occur during heart failure and arrhythmia development. These insights have led to the development of several therapeutic agents, including elamipretide, that elucidate the mechanism of action of established cardiac therapies, such as β-blockers, and target metabolic pathways despite an incomplete understanding of the kinetics of such interventions.

[0004] Myocardial electromechanical rhythms have long been hypothesized to drive cyclic changes in cardiomyocyte metabolism. Alterations in cellular metabolism due to dyslipidemia or insulin resistance are thought to contribute to abnormal ion homeostasis that increases susceptibility to arrhythmic events. Unfortunately, differences in ion channel kinetics, contraction velocity, and metabolism often hinder our ability to translate findings from small animal models to patients. These differences between animal and human models result in unique responses to pathological events at the molecular and metabolic levels.

[0005] Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes provide a more relevant model of human cardiac metabolism and physiology. Recent studies have demonstrated the utility of transitioning to three-dimensional (3D) cardiac tissue, resulting in more mature tissue function, and higher structural complexity that captures key aspects of cardiac metabolism. Other studies have increased the tissue complexity by adding endothelial vascularization, epicardial cell layers, cardiac fibroblasts, or internal cavities. In parallel, several groups have developed constructs that enable real-time sensing of contractile dynamics using heteropolar wires, 3D printed strain sensors, and microelectrode arrays, offering new opportunities to study aspects of cardiac physiology in a human-relevant system.

[0006] There remains a great need for microphysiological metamaterials that combine cells and sensors, such as to advance our understanding of human physiology, including the behavior of cardiomyocytes. Summary of the Invention [Problem to be solved by the invention]

[0007] The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. [681870]).

[0008] The present invention is based in part on the discovery that in some embodiments, the vascularization of cardiac organoids generates anisotropic stress, resulting in complex multi-chamber structure.These cardiac organoids contain pacemaker-like cell clusters, fibroblasts, epicardial shells, and endocardium, while showing in vivo-like gene expression and function.The integration of the cardiac organoids disclosed herein with a dual photomultiplier tube (PMT) sensor platform allows for simultaneous real-time measurement of oxygen, field potential, and contraction with a resolution of more than 10Hz.

[0009] Metabolic imbalances are important drivers and markers of cardiac disease. However, current methods for measuring metabolism are slow, taking minutes to hours to quantify metabolic fluxes such as glycolysis, mitochondrial respiration, or fatty acid oxidation. Thus, drug development is limited to understanding and treating systemic metabolic diseases in the context of cardiac health. As a non-limiting example, high glucose or dyslipidemia are chronic conditions that ultimately affect cardiac function, and thus treatment with drugs such as empagliflozin or statins is indicated. The electrical metabolic mechanical sensing method developed in this application allows for rapid measurement of metabolic fluxes that control cardiac rhythm. In fact, arrhythmias occur in 25% of adults over 40 years of age, and their exact causes are unknown. Thus, the method described in this patent can be used to develop new drugs and therapeutics to treat multiple types of cardiac arrhythmias as well as ischemic injuries.

[0010] By simultaneous electrical-metabolic-mechanical sensing, we can demonstrate that mitochondrial function in human cardiac organoids is synchronized with their electrical activity, not their mechanical action, as previously theorized.We demonstrate that any type of inhibition of mitochondrial calcium uniporter (MCU) causes arrhythmia, and that this arrhythmia can be reversed by either (1) blocking the interaction of drugs with MCU protein, or (2) directly or indirectly increasing MCU activity.

[0011] As a non-limiting example, we have shown that inhibition of the mitochondrial calcium uniporter by the chemotherapeutic agent mitoxantrone disrupts this electrical mitochondrial coupling, thereby resulting in arrhythmias. We partially reverse this effect by co-administration of metformin, suggesting a combination therapy that may indirectly activate the MCU and thereby block chemotherapy-induced arrhythmias.

[0012] Chemotherapy-induced arrhythmia is a complication of cancer treatment that leads to a significant increase in morbidity and mortality. Atrial fibrillation, ventricular ectopic beats, and prolonged QTc are the most common arrhythmias suffered by cancer patients undergoing chemotherapy. The mechanism of chemotherapy-induced arrhythmia was poorly understood until the present inventors demonstrated that it is caused by the disruption of mitochondrial metabolism using a simultaneous electrical-metabolic-mechanical sensing platform. [Means for solving the problem]

[0013] According to a first aspect, there is provided a method of treating a disease or disorder characterized by electrical mitochondrial desynchronization in a subject in need thereof, the method comprising: identifying that the disease or disorder is characterized by electrical mitochondrial desynchronization in the subject; administering to the subject: a. regulating mitochondrial calcium concentration in diseased or disordered tissue in a subject; b. modulating mitochondrial calcium channel activity in a tissue; or C. A combination of these administering a therapeutically effective amount of an agent that enables Thereby treating a disease or disorder characterized by electrical mitochondrial desynchronization; In one embodiment, a method is provided for treating a disease or disorder characterized by electrical mitochondrial desynchronization in a subject in need thereof, comprising:

[0014] According to some embodiments, the desynchronization comprises decreased mitochondrial calcium concentration or mitochondrial calcium channel activity compared to healthy controls and the modulation is increased, or the desynchronization comprises elevated mitochondrial calcium concentration or mitochondrial calcium channel activity compared to healthy controls and the modulation is decreased.

[0015] According to some embodiments, modulating mitochondrial calcium concentration and / or mitochondrial calcium channel activity comprises modulating mitochondrial calcium uniporter (MCU) activity.

[0016] According to some embodiments, modulating comprises administering an agent selected from metformin, kaempferol, spermine, A-769662, AICAR, IND1316, PF06409577, ZLN024, erastin, honokiol, ezetimibe, disulfiram, efsevin, and spermidine.

[0017] According to some embodiments, the disease or disorder is selected from the group consisting of arrhythmia, cardiomyopathy, seizures, epilepsy, motor neuron spasms, muscle weakness, muscle atrophy, channelopathies, catecholaminergic polymorphic ventricular tachycardia (CPVT), myopathy with extrapyramidal signs (MPXPS), Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (AML), hereditary spastic paraplegia, ischemia-reperfusion injury, ischemic heart disease, rare mitochondrial encephalomyopathy, sagittal sinus thrombosis, intracranial sinus thrombosis, Stormer-Ken syndrome, generalized epilepsy febrile seizures plus, optic atrophy 3, autosomal dominant, generalized epilepsy febrile seizures plus, type 6, palmoplantar keratoderma, non-epidermolytic, and eastern equine encephalitis, and optionally, the arrhythmia is selected from cancer therapy-induced arrhythmia (CTIA).

[0018] According to some embodiments, the desynchronization comprises an increase in mitochondrial calcium concentration or mitochondrial calcium channel activity, and the disease or disorder is selected from mitochondrial encephalomyopathy, sagittal sinus thrombosis, intracranial sinus thrombosis, Stormer-Ken syndrome, generalized epilepsy febrile seizures plus, optic atrophy 3, autosomal dominant, generalized epilepsy febrile seizures plus, type 6, palmoplantar keratoderma, non-epidermolytic, Eastern equine encephalitis, and CTIA, and optionally the cancer treatment is doxorubicin.

[0019] According to some embodiments, the desynchronization comprises a decrease in mitochondrial calcium concentration or mitochondrial calcium channel activity, and the disease or disorder is selected from arrhythmias, cardiomyopathy, seizures, epilepsy, motor neuron spasms, muscle weakness, muscle atrophy, channelopathies, CPVT, MPXPS, Alzheimer's disease, Huntington's disease, Parkinson's disease, AML, hereditary spastic paraplegia, ischemia-reperfusion injury, ischemic heart disease, rare, and CTIA.

[0020] According to some embodiments, the disease is CPVT and modulating comprises administering an agent selected from spermine, spermidine, metformin, erastin, A-769662, AICAR, IND1316, PF06409577, and ZLN024.

[0021] According to some embodiments, the disease or disorder is caused by administration of an agent that causes cardiac side effects, and the agent is selected from a calcium signaling targeting agent, a calcium channel blocker, and an anti-neoplastic agent, and optionally the agent is selected from the agents provided in Table 1.

[0022] According to some embodiments, the verifying comprises: a. determining a mitochondrial calcium concentration in a sample obtained from a subject, a concentration above a predetermined threshold indicating desynchronization, optionally the predetermined threshold being a calcium concentration in a healthy subject or in a subject suffering from a disease or disorder not characterized by electrical mitochondrial desynchronization; b. observing in the patient an arrhythmic or proarrhythmic symptom that is unresponsive to an antiarrhythmic therapy that targets electrical activity via membrane channels, thus indicating desynchronization, optionally wherein the antiarrhythmic therapy is selected from sodium channel blockers, beta blockers, potassium channel blockers, non-dihydropyridine calcium channel blockers, adenosine, and digoxin; c. abnormal readings indicate desynchronization, optionally the abnormal readings include at least one of late potentials, reduced R waves, and increased R / R ratios; d. confirming exposure to agents known to cause electrical mitochondrial desynchronization, optionally the agents being agents selected from those provided in Table 1 and toluene, trichloroethane, xylene, heptane, hexane, ethyl ether trichloroethylene, trichlorotrifluoroethane, carbon monoxide, carbon disulfide, pesticides, bisphenol A (BPA), methane-derived halogenated hydrocarbons, organic nitrates, arsenic, cadmium, cobalt, organic solvents, and metals; e. Ascertaining a medical history including a diagnosis of symptoms indicative of electrical mitochondrial desynchronization; Includes at least one of the following:

[0023] According to another embodiment, a multi-chamber cardiac organoid is provided comprising cardiomyocytes and endothelial cells and at least two synchronously beating chambers.

[0024] According to some embodiments, all chambers beat synchronously or the organoids beat biphasically.

[0025] According to some embodiments, the organoids comprise pacemaker-like cell clusters, and optionally the pacemaker-like cell clusters are potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and short stature homeobox 2 (SHOX2) positive.

[0026] According to some embodiments, the organoid comprises an outer epicardium, and optionally, the epicardium comprises cells positive for Wilms' tumor-1 (WT1) and T-box transcription factor 18 (TBX18).

[0027] According to some embodiments, the organoid comprises an inner endocardium, and optionally, the endocardium comprises cells positive for platelet endothelial cell adhesion molecule (PECAM-1).

[0028] According to some embodiments, the synchronized beats include a basal beat cycle of 50 to 90 beats per minute (bpm).

[0029] According to some embodiments, the organoids comprise vascular structures, circumferentially aligned cardiomyocytes surrounding a hollow chamber, elongated cardiomyocytes organized in a sarcoid pattern, capillaries within the walls of the chamber, and cardiac fibroblast-like cells, optionally wherein the fibroblast-like cells are periostin (POSTN) and / or vimentin positive.

[0030] According to some embodiments, the organoids comprise at least one parameter that is increased compared to isolated cardiomyocytes in culture or fetal heart tissue in culture, the parameter being selected from basal respiration, oxidative phosphorylation, mitochondrial maximum capacity, and expression of at least a factor selected from the group consisting of TNNT2, TNNI3, Cx43, MYH7, AKAP6, GJA5, JPH2, SLC8A1, ATP2A2, CACNA1C, RYR2, CASQ2, PLN, CAMK2B, TRDN, CAV3, BIN1, AMP2, SCN5A, KIR2.1, ITPR3, HCN2, SCN1B, HCN1, KCNJ8, KCNH2, PRKAA1, CPT1A, TFAM, PPARGC1A, PPA1, PPP2R4, SLC2A4, MAPK1, PRKACA, alpha 1A, alpha 1B, SCN4B, KCNE1.

[0031] According to another aspect, a method for producing a multi-chamber cardiac organoid comprising at least two synchronously beating chambers is provided, comprising co-culturing a mass of cardiomyocytes and endothelial cells in a geometrically confined culture space to generate an anisotropic stress gradient in the cell mass, thereby producing a multi-chamber cardiac organoid.

[0032] According to some embodiments, the method includes culturing approximately 6.8 x 10^4 cells in a microwell comprising a diameter of 1 to 1.2 mm.

[0033] According to some embodiments, the co-culture comprises a cardiomyocyte to endothelial cell ratio of 1.5:1 to 2.5:1.

[0034] According to another aspect, there is provided a multi-chamber cardiac organoid comprising at least two synchronously beating chambers produced by the method of the invention.

[0035] According to another aspect, a method for evaluating cardiac cell function is provided, comprising exposing the multi-chamber cardiac organoid of the present invention to a condition and measuring at least one parameter of the multi-chamber cardiac organoid.

[0036] According to some embodiments, the condition is selected from the application of a drug or chemical, hypoxic conditions, circulatory conditions, altered metabolite exposure, altered hormone exposure, and genetic mutations of cells within the organoid.

[0037] According to some embodiments, the at least one parameter is electro-mitochondrial synchronization.

[0038] According to another aspect, there is provided a sensing system comprising: An illumination source; a first photomultiplier tube (PMT) sensor; A second PMT sensor; and A controller, controlling the illumination source to illuminate the microparticles embedded in the tissue or cell aggregate with a photon beam having a first wavelength; detecting, by a first PMT sensor, a first signal indicative of photons reflected from the particulate at a first wavelength; A second PMT sensor detects a second signal indicative of emission from the microparticle at a second wavelength, the microparticle including an excitable molecule that is quenchable by a cofactor. measuring a shift between a frequency of the first signal and a frequency of the photon beam, determining a background noise based on the measured shift, and reducing the background noise from the second signal. Calculating the temporal cofactor consumption of the tissue or cell aggregates based on the background noise-reduced second signal; A controller configured as follows: A sensing system is provided that includes:

[0039] According to some embodiments, temporal cofactor consumption is indicative of oxygen levels in a tissue or cell aggregate.

[0040] According to some embodiments, the controller is further configured to detect a change in an intensity of the first signal and calculate a relative displacement of the microparticle based on the detected change.

[0041] According to some embodiments, the detected change in intensity of the signal indicates a relative displacement of the microparticle, optionally the displacement being measured in an axis perpendicular to the photon beam.

[0042] According to some embodiments, the controller is further configured to sense a field potential of the tissue or cell aggregate from the array of microelectrodes simultaneously with detecting the first signal and the second signal to measure electrical activity of the tissue or cell aggregate.

[0043] According to another aspect, there is provided a method for assessing cellular function, comprising the steps of: a. placing a tissue, organoid or cell aggregate in a sensing system of the present invention; b. applying a condition to the tissue, organoid, or cell aggregate; c. Measuring at least cofactor consumption in tissues, organoids or cell aggregates; thereby evaluating cellular function; and A method is provided that includes:

[0044] According to some embodiments, the sensing system includes a controller further configured to sense a field potential of the tissue or cell aggregate from the array of microelectrodes to measure electrical activity of the tissue or cell aggregate simultaneously with detecting the first signal and the second signal, wherein measuring includes measuring cofactor consumption, displacement and field potential in the tissue, organoid or cell aggregate, wherein a significant deviation in displacement, cofactor consumption and field potential after applying the condition compared to the displacement, cofactor consumption and field potential before applying the condition or compared to a control untreated tissue, organoid or cell aggregate indicates electrical mitochondrial desynchronization.

[0045] According to some embodiments, the cardiac or cerebral organoids are placed in a sensing system.

[0046] According to some embodiments, applying the condition is selected from application of a drug or chemical, application of hypoxic conditions, application of circulatory conditions, altered metabolite exposure, altered hormone exposure, and genetic mutation of cells in the tissue, organoid or aggregate.

[0047] According to another aspect, a. regulating mitochondrial calcium concentration in diseased or disordered tissue in a subject; b. modulating mitochondrial calcium channel activity in a tissue; or C. A combination of these 1. A method for selecting a subject suffering from a disease or disorder suitable for treatment with an agent capable of: Methods are provided that include determining the presence of electrical mitochondrial desynchronization in a subject, the presence of desynchronization indicating that the subject is suitable for treatment.

[0048] According to some embodiments, the determining comprises: a. determining a mitochondrial calcium concentration in a sample obtained from a subject, a concentration above a predetermined threshold indicating desynchronization, optionally the predetermined threshold being a calcium concentration in a healthy subject or in a subject suffering from a disease or disorder not characterized by electrical mitochondrial desynchronization; b. observing in the patient an arrhythmic or proarrhythmic symptom that is unresponsive to an antiarrhythmic therapy that targets electrical activity via membrane channels, thus indicating desynchronization, optionally wherein the antiarrhythmic therapy is selected from sodium channel blockers, beta blockers, potassium channel blockers, non-dihydropyridine calcium channel blockers, adenosine, and digoxin; c. An EKG, EEG, or EMG, wherein the abnormal readings indicate desynchronization, and optionally the abnormal readings include at least one of late potentials, reduced R waves, and increased R / R ratio; d. confirming exposure to an agent known to cause electrical mitochondrial desynchronization, optionally the agent is an agent selected from those provided in Table 1 and toluene, trichloroethane, xylene, heptane, hexane, ethyl ether trichloroethylene, trichlorotrifluoroethane, carbon monoxide, carbon disulfide, pesticides, methane-derived halogenated hydrocarbons, organic nitrates, arsenic, cadmium, cobalt, organic solvents, and metals; e. Ascertaining a medical history including a diagnosis of symptoms indicative of electrical mitochondrial desynchronization; Includes at least one of the following:

[0049] According to another aspect, a multi-chamber cardiac organoid is provided comprising cardiomyocytes and endothelial cells within a geometrically confined compartment, wherein the chamber comprises a chamber wall capable of beating.

[0050] According to another aspect, a method of testing a drug is provided, comprising contacting the multi-chamber cardiac organoid of the invention with the drug.

[0051] According to another aspect, there is provided a method for treating a disease or disorder characterized by electrical mitochondrial desynchronization in a subject in need thereof, comprising: a. an increase in mitochondrial calcium concentration in diseased or disordered tissue in a subject; b. Increased mitochondrial calcium channel activity in the tissue; or c. combinations thereof; Thereby, treating a disease or disorder characterized by electrical mitochondrial desynchronization. Methods are provided that include administering to a subject a therapeutically effective amount of an agent that enables

[0052] According to another aspect, there is provided a method of treating a disease in a subject in need thereof with a first agent that produces a cardiac side effect, comprising: a. a first agent; and b. a second agent that increases mitochondrial calcium concentration in cardiac tissue, increases mitochondrial calcium channel activity in cardiac tissue, or both. and administering thereby treating a disease in a subject; and A method is provided that includes:

[0053] According to another aspect, a method for producing cardiac organoids is provided, comprising co-culturing cardiomyocytes and endothelial cells under conditions such that the cells are geometrically confined to produce cardiac organoids.

[0054] According to another aspect, there is provided a method for measuring a property of a tissue or cell aggregate, comprising the steps of: illuminating a microparticle embedded in the tissue or cell aggregate with a photon beam having a first wavelength and detecting, by a first sensor, a first signal indicative of photons reflected from the microparticle at the first wavelength; detecting a change in intensity of the first signal; calculating a relative displacement of the microparticle based on the detected change; A method is provided that includes:

[0055] According to another aspect, there is provided a sensing system comprising: An illumination source; a first photomultiplier tube (PMT) sensor; A controller, controlling an illumination source to illuminate the microparticle embedded in the tissue or cell aggregate with a photon beam having a first wavelength; detecting a first signal indicative of photons reflected from the microparticle at the first wavelength by a first PMT sensor; detecting a change in the intensity of the first signal; and calculating a relative displacement of the microparticle based on the detected change; A controller configured to: A sensing system is provided that includes:

[0056] According to another aspect, there is provided a method of testing an agent for treating a cardiac side effect, the method comprising: a. placing a cardiac organoid within a sensing system of the present invention; b. adding a therapeutic agent to the cardiac organoids; c. Measuring displacement, cofactor consumption, and field potential within cardiac organoids; A significant deviation in any one of the displacement, cofactor consumption, and field potential after addition of the therapeutic agent compared to the displacement, cofactor consumption, and field potential before addition of the therapeutic agent or in a control untreated cardiac organoid indicates a cardiac side effect caused by the therapeutic agent; thereby testing therapeutic agents for cardiac side effects; A method is provided, comprising:

[0057] According to some embodiments, the endothelial cells are microvascular endothelial cells.

[0058] According to some embodiments, the cardiomyocytes are derived or produced from induced pluripotent stem cells.

[0059] According to some embodiments, the multi-chamber cardiac organoid further comprises a vasculature.

[0060] According to some embodiments, the multi-chamber cardiac organoid comprises at least two chambers.

[0061] According to some embodiments, the multi-chamber cardiac organoids comprise circumferentially aligned cardiomyocytes surrounding a hollow chamber.

[0062] According to some embodiments, the multi-chamber cardiac organoids comprise elongated cardiomyocytes organized into a sarcoid pattern.

[0063] According to some embodiments, the multi-chamber cardiac organoids contain capillaries within the walls of the chambers.

[0064] According to some embodiments, the multi-chamber cardiac organoids comprise cardiac fibroblast-like cells.

[0065] According to some embodiments, the fibroblast-like cells are POSTN positive cells.

[0066] According to some embodiments, the multi-chamber cardiac organoids comprise pacemaker-like cell clusters.

[0067] According to some embodiments, the pacemaker-like cell clusters are HCN4 and SHOX2 positive.

[0068] According to some embodiments, the multi-chamber cardiac organoid comprises an outer epicardium.

[0069] According to some embodiments, the epicardium comprises cells positive for WT1 and TBX18.

[0070] According to some embodiments, the chamber wall comprises PECAM-1 positive endocardial-like cells.

[0071] According to some embodiments, the multi-chamber cardiac organoids are capable of synchronous beating.

[0072] According to some embodiments, the synchronized beating persists for at least one week in culture.

[0073] According to some embodiments, the pulsation is at least 50 beats per minute (bpm).

[0074] According to some embodiments, the multi-chamber cardiac organoids comprise increased expression of at least one factor selected from KCNJ2, KCNJ8, TMNI3, MYH7, AKAP6, PPKAA2, PGC1A, RAR2, CASQ2 and CAV3 compared to isolated cardiomyocytes and / or fetal cardiac tissue.

[0075] According to some embodiments, the multi-chamber cardiac organoids comprise increased expression of 2 to 10 genes selected from the group consisting of KCNJ2, KCNJ8, TMNI3, MYH7, AKAP6, PPKAA2, PGC1A, RAR2, CASQ2 and CAV3 compared to isolated cardiomyocytes and / or fetal cardiac tissue.

[0076] According to some embodiments, the multi-chamber cardiac organoids comprise at least one of increased basal respiration, oxidative phosphorylation, or mitochondrial maximum capacity compared to cardiomyocytes in culture.

[0077] According to some embodiments, the multi-chamber cardiac organoids are capable of generating a physiological response to a therapeutic agent.

[0078] According to some embodiments, the therapeutic agent is epinephrine.

[0079] According to some embodiments, the therapeutic agent is amiodarone.

[0080] According to some embodiments, the method further comprises testing the physiological output of the multi-chamber cardiac organoid after contacting.

[0081] According to some embodiments, the method further comprises comparing the physiological output to an output measured before the contact.

[0082] According to some embodiments, the testing is to test for negative cardiac side effects.

[0083] According to some embodiments, the side effect is arrhythmia.

[0084] According to some embodiments, the drug is an anti-cancer drug.

[0085] According to some embodiments, the drug is a calcium signaling targeting agent.

[0086] According to some embodiments, the drug is a calcium channel blocker.

[0087] According to some embodiments, the method further comprises inducing a cardiac defect, condition or disease in the multi-chamber cardiac organoid prior to contacting, and the drug is a therapeutic agent designed to treat the defect, condition or disease.

[0088] According to some embodiments, the condition is arrhythmia.

[0089] According to some embodiments, increasing mitochondrial calcium activity or increasing mitochondrial calcium chancer activity comprises increasing mitochondrial activity.

[0090] According to some embodiments, increasing mitochondrial calcium channel activity comprises increasing mitochondrial calcium uniporter (MCU) activity.

[0091] According to some embodiments, the agent that increases MCU activity is an agent that blocks the interaction of the drug with MCU, where the drug inhibits MCU activity.

[0092] According to some embodiments, the administered agent is an MCU activator.

[0093] According to some embodiments, the MCU activator is metformin.

[0094] According to some embodiments, the disease or disorder is selected from arrhythmia, cardiomyopathy, seizures, epilepsy, motor neuron spasms, muscle weakness and muscle atrophy, and optionally, the arrhythmia is cancer therapy induced arrhythmia (CTIA).

[0095] According to some embodiments, the first agent is a calcium signaling targeting agent.

[0096] According to some embodiments, the first agent is a calcium channel blocker.

[0097] According to some embodiments, the disease is cancer and the first agent is an anti-neoplastic agent.

[0098] According to some embodiments, the disease is an inflammatory disease and the first agent is an anti-inflammatory agent.

[0099] According to some embodiments, the disease is a disease of the central nervous system (CNS) and the first agent is a CNS agent.

[0100] According to some embodiments, the disease is a gastrointestinal disease and the first agent is a gastrointestinal agent.

[0101] According to some embodiments, the disease is a reproductive or urinary tract disease and the first agent is a urogenital agent.

[0102] According to some embodiments, the disease is an allergic reaction and the first agent is an anti-allergy agent.

[0103] According to some embodiments, the disease is an infectious disease and the first agent is an anti-infective agent.

[0104] According to some embodiments, the disease is a cardiovascular disease and the first agent is a cardiovascular agent.

[0105] According to some embodiments, the first agent is selected from the agents provided in Table 1.

[0106] According to some embodiments, increasing mitochondrial calcium channel activity comprises increasing MCU activity.

[0107] According to some embodiments, the second agent is metformin.

[0108] According to some embodiments, the subject does not suffer from a metabolic disorder, is not being treated for metabolic syndrome, or both.

[0109] According to some embodiments, the metabolic disorder is diabetes or hyperglycemia.

[0110] According to some embodiments, the condition is a concentration of about 6.8x10^4 cells in a 1-1.2 mm diameter microwell.

[0111] According to some embodiments, the co-culture comprises a cardiomyocyte to endothelial cell ratio of 1.5:1 to 2.5:1.

[0112] According to some embodiments, the endothelial cells are microvascular cardiac endothelial cells.

[0113] According to some embodiments, the culture includes the addition of vascular endothelial growth factor (VEGF).

[0114] According to some embodiments, the culturing is carried out for a time sufficient for the formation and synchronous beating of multiple hollow chambers surrounded by cardiomyocytes.

[0115] According to some embodiments, the method is a method for producing the cardiac organoids of the invention.

[0116] According to some embodiments, the culture is carried out for a time sufficient to form organoids characterized by the characteristics of the cardiac organoids of the present invention.

[0117] According to some embodiments, the detected change in intensity of the signal is proportional to the relative displacement of the microparticle.

[0118] According to some embodiments, the displacement is measured in an axis perpendicular to the photon beam.

[0119] According to some embodiments, the method comprises: detecting, by a second sensor, a second signal indicative of emission from a microparticle embedded in the tissue or cell aggregate at a second wavelength, the microparticle comprising an excitable molecule that is quenchable by a cofactor; Calculating the temporal cofactor consumption of the tissue or cell aggregate based on the first and second signals; Includes.

[0120] According to some embodiments, the temporal cofactor consumption correlates with the difference in frequency of the first signal and the second signal.

[0121] According to some embodiments, the temporal cofactor consumption is the oxygen level of a tissue or cell aggregate.

[0122] According to some embodiments, the method further comprises filtering the second signal using a parameter of the photon beam.

[0123] According to some embodiments, the filtering comprises: Measuring a shift between a frequency of the second signal and a frequency of the photon beam; determining a background noise based on the measurement shift; and Reducing background noise from the second signal; and Includes.

[0124] According to some embodiments, the method comprises: Sensing the field potential of the tissue or cell aggregate from an array of microelectrodes to simultaneously measure electrical activity of the tissue or cell aggregate with optical measurements. Includes.

[0125] According to some embodiments, the method further comprises comparing a frequency of the first signal, the second signal, and the tissue field potential.

[0126] According to some embodiments, if the comparison between the frequencies results in a deviation below a threshold, the comparison is indicative of a healthy tissue or cell aggregate.

[0127] According to some embodiments, the detected change in intensity of the signal indicates a relative displacement of the microparticles.

[0128] According to some embodiments, the displacement is measured in an axis perpendicular to the photon beam.

[0129] According to some embodiments, the system comprises: Second PMT Sensor Including, The controller is detecting a second signal from a second PMT sensor indicative of emission from a microparticle embedded in the tissue or cell aggregate at a second wavelength, the microparticle comprising an excitable molecule that is quenchable by a cofactor; Calculating the temporal cofactor consumption of the tissue or cell aggregates based on the first and second signals The method is further configured as follows.

[0130] According to some embodiments, the temporal cofactor consumption correlates with the difference in frequency of the first signal and the second signal.

[0131] According to some embodiments, the temporal cofactor consumption is the oxygen level of a tissue or cell aggregate.

[0132] According to some embodiments, the controller is further configured to filter the second signal using a parameter of the photon beam.

[0133] According to some embodiments, the filtering comprises: Measuring a shift between a frequency of the second signal and a frequency of the photon beam; determining a background noise based on the measurement shift; and Reducing background noise from the second signal; and Includes.

[0134] According to some embodiments, the controller: Concurrently with detecting the first signal, the device is further configured to sense a field potential of the tissue or cell aggregate from the array of microelectrodes to measure electrical activity of the tissue or cell aggregate.

[0135] According to some embodiments, the controller: The device is further configured to compare a frequency of the first signal, the second signal, and the tissue field potential.

[0136] According to some embodiments, if the comparison between the frequencies results in a deviation below a threshold, the comparison is indicative of a healthy tissue or cell aggregate.

[0137] According to some embodiments, the cardiac organoids are cardiac organoids that are capable of beating.

[0138] According to some embodiments, the cardiac organoid is a multi-chamber cardiac organoid of the invention.

[0139] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0140] [Figures 1A-1O]Figures, graphs, photomicrographs and heatmaps showing the production of vascularized cardiac organoids derived from human induced pluripotent stem cells (hiPSCs). Figure 1A is a scheme describing the formation of vascularized cardiac organoids derived from hiPSCs. Cardiomyocytes are differentiated from hiPSCs over 10 days, dissociated and mixed with microvascular endothelial cells in a 3D scaffold. The cells form beating organoids in 4 days in the well and develop multiple chambers under anisotropic stress over 25 days. Figure 1B is a representative time-lapse sequence of bright-field images showing the formation of vascularized cardiac organoids embedded in oxygen sensors. Single masses form on day 4, beat by day 10, and obtain smooth external and uniformly synchronized beating by day 25. Bar = 250 μm. Figure 1C: Left: Finite element model of von Mises stress distribution in 3D cardiac organoids formed on a solid surface (open organoids), geometrically confined in microwells, or vascularized and confined in microwells. Figure 1D: Cross-sectional stress profile and DNA staining showing uniform stress in open organoids resulting in cardiac spheroids, radial stress gradient in geometrically confined organoids resulting in single chamber formation. Anisotropic stress distribution in vascularized organoids resulting in multi-chamber cardiac organoid formation. Bar = 200 μm. Figure 1E: Confocal cross-sections and expression distribution of mechanical stress markers Lamin A / C (Lamin) and YAP1 in cardiac organoids. Open organoids show uniform distribution of both stress markers, whereas geometrically confined organoids show the predicted circumferential stress. Anisotropic stress distribution in vascularized organoids resulting in multi-chamber cardiac organoid formation. Bar = 100 μm. FIG. 1F: Confocal section of vascularized cardiac organoid showing circumferentially aligned cardiac fibers (α-actinin) surrounding a vertical-like chamber embedded with a vascular network of cardiac endothelial cells (CECs). Bar = 50 μm. Confocal section of organoid wall showing aligned cardiac fibers (cTnT) perforated with patent microvasculature capillaries (CECs). Bar = 50 μm. Scanning electron micrograph of vascularized cardiac organoid demonstrating lumen formation. Bar = 10 μm.FIG. 1G is an immunofluorescence of cardiac troponin T (cTnT) and α-actinin fibers. The fibers form long sarcomeres aligned with the cardiac tissue, a phenotype associated with mature cardiac tissue. Bar = 10 μm. FIG. 1H is a confocal section of a vascularized cardiac organoid showing a myocardium-like layer woven with POSTN cardiac fibroblast-like cells and isolated cell clusters expressing pacemaker-related markers HCN4 and SHOX2. Bar = 100 μm. FIG. 1I is a confocal section of a vascularized cardiac organoid showing circumferentially oriented epicardial cells on the cardiac organoid surface expressing the transcription factors Wilms' tumor gene 1 (WT1) and TBX18 embedded in a vascular network of cardiac endothelial cells (CECs). Bar = 100 μm. FIG. 1J is a confocal section further showing that the organoid cavity is lined with PECAM-1 endocardial-like cells. Bar = 100 μm. Figure 1K shows clustering of RNA-Seq data from iPS-derived and fetal cardiomyocytes compared to vascularized cardiac organoids and adult cardiomyocytes, RNA sequencing of cardiac organoids, and expression signatures associated with endocardium, epicardium, cardiac fibroblasts, and pacemaker cells. Figure 1L shows representative time-lapse sequence of confocal images showing formation of obvious vascular networks. Confocal microscopy shows distribution of GFP-expressing cardiac endothelial cells (CECs) in organoids. By day 10, vascular networks are evident. Figure 1M shows confocal cross-sections of vascularized cardiac organoids reveal circumferentially aligned cardiac fibers (α-actinin) surrounding vertical-like chambers, embedded with a vascular network of cardiac endothelial cells. Figure 1N shows quantification of chamber formation in three stress regimes. Multiple chambers occur 87% of the time under geometric confinement and vasculature. Figure 1O shows confocal sections of vascularized cardiac organoids stained for TUNEL to indicate apoptosis and carbonic anhydrase ix (CA-IX) expression, considered an endogenous biomarker of hypoxia, to determine upregulated hypoxic regions. Immunofluorescence staining showed faint expression in TUNEL and CA-IX. Bar = 100 μm. [Fig. 2A-2F]Heatmaps, graphs, photomicrographs, tables, and diagrams showing the functional characteristics of human cardiac organoids. Figure 2A shows the transcriptome analysis of iPS-derived and fetal cardiomyocytes compared to vascularized cardiac organoids and adult cardiomyocytes. Figure 2B shows that principal component analysis (PCA) of gene expression patterns of genes between cardiac organoids and adult cardiomyocytes sufficiently distinguishes adult and fetal cardiac tissues, with clusters 2 and 3 clustering cardiac organoids with adult tissues. Figure 2C shows that vascularized cardiac organoids exhibit spontaneous beating of 66±5 beats per minute (bpm). Organoids retain synchronized beating under pharmacological stimulation, resulting in physiological-like responses to drugs. Stimulation with 100 μM epinephrine increased the contraction rate to 88 ± 7 bpm, an 18% increase in relative contraction, whereas stimulation with 10 μM amiodarone decreased the rate to 52 ± 4 bpm, a 28% decrease in contraction. Figure 2D is a diagram of a Seahorse MitoStress study of cardiac organoids compared to hiPSC-derived cardiomyocytes. Reconstitution of cardiomyocytes into vascularized cardiac organoids increased oxidative phosphorylation by 85% and maximal respiratory capacity by 58% (n = 3, p < 0.05), whereas glycolysis was unaffected. Figure 2E is a diagram of a confocal section of vascularized cardiac organoids embedded with oxygen phosphorescence sensors after 14 days in culture. Figure 2F is a diagram of intracellular metabolic fluxes of vascularized cardiac organoids during the cardiac cycle. Glucose utilization and calculated ATP production are shown as nmol / min / 106 cells. p<0.05, **p<0.01, **p<0.001. Error bars represent ± standard error of the mean (SE). Significance was determined using a one-tailed heteroscedastic Student's test. Bars = 250 μm. [Figure 3A-3L]Diagrams, images, micrographs, and graphs showing that an integrated optoelectronic sensor enables real-time simultaneous measurement of cardiac metabolism, contraction, and action potential. Figure 3A is a schematic of the integrated metabolic electromechanical sensor chip. A lifetime-based Ru-CPOx phosphorescent sensor is embedded in a cardiac organoid and probed by two frequency LED modulation. Oxygen is measured as a phase shift in the emission signal (PMT), and a second detector is used for noise reduction and measurement of tissue contraction (cPMT). Nanofabricated gold microelectrode arrays (MEAs) are used to record electrophysiological activity. All measurements are synchronized and processed in real-time by a single microprocessor. Figure 3B is a schematic of the optical measurements. Oxygen concentration (left) is measured by phosphorescent quenching of the signal by ambient triplet oxygen, resulting in reduced decay time and a shorter phase shift. Contraction velocity (right) is measured by monitoring sub-second time changes in the mean phosphorescence intensity, which correlates with cardiac contraction. Figure 3C is an exploded view and image of the sensor-integrated heart-on-a-chip platform. The device consists of a 3D-printed casing supporting a PDMS microwell grid laminated onto a nanofabricated gold-on-glass MEA. The device is sealed by a 3D-printed holder containing connectors to the MEA leads. The MEA-integrated chip contains 45 gold electrodes on a 25 x 25 mm glass chip. The PDMS microwell array supports nine cardiac organ-bearing scaffolds, each with five recording electrodes. Figure 3D is a representative image of an oxygen sensor embedded in a cardiac organoid formed within an MEA-integrated scaffold. The gold electrodes of the MEA are visible as the shaded areas. Bar = 200 μm. Figure 3E is a scanning electron micrograph showing a 30-day-old cardiac organoid formed on an integrated scaffold. Bar = 200 μm. Figures 3F-H are simultaneous measurements and fast Fourier transform (FFT) analysis (see Figure 9A) of (Figure 3F) contraction, (Figure 3G) field potential, and (Figure 3H) interstitial oxygen in cardiac organoids during spontaneous beating. Interstitial oxygen concentration exhibits oscillatory behavior during the cardiac cycle, resulting in a distinct single frequency peak in FFT analysis that correlates with the mechanical and electrical behavior of cardiac tissue.Figures 3I-J are representative graphs of (Figure 3I) contraction, (Figure 3J) interstitial oxygen behavior of cardiac organoids after stimulation with 100 μM epinephrine. Epinephrine increases the contractility and contraction rate of vascularized cardiac organoids over time while decreasing the average interstitial oxygen concentration (dotted line) within the organoids and increasing their oscillation frequency. This analysis demonstrates that contractile behavior correlates with oxygen behavior (see Figures 9A-D). Figure 3K is a representative graph of contraction, field potential, and interstitial oxygen behavior, as well as fast Fourier transform (FFT), of cardiac organoids after treatment with 10 μM of the myosin II inhibitor blebbistatin (see Figure 9B). Blebbistatin inhibits contraction of vascularized cardiac organoids but does not affect the frequency and intensity of FP or interstitial oxygen oscillations, suggesting the presence of electrical mitochondrial coupling without actomyosin activity. Figure 3L is a representative graph of contraction, field potential, and interstitial oxygen behavior of cardiac organoids after treatment with 25 μM of the Nav channel inhibitor tetrodotoxin (TTX). Exposure to TTX resulted in a complete loss of field potential generation and associated mechanical contraction. Oxygen oscillations decayed simultaneously with the field potential. [Figure 4A-4G]Micrographs and graphs showing that disruption of electrical mitochondrial coupling induces arrhythmic behavior. Figure 4A: Immunofluorescent TMRE staining for mitochondrial membrane potential (MMP) and corresponding frequency heatmap in 2D cultured hiPSC-derived cardiomyocytes. Contraction velocity and corresponding mitochondrial membrane potential oscillations captured by live fluorescence microscopy (see Materials and Methods). Mitochondrial membrane potential of beating 2D hiPSC-derived cardiomyocytes oscillated at the frequency of contraction, whereas adjacent non-beating cells did not (n=5). Bar=25 μm. Figure 4B: Kinetic measurements of dynamic changes in cell contraction and mitochondrial calcium [Ca2+]m after treatment with 10 μM mitochondrial calcium uniporter (MCU) inhibitor KB-R7943. [Ca2+]m was measured using Rhod-2AM under a stable mitochondrial dye stain (MitoTracker). Contraction was measured by visual analysis (see Materials and Methods). Acute MCU inhibition dramatically reduces cardiomyocyte contractility and [Ca2+]m oscillation amplification without affecting contraction velocity. Figure 4C: Phase images (n=4) of hiPSC-derived vascularized cardiac organoids in an integrated metabolic electromechanical sensor chip. Bar=100 μm. Figure 4D: Real-time measurements of interstitial oxygen content and intracellular metabolic fluxes of vascularized cardiac organoids exposed to 10 μM of the MCU inhibitor KB-R7943. Fluxes were calculated at 0, 15 and 25 min after exposure to 10 μM of KB-R7943. Glucose utilization and calculated ATP production are shown as nmol / min / 106 cells (see Materials and Methods). Interstitial oxygen content suggests a progressive decrease in oxygen uptake in cardiac organoids caused by MCU inhibition, leading to a decrease in ATP production and an increase in the ATP gap. Figure 4E shows representative kinetic measurements of contraction, field potential, and interstitial oxygen content in cardiac organoids exposed to 10 μM KB-R7943 (n=9). Figure 4F shows simultaneous kinetic measurements of contraction frequency, oxygen oscillation frequency, and field potential oscillation frequency after MCU inhibition. MCU inhibition resulted in a correlated increase in all oscillation rates.In contrast to oscillation rate, MCU inhibition progressively decreased the magnitude of cardiomyocyte contraction, electrical activity, and oxygen uptake, resulting in arrhythmogenic cardiac behavior (n=9). Figure 4G shows representative changes in single-contraction measurements of contraction, field potential, and interstitial oxygen content upon treatment with 10 μM KB-R7943. MCU inhibition caused uncoupling between mitochondrial activity and electromechanical activity, resulting in arrhythmogenic cardiac behavior. [Figure 5A-5E] Micrographs, graphs, diagrams and illustrations showing that CRISPR / Cas9 knockout of MCU disrupts electrical mitochondrial coupling and induces arrhythmic behavior. Figure 5A is a diagram of kinetic Rhod-2AM measurement of mitochondrial membrane potential (MMP) in beating 2D cultured hiPSC-derived cardiomyocytes. Non-targeting sgRNA had no effect on [Ca2+]m, showing a dominant frequency of 0.8 Hz, whereas MCU knockout (MCUKO) showed a 50% reduction in [Ca2+]m and oscillation magnitude while increasing the oscillation rate to 1.3 Hz (n=7, p<0.01). Figure 5B is a diagram of immunofluorescence staining of MCU in non-homogeneous MCUKO chimeric vascularized cardiac organoids. Immunofluorescence staining showed a significant reduction in MCU expression. Bar=100 μm. Figure 5C-D are diagrams of (Figure 5C) representative kinetic measurements, (Figure 5D) dynamic analysis of contraction, field potential, and interstitial oxygen content in non-homogeneous MCUKO or sgRNA chimeric cardiac organoids (n=9, p<0.001). Disruption of MCU expression in organoids reduced organoid contractility, decreased the magnitude of oxygen oscillations, and showed obvious arrhythmogenic behavior in organoid field potential. Figure 5E is a scheme showing changes in the interplay between electrical, mitochondrial, and mechanical activity in cardiomyocytes by MCU inhibition. Electrical-mitochondrial coupling underlies this coordination, and its uncoupling by MCU inhibition results in uncoordinated activity and arrhythmogenic behavior. *p<0.05, **p<0.01, ***p<0.001. Error bars represent ±SE. Significance was determined using a two-tailed heteroscedastic Student's test. [Figure 6A-6F]Tables, figures and graphs showing that mitoxantrone inhibition of electrical mitochondrial coupling and the resulting arrhythmias is partially reversed by metformin. Figure 6A is a diagram of the structure, clinical indication and maximum physiological concentration (Cmax) of mitoxantrone and metformin. Figure 6B is a scheme showing electrical mitochondrial coupling disruption in cardiomyocytes exposed to mitoxantrone and the recovery resulting from the addition of metformin. MCU inhibition by mitoxantrone causes insufficient ATP production during depolarization, resulting in arrhythmogenic behavior. The addition of metformin improves calcium entry into mitochondria during depolarization, allowing more ATP to be produced. Figures 6C-6D show (Figure 6C) representative recordings, (Figure 6D) average mitochondrial calcium measured using live imaging of Rhod-2AM dye in different hiPSC-derived cardiomyocyte cultures treated with 10 μM MCU inhibitor mitoxantrone or DMSO (control) followed by treatment with 10 μM mitoxantrone (mitoxantrone) or 10 μM mitoxantrone and 100 μM AMP-activated protein kinase activator metformin (mitoxantrone + metformin; n=4). Mitoxantrone alone reduced mitochondrial calcium by 79% (p<0.001). Addition of 100 μM metformin to mitoxantrone treatment resulted in a 3.4-fold increase in average mitochondrial calcium content (p<0.001). FIG. 6E is a representative simultaneous kinetic measurement of contraction, field potential, and interstitial oxygen content in cardiac organoids exposed to DMSO (control), 10 μM mitoxantrone (mitoxantrone), or 10 μM mitoxantrone and 100 μM metformin (mitoxantrone+metformin). Mitoxantrone caused irregular cardiac contraction and field potential. Co-treatment with metformin reversed the effect, showing regular cardiac contraction and field potential. FIG. 6F is a representative change in single contraction measurements of contraction, field potential, and interstitial oxygen content by treatment with 10 μM mitoxantrone or co-treatment with 10 μM mitoxantrone and 100 μM metformin.MCU inhibition with mitoxantrone caused electrical mitochondrial uncoupling, resulting in arrhythmic cardiac behavior. Co-treatment with metformin and mitoxantrone rescued arrhythmic cardiac behavior and indicates coordinated cardiac behavior. *p<0.05, **p<0.01, ***p<0.001. Error bars represent ±SE. Significance was determined using a two-tailed heteroscedastic Student's test. [Figure 7A-7C] Schematics and micrographs showing the generation of multi-chamber vascularized cardiac organoids. Finite element models of (Figure 7A) von Mises stress distribution, (Figure 7B) Gaussian displacement in 3D cardiac organoids formed on a solid surface (open organoids), geometrically confined in microwells, or vascularized and confined in microwells. The finite element models show the gradient of mechanical stress that contributes to the formation of the heart chambers. Figure 7C shows confocal sections of organoids stained for mechanical stress markers Lamin A / C (Lamin) and YAP1. Open organoids show a uniform distribution of both stress markers, whereas geometrically confined organoids show the predicted circumferential stress. Anisotropic stress distribution in vascularized organoids leads to multi-chamber cardiac organoid formation. Bar = 100 μm. [Figure 8A-8D]Figure 8A includes diagrams and graphs showing the establishment of an integrated 2-PMT heart-on-a-chip platform. Figure 8A is a diagram of a scheme and a typical measurement showing the advantages of using a 2-PMT system over a single PMT system. The addition of a second detector (cPMT) measuring the excitation signal reduces noise and allows for accurate measurements with sub-second resolution. The second PMT also allows for emission-independent measurements of tissue contraction (see Materials and Methods). Figure 8B is an illustration of calibrated measurements of the reflected signal measured by the second PMT at different displacements. Curve fitting reveals a sigmoidal relationship between emission intensity, measured by peak-to-peak voltage (VP-P), and sensor displacement. Heart displacement was measured by oxygen beads embedded within cardiac organoids during contractions where the beads move different distances from the focus. The sigmoidal fit shows a correlation with R-squared: 0.9835 and RMSE less than 4. Figure 8C is a schematic showing the fabrication process of MEAs using lift-off lithography techniques. FIG. 8D is a diagram of a PDMS microscaffold attached to an MEA transparent chip supporting the formation of nine cardiac organoids. The PDMS scaffold was fabricated using a laser cutting CNC machine and covalently bonded on top of the MEA chip using oxygen plasma activation. FIG. 8A is a diagram of a prior art system and a system according to some embodiments of the present invention. Schematic diagram of the advantages of using a 2-PMT system.Oxygen is measured by the phase shift between the emission signal detected by a photomultiplier tube (PMT) and the excitation signal. Thus, adding a second detector (cPMT) that measures the excitation signal reduces noise and allows for accurate measurements with sub-second resolution. The second PMT also allows for emission-independent measurements of tissue contraction. Figure 8B shows a sigmoidal curve fitting using a 2-PMT system to examine the relationship between emission intensity measured in peak-to-peak voltage (VP-P) from an oxygen bead and its displacement. Cardiac displacement was measured by oxygen beads embedded within cardiac organoids during contraction where the beads move different distances from the focus. The sigmoidal fit shows a correlation of R-squared: 0.9835 and RMSE less than 4. Figure 8E is a flow chart of a method for measuring properties of tissue or cell aggregates according to some embodiments of the present invention. [Figure 9A-9D] Figures 9A and 9B include non-limiting schemes and graphs showing real-time metabolic measurements of vascularized cardiac organoids. Figure 9A shows simultaneous measurements of contraction, field potential, and interstitial oxygen in cardiac organoids during spontaneous beating. Interstitial oxygen concentration shows oscillatory behavior during the cardiac cycle, resulting in a clear single-frequency peak in the FFT analysis (Figure 9B) that correlates with the mechanical and electrical behavior of cardiac tissue. Figure 9C is a schematic diagram of an electrophysiological recording system connected to an MEA to simultaneously track spontaneous cardiac field potentials (FP) in real time. An integrated signal conditioning circuit (AD8232) with a 2-pole adjustable high-pass filter, a 3-pole adjustable low-pass filter, adjustable gain, and a medical device amplifier was used to amplify and filter the field potential. An Arduino MEGA 2560® microcontroller was used as an analog-to-digital converter (ADC). Figure 9D shows representative raw and filtered field potential measurements of cardiac organoids. A finite impulse response filter was applied in real time using custom-written MATLAB code (Methods). Periodically, anti-aliasing filters were used to remove noise with frequencies higher than 8 Hz generated by the electromagnetic fields of different equipment in the workspace. [Figure 10A-10G] Graphs showing real-time metabolic measurements of vascularized cardiac organoids under epinephrine stimulation. FIG. 10A is an analysis of the kinetic behavior of contraction rate of organoids during prolonged stimulation with 100 μM epinephrine. The kinetic analysis suggests that epinephrine stimulation leads to a sigmoidal change in organoid contraction rate. FIG. 10B-FIG. 10C are representative relationship graphs of (FIG. 10B) contraction amplification (contractility)-contraction rate, (FIG. 10C) interstitial oxygen content-contraction rate during prolonged epinephrine stimulation. The analysis suggests a correlation between increased cardiac organoid contractility and oxygen consumption. FIG. 10D is a representative frequency histogram of interstitial oxygen measurements after 0, 15, and 90 min of stimulation with 100 μM epinephrine. The analysis shows that the increase in oxygen consumption correlates with an increase in interstitial oxygen content variability, which correlates with the increase in measured oxygen amplification. Figures 10E-10F are representative correlation analyses of (Figure 10E) oxygen oscillation frequency and contraction frequency, (Figure 10F) oxygen oscillation amplification and contractility revealing a direct linear correlation between interstitial oxygen oscillatory behavior and organoid contractility. Figure 10G is a representative graph of a fast Fourier transform (FFT) analysis showing contraction, field potential, and interstitial oxygen frequency behavior of cardiac organoids after treatment with 10 µM of the myosin II inhibitor blebbistatin. Blebbistatin treatment blocked cardiac contraction and sensor movement but did not affect field potential or oxygen oscillation frequency and strength. [Figures 11A-11L]Figure 11 includes micrographs, graphs, and heat maps showing that live mitochondrial imaging reveals oscillations in mitochondrial membrane potential. Figure 11A is an immunofluorescence micrograph of mitochondrial membrane potential (ΔΨm) measured using live imaging of TMRE dye. Figure 11B is an illustration of kinetic analysis of mitochondrial membrane potential (ΔΨm) using TMRE. Mitochondrial membrane potential of hiPSC-derived cardiomyocytes oscillates at the frequency of contraction. Mitochondrial membrane potential of non-beating cells did not oscillate and was generally low. Figure 11C-D are rainbow heat maps of heat map micrographs showing (Figure 11C) the mean mitochondrial membrane potential, (Figure 11D) the predominant oscillation frequency of ΔΨm. The images show a correlation between areas with high mean mitochondrial membrane potential and the oscillation frequency. Figure 11E is an immunofluorescence micrograph of mitochondrial membrane potential (ΔΨm) measured using live imaging of JC-1 dye. Figure 11F is an illustration of the kinetic analysis of mitochondrial membrane potential (ΔΨm) after aggregation of JC-1 dye. Mitochondrial membrane potential showed a clear polarization peak in contracting cells, whereas non-beating cells did not oscillate, showing an overall decline in mitochondrial membrane potential. Figures 11G-H are rainbow heat maps (see Materials and Methods) of the mean mitochondrial membrane potential (Figure 11G) and the major oscillation frequency of ΔΨm (Figure 11H) measured using JC-1. Similar to the behavior measured by TMRE, the JC-1 heat maps suggest a correlation between regions with high mean mitochondrial membrane potential and oscillation frequency. Figure 11I shows that live imaging of hiPSC-derived cardiomyocytes in 2D culture showed rapid oscillations of mitochondrial calcium [Ca2+]m in beating cells, which were precisely correlated with the contraction frequency of the cells. Figure 11J is an immunofluorescence micrograph of the dynamic changes in mitochondrial calcium content after treatment with 10 μM mitochondrial calcium uniporter (MCU) inhibitor KB-R7943. Mitochondrial calcium was measured using live imaging of Rhod-2AM dye, mitochondrial content was measured by MitoTracker staining, and contraction was measured using a visual contraction assay (see Materials and Methods).FIG. 11K is a representative single-twitch measurement of mitochondrial calcium and cardiomyocyte contraction after treatment with 10 μM KB-R7943. Acute MCU inhibition reduces the magnitude of cardiomyocyte contraction and its dependence on efficient oscillations of mitochondrial calcium. FIG. 11L is a dynamic analysis of mitochondrial calcium amplification and contractility of cardiomyocytes after treatment with 10 μM KB-R7943. This analysis suggests that the magnitude of mitochondrial calcium oscillations correlates with contractility, suggesting that mitochondrial calcium oscillations are essential to maintain the magnitude of contraction. Bar = 25 μm. [Figures 12A-12C] Figures 12A and 12B include graphs and micrographs showing that CRISPR / Cas9 knockout of MCU disrupts electrical mitochondrial coupling and induces arrhythmic behavior. Figure 12A shows kinetic Rhod-2AM measurements of mitochondrial membrane potential (MMP) in beating 2D-cultured hiPSC-derived cardiomyocytes. Non-targeting sgRNA had no effect on [Ca2+]m, showing a dominant frequency of 0.8 Hz, whereas MCU knockout (MCUKO) showed a 35-50% reduction in [Ca2+]m and oscillation magnitude while increasing the oscillation rate to 1.3-1.4 Hz. Figures 12B-C show that (Figure 12B) RT-qPCR, (Figure 12C) immunofluorescence confocal microscopy demonstrated a significant reduction in MCU expression at the mRNA and protein levels. *p<0.05, **p<0.01, ***p<0.001. Error bars represent ±SE. Significance was determined using a two-tailed heteroscedastic Student's test. Bar = 100 μm. [Figures 13A-13E]Micrographs, graphs and schematics showing validation of electrical mitochondrial coupling in an ex vivo pig model. Figure 13A is a schematic showing the use of stimulated porcine left ventricle dissected tissue (see Materials and Methods) with an oxygen sensor embedded and placed on a multi-electrode array as an ex vivo validation model. Figure 13B is a photograph of sectioned porcine tissue (green) on an electrode array. Bar = 5 mm. Figure 13C is a scanning electron micrograph showing an oxygen sensor (pseudocolor) attached to a pig heart tissue section. Bar = 25 μm. Figure 13D is a representative simultaneous kinetic measurement of contraction, field potential and interstitial oxygen content in pig heart tissue exposed to DMSO (control), 10 μM blebbistatin, 10 μM mitoxantrone, or 10 μM mitoxantrone and 100 μM metformin (mitoxantrone + metformin). The myosin II inhibitor blebbistatin blocked cardiac contractions without affecting field potential and oxygen oscillation frequency. Exposure to mitoxantrone induced arrhythmogenic behavior, increasing the beating frequency of porcine tissue to 1-2 Hz. Concomitant treatment with metformin partially reversed this effect in porcine tissue, decreasing the beating frequency to 1.4 Hz. Figure 13E shows the kinetics of inhibition of mitochondrial activity as measured by continuous interstitial oxygenation in porcine cardiac tissue exposed to mitoxantrone. Treatment with metformin 50 min after exposure shows a progressive recovery of function and reversal of the effects of mitoxantrone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] In some embodiments, the present invention provides the method for treating the disease or disorder characterized by mitochondrial electrical dyssynchrony.Also provided is a multi-chambered cardiac organoid comprising endothelial cells, cardiomyocytes and at least two chambers that beat synchronously.Also provided are the method for making and using multi-chambered cardiac organoid, and the sensing system and their use.

[0142] Cardiac Organoids According to a first aspect, a cardiac organoid is provided.

[0143] As used herein, "organoid" refers to a simplified version of an organ produced in vitro. In some embodiments, organoids are smaller than in vivo organs. In some embodiments, cardiac organoids are cardiac organoids of the present invention. In some embodiments, organoids have a microanatomy or cellular organization similar to that of an organ. In some embodiments, the cellular organization is a 2D tissue. In some embodiments, the cellular organization is a 3D cellular organization. In some embodiments, organoids function similar to an organ. In some embodiments, organoids have gene expression similar to that of an organ. In some embodiments, the gene expression is a gene expression profile. In some embodiments, organoids beat similar to an organ. In some embodiments, the cells of an organoid are synchronized similar to the cells of an organ. In some embodiments, organoids show a response to calcium similar to that of an organ. In some embodiments, organoids have mitochondria that function similar to that of an organ. In some embodiments, organoids transmit signals similar to that of an organ. In some embodiments, organoids respond to drugs or compounds similar to that of an organ. In some embodiments, organoids are useful for testing drugs to be used on an organ. In some embodiments, the organoids are useful for testing the side effects of drugs or compounds on the organ. In some embodiments, the organoids are useful for modeling diseases of the organ. In some embodiments, the organ is a heart. In some embodiments, the organ is a portion of a heart. In some embodiments, the portion is a ventricle. In some embodiments, the portion is an atrium. In some embodiments, the organoids are self-renewing. In some embodiments, the organoids are not immortalized.

[0144] In some embodiments, the organoids are not genetically engineered. In some embodiments, the organoids comprise diploid cells. In some embodiments, the organoids consist of diploid cells. In some embodiments, the organoids lack aneuploid cells. In some embodiments, the organoids comprise cardiomyocytes. In some embodiments, the cardiomyocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the cardiomyocytes are obtained from PSCs. In some embodiments, the cardiomyocytes are human cardiomyocytes. In some embodiments, the iPSCs are human iPSCs. Methods for differentiating pluripotent stem cells into cardiomyocytes are well known in the art, and any such method can be used. Further exemplary methods are provided below. In some embodiments, the iPSCs are differentiated into cardiomyocytes.

[0145] As used herein, the term "cardiomyocyte" refers to cardiac muscle cells that make up the myocardium (heart muscle). Each cardiomyocyte contains a myofibril, a specialized organelle made up of long chains of sarcomeres, the basic contractile unit of a muscle cell. Cardiomyocytes exhibit striations similar to those on skeletal muscle cells. Unlike multinucleated skeletal cells, the majority of cardiomyocytes contain only one nucleus, although they may have as many as four nuclei. Cardiomyocytes have high mitochondrial density, can rapidly produce adenosine triphosphate (ATP), and are highly resistant to fatigue.

[0146] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs derived from non-PSC cells (i.e., from cells that are differentiated in comparison to PSCs). iPSCs can be derived from several different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology and grow as flat colonies with large nuclear-cytoplasmic ratios, defined borders, and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known to those of skill in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Application Publication Nos. 20090047263, 20090068742, 20090191159, 20090227032, 20090246875, and 20090304646. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art for reprogramming somatic cells to become pluripotent stem cells (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.).

[0147] In some embodiments, the organoid comprises endothelial cells. In some embodiments, the endothelial cells are microvascular endothelial cells. In some embodiments, the endothelial cells are cardiac endothelial cells. In some embodiments, the cardiac organoid comprises endothelial cells in a geometrically confined compartment. In some embodiments, the cells of the organoid are in a geometrically confined compartment. In some embodiments, the organoid comprises a mixture of cardiomyocytes and endothelial cells. In some embodiments, the mixture comprises a ratio of about 2:1 between cardiomyocytes and endothelial cells.

[0148] In some embodiments, organoid comprises at least 100, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 68000, 70000, 75000, 80000, 85000, 90000, 95000 or 100000 cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, organoid comprises at least 68000 cells. In some embodiments, organoid comprises at least 75000 cells. In some embodiments, the organoids comprise up to 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 125,000, 150,000, 175,000, 200,000, 300,000, 400,000, 500,000, 1000,000, 100,000,000, or 100,000,000 cells. Each possibility represents a separate embodiment of the present invention.

[0149] In some embodiments, the cardiac organoids are multi-chamber cardiac organoids. In some embodiments, the organoids are multi-chamber organoids. In some embodiments, the multi-chambers include at least two chambers. In some embodiments, the multi-chambers include at least three chambers. In some embodiments, the multi-chambers include at least four chambers. In some embodiments, the chambers include a chamber wall. In some embodiments, the chamber wall includes the endocardium. In some embodiments, the chamber wall is the endocardium. In some embodiments, the chamber wall can pulsate. In some embodiments, the organoids are pulsating organoids. In some embodiments, the pulsation is a spontaneous pulsation. In some embodiments, the chamber wall can contract. In some embodiments, the pulsation or contraction is in response to a stimulus. In some embodiments, the stimulus is an endogenous stimulus. In some embodiments, the stimulus is an exogenous stimulus. In some embodiments, the stimulus is an endocrine or hormonal stimulus. In some embodiments, the stimulus is a chemical stimulus. In some embodiments, the stimulus is an electrical stimulus. In some embodiments, the organoids pulsate spontaneously and pulsate or change pulsation in response to a stimulus. In some embodiments, the beating is synchronous.

[0150] In some embodiments, the cardiac organoids further comprise a vascular structure or a plurality thereof. In some embodiments, the cardiac organoids comprise endothelial cells. In some embodiments, the addition of endothelial cells forms a vascular structure. In some embodiments, the structure naturally forms from the added endothelial cells without further stimulation. In some embodiments, the structure naturally forms from the added endothelial cells after the addition of a pro-angiogenic stimulus. Pro-angiogenic proteins are known in the art, and any may be used. In some embodiments, the stimulus is a protein. In some embodiments, the protein is VEGF. In some embodiments, the VEGF is VEGF-A. In some embodiments, the vascular structure comprises or is selected from arteries, arterioles, capillaries, veins, blood vessels, venules, sinuses, or any combination thereof. In some embodiments, the vascular structure comprises capillaries. In some embodiments, the walls of the chamber comprise capillaries. In some embodiments, the organoids comprise an anisotropic stress gradient. In some embodiments, the organoids comprise regions of low stress. In some embodiments, the vasculature forms in response to anisotropic stress.

[0151] In some embodiments, the cardiac organoid comprises circumferentially aligned cardiomyocytes. In some embodiments, the circumferentially aligned cardiomyocytes surround a chamber. In some embodiments, the chamber is hollow. In some embodiments, the chamber comprises a fluid. In some embodiments, the fluid is a culture medium. In some embodiments, the chamber is surrounded by cardiomyocytes. In some embodiments, the chamber is layered with cardiomyocytes. In some embodiments, the wall of the chamber comprises circumferentially aligned cardiomyocytes. In some embodiments, the circumference is the circumference of the chamber. In some embodiments, the circumference is the circumference of the cardiomyocytes. In some embodiments, the chamber of the cardiac organoid comprises at least one capillary in the wall surrounding the chamber. In some embodiments, the chamber is surrounded by the endocardium. In some embodiments, the endocardium is surrounded by the epicardium.

[0152] In some embodiments, the cardiac organoid comprises elongated cardiomyocytes. In some embodiments, the cardiomyocytes are elongated cardiomyocytes. In some embodiments, the cardiomyocytes are organized in a sarcomere pattern. In some embodiments, the cardiomyocytes are organized in a sarcomere pattern. As used herein, the term "sarcomere pattern" refers to an organization similar to or essentially the same as a sarcomere, e.g., the smallest functional unit of striated muscle or muscle tissue. In some embodiments, the cardiomyocytes are alpha-actinin positive. In some embodiments, the cardiomyocytes are cardiac troponin positive. The structure of sarcoma and how to determine it, such as by histology and / or microscopy, are common and will be clear to those skilled in the art.

[0153] In some embodiments, cardiac organoids comprise at least one fibroblast-like cell. In some embodiments, the fibroblast-like cell is a fibroblast. In some embodiments, the fibroblast-like cell is a cardiac fibroblast-like cell. In some embodiments, cardiac organoids comprise cardiac fibroblast-like cells. In some embodiments, the cardiomyocyte layer comprises fibroblast-like cells. In some embodiments, the chamber wall comprises fibroblast-like cells. Fibroblast-like cells exhibit structural features and antigen profiles that are associated with their specific location and function. Non-limiting examples of fibroblast-like cells include, but are not limited to, myofibroblasts, perineural sheath cells, ITO cells, endocrine fibroblast-like cells, intestinal perithelium and villus fibroblasts, myenteric plexus fibroblast-like cells, lymphoid organ dendritic cells, and fibroblasts of different sites, including tendons, dermis and cornea.

[0154] In some embodiments, the fibroblast-like cells are periostin (POSTN) positive cells. In some embodiments, the fibroblast-like cells are characterized by POSTN expression. In some embodiments, the fibroblast-like cells contain POSTN mRNA, its protein product, or both. Methods for identifying fibroblast-like cells and markers therefor are well known to those of skill in the art. In some embodiments, the positive is protein positive. In some embodiments, the positive is mRNA positive. In some embodiments, the positive is expression positive.

[0155] In some embodiments, the cardiac organoid comprises pacemaker-like cells. In some embodiments, the pacemaker-like cells are in clusters. In some embodiments, the cardiac organoid comprises pacemaker-like cell clusters. In some embodiments, the pacemaker-like cells are potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) positive. In some embodiments, the pacemaker-like cell clusters are HCN4 positive. In some embodiments, the pacemaker-like cells are short stature homeobox 2 (SHOX2) positive. In some embodiments, the pacemaker-like cell clusters are SHOX2 positive. In some embodiments, the pacemaker-like cells are HCN4 and SHOX2 double positive. In some embodiments, the pacemaker-like cell clusters are HCN4 and SHOX2 double positive. In some embodiments, the pacemaker-like cell clusters comprise HCN4 mRNA, its protein product, or both, SHOX2 mRNA, its protein product, or both, or any combination thereof. In some embodiments, the pacemaker-like cells are pacemaker cells.

[0156] In some embodiments, the fibroblast-like cells are in contact with the pacemaker-like cells. In some embodiments, the cardiomyocytes are in contact with the pacemaker-like cells. In some embodiments, the contact with the pacemaker-like cells is within the cardiomyocyte layer. In some embodiments, the chamber wall comprises the pacemaker-like cells.

[0157] Methods for determining "positive" for the factors or expression described herein are common and will be apparent to one of skill in the art. Non-limiting examples of methods for determining expression include, but are not limited to, PCR, RT-PCR, quantitative RT-PCR, Northern blot, RNA in situ hybridization, dot blot, Western blot, etc.

[0158] In some embodiments, the cardiac organoid comprises epicardium. In some embodiments, the epicardium is the outer epicardium. In some embodiments, the outer epicardium is the outer shell. In some embodiments, the chamber and the chamber wall are surrounded by the epicardium. In some embodiments, the myocardial cell layer is surrounded by the epicardium. In some embodiments, the epicardium comprises cells positive for Wilms' tumor-1 (WT1). In some embodiments, the epicardium comprises cells positive for T-box transcription factor 18 (TBX18). In some embodiments, the epicardium comprises cells positive for WT1 and TXB18. In some embodiments, the epicardial cells comprise WT1 mRNA, its protein product, or both, TBX18 mRNA, its protein product, or both, or any combination thereof.

[0159] In some embodiments, the chamber wall comprises endocardial-like cells. In some embodiments, the chamber wall comprises endocardium. In some embodiments, the endocardial-like cells are platelet endothelial cell adhesion molecule (PECAM-1) positive. In some embodiments, the endocardium comprises cells positive for PECAM-1. In some embodiments, the endocardium is inner endocardium. In some embodiments, the organoid comprises inner endocardium. In some embodiments, the organoid comprises inner endocardium and outer epicardium. In some embodiments, the chamber wall comprises PECAM-1 mRNA, its protein product, or both. In some embodiments, the organoid comprises an endocardial-like cell layer. In some embodiments, the chamber is laminated with an endocardial-like cell layer. In some embodiments, a portion of the chamber is laminated with an endocardial-like cell layer.

[0160] In some embodiments, the organoid comprises a ring of high stress cells. In some embodiments, the high stress cells are high stress regions. In some embodiments, the high stress is characterized by Ramanin A / C (LMNA). In some embodiments, the organoid comprises a gradient of Yes-associated protein 1 (YAP1). In some embodiments, the gradient surrounds a central cavity or chamber.

[0161] In some embodiments, the cardiac organoids disclosed herein can beat. In some embodiments, the cardiac organoids disclosed herein are characterized by being capable of synchronous beating. In some embodiments, the cardiac organoids beat synchronously. In some embodiments, the cardiac organoids comprise chambers that beat synchronously. In some embodiments, at least two chambers of the organoid beat synchronously. In some embodiments, all chambers of the organoid beat synchronously. In some embodiments, the organoids produce polyphasic beating. In some embodiments, the organoids comprise polyphasic beating. In some embodiments, the polyphasic is biphasic. In some embodiments, the organoids comprise at least a first two chambers that beat synchronously and at least a second two chambers that beat synchronously. In some embodiments, the first two chambers and the second two chambers are not synchronous. In some embodiments, the first two chambers and the second two chambers beat at different times. In some embodiments, the beating at different times comprises the synchronization of two chamber groups, so that when one beats, the other does not beat, and vice versa. In some embodiments, the beating is spontaneous beating. In some embodiments, synchronized beating refers to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the cells or contracting cells of the cardiac organoid disclosed herein contract at the same time. Each possibility represents a separate embodiment of the present invention. In some embodiments, the synchronized contraction is in response to a stimulus. In some embodiments, the cells all respond to the same stimulus.

[0162] In some embodiments, the synchronized beating persists in culture for at least 1, 2, 3, 4, 5 weeks or more, or any values ​​and ranges therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the organoids are capable of synchronized beating in culture for at least 1, 2, 3, 4, 5 weeks or more, or any values ​​and ranges therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the synchronized beating persists in culture for 1-10 weeks, 2-12 weeks, 3-15 weeks, 4-10 weeks, 5-25 weeks. Each possibility represents a separate embodiment of the present invention. In some embodiments, the synchronized beating persists in culture for at least 1 week.

[0163] In some embodiments, the pulsation is at a rate of at least 20 beats per minute (bpm), at least 30 bpm, at least 35 bpm, at least 40 bpm, at least 45 bpm, at least 50 bpm, at least 55 bpm, at least 60 bpm, at least 65 bpm, at least 70 bpm, at least 75 bpm, at least 80 bpm, at least 85 bpm, at least 88 bpm, at least 90 bpm, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the pulsation is at a rate of at least 50 bpm. In some embodiments, the pulsation is at a rate of at least 52 bpm. In some embodiments, the pulsation is at a rate of at least 60 bpm. In some embodiments, the pulsation is at a rate of at least 65 bpm. In some embodiments, the pulsation is about 66 bpm. In some embodiments, the pulsation is at a rate of at least 88 bpm. In some embodiments, the pulsation is about 88 bpm. In some embodiments, the organoid pulsates at a rate similar to the organ. In some embodiments, the organoid beating rate is the unstimulated beating rate.It will be understood that the addition of stimulants or other agents can increase or decrease the beating rate, but the standard / untreated beating rate is as indicated.

[0164] In some embodiments, the pulse is 20-90 bpm, 30-90 bpm, 40-90 bpm, 50-90 bpm, 60-90 bpm, 55-90 bpm, 45-90 bpm, 20-88 bpm, 30-88 bpm, 40-88 bpm, 50-88 bpm, 60-88 bpm, 55-88 bpm, 45-88 bpm, 20-80 bpm, 30-80 bpm, 40-80 bpm, 50-80 bpm, 60-80 bpm, 55-80 bpm, 45-80 bpm, 35-90 bpm, 55-80 bpm, or 55-75 bpm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the pulse is 40-80 bpm. In some embodiments, the pulse is 50-80 bpm. In some embodiments, the pulsation is between 40 and 90 bpm. In some embodiments, the pulsation is between 50 and 90 bpm. In some embodiments, the organoids respond to treatment with a stimulant by increasing the pulsation rate. In some embodiments, the organoids respond to treatment with a stimulant by increasing contraction amplification. Stimulants that increase heart rate are known in the art, and any such stimulant can be used. In some embodiments, the stimulant is epinephrine. In some embodiments, the organoids respond to treatment with a potassium channel blocker by decreasing the pulsation rate. In some embodiments, the organoids respond to treatment with a potassium channel blocker by decreasing the contraction amplification. In some embodiments, the organoids respond to treatment with an antiarrhythmic drug by decreasing the pulsation rate. In some embodiments, the organoids respond to treatment with an antiarrhythmic drug by decreasing the contraction amplification. Drugs that reduce heart rate are known in the art, and any such drug can be used. In some embodiments, the drug that reduces heart rate and / or contraction amplification is amiodarone.

[0165] In some embodiments, the cardiac organoids disclosed herein comprise cells comprising increased expression of at least one of the factors provided in Figure 2A. In some embodiments, the cardiac organoids disclosed herein comprise cells comprising increased expression of at least one of the factors provided in Figure 2A. In some embodiments, the cardiac organoids disclosed herein comprise cells comprising increased expression of at least one of the factors provided in Figure 2A. In some embodiments, the cardiac organoids disclosed herein comprise cells comprising increased expression of at least one of the factors provided in Figure 2A. (CACNA1C), ryanodine receptor 2 (RYR2), calsequestrin 2 (CASQ2), phospholamban (PLN also known as PLB), calcium / calmodulin-dependent protein kinase type II beta chain (CAMK2B), triadin (TRDN), caveolin (CAV3), Myc box-dependent interacting protein 1 (BIN1), amphiphilin (AMP2 also known as AMPH), sodium channel protein type 5 subunit alpha (SCN5A), potassium voltage-gated channel subfamily J member 2 (KIR2 also known as KCNJ2).1), inositol 1,4,5-triphosphate receptor, type 3 (ITPR3), potassium / sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), sodium channel subunit beta-1 (SCN1B), potassium / sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 1 (HCN1), potassium maladaptive rectifier channel, subfamily J, member 8 (KCNJ8), potassium voltage-gated channel subfamily H member 2 (KCNH2, also known as hERG), 5'-AMP-activated protein kinase catalytic subunit alpha-1 (PRKAA1), carnitine palmitoyltransferase I (CPT1A), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1-alf and / or α-terminally activated protein kinase A (PPARGC1A), inorganic pyrophosphatase 1 (PPA1), serine / threonine protein phosphatase 2A regulatory subunit B (PPP2R4), glucose transporter type 4 (SLC2A4, also known as GLUT4), mitogen-activated protein kinase 1 (MAPK1, also known as ERK2), catalytic subunit alpha of protein kinase A (PRKACA), alpha-1A adrenergic receptor (α1A, also known as ADRA1A), alpha-1B adrenergic receptor (α1B, also known as ADRA1B), sodium channel beta subunit 4 (SCN4B), and potassium voltage-gated channel subfamily E member 1 (KCNE1). In some embodiments, the cardiac organoids disclosed herein express TNNT2, TNNI3, Cx43, MYH7, AKAP6, GJA5, JPH2, SLC8A1, ATP2A2, CACNA1C, RYR2, CASQ2, PLN, CAMK2B, TRDN, CAV3, BIN1, AMP2, SCN5A, KIR2.The organoids are characterized or comprise an increased expression of at least one factor selected from: 1, ITPR3, HCN2, SCN1B, HCN1, KCNJ8, KCNH2, PRKAA1, CPT1A, TFAM, PPARGC1A, PPA1, PPP2R4, SLC2A4, MAPK1, PRKACA, alpha 1A, alpha 1B, SCN4B, and KCNE1. In some embodiments, the increase is compared to isolated cardiomyocytes. In some embodiments, the increase is compared to cardiomyocyte culture. In some embodiments, the cardiomyocytes are the same cardiomyocytes used to produce the organoids. In some embodiments, the cardiomyocytes are derived from iPSCs. In some embodiments, the increase is compared to fetal heart tissue. In some embodiments, the organoids comprise gene expression more similar to adult myocardium than fetal myocardium. In some embodiments, cardiac organoids disclosed herein comprise cells that contain increased expression of at least one, two, three, four, five, six, seven, eight, nine, or all of TNNT2, TNNI3, Cx43, MYH7, AKAP6, GJA5, JPH2, SLC8A1, ATP2A2, CACNA1C, RYR2, CASQ2, PLN, CAMK2B, TRDN, CAV3, BIN1, AMP2, SCN5A, KIR2.1, ITPR3, HCN2, SCN1B, HCN1, KCNJ8, KCNH2, PRKAA1, CPT1A, TFAM, PPARGC1A, PPA1, PPP2R4, SLC2A4, MAPK1, PRKACA, alpha 1A, alpha 1B, SCN4B, and KCNE1. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cardiac organoids disclosed herein express TNNT2, TNNI3, Cx43, MYH7, AKAP6, GJA5, JPH2, SLC8A1, ATP2A2, CACNA1C, RYR2, CASQ2, PLN, CAMK2B, TRDN, CAV3, BIN1, AMP2, SCN5A, KIR2.In some embodiments, the method is characterized by or includes increased expression of at least one, two, three, four, five, six, seven, eight, nine, or all of: 1, ITPR3, HCN2, SCN1B, HCN1, KCNJ8, KCNH2, PRKAA1, CPT1A, TFAM, PPARGC1A, PPA1, PPP2R4, SLC2A4, MAPK1, PRKACA, alpha1A, alpha1B, SCN4B, and KCNE1. Each possibility represents a separate embodiment of the present invention.

[0166] In some embodiments, the increased expression is an increase in mRNA expression. In some embodiments, the increased expression is an increase in protein expression. In some embodiments, the increased expression comprises an increase in the level or amount of an mRNA molecule, a protein molecule, or both, or at least one factor disclosed herein. In some embodiments, the increase is at least 10, 15, 20, 23, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 40, or 500% increase, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is between 10 and 1,000%, between 150 and 900%, between 200 and 990%, between 300 and 1,200%, between 500 and 975%, or between 600 and 1,100%, with each possibility representing a separate embodiment of the present invention.

[0167] In some embodiments, the cardiac organoids produce a physiological response to a condition. In some embodiments, the condition is an induced condition. In some embodiments, the condition is a physiological condition or parallel to a physiological condition. In some embodiments, the condition is application, administration, or contact with a therapeutic agent. In some embodiments, the therapeutic agent is a drug. In some embodiments, the condition is application, administration, or contact with a chemical. In some embodiments, the chemical is a harmful chemical. In some embodiments, the chemical is a toxin. In some embodiments, the chemical is a solvent. In some embodiments, the solvent is an organic solvent. Examples of chemicals that may cause cardiac diseases / disorders or side effects can be found, for example, in Kurppa et al., 1984 "Chemical exposures at work and cardiovascular morbidity", Scand. J. Work Environ. Health 10:381-388, Assadi, 2017, "Electrocardiographic changes and exposure to solvents", J Arrhythm., Dec 14; 34(1):65-70 and Tsutsumi, 2015, "Prevention and management of work-related cardiovascular disorders", Int J Occup Med Environ Health; 28(1):4-7, which are incorporated by reference in their entireties.

[0168] In some embodiments, the condition is hypoxia. In some embodiments, the condition is a hypoxic condition. Hypoxia is a situation of low oxygen for cells. In some embodiments, hypoxia includes oxygen levels below 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.75, 0.5.0.25, or 0.1%. Each possibility represents a separate embodiment of the present invention. In some embodiments, hypoxia includes 2% or less oxygen. In some embodiments, the condition is a circulatory condition. In some embodiments, the condition is an application of circulation. In some embodiments, the circulation is irregular or abnormal circulation. In some embodiments, the irregular circulation is low circulation. In some embodiments, the irregular circulation is high circulation. In some embodiments, the circulation is high, low, regular, and abnormal compared to circulation in a healthy heart. In some embodiments, the low circulation includes low fluid flow over the organoid, and / or the high circulation includes high fluid flow over the organoid. In some embodiments, the low circulation is an ischemic condition. In some embodiments, the condition is an ischemic condition. In some embodiments, the low circulation includes low nutrients. In some embodiments, the nutrient is glucose. In some embodiments, the nutrient is an amino acid. In some embodiments, the condition is exposure to a metabolite. In some embodiments, the condition is a change in metabolite exposure. In some embodiments, the condition is withdrawal of a metabolite. In some embodiments, the metabolite is glucose. In some embodiments, the condition is exposure to a hormone. In some embodiments, the condition is a change in hormone exposure. In some embodiments, the condition is withdrawal of a hormone. In some embodiments, the condition is a genetic mutation in the cells of the organoid. In some embodiments, the condition mimics diabetes, ischemia, a genetic disease, or any other condition, disease, or injury. In some embodiments, the cardiac organoid generates essentially the same physiological response to conditions as cardiac tissue or the heart. In some embodiments, the cardiac organoid disclosed herein is a cardiac mimic organoid.

[0169] In some embodiments, cardiac organoids disclosed herein comprise cells that comprise increased oxidative phosphorylation. In some embodiments, cardiac organoids disclosed herein are characterized by or comprise increased oxidative phosphorylation. In some embodiments, cardiac organoids disclosed herein comprise cells that comprise increased basal respiration. In some embodiments, cardiac organoids disclosed herein are characterized by or comprise increased basal respiration. In some embodiments, cardiac organoids disclosed herein comprise cells that comprise increased mitochondrial maximum capacity. In some embodiments, cardiac organoids disclosed herein are characterized by or comprise increased mitochondrial maximum capacity. In some embodiments, basal respiration is increased by at least 30%. In some embodiments, basal respiration is increased by at least 35%. In some embodiments, basal respiration is increased by about 35%. In some embodiments, oxidative phosphorylation is increased by at least 80%. In some embodiments, oxidative phosphorylation is increased by at least 85%. In some embodiments, oxidative phosphorylation is increased by about 85%. In some embodiments, mitochondrial maximum capacity is increased by at least 90%. In some embodiments, mitochondrial maximal capacity is increased by at least 100%. In some embodiments, mitochondrial maximal capacity is increased by at least 200%. In some embodiments, mitochondrial maximal capacity is increased by about 2-fold. In some embodiments, mitochondrial maximal capacity is increased by about 100%.

[0170] In some embodiments, the organoid further comprises an oxygen sensing particle. In some embodiments, the particle is a bead. In some embodiments, the particle is synthetic. In some embodiments, the particle is non-organic. In some embodiments, the particle is embedded in the organoid. In some embodiments, the organoid further comprises a sensor. In some embodiments, the sensor is an electrochemical sensor. In some embodiments, the sensor is a glucose sensor. In some embodiments, the sensor is a lactate sensor. In some embodiments, the sensor is a glutamine sensor. In some embodiments, the sensor is configured to measure or sense at least one of glucose, lactate, or glutamine. In some embodiments, the organoid is characterized by fatty acid oxidation as a primary metabolic pathway. In some embodiments, the organoid is characterized by changes in interstitial oxygen with a resolution of less than 1 second.

[0171] Manufacturing method According to another aspect, a method of producing cardiac organoids is provided.

[0172] According to another aspect, there is provided a cardiac organoid produced by the method of the invention.

[0173] In some embodiments, the method comprises co-culturing cardiomyocytes and endothelial cells. In some embodiments, the co-culturing is co-culturing a cluster of cardiomyocytes and endothelial cells. In some embodiments, the endothelial cells are microvascular cells. In some embodiments, the endothelial cells are cardiac cells. Microvascular cardiac endothelial cells are commercially available or can be produced by any method known in the art. In some embodiments, the cardiomyocytes are derived from iPSCs. In some embodiments, the organoids are human organoids and the cardiomyocytes are human cells. In some embodiments, the organoids are human organoids and the endothelial cells are human cells. In some embodiments, the method further comprises differentiating the iPSCs into cardiomyocytes. In some embodiments, the differentiation is as described below. It will be understood that any differentiation method known in the art can be used.

[0174] In some embodiments, cardiomyocytes and endothelial cells are co-cultured at a ratio of about 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Each possibility represents a separate embodiment of the invention. In some embodiments, cardiomyocytes and endothelial cells are co-cultured at a ratio of about 2:1. In some embodiments, cardiomyocytes and endothelial cells are co-cultured in a ratio of at least 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. Each possibility represents a separate embodiment of the invention. In some embodiments, cardiomyocytes and endothelial cells are co-cultured in a ratio of at least 2:1. In some embodiments, the cardiomyocytes and endothelial cells are mixed at a ratio of 1:1 to 3:1, 1.1:1 to 3:1, 1.2:1 to 3:1, 1.3:1 to 3:1, 1.4:1 to 3:1, 1.5:1 to 3:1, 1.6:1 to 3:1, 1.7:1 to 3:1, 1.8:1 to 3:1, 1.9:1 to 3:1, 2:1 to 3:1, 1:1 to 2.9:1, 1. 1:1~2.9:1, 1.2:1~2.9:1, 1.2.9:1~2.9:1, 1.4:1~2.9:1, 1.5:1~2.9:1, 1.6:1~2.9:1, 1.7:1~2.9:1, 1.8:1~2.9:1, 1.9:1~2.9:1, 2:1~2.9:1, 1:1~2.8:1, 1.1:1~2.8:1, 1.2 :1~2.8:1, 1.3:1~2.8:1, 1.4:1~2.8:1, 1.5:1~2.8:1, 1.6:1~2.8:1, 1.7:1~2.8:1, 1.8:1~2.8:1, 1.9:1~2.8:1, 2:1~2.8:1, 1:1~2.7:1, 1.1:1~2.7:1, 1.2:1~2.7:1, 1.3:1 ~2.7:1, 1.4:1~2.7:1, 1.5:1~2.7:1, 1.6:1~2.7:1, 1.7:1~2.7:1, 1.8:1~2.7:1, 1.9:1~2.7:1, 2:1~2.7:1, 1:1~2.6:1, 1.1:1~2.6:1, 1.2:1~2.6:1, 1.3:1~2.6:1, 1.4:1~2.6:1, 1.5:1~2.6:1, 1.6:1~2.6:1, 1.7:1~2.6:1, 1.8:1~2.6:1, 1.9:1~2.6:1, 2:1~2.6:1, 1:1~2.5:1, 1.1:1~2.5:1, 1.2:1~2.5:1, 1.3:1~2.5:1, 1.4:1~2.5:1, 1.5:1~2.5:1, 1.6:1~2.5:1, 1.7:1~2.5:1, 1.8:1~2.5:1, 1.9:1~2.5:1, 2:1~2.5:1, 1:1 ~2.4:1, 1.1:1~2.4:1, 1.2:1~2.4:1, 1.3:1~2.4:1, 1.4:1~2.4:1, 1.5:1~2.4:1, 1.6:1~2.4:1, 1.7:1~2.4:1, 1.8:1~2.4:1, 1.9:1~2.4:1, 2:1~2.4:1, 1:1~2.3:1, 1.1:1~2.3:1, 1.2:1~2.3:1, 1.3:1~2.3:1, 1.4:1~2.3:1, 1.5:1~2.3:1, 1.6:1~2.3:1 , 1.7:1~2.3:1, 1.8:1~2.3:1, 1.9:1~2.3:1, 2:1~2.3:1, 1:1~2.2:1, 1.1:1~2.2:1, 1.2:1~2.2:1, 1.3:1~2.2:1, 1.4:1~2.2:1, 1.5:1~2.2:1, 1.6:1~2.2:1, 1.7:1~2.2:1, 1.8:1~2.2:1, 1.9:1~2.2:1, 2:1~2.2:1, 1:1~2.1:1, 1.1:1~2.1:1, 1.2:1~2. The co-culture is performed at a ratio of 1:1, 1.3:1 to 2.1:1, 1.4:1 to 2.1:1, 1.5:1 to 2.1:1, 1.6:1 to 2.1:1, 1.7:1 to 2.1:1, 1.8:1 to 2.1:1, 1.9:1 to 2.1:1, 2:1 to 2.1:1, 1:1 to 2:1, 1.1:1 to 2:1, 1.2:1 to 2:1, 1.3:1 to 2:1, 1.4:1 to 2:1, 1.5:1 to 2:1, 1.6:1 to 2:1, 1.7:1 to 2:1, 1.8:1 to 2:1, or 1.9:1 to 2:1. Each possibility represents a separate embodiment of the invention. In some embodiments, the cardiomyocytes and endothelial cells are co-cultured at a ratio of 1.5:1 to 2.5:1. In some embodiments, the co-culture is a mixture.

[0175] In some embodiments, the co-culture is in a 3D scaffold. In some embodiments, the co-culture is in a medium sufficient for the formation of a 3D cellular structure. In some embodiments, the co-culture is in a cell culture matrix. In some embodiments, the co-culture is in a basement membrane matrix. In some embodiments, the matrix is ​​solubilized. In some embodiments, the matrix is ​​Matrigel. In some embodiments, the Matrigel is growth factor reduced Matrigel. In some embodiments, the Matrigel is devoid of growth factors. In some embodiments, the growth factors are supplemental growth factors. In some embodiments, the cells are suspended in the matrix / scaffold.

[0176] In some embodiments, the co-cultured cells are about 6.8×10 4 In some embodiments, the co-cultured cells are at a density of about 1, 2, 3, 4, 4.5, 5, 5.5, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 8, 9, or 10×10 cells / μl. 4 Each possibility represents a separate embodiment of the present invention. In some embodiments, the co-cultured cells are at a density of about 6.8×10 4 In some embodiments, the co-cultured cells are seeded at a density of at least 1, 2, 3, 4, 4.5, 5, 5.5, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 x 10 cells / μl. 4 The co-cultured cells are at a density of up to 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100×10 4 Each possibility represents a separate embodiment of the present invention.

[0177] In some embodiments, the co-culture is performed in a microwell. In some embodiments, the co-culture is transferred to the microwell. In some embodiments, the co-culture is allowed to grow in the microwell. In some embodiments, the co-culture is allowed to form organoids in the microwell. In some embodiments, the co-culture is allowed to fuse in the microwell. In some embodiments, a mixture of cardiomyocytes and endothelial cells is co-cultured in the microwell. In some embodiments, the microwell is sized to allow for growth of cells at a seeding density. In some embodiments, the microwell is sized to allow for growth of a given number of cells. In some embodiments, the microwell comprises a diameter of about 1 mm. In some embodiments, the microwell comprises a diameter of about 1.2 mm. In some embodiments, the microwell comprises a diameter of about 1.5 mm. In some embodiments, the microwell comprises a diameter of 1-1.5 mm. A 1-1.5 mm microwell is a sufficient size for 7.48 x 10^4 cells in approximately 1.1 μl of Matrigel, however, it will be understood by one of skill in the art that the size of the organoids can be scaled up or down by proportionally increasing the volume of the microwell and the volume of scaffold / cells.

[0178] In some embodiments, the co-culture is in a geometrically confined space. In some embodiments, the geometrically confined space is a microwell. In some embodiments, the co-culture in the geometrically confined space is such that an anisotropic stress gradient is generated for the cells. In some embodiments, the co-culture generates an anisotropic stress gradient for the cells. In some embodiments, the cells are in a cell mass. In some embodiments, the method comprises generating an anisotropic stress gradient for the cells. In some embodiments, the anisotropic stress gradient is generated by the co-culture in the geometrically confined space. In some embodiments, the space confines the growth of the cells. In some embodiments, confining the cells comprises the cells growing in contact with the walls of the space. It will be understood by those skilled in the art that the size of the culture well / dish must be calibrated to the number of cells added to confine the forming organoids. If too few cells are added, the culture will not be confined and will therefore form a single cell mass and will not form a multi-chamber structure due to uniform stress. Too many cells and organoids will not be able to properly organize structurally as described herein. The same is true for cells in a scaffold (e.g., Matrigel). The size of the well must be calibrated to the volume of scaffolds to be added.

[0179] In some embodiments, the cells in the matrix / scaffold are cultured in a medium. In some embodiments, the medium is a culture medium. In some embodiments, the medium is a tissue culture medium. In some embodiments, the medium is RPMI or an equivalent medium. In some embodiments, the RPMI is RPMI-1640. Tissue culture media are well known and one of skill in the art would understand which media are equivalent. In some embodiments, the medium is a medium for non-adherent cells. In some embodiments, the medium is a non-adherent cell medium. In some embodiments, the medium is supplemented with B27. In some embodiments, the medium is not supplemented with insulin. In some embodiments, the medium is supplemented with a growth factor. In some embodiments, the growth factor is a vascular endothelial growth factor (VEGF). In some embodiments, the VEGF is VEGF-A. In some embodiments, the growth factor is at a concentration of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the growth factor is at a concentration of at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the growth factor is at a concentration of up to 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the growth factor is at a concentration of about 5 ng / ml. In some embodiments, the growth factor is at a concentration of 2.5-7.5 ng / ml. In some embodiments, the growth factor is at a concentration of 3-7 ng / ml. In some embodiments, the growth factor is at a concentration of 4-6 ng / ml. In some embodiments, the growth factor is at a concentration of 4.25-5.75 ng / ml. In some embodiments, the growth factor is at a concentration of 4.5-5.5 ng / ml.

[0180] In some embodiments, the cells are cultured for a sufficient time for them to start beating. In some embodiments, the beating is a synchronous beating. In some embodiments, the beating is a spontaneous beating. In some embodiments, the beating is a beating as produced by the organoid of the present invention. In some embodiments, the cells are cultured for a sufficient time for producing the organoid of the present invention. In some embodiments, the cells are cultured for a sufficient time for producing an organoid that exhibits or comprises at least one characteristic of the organoid of the present invention. The characteristics of the organoid of the present invention are described herein above. In some embodiments, the cardiac organoid is the cardiac organoid of the present invention. In some embodiments, the method is a method for producing the organoid of the present invention.

[0181] How to use According to another aspect, a method for testing a therapeutic agent or compound is provided, comprising contacting the cardiac organoids of the invention with the therapeutic agent or compound.

[0182] According to another aspect, a method for assessing cardiac cell function is provided, comprising exposing the cardiac organoids of the present invention to a condition, thereby assessing cardiac cell function.

[0183] In some embodiments, the method is a diagnostic method. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is a non-patient specific method. In some embodiments, the method is a culture method. In some embodiments, the method is a method of determining efficacy. In some embodiments, the method is a method of determining side effects. In some embodiments, the method is a method of determining dosage. In some embodiments, the dosage is a therapeutically effective dosage. In some embodiments, the contacting is a therapeutically effective dosage.

[0184] In some embodiments, the method further comprises testing the physiological output of the cardiac organoid after contact or exposure. In some embodiments, the method further comprises measuring at least one parameter of the cardiac organoid after contact or exposure. In some embodiments, the parameter is output. In some embodiments, the parameter is electrical mitochondrial synchronization. In some embodiments, the output is mechanical output. In some embodiments, the output is electrical output. In some embodiments, the output is beating rate. In some embodiments, the output is respiration. In some embodiments, the output is oxidative phosphorylation. In some embodiments, the output is mitochondrial maximum capacity. In some embodiments, the output is contractile rhythm. In some embodiments, the rhythm is sinus rhythm. In some embodiments, the output is gene expression. In some embodiments, the output is protein expression.

[0185] In some embodiments, the method further comprises comparing the physiological output to an output measured before the contact or exposure. In some embodiments, the testing comprises comparing. In some embodiments, an increase is indicative of the effectiveness of the agent or compound. In some embodiments, a decrease is indicative of the effectiveness of the agent or compound. In some embodiments, an increase is indicative of a side effect of the agent or compound. In some embodiments, a decrease is indicative of a side effect of the agent or compound.

[0186] In some embodiments, the method comprises testing the physiological output of the cardiac organoids before contacting or exposing. In some embodiments, the method comprises testing the physiological output of the cardiac organoids after contacting. In some embodiments, the method comprises testing the physiological output of the cardiac organoids before contacting and testing the physiological output of the cardiac organoids after contacting and comparing both tests.

[0187] In some embodiments, the test is or includes a test for a negative cardiac side effect. In some embodiments, the side effect is a negative side effect. In some embodiments, the negative side effect is a negative cardiac side effect. In some embodiments, the negative cardiac side effect is electrical mitochondrial desynchronization. In some embodiments, the negative cardiac side effect is an arrhythmia. In some embodiments, the negative cardiac side effect includes an arrhythmia. As used herein, the term "arrhythmia" refers to or includes any abnormal or irregular rate or rhythm of the heartbeat. In some embodiments, the arrhythmia is selected from hyperventricular beats, supraventricular tachycardia, ventricular arrhythmia, or bradyarrhythmia. In some embodiments, the hyperventricular beats include any one of supraventricular premature beats, ventricular premature beats, and junctional premature beats. In some embodiments, the supraventricular tachycardia includes any one of atrial fibrillation, atrial flutter, and paroxysmal supraventricular tachycardia. In some embodiments, the ventricular arrhythmia includes any one of ventricular fibrillation and ventricular tachycardia. In some embodiments, the bradyarrhythmia includes any one of sinus node dysfunction-induced bradyarrhythmia and atrioventricular conduction disorder-induced bradyarrhythmia. In some embodiments, the arrhythmia is a therapy-induced arrhythmia. In some embodiments, the therapeutic agent is an anti-cancer therapeutic agent. In some embodiments, the anti-cancer therapeutic agent is a cancer therapy. In some embodiments, the arrhythmia is a cancer therapy-induced arrhythmia (CTIA). In some embodiments, the cancer therapy is doxorubicin. In some embodiments, the cancer therapy is selected from the anti-cancer agents provided in Table 1.

[0188] Therapeutic agents or conditions that induce cardiac side effects are well known in the art, and the subject may be taking any such therapeutic agent or exposed to any such condition. In some embodiments, the subject is taking a therapeutic agent that induces cardiac side effects. In some embodiments, the subject is exposed to a condition that induces cardiac side effects. In some embodiments, the test is to test an agent that has or is known to cause cardiac side effects. In some embodiments, the test is to test a condition that is known to cause cardiac side effects. Examples of agents that have cardiac side effects can be found, for example, in Mamoshina et al., 2021 "Toward a broader view of mechanisms of drug cardiotoxicity", Cell Reports Medicine, Mar 16;2(3):100216, which is incorporated herein by reference in its entirety. In some embodiments, the therapeutic agent is selected from those provided in Table 1. In some embodiments, the therapeutic agent is an antineoplastic agent. In some embodiments, the antineoplastic agent is an anti-cancer agent. In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the therapeutic agent is a central nervous system agent. In some embodiments, the therapeutic agent is a gastrointestinal agent. In some embodiments, the therapeutic agent is a genitourinary system agent. In some embodiments, the therapeutic agent is an anti-allergy agent. In some embodiments, the therapeutic agent is an anti-infective agent. In some embodiments, the therapeutic agent is a cardiovascular agent. Examples of these agents with cardiac side effects can be found in Table 1.

[0189] [Table 1] TIFF2024537643000003.tif239159TIFF2024537643000004.tif25159

[0190] In some embodiments, the therapeutic agent is 5-fluorouracil, arsenic trioxide, bevacizumab, bortezomib, cisplatin, cytarabine, daunorubicin, dasatinib, docetaxel, doxorubicin, idarubicin, imatinib, ipilimumab, lapatinib, nilotinib, nivolumab, paclitaxel, romidopsin, sorafenib, sunitinib, trastuzumab, vandetanib, vinblastine, diclofenac, etoricoxib, ibuprofen, indomethacin, naproxen, rofecoxib, central nervous system agents, benfluorex, bupivacaine, chlorphentermine, clozapine, cocaine, dexfenfluramine, ergotamine, fenfluramine, fluoxetine, haloperidol. In some embodiments, the therapeutic agent is selected from the group consisting of levomethadyl acetate, lidocaine, methysergide, pergolide, phentermine, propoxyphene, sertindole, sibutramine, thioridazine, venlafaxine, ziprasidone, cispapride, loperamide, omeprazole, tegaserod, terodiline, astemizole, diphenhydramine, terfenadine, azidothymidine, azithromycin, clarithromycin, erythromycin, grepafloxacin, sofobuvir, sparfloxacin, pentamidine, buflomedil, dofetilide, encainide, lidoflazine, mibefradil, orciprenaline, prenylamine, probucol, alogliptin, clobutinol, rosiglitazone, and saxagliptin. In some embodiments, the therapeutic agent interacts with the MCU. In some embodiments, the interaction with the MCU inhibits MCU activity. In some embodiments, the MCU activity is calcium transport, hi some embodiments, the Therapeutic Agent interacts with a calcium channel.In some embodiments, the therapeutic agent that interacts with the MCU is selected from sorafenib, sunitinib, vandetanib, bupivacaine, cocaine, fluoxetine, haloperidol, levomethadyl, propoxyphene, sertindole, thioridazine, venlafaxine, ziprasidone, cisapride, loperamide, terodiline, astemizole, diphenhydramine, terfenadine, azithromycin, mitoxantrone, clarithromycin, erythromycin, grepafloxacin, sofosbuvir, sparfloxacin, pentamidine, buflomedil, dofetilide, encainide, lidoflazine, mibefradil, orciprenaline, prenylamine, probucol, and clobutinol.

[0191] In some embodiments, the cardiac side effect is electrical mitochondrial desynchronization. In some embodiments, the cardiac side effect is arrhythmia. In some embodiments, the arrhythmia is ventricular arrhythmia. In some embodiments, the arrhythmia is supraventricular arrhythmia. In some embodiments, the arrhythmia is hereditary arrhythmia. In some embodiments, the arrhythmia is bradycardia. In some embodiments, the arrhythmia is tachycardia. In some embodiments, the arrhythmia is valvular atrial fibrillation. In some embodiments, the cardiac side effect is ischemia. In some embodiments, the ischemia is myocardial ischemia. In some embodiments, the cardiac side effect is heart failure. In some embodiments, the heart failure is systolic heart failure. In some embodiments, the cardiac side effect is QT prolongation. In some embodiments, the cardiac side effect is tachycardia. In some embodiments, the tachycardia is ventricular tachycardia. In some embodiments, the cardiac side effect is bradycardia. In some embodiments, the cardiac side effect is left ventricular dysfunction. In some embodiments, the cardiac side effect is myocarditis. In some embodiments, the myocarditis is fatal myocarditis. In some embodiments, the cardiac side effect is myocardial infarction. In some embodiments, the cardiac side effect is long QT. In some embodiments, the cardiac side effect is left ejection fraction. In some embodiments, the cardiac side effect is a thrombotic event. In some embodiments, the cardiac side effect is hypertension. In some embodiments, the cardiac side effect is valvular heart disease. In some embodiments, the cardiac side effect is myocardial depression. In some embodiments, the cardiac side effect is pulmonary heart disease. In some embodiments, the cardiac side effect is myocarditis. In some embodiments, the cardiac side effect is cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy. In some embodiments, the cardiac side effect is left ventricular hypertrophy. In some embodiments, the cardiac side effect is torsades de pointes (TdP). In some embodiments, the cardiac side effect is sudden cardiac death. In some embodiments, the cardiac side effect is cardiac arrest. In some embodiments, the cardiac side effect is long QT syndrome. In some embodiments, the cardiac side effect is ventricular fibrillation. In some embodiments, the cardiac side effect is palpitations.

[0192] In some embodiments, the therapeutic agent is a drug. In some embodiments, the therapeutic agent is a small molecule. In some embodiments, the therapeutic agent is a biologic. In some embodiments, the therapeutic agent is a cardiac therapeutic agent. In some embodiments, the therapeutic agent is a non-cardiac therapeutic agent. In some embodiments, the therapeutic agent is a therapeutic agent suspected of causing cardiac side effects. In some embodiments, the therapeutic agent is a systemically administered agent. In some embodiments, the therapeutic agent is a drug formulated for systemic administration. In some embodiments, the therapeutic agent is a drug contemplated for systemic administration.

[0193] In some embodiments, the therapeutic agent is an anti-cancer therapeutic agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is mitoxantrone. In some embodiments, the therapeutic agent is suitable for treating, preventing, or ameliorating cardiac disease or a condition or symptom associated therewith. In some embodiments, the therapeutic agent is a calcium signaling targeting agent. In some embodiments, a calcium signaling targeting agent includes any compound capable of modulating intracellular calcium transport, mobilization, exchange, or any combination thereof. In some embodiments, modulating includes increasing or enhancing. In some embodiments, modulating includes decreasing or inhibiting.

[0194] In some embodiments, the calcium signaling targeting agent decreases calcium flux across the mitochondrial membrane. In some embodiments, the calcium signaling agent decreases the frequency of calcium oscillations across the mitochondrial membrane. In some embodiments, the calcium signaling agent decreases the rate of calcium flux, oscillations, mobilization, translocation, or any combination thereof across the mitochondrial membrane. In some embodiments, the mitochondrial membrane comprises the inner mitochondrial membrane, the outer mitochondrial membrane, or both.

[0195] In some embodiments, the anti-cancer agent is or includes a calcium signaling targeting agent. In some embodiments, the agent includes or is a calcium channel blocker. In some embodiments, the anti-cancer agent induces, stimulates, enhances, promotes, or any combination thereof, arrhythmia. In some embodiments, the anti-cancer agent is suspected of inducing CTIA. The types of calcium channel blockers will be apparent to those skilled in the art. Non-limiting examples of such calcium channel blockers include, but are not limited to, amlodipine (Norvasc), diltiazem (Cardizem, Tiazac, etc.), felodipine, isradipine, nicardipine, nifedipine (Procardia), nisoldipine (Sular), and verapamil (Karan SR, Vereran), to name a few. In some embodiments, the increase in mitochondrial calcium includes an increase in mitochondrial activity. In some embodiments, the mitochondrial activity includes oxidative phosphorylation.

[0196] In some embodiments, the method further comprises inducing a cardiac defect, condition or disease, or a mimic thereof, in the cardiac organoid before contacting. In some embodiments, inducing comprises genetic modification of the cells of the organoid. In some embodiments, the therapeutic agent is designed to treat the induced defect, condition or disease, or a mimic thereof. In some embodiments, the method is a method for testing the efficacy of the therapeutic agent. It will be understood by those skilled in the art that a condition or disease can be induced in the organoid to test an agent designed to treat the condition or disease. In some embodiments, the condition or disease is or includes arrhythmia. In some embodiments, arrhythmia is induced by contacting the organoid with mitoxantrone.

[0197] According to another aspect, a method of treating arrhythmia in a subject is provided.

[0198] According to another aspect, a method of treating a disease or disorder in a subject is provided.

[0199] In some embodiments, the disease or disorder is characterized by electrical mitochondrial desynchronization. In some embodiments, the symptoms of the disease or disorder are caused by electrical mitochondrial desynchronization. As described for the first time below, mitochondrial function and electrical activity of tissues can be uncoupled. This desynchronization leads to impaired cellular function. It is well known that mitochondrial dysfunction (e.g., energy output) can cause disease and disorders, and ion levels are known to affect mitochondrial function, but it was not previously known that ion flux to cells and mitochondria must be kept in lockstep to coordinate oxygen consumption with other cellular functions and maintain cellular homeostasis. Thus, impairment of normal biological function can occur even when both energy output from mitochondria and electrical activity of mitochondria and tissues appear normal individually, but the two are no longer synchronized.

[0200] In some embodiments, the disease is selected from the group consisting of arrhythmia, cardiomyopathy, seizures, epilepsy, motor neuron spasms, muscle weakness, muscle atrophy, channelopathy, catecholaminergic polymorphic ventricular tachycardia (CPVT), myopathy with extrapyramidal signs (MPXPS), Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (AML), hereditary spastic paraplegia, ischemia-reperfusion injury, ischemic heart disease, rare mitochondrial encephalomyopathy, sagittal sinus thrombosis, intracranial sinus thrombosis, Stormer-Ken syndrome, generalized epilepsy febrile seizures plus, optic atrophy 3, autosomal dominant, generalized epilepsy febrile seizures plus, type 6, palmoplantar keratoderma, non-epidermolytic, and eastern equine encephalitis. It will be understood by those skilled in the art that the above listed diseases / conditions are examples of diseases / conditions that can be characterized by electrical mitochondrial desynchronization. However, not all cases of these diseases / conditions are so characterized (i.e., there is no desynchronization component to how the disease / condition manifests in some subjects), so one of skill in the art must determine whether desynchronization is present in any given subject. In some embodiments, the arrhythmia is cancer therapy-induced arrhythmia (CTIA). In some embodiments, the cancer treatment is doxorubicin. In some embodiments, the cancer treatment is selected from the anti-cancer agents provided in Table 1. Channelopathies are well known in the art and are summarized, for example, in Kim, 2014, "Channelopathies", Korean J. Pediatr.; 57(1):1-18, which is incorporated herein by reference in its entirety.

[0201] In some embodiments, the method further comprises confirming electrical mitochondrial desynchronization. In some embodiments, the method further comprises confirming that the disease or disorder is characterized by electrical mitochondrial desynchronization. In some embodiments, the method further comprises confirming electrical mitochondrial desynchronization in the subject. In some embodiments, the method further comprises confirming a disease or disorder manifested by electrical mitochondrial desynchronization in the subject. It will be understood by those skilled in the art that while many diseases may have components, symptoms, or causes that are electrical mitochondrial in nature, not all symptoms of a disease do. Thus, the method may comprise determining that electrical mitochondrial desynchronization is present in this particular subject suffering from a disease. In some embodiments, the confirming is in the subject. In some embodiments, the confirming is in a tissue of the subject. In some embodiments, the tissue is electrical tissue. Examples of electrical tissue include, but are not limited to, neurons and cardiac tissue. In some embodiments, the tissue is cardiac tissue. In some embodiments, the tissue is neuronal tissue. In some embodiments, the tissue is neuronal. In some embodiments, the tissue is central nervous system tissue. In some embodiments, the tissue is brain. In some embodiments, the tissue is peripheral nervous system tissue. In some embodiments, the tissue is diseased tissue. In some embodiments, the tissue is a disease or disorder tissue. In some embodiments, the tissue is from a subject. In some embodiments, the tissue is from cells from the subject. In some embodiments, "derived from" includes "grown from". In some embodiments, "derived from" includes "differentiated from". In some embodiments, the tissue is a biopsy. In some embodiments, the tissue is a tissue that has been contacted with a drug or agent. In some embodiments, the subject has been administered a drug or agent. In some embodiments, the method further comprises selecting a subject suffering from a disease or disorder. In some embodiments, the method further comprises determining that a symptom of the disease or disorder in the subject is caused by electrical mitochondrial desynchronization.

[0202] In some embodiments, the desynchronization is a desynchronization of mitochondrial function and electrical activity. In some embodiments, the electrical activity is electrical activity in mitochondria. In some embodiments, the electrical activity is electrical activity in tissue. In some embodiments, the tissue is a tissue containing mitochondria. Any method for simultaneously determining both electrical activity and mitochondrial function can be used to assess synchronization. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization is in vitro. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization is in vivo. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization is ex vivo. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization is a method of the invention. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization is a method described below. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization comprises evaluating the tissue in a system of the invention. In some embodiments, the method for confirming / determining electrical mitochondrial desynchronization comprises measuring electrical mitochondrial desynchronization using a system of the invention. In some embodiments, the confirming / determining comprises measuring mitochondrial activity. In some embodiments, the mitochondrial activity comprises oxygen consumption. In some embodiments, the mitochondrial activity comprises mitochondrial calcium levels. In some embodiments, the determining / determining comprises measuring a field potential in the tissue and contraction in the tissue. In some embodiments, the determining / determining comprises measuring a field potential in the tissue and oxygen consumption in the tissue. In some embodiments, the determining / determining comprises measuring a field potential in the tissue and calcium levels in the tissue. In some embodiments, the determining / determining comprises measuring oxygen consumption in the tissue and contraction in the tissue. In some embodiments, the determining / determining comprises measuring calcium levels in the tissue and contraction in the tissue.In some embodiments, the identifying / determining comprises measuring the field potential in the tissue, the oxygen consumption in the tissue, and the contraction in the tissue. In some embodiments, the identifying / determining comprises measuring the field potential in the tissue, the calcium level in the tissue, and the contraction in the tissue. In some embodiments, the method of identifying / determining electrical mitochondrial desynchronization comprises measuring the oxygen consumption in the tissue and the calcium level in the tissue using a system of the invention. In some embodiments, the method of identifying / determining electrical mitochondrial desynchronization comprises measuring the contraction of the tissue and the oxygen consumption of the tissue using a system of the invention. In some embodiments, the measuring is a simultaneous measurement.

[0203] Besides the methods provided herein for confirming / determining electrical mitochondrial desynchronization, other methods known in the art can be used for this purpose. For example, typical and well-known methods of measuring electrical output can be used. These include EKG, EMG, EEG, ECG, and EOG. Desynchronization can be seen only in these measurements and manifests as late potentials, reduced R waves, increased R / R ratios, and other abnormalities. Additional methods can also be used, such as those described in Saminathan et al., 2021, “A DNA-based voltmeter for organelles”, Nature Nanotechnology, 16, 96-103, which is incorporated herein by reference in its entirety. Mitochondrial function can also be assessed in tissues using oxygen or calcium functional staining, high-resolution microscopy, or in vivo calcium measurements. In particular, mitochondrial calcium levels can be assessed by mitochondrial calcium-selective dyes such as Rhod-2AM, or tracked by NMR / IR scanning. Generally, methods for measuring mitochondrial calcium in vivo can be found, for example, in Pozzan and Rudolf, 2009, "Measurement of mitochondrial calcium in vivo", Biochim Biophys Acta., Nov;1787(11):1317-23, and Serrat et al., 2022, "Imaging mitochondrial calcium dynamics in the central nervous system", J. Neuroscience Methods, vol 373, May;209560, which are incorporated by reference in their entireties.Simultaneous measurement methods such as measuring oxygen consumption by protoporphyrin IX-triplet state lifetime technology, NMR or other optical methods, and electrical activity such as by EEG / EMG / ECG are known and can be found, for example, in Campbell and Marcinek, 2017, “Evaluation of in vivo mitochondrial bioenergetics in skeletal muscle using NMR and optical methods”, Biochim Biophys Acta, Apr;1862(4):716-724, which is incorporated herein by reference in its entirety. Genetic screening of mitochondrial genes and discovery of calcium channel mutations can indicate desynchronizing properties. Similarly, the use of drugs with known desynchronizing effects, such as cancer treatments that cause arrhythmias (CTIA), is another indicator that the condition is characterized by desynchronization.

[0204] In some embodiments, ascertaining / determining comprises determining mitochondrial calcium concentration in a sample obtained from the subject. In some embodiments, determining is measuring. In some embodiments, the sample is a fluid sample. In some embodiments, the sample comprises cells. In some embodiments, the cells are cardiac cells. In some embodiments, the cells are diseased cells. In some embodiments, the sample is an acellular sample. In some embodiments, the fluid is a bodily fluid. In some embodiments, the fluid is selected from blood, serum, plasma, tumor fluid, gastric fluid, intestinal fluid, saliva, bile, tumor fluid, breast milk, urine, interstitial fluid, cerebrospinal fluid, and stool. In some embodiments, the fluid is blood. In some embodiments, the blood is peripheral blood. In some embodiments, a calcium concentration above a predetermined threshold is indicative of desynchronization. In some embodiments, the predetermined threshold is a calcium concentration in a subject not suffering from electrical mitochondrial desynchronization. In some embodiments, the predetermined threshold is a calcium concentration in a healthy subject. In some embodiments, the predetermined threshold is a calcium concentration in a subject suffering from a disease or disorder but not characterized by electrical mitochondrial desynchronization.

[0205] In some embodiments, "beyond" is "above." In some embodiments, "beyond" is "below." One of ordinary skill in the art will understand that if a threshold is a maximum allowed value, then exceeding it will be above it, and if a threshold is a minimum allowed value, then exceeding it will be below it. In some embodiments, exceeding a threshold is significantly exceeding. In some embodiments, significantly is statistically significant. In some embodiments, exceeding a threshold is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%. Each possibility represents a separate embodiment of the present invention. In some embodiments, "exceeding" is at least 10%. In some embodiments, "exceeding" is at least 30%.

[0206] In some embodiments, the confirming / determining includes observing an arrhythmic or proarrhythmic symptom in the subject. In some embodiments, the symptom is arrhythmia. In some embodiments, the symptom is unresponsive to antiarrhythmic therapy. In some embodiments, the presence of the symptom indicates desynchronization. In some embodiments, the antiarrhythmic therapy targets electrical activity through a membrane channel. In some embodiments, the membrane channel is not a calcium channel. In some embodiments, the membrane channel is not a calcium channel other than a non-dihydropyridine calcium channel. In some embodiments, the calcium channel is a non-mitochondrial calcium channel. In some embodiments, the calcium channel is a mitochondrial calcium channel. It will be understood by those skilled in the art that if calcium channel blockers are not effective, activation of calcium channels is required as described in the methods provided herein. In some embodiments, the antiarrhythmic therapy is a sodium channel blocker. Sodium channel blockers can prevent sodium from entering cells and thus slow the electrical impulses of the heart muscle. Examples of sodium channel blockers include, but are not limited to, disopyramide, flecainide, mexiletine, propafenone, and quinidine. In some embodiments, the antiarrhythmic therapy is a beta blocker. Beta blockers often slow the heart rate by blocking hormones such as adrenaline. Examples of beta blockers include, but are not limited to, acebutolol, atenolol, bisoprolol, metoprolol, nadolol, and propranolol. In some embodiments, the antiarrhythmic treatment is a potassium channel blocker. Potassium channel blockers can prevent potassium from entering cells, thus slowing down electrical impulses in the heart. Examples of potassium channel blockers include, but are not limited to, amiodarone, bretylium, dofetilide, dronedarone, ibutilide, and sotalol. In some embodiments, the antiarrhythmic treatment is a non-dihydropyridine calcium channel blocker.Non-dihydropyridine calcium channel blockers can prevent calcium from entering heart cells, decreasing heart rate and contraction. Examples of non-dihydropyridine calcium channel blockers include, but are not limited to, diltiazem and verapamil. In some embodiments, the antiarrhythmic therapy is adenosine. Adenosine blocks / delays electrical impulses in the atrioventricular node. In some embodiments, the antiarrhythmic therapy is digoxin. Digoxin slows heart rate and increases heart contractility.

[0207] In some embodiments, the checking / determining includes performing an electrical test selected from an EKG, EEG, and EMG. In some embodiments, the checking / determining includes performing an electrocardiogram (EKG or ECG). In some embodiments, the checking / determining includes performing an electroencephalogram (EEG). In some embodiments, the checking / determining includes performing an electromyogram (EMG). In some embodiments, the checking / determining includes performing an electrooculogram (EOG). In some embodiments, the abnormal electrical readout is indicative of desynchronization. In some embodiments, the abnormal readout is compared to a readout from a healthy subject. In some embodiments, the abnormal readout is compared to a readout from a subject suffering from a disease or disorder but not characterized by electrical mitochondrial synchronization. In some embodiments, the abnormal readout includes a late potential. In some embodiments, the abnormal readout includes a reduced R wave. In some embodiments, the abnormal readout includes an increased R / R ratio.

[0208] In some embodiments, the identifying / determining includes identifying exposure to an agent known to cause electrical mitochondrial desynchronization. In some embodiments, the agent is a drug. In some embodiments, the agent is a chemical. In some embodiments, the agent is a pesticide. In some embodiments, the chemical is a solvent. In some embodiments, the chemical is an insecticide. In some embodiments, the agent is selected from those provided in Table 1. In some embodiments, the chemical agent is selected from toluene, trichloroethane, xylene, heptane, hexane, ethyl ether trichloroethylene, and trichlorotrifluoroethane. Other examples of agents include, but are not limited to, carbon monoxide, carbon disulfide, insecticides, methane-derived halogenated hydrocarbons, caffeine, bisphenol A, organic nitrates, arsenic, cadmium, cobalt, organic solvents, and metals.

[0209] According to another aspect, a method of treating a cell proliferation associated disorder in a subject is provided.

[0210] According to another aspect, there is provided a method of treating a disease in a subject, comprising administering a first agent and a second agent, wherein the disease is treatable by the first agent.

[0211] According to another aspect, there is provided a method of treating a cell proliferation associated disorder in a subject, the method comprising administering a first agent and a second agent.

[0212] In some embodiments, the first agent produces a cardiac side effect. In some embodiments, the side effect is an adverse side effect. In some embodiments, the first agent is suspected or likely to produce a cardiac side effect. In some embodiments, the disease is cancer and the first agent is an anti-neoplastic agent. In some embodiments, the disease is an inflammatory disease and the first agent is an anti-inflammatory agent. In some embodiments, the disease is a disease of the central nervous system (CNS) and the first agent is a CNS agent. In some embodiments, the CNS disease is a brain disease. In some embodiments, the CNS disease is a neuronal disease. In some embodiments, the disease is a gastrointestinal disease and the first agent is a gastrointestinal agent. In some embodiments, the disease is a reproductive or urinary tract disease and the first agent is a urogenital agent. In some embodiments, the disease is an allergic reaction and the first agent is an anti-allergic agent. In some embodiments, the disease is an infectious disease and the first agent is an anti-infective agent. In some embodiments, the anti-infective agent is an antibiotic. In some embodiments, the anti-infective agent is an antiviral agent. In some embodiments, the anti-infective agent is a vaccine. In some embodiments, the disease is a cardiovascular disease and the first agent is a cardiovascular agent. In some embodiments, the first agent is selected from the agents provided in Table 1.

[0213] Examples of inflammatory diseases include, but are not limited to, autoimmune diseases, rheumatoid arthritis, inflammatory bowel syndrome (IBS), inflammatory bowel disease (IBD), colitis, Crohn's disease, ankylosing spondylitis, antiphospholipid syndrome, gout, myositis, scleroderma, lupus, Sjogren's syndrome, and vasculitis, to name a few. Examples of CNS diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), muscular dystrophy, paralysis, epilepsy, multiple sclerosis (MS), neurofibromatosis, and sciatica, to name a few. Examples of gastrointestinal diseases include, but are not limited to, IBS, IBD, acid reflux, gallstones, hemorrhoids, intestinal cancer, colon polyps, and diverticular disease, to name a few. Examples of diseases of the reproductive or urinary tract include, but are not limited to, urinary tract infections, testicular cancer, renal failure, endometrial ablation, erectile dysfunction, incontinence, kidney stones, prostate cancer, sickle cell nephropathy, and yeast infections, to name a few. Examples of allergic reactions include, but are not limited to, food allergies, asthma, environmental allergies, and drug allergies, to name a few. Examples of infections include, but are not limited to, bacterial infections, viral infections, and parasitic infections. Examples of cardiovascular diseases include, but are not limited to, heart attacks, strokes, heart failure, arrhythmias, and palpitations, to name a few.

[0214] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that modulates mitochondrial calcium concentration. In some embodiments, the treatment comprises administering to the subject a therapeutically effective amount of an agent that modulates mitochondrial calcium concentration. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that modulates mitochondrial calcium channel activity. In some embodiments, the treatment comprises administering to the subject a therapeutically effective amount of an agent that modulates mitochondrial calcium channel activity. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that modulates mitochondrial calcium uniporter (MCU) activity. In some embodiments, the treatment comprises administering to the subject a therapeutically effective amount of an agent that modulates MCU activity. In some embodiments, the mitochondrial calcium channel is MCU. In some embodiments, the mitochondrial calcium channel is a voltage-dependent anion channel (VDAC). In some embodiments, the mitochondrial calcium channel is not a non-dihydropyridine calcium channel. Examples of mitochondrial calcium channels include, but are not limited to, MCU and VDAC. Other examples of mitochondrial calcium channels include uptake permeability mode (RaM), ryanosine receptor (mRyR or RyR1), mitochondrial rapid exchanger (mHCX), DAG-activated transport pore (mPTP), Na+ / Ca2+ exchanger (mNCX) and H+ / Ca2 cation channel (DCC) well known in the art. Other examples can be found, for example, in Malli et al., 2014, "Mitochondrial Ca2+channels: great unknowns with important functions", FEBS Lett. May 17; 584(10): 1942-1947, which is incorporated herein by reference in its entirety. In some embodiments, the increase is in a tissue. In some embodiments, the tissue is a tissue of a subject. In some embodiments, the increase is in cardiac tissue. In some embodiments, the cardiac tissue is cardiac tissue of a subject. In some embodiments, modulating is modulating mitochondrial activity.In some embodiments, the modulation of activity occurs within a tissue.

[0215] In some embodiments, modulating MCU activity comprises inhibiting an interaction of the drug with the MCU. In some embodiments, the interaction is binding. In some embodiments, the drug inhibits MCU activity. In some embodiments, the drug activates MCU activity. In some embodiments, the interaction of the drug with the MCU inhibits or activates MCU activity. In some embodiments, the agent inhibits or induces an interaction of the drug with the MCU. In some embodiments, the inhibition is blocking. In some embodiments, the induction is triggering. In some embodiments, the agent competes with the MCU for binding to the drug. In some embodiments, the agent is a peptide that binds to the drug. In some embodiments, the agent is a small molecule that binds to the drug. In some embodiments, the drug that interacts with the MCU is selected from sorafenib, sunitinib, vandetanib, bupivacaine, cocaine, fluoxetine, haloperidol, levomethadyl, propoxyphene, sertindole, thioridazine, venlafaxine, ziprasidone, cisapride, loperamide, terodiline, astemizole, diphenhydramine, terfenadine, azithromycin, mitoxantrone, clarithromycin, erythromycin, grepafloxacin, sofosbuvir, sparfloxacin, pentamidine, buflomedil, dofetilide, encainide, lidoflazine, mibefradil, orciprenaline, prenylamine, probucarin, and clobutinol. In some embodiments, the administration of the drug treats arrhythmia. In some embodiments, the drug is a second drug.

[0216] In some embodiments, modulating comprises administering an agent selected from metformin, kaempferol, spermine, A-769662, AICAR, IND1316, PF06409577, ZLN024, erastin, honokiol, ezetimibe, disulfiram, efsevin, and spermidine. In some embodiments, modulating comprises administering an agent selected from metformin, spermine, A-769662, AICAR, IND1316, PF06409577, ZLN024, and spermidine. In some embodiments, the agent is metformin. In some embodiments, the agent is selected from metformin, spermidine, and spermine.

[0217] In some embodiments, the method includes administering to the subject a therapeutically effective amount of an agent capable of modulating mitochondrial calcium concentration in cardiac tissue of the subject, thereby treating a cardiac disease or disorder characterized by electrical mitochondrial desynchronization. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an agent capable of modulating mitochondrial calcium concentration in neural tissue of the subject, thereby treating a neurological disease or disorder characterized by electrical mitochondrial desynchronization. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an agent capable of modulating mitochondrial calcium concentration in cardiac tissue of the subject, thereby treating an arrhythmia. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an agent capable of modulating mitochondrial calcium channel activity in cardiac tissue of the subject, thereby treating a cardiac disease or disorder characterized by electrical mitochondrial desynchronization. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an agent capable of modulating mitochondrial calcium channel activity in neural tissue of the subject, thereby treating a neurological disease or disorder characterized by electrical mitochondrial desynchronization. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an agent capable of modulating mitochondrial calcium channel activity in cardiac tissue of the subject, thereby treating an arrhythmia. In some embodiments, the agent is a second agent.

[0218] In some embodiments, the modulation is increased. In some embodiments, the modulation is decreased. In some embodiments, the increase is increased above a predetermined threshold. In some embodiments, the decrease is decreased below a predetermined threshold. In some embodiments, the increase is a statistically significant increase. In some embodiments, the decrease is a statistically significant decrease. In some embodiments, the predetermined threshold is a level or activity in a healthy subject. In some embodiments, the desynchronization comprises a decrease in mitochondrial calcium concentration and the modulation is increased. In some embodiments, the desynchronization comprises a decrease in mitochondrial calcium channel activity and the modulation is increased. In some embodiments, the decrease is a significant decrease. In some embodiments, the decrease is compared to a healthy control. In some embodiments, the decrease is compared to a subject suffering from a disease but not characterized by desynchronization. In some embodiments, the desynchronization comprises an increase in mitochondrial calcium concentration and the modulation is decreased. In some embodiments, the desynchronization comprises an increase in mitochondrial calcium channel activity and the modulation is decreased. In some embodiments, the increase is a significant increase. In some embodiments, the increase is compared to a healthy control. In some embodiments, the increase is as compared to subjects who have the disease but are not characterized by desynchronization.

[0219] In some embodiments, the desynchronization comprises increased mitochondrial calcium concentration or mitochondrial calcium channel activity, and the disease or disorder is selected from mitochondrial encephalomyopathy, sagittal sinus thrombosis, intracranial sinus thrombosis, Stormer-Ken syndrome, generalized epilepsy febrile seizures plus, optic atrophy 3, autosomal dominant, generalized epilepsy febrile seizures plus, type 6, palmoplantar keratoderma, non-epidermolytic, Eastern equine encephalitis, and CTIA. In some embodiments, the CTIA is doxorubicin-induced arrhythmia. In some embodiments, the desynchronization comprises decreased mitochondrial calcium concentration or increased mitochondrial calcium channel activity, and the disease or disorder is selected from arrhythmia, cardiomyopathy, seizures, epilepsy, motor neuron spasms, muscle weakness, muscle atrophy, channelopathy, CPVT, MPXPS, Alzheimer's disease, Huntington's disease, Parkinson's disease, AML, hereditary spastic paraplegia, ischemia-reperfusion injury, ischemic heart disease, rare, and CTIA.

[0220] In some embodiments, the disease or disorder is a genetic disease or disorder. In some embodiments, the genetic disease or disorder induces arrhythmia and / or cardiac dysfunction. Examples of such genetic diseases include, but are not limited to, CPTV and MPXPS. In some embodiments, the disease or disorder is CPVT, and modulating comprises administering an agent selected from spermine, spermidine, metformin, elastin, A-769662, AICAR, IND1316, PF06409577, and ZLN024. In some embodiments, the genetic disorder or genetic disease induces neurotrophic disorders. Examples of such genetic diseases include, but are not limited to, rare mitochondrial encephalomyopathy, sagittal sinus thrombosis, intracranial sinus thrombosis, Stormer-Ken syndrome, generalized epilepsy febrile convulsions plus, optic atrophy 3, autosomal dominant, generalized epilepsy febrile convulsions plus, type 6, palmoplantar keratoderma, non-epidermolytic, and eastern equine encephalitis.

[0221] In some embodiments, the disease or disorder is a symptom of a disease or disorder. In some embodiments, the disease or disorder is a cardiac disease or disorder. In some embodiments, the cardiac disease or disorder is selected from arrhythmia and cardiomyopathy. In some embodiments, the disease or disorder is arrhythmia. In some embodiments, the disease or disorder is a nervous system disease or disorder. In some embodiments, the disease or disorder is a neuronal disease or disorder. In some embodiments, the nervous system is the central nervous system (CNS). In some embodiments, the nervous system is the peripheral nervous system (PNS). In some embodiments, the nervous system is the CNS, the PNS, or both. In some embodiments, the CNS disease or disorder is selected from seizures and epilepsy. In some embodiments, the seizures are selected from central seizures and peripheral seizures. In some embodiments, the PNS disease or disorder is selected from seizures, motor neuron spasms, muscle weakness, and muscle atrophy. In some embodiments, the disease or disorder is a drug-induced disease or disorder. In some embodiments, the disease or disorder is a drug-induced symptom or side effect. In some embodiments, the drug is an anti-cancer drug.

[0222] In some embodiments, the method comprises administering to a subject a therapeutically effective amount of Ca 2+ In some embodiments, the method includes administering to the subject an agent capable of increasing mitochondrial calcium channel signaling. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a mitochondrial calcium channel activator. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an MCU activator. As used herein, the term "MCU activator" refers to an agent capable of increasing mitochondrial Ca 2+ In some embodiments, the MCU activator is an activator of the Ca2+ receptor in the mitochondrial matrix. 2+ In some embodiments, the MCU activator alters the frequency or rate of Ca oscillations between the mitochondrial matrix and the mitochondrial intermembrane space. In some embodiments, the MCU activator increases the concentration or effective concentration of Ca. 2+An agent capable of increasing signal transduction is metformin. In some embodiments, the MCU activator is or includes metformin.

[0223] In some embodiments, the cell proliferation associated disease comprises cancer. In some embodiments, the cell proliferation associated disease is cancer. As used herein, the term "cancer" refers to any disease characterized by abnormal cell proliferation. In some embodiments, cancer is further characterized by the possibility or ability to invade other parts of the body beyond the area where the abnormal cell proliferation occurs. In some embodiments, the cancer is selected from breast cancer, cervical cancer, cervical canal cancer, colon cancer, lymphoma, esophageal cancer, brain cancer, head and neck cancer, renal cancer, meningeal cancer, glioma, glioblastoma, Langerhans cell cancer, lung cancer, mesothelioma, ovarian cancer, pancreatic cancer, neuroendocrine cancer, prostate cancer, skin cancer, gastric cancer, tenosynovial cancer, tongue cancer, thyroid cancer, uterine cancer, and testicular cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a tumor.

[0224] In some embodiments, the method includes administering an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is a calcium signaling targeting agent. In some embodiments, the anti-cancer agent is a calcium channel blocker. In some embodiments, the anti-cancer agent induces CTIA. In some embodiments, the anti-cancer agent causes CTIA. In some embodiments, the anti-cancer agent is a first agent.

[0225] In some embodiments, the subject is a subject in need of the methods of the invention. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is suffering from a proliferation-related disease. In some embodiments, the subject is suffering from arrhythmia. In some embodiments, the subject is at risk of developing cancer. In some embodiments, the subject is suspected of having cancer. In some embodiments, the subject is genetically predisposed to cancer. In some embodiments, the subject has cancer. In some embodiments, the subject is undergoing cancer treatment. In some embodiments, the cancer treatment comprises chemotherapy. In some embodiments, the subject is suffering from CTIA.

[0226] In some embodiments, the subject does not suffer from a metabolic disorder or any disease or condition associated therewith. In some embodiments, the metabolic disorder or any disease or condition associated therewith is diabetes, hyperglycemia, or both. In some embodiments, the subject does not suffer from diabetes or any disease associated with metabolic syndrome. In some embodiments, the subject is not treated with metformin to treat metabolic syndrome or any disease or condition associated therewith, such as, but not limited to, diabetes. In some embodiments, the subject does not suffer from a disease or condition treatable with metformin. In some embodiments, the subject does not suffer from a disease or condition treatable by increasing mitochondrial calcium concentration. In some embodiments, the subject does not suffer from a disease or condition treatable by increasing MCU activity. In some embodiments, the subject does not suffer from a disease or condition treatable with an MCU activator.

[0227] In some embodiments, the method further comprises selecting a subject suitable for treatment. In some embodiments, the treatment is a treatment by the method of the present invention. In some embodiments, the method further comprises selecting a subject suitable for treatment with metformin. In some embodiments, the method further comprises selecting a subject suitable for treatment with an agent selected from metformin, kaempferol, spermine, A-769662, AICAR, IND1316, PF06409577, ZLN024, erastin, honokiol, ezetimibe, disulfiram, efsevin and spermidine. In some embodiments, the selecting comprises excluding subjects suffering from or at high risk of developing metabolic syndrome or any disease or condition associated therewith, such as diabetes.

[0228] According to another aspect, a. modulating mitochondrial calcium concentration in a tissue of a subject; b. modulating mitochondrial calcium channel activity in a tissue; or C. A combination of these 1. A method for selecting a subject suitable for treatment with an agent capable of: Methods are provided which include determining the presence of electrical mitochondrial desynchronization in a subject, the presence of said desynchronization indicating that the subject is suitable for treatment. In some embodiments, the subject is suffering from a disease or disorder. In some embodiments, the disease or disorder is known to potentially involve or may be characterized by electrical mitochondrial desynchronization. In some embodiments, the tissue is a diseased tissue. In some embodiments, the tissue is a diseased or disordered tissue. In some embodiments, the tissue is a diseased or disordered tissue in the subject.

[0229] Arrays and Systems Reference is now made to FIG. 8A, which is a diagram of a prior art sensing system and a system according to some embodiments of the present invention. The prior art system 10 may include an illumination source 110 configured to emit an LED laser emitting light at a first wavelength, e.g., 532 nm. The system 10 may further include a PMT sensor 140 configured to detect a signal indicative of an emission from a microparticle embedded in the tissue or cell aggregate 5 at a second wavelength, e.g., 605 nm. In some embodiments, the microparticle comprises an excitable molecule that is quenchable by a cofactor. In some embodiments, the controller (not shown) is configured to calculate a temporal cofactor consumption of the tissue or cell aggregate based on the first and second signals, e.g., oxygen levels of the tissue or cell aggregate 5. In some embodiments, the cofactor is oxygen.

[0230] According to some embodiments of the present invention, the system 100 may include an illumination source 110 configured to emit light (photon beam 115) at a first wavelength, for example an LED laser emitting light at 532 nm, a first PMT sensor 120, and a controller 130. In some embodiments, the first PMT sensor 120 may be configured to detect photons at the first wavelength reflected from the tissue or cell aggregates 5. In some embodiments, the system 100 may further include a second PMT sensor 140 configured to detect a signal indicative of an emission from a microparticle embedded in the tissue or cell aggregates 5 at a second wavelength. In some embodiments, the system 100 may include an optical element 125 configured to direct reflected photons to the first PMT sensor 120 and direct emitted photons to the second PMT sensor 140.

[0231] The controller 130 may be configured to control the illumination source 110 to emit light at a first wavelength and receive signals from the first PMT sensor 120 and the second PMT sensor 140. The controller 130 may be any suitable computing device capable of controlling the illumination source 110 and receiving signals from the PMT sensors 120 and 140. The controller 130 may include a processing unit (e.g., a CPU), a memory, and any input / output device(s). The controller 130 may be configured to perform methods according to some embodiments of the present invention, such as the method of FIG. 8E.

[0232] Please refer to Figure 8E, which is a flow chart of a method for measuring a property of a tissue or cell aggregate according to some embodiments of the present invention. In step 810, microparticles embedded in the tissue or cell aggregate 5 may be illuminated with a photon beam having a first wavelength. For example, the controller 130 may control the illumination source 110 to emit a photon beam having a wavelength of 532 nm (e.g., an LED laser as shown in Figure 8A).

[0233] In step 820, a first signal indicative of photons reflected from the microparticle may be detected by a first sensor (e.g., first PMT sensor 120) at a first wavelength. For example, controller 130 may receive a first signal 155 indicative of emission from a microparticle embedded in tissue or cell aggregate 5 at a wavelength of 605 nm from first PMT sensor 120, as shown in FIG. 8A.

[0234] In step 830, a second signal indicative of emission from a microparticle comprising an excitable molecule that can be quenched by a cofactor can be detected at a second wavelength by a second sensor (e.g., a second cPMT sensor 140). For example, photons having a wavelength of 605 nm can be reflected from a microparticle in the tissue or cell aggregate 5 and detected by the second PMT sensor 140 and controller 130.

[0235] In step 840, a shift between the frequency of the first signal and the frequency of the photon beam is measured, and background noise is determined based on the measured shift. Background noise is reduced from the second signal. For example, the second signal 160 may be used to reduce noise of a signal to generate a clean smooth signal 150. In some embodiments, the raw signal may be signal 155, and signal 150 is a filtered signal. In some embodiments, filtering may include measuring a shift between the frequency of the second signal 160 and the frequency of the first signal 155, determining background noise based on the measured shift, and reducing background noise from the second signal to receive the filtered signal 160.

[0236] In step 850, the temporal cofactor consumption of the tissue or cell aggregate can be calculated based on the background noise reduced second signal. In some embodiments, the temporal cofactor consumption can be the oxygen level in the tissue or cell aggregate 5.

[0237] In some embodiments, the change in the intensity of the first signal over time can be detected. In some embodiments, the relative displacement of the microparticle can be calculated based on the detected change. In some embodiments, the detected change in the intensity of the signal is proportional to the relative displacement of the microparticle, for example, the focal distance can be measured by measuring the movement of the xy table (e.g., mechanical stage), holding system 100 (e.g., in micrometers) when the focus of the microscope lens moves away from the microparticle in the tissue or cell aggregate 5 due to the displacement. The xy table is moved to refocus the lens on the microparticle, and the length of the movement is measured. For example, the distance between two consecutive maxima in the signal 160 is proportional to the relative displacement. In some embodiments, the displacement is measured on an axis perpendicular to the photon beam 115. In some embodiments, the displacement is the contraction of the organoid. In some embodiments, the displacement is the pulsation of the organoid. It will be understood by those skilled in the art that any measure of contraction can be measured by displacement, including but not limited to the frequency of contraction and the magnitude of contraction.

[0238] In some embodiments, the method may further include sensing the field potential of the tissue or cell aggregate from an array of microelectrodes (not shown) to measure electrical activity of the tissue or cell aggregate simultaneously with detecting the first signal (as shown in the example of FIG. 3G). In some embodiments, the method may further include comparing the frequencies of the first signal, the second signal, and the tissue field potential. Non-limiting examples of such comparisons when the tissue is simulating a beating heart are shown in FIG. 3F (when the displacement indicates a heartbeat), 3G (when the electrical activity indicates thermal electrical activity), and 3H (when the oxygen level indicates respiration). In some embodiments, if the comparison between the frequencies results in a deviation below a threshold, the comparison indicates a healthy tissue or cell aggregate, e.g., a healthy heart.

[0239] According to another aspect, a system is provided that includes an illumination source, a first photomultiplier tube (PMT) sensor, and a controller.

[0240] In some embodiments, the controller is configured to control the illumination source to illuminate the microparticle embedded in the tissue or cell aggregate with the photon beam. In some embodiments, the photon beam has a first wavelength. In some embodiments, the controller is configured to control the illumination source to illuminate the tissue or cell aggregate with the photon beam. In some embodiments, the controller is configured to detect a first signal indicative of photons reflected from the microparticle. In some embodiments, the detecting the first signal is by a first PMT sensor. In some embodiments, the first signal is at a first wavelength. In some embodiments, the photons reflected from the microparticle are at a first wavelength. In some embodiments, the controller is configured to detect a change in intensity of the first signal. In some embodiments, the controller is configured to calculate a displacement of the microparticle. In some embodiments, the displacement is a relative displacement. In some embodiments, the calculation is based on the detected change. In some embodiments, the detected change in intensity of the first signal is indicative of a relative displacement of the microparticle. In some embodiments, the displacement is measured on an axis perpendicular to the photon beam. In some embodiments, the displacement is a measure of movement of the tissue or cell aggregate. In some embodiments, the movement is a contraction.

[0241] In some embodiments, the system is a sensing system. In some embodiments, the sensing is sensing a parameter in tissue or cell aggregate. In some embodiments, the system comprises a container for tissue or cell aggregate. In some embodiments, the cell aggregate is an organoid. In some embodiments, the organoid is a cardiac organoid. In some embodiments, the organoid is a brain organoid. In some embodiments, the organoid is the cardiac organoid of the present invention. The method of making brain organoid is well known in the art, and any such method can be used to make the organoid that is sensed.

[0242] In some embodiments, the system further comprises a second PMT sensor. In some embodiments, the controller is configured to detect a second signal. In some embodiments, the second signal is detected by the second PMT sensor. In some embodiments, the second signal is indicative of an emission from a microparticle embedded in the tissue or cell aggregate. In some embodiments, the second signal is at a second wavelength. In some embodiments, the second wavelength is a wavelength different from the first wavelength. In some embodiments, the first and second signals are sufficiently different so as not to overlap. In some embodiments, the first and second signals are at different band passes. In some embodiments, the first signal comprises a wavelength of about 532 nm. In some embodiments, the photon beam comprises a wavelength of about 532 nm. In some embodiments, the photon beam and the first signal comprise the same wavelength. In some embodiments, the second signal comprises a wavelength of about 605 nm. In some embodiments, the microparticle comprises an excitable molecule or moiety that is quenchable by a cofactor. In some embodiments, the control is configured to calculate temporal cofactor consumption in the tissue of the cell aggregate. In some embodiments, the calculation is based on the second signal. In some embodiments, the calculation is based on the second signal and the first signal.

[0243] In some embodiments, the cofactor is a biological compound. In some embodiments, the compound is produced in a tissue or cell aggregate. In some embodiments, the cofactor is oxygen (O2). In some embodiments, the excitable molecule or moiety is excitable by a photon beam. In some embodiments, upon excitation, the excitable molecule or moiety emits light at a second wavelength. In some embodiments, the excitable molecule or moiety is phosphorescent. In some embodiments, the microparticle is an oxygen sensor. Quenchable oxygen sensors that emit detectable signals are known in the art, and any such sensor can be used. In some embodiments, the quenchable sensor comprises an oxygen quenchable luminescent dye. Such dyes are known in the art. In some embodiments, the quenchable sensor comprises ruthenium. Such examples can be found in McEvoy et al., 1996, “Dissolved oxygen sensor based on fluorescence quenching of oxygen-sensitive ruthenium complexes immobilized in sol-gel-derived porous silica coatings”, doi.org / 10.1039 / AN9962100785, Wang et al., 2014, “Optical methods for sensing and imaging oxygen: materials, spectroscopies and applications”, hem. Soc. Rev., 2014, 43, 3666-3761, and Achatz et al., 2010, “Luminescent Sensing of Oxygen Using a Quenchable Probe and Upconverting Nanoparticles”, Angew Chem Int Ed Engl. 2011 Jan 3;50(1):260-3, which are incorporated by reference in their entireties. In some embodiments, the temporal cofactor consumption is the cofactor level in a tissue or cell aggregate.In some embodiments, temporal cofactor consumption is proportional to the cofactor level in the tissue or cell aggregate.

[0244] In some embodiments, the controller is configured to filter the second signal. In some embodiments, the filtering is by a parameter of the photon beam. In some embodiments, the filtering is by the first signal. In some embodiments, the filtering is by a difference between the photon beam and the first signal. In some embodiments, the difference is a shift between the photon beam and the first signal. In some embodiments, the shift is a shift in frequency. In some embodiments, the shift is a shift in wavelength. In some embodiments, the filtering includes measuring a shift between a frequency of the first signal and a frequency of the photon beam. In some embodiments, the filtering includes measuring a shift between a wavelength of the first signal and a wavelength of the photon beam. In some embodiments, the filtering includes determining a background based on the measured shift. In some embodiments, the background is background noise. In some embodiments, the filtering includes reducing the background from the second signal. In some embodiments, the filtering includes filtering the background from the second signal. In some embodiments, the filtering includes cleaning the second signal.

[0245] In some embodiments, the controller is further configured to sense a field potential of the tissue or cell aggregate. In some embodiments, the field potential is from an array of microelectrodes. In some embodiments, the array is within the tissue or cell aggregate. In some embodiments, the array is within a container that holds the tissue or cell aggregate. In some embodiments, the microelectrodes are for measuring electrical activity of the tissue or cell aggregate. In some embodiments, the tissue or cell aggregate is of the tissue or cell aggregate. In some embodiments, the sensed field potential is performed simultaneously with the detection of the first signal. In some embodiments, the sensed field potential is performed simultaneously with the detection of the second signal. In some embodiments, the sensed field potential is performed simultaneously with the detection of the first signal and the second signal. In some embodiments, the controller is configured to compare the frequency of the first signal and the second signal. In some embodiments, the controller is configured to compare the frequency of the first signal and the field potential. In some embodiments, the controller is configured to compare the frequency of the second signal and the field potential. In some embodiments, if the comparison between the frequencies results in a deviation below a predetermined threshold, the low deviation indicates a healthy tissue or cell aggregate. In some embodiments, if the comparison between the frequencies results in a deviation greater than a predetermined threshold, the high deviation is indicative of the tissue or cell aggregate having a disease or disorder. In some embodiments, the disease or disorder comprises electrical mitochondrial desynchronization. In some embodiments, the high deviation is indicative of electrical mitochondrial desynchronization.

[0246] Another aspect provides the use of the system of the invention in performing measurements in tissues or cell aggregates.

[0247] In another aspect, there is provided a method for assessing cellular function, comprising the steps of: a. placing a tissue, organoid or cell aggregate in a system of the invention; b. applying a condition to the tissue, organoid, or cell aggregate; c. Measuring at least cofactor consumption in tissues, organoids or cell aggregates; thereby evaluating cellular function; and A method is provided that includes:

[0248] In some embodiments, the method includes placing the tissue or cell aggregate in the system. In some embodiments, the tissue or cell aggregate is an organoid. In some embodiments, the organoid is a cardiac organoid. In some embodiments, the cardiac organoid can beat. In some embodiments, the cardiac organoid is multi-chambered. In some embodiments, the cardiac organoid is a cardiac organoid of the present invention.

[0249] In some embodiments, measuring includes measuring cofactor consumption, displacement and field potential. In some embodiments, at least one significant deviation of displacement, cofactor consumption and field potential after applying the condition indicates electric mitochondrial desynchronization. In some embodiments, at least two significant deviations of displacement, cofactor consumption and field potential after applying the condition indicate electric mitochondrial desynchronization. In some embodiments, all significant deviations of displacement, cofactor consumption and field potential after applying the condition indicate electric mitochondrial desynchronization. In some embodiments, the deviation is a deviation from a control. In some embodiments, the control is the displacement, cofactor consumption and / or field potential before applying the condition. In some embodiments, the control is the displacement, cofactor consumption and / or field in a control tissue, organoid or cell aggregate. In some embodiments, the control is a healthy control. In some embodiments, the control is an untreated control. In some embodiments, the control is a control without electric mitochondrial desynchronization.

[0250] According to another aspect, a method for testing a therapeutic agent for a cardiac side effect is provided, the method comprising placing cardiac organoids in a system and adding a therapeutic agent, thereby testing the therapeutic agent for a cardiac side effect.

[0251] According to another aspect, a method of testing a cardiovascular drug is provided, the method comprising placing cardiac organoids with a system and adding a cardiovascular drug, thereby testing the cardiovascular drug.

[0252] In some embodiments, the agent is not a cardiovascular drug. In some embodiments, the agent is a cardiovascular drug. In some embodiments, the cardiovascular drug is a cardiac drug. In some embodiments, the method further comprises measuring at least one of displacement, cofactor consumption, and field potential in said cardiac organoid. In some embodiments, at least two of displacement, cofactor consumption, and field potential are measured. In some embodiments, all of displacement, cofactor consumption, and field potential are measured. In some embodiments, the displacement is contraction of the cardiac organoid. In some embodiments, the cofactor consumption is metabolism in the cardiac organoid. In some embodiments, the cofactor is oxygen. In some embodiments, the metabolism is respiration. In some embodiments, the metabolism comprises mitochondrial function. In some embodiments, the field potential is field potential duration. In some embodiments, the field potential is field potential amplification. In some embodiments, the field potential is spontaneously generated field potential. In some embodiments, the field potential comprises electrical activity in the organoid.

[0253] In some embodiments, a deviation in any one of the displacement, cofactor consumption, and field potential indicates a cardiac side effect. In some embodiments, the deviation is an imbalance between the displacement, cofactor consumption, and field potential. In some embodiments, the deviation is a change that is not compensated for by other conditions. It will be understood that a therapeutic agent can increase the displacement, consumption, and potential all in equal proportions. This does not constitute a deviation because all measures are still proportional, but simply increased. Thus, the deviation is an imbalance between the three measures. In some embodiments, a deviation in any one of the displacement, cofactor consumption, and field potential indicates an effect of the agent. In some embodiments, the deviation is a significant deviation. In some embodiments, the significance is statistically significant. In some embodiments, the significance is a deviation greater than a pre-determined threshold. In some embodiments, the deviation is after administration of the drug compared to before administration of the drug. In some embodiments, the deviation is after administration of the drug compared to a control where the drug was not administered. In some embodiments, the control is an untreated control. In some embodiments, the deviation is any two of the displacement, cofactor consumption, and field potential. In some embodiments, the deviation is all of displacement, cofactor consumption, and field potential.It will be understood that any change in displacement, cofactor consumption, and field potential indicates that the drug affects cardiac organoid and therefore affects the subject's heart.In some embodiments, the deviation is an imbalance between displacement, cofactor consumption, and field potential.In some embodiments, the imbalance is between contraction and mitochondrial function.In some embodiments, the imbalance indicates arrhythmia.In some embodiments, the imbalance indicates the therapeutic effect on contraction rhythm.

[0254] general Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0255] In the description, unless otherwise stated, adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of one or more features of an embodiment of the present invention are understood to mean that the condition or characteristic is defined within a tolerance allowed for the operation of the embodiment for its intended use. Unless otherwise indicated, the word "or" in this specification and claims is considered to be an inclusive "or" rather than an exclusive "or" and indicates at least one, or any combination, of the items with which it is associated.

[0256] In the description and claims of this application, the verbs "comprise", "include", and "have" and their conjugations are used to indicate that the object or objects of the verb are not necessarily an exhaustive list of components, elements or parts of the subject or subjects of the verb.

[0257] Other terms used herein are meant to be defined according to their known meanings in the art.

[0258] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0259] As used herein, the term "about" when combined with a value refers to ±10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm +- 100 nm.

[0260] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides, a reference to a "polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for use of exclusive language such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0261] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by one of ordinary skill in the art that virtually any disjunction and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0262] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination was individually and explicitly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0263] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0264] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0265] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, see, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley&Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York, all of which are incorporated by reference. New York (1998); methodologies described in U.S. Patent Nos. 4,666,828, 4,683,202; 4,801,531, 5,192,659, and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, JE, ed. (1994); “Culture of Animal Cells-A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996). Other general references are provided throughout this document.

[0266] Materials and Methods cell culture Human induced pluripotent stem cell lines: UN-1, ACS-1021 and ACS-1028 were cultured on growth factor reduced Matrigel (BD Biosciences, San Jose, CA) in mTeSR-1 medium (StemCell Technologies, Canada) with daily medium changes. Cells were obtained and tested for mycoplasma contamination using PCR. Cells were serially passaged using 0.5x TrypLE enzyme (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.5 mM EDTA (Invitrogen, Thermo Fisher Scientific).

[0267] HEK293T cells (ATCC, USA) were seeded in 10 cm cell culture plates at a density of 4×10^6 cells / plate. The cells were maintained in 293T medium consisting of DMEM high glucose (4.5 g / l; Merck, USA) supplemented with 10% FBS (BI, Israel), 1×NEAA (BI, Israel) and 2 mM L-alanine-L-glutamine (BI, Israel).

[0268] Primary rat cardiac microvascular endothelial cells (CECs; Vec Technologies, USA) were cultured on gelatin-coated flasks using endothelial cell growth medium-2MV (Lonza, Switzerland) according to the manufacturer's instructions.

[0269] All cells were cultured under standard conditions at 37°C and 5% CO2 in a humidified incubator.

[0270] finite element analysis Finite element models were created using the Multiphysics static structural model in SolidWorks® 2018. Numerical simulations and investigations were performed for several conditions: an open myocardial volume on a low-adhesion plate surface, a homogeneous geometrically confined myocardial volume embedded in a soft matrix, and a heterogeneous geometrically confined myocardial volume embedded in a soft matrix and patterned by a rigid microvascular structure. The initial mass had a Poisson's ratio of 0.4, a density of 1.06 g / ml, and a density of 1 Pa / m 2 The tissue was simulated as a sphere of diameter 500 μm with tissue parameters defined by an isotropic spring constant of 1 Pa / m. A physiological Young's modulus of 4 kPa was chosen between the 1.25 kPa measured for cardiomyocytes and 10–20 kPa for adult tissue. 2 isotropic spring foundation was applied to its underside to simulate the bottom of the glass plate.

[0271] The confinement was achieved by a 1.5 mm diameter silicone well (Young's modulus 800 kPa, Poisson's ratio 0.45, density 0.965 g / ml, isotropic spring constant 1 Pa / m 2 ) with a Young's modulus of 1 kPa, a Poisson's ratio of 0.5, a density of 1.03 g / ml, and a saturation coefficient of 1 Pa / m 2 The microvascular structures were simulated with a Poisson's ratio of 0.4, a density of 1.03 g / ml, and a surrounding matrix defined by an isotropic spring constant of 1 Pa / m 2The load was simulated by a random pattern on a microvessel-like structure of 30 µm in diameter defined by an isotropic spring constant of 10 kPa. A physiological Young's modulus of 50 kPa was chosen, between the 10 kPa estimated for capillaries and 300 kPa for arteries. A static load model was applied using an isotropic contraction of 350 µN causing a Gaussian displacement fitted to the measured curvature, resulting in a radial displacement of 5-15 µm (Figure 7B). The mesh used minimum and maximum element sizes of 150 and 165 µm, respectively.

[0272] Cardiac differentiation of human iPSCs Human induced pluripotent stem cells (hiPSCs) were seeded on Matrigel in mTeSR-1 medium and allowed to reach 80-90% confluence. Cardiac differentiation was performed as previously described. Briefly, a basal medium named CDM3 was used (RPMI-1640, 500 μg / mL recombinant human serum albumin, 213 μg / mL L-ascorbic acid 2-phosphate and 1% penicillin / streptomycin). On day 0 of differentiation, the medium was replaced with CDM3 supplemented with 6 μmol / L CHIR99021 (Stemgent) for 2 days. On day 2 of differentiation, the culture medium was switched to CDM3 medium supplemented with 2 μM Wnt-C59 (Selleckchem) for an additional 2 days. From day 4 onwards, cells were cultured using RPMI supplemented with insulin-free B27 (Gibco, USA).

[0273] Generation of GFP-reporting CECs Plasmids encoding a GFP reporter upstream of a minimal CMV promoter were purchased from System Biosciences and verified in-house. Plasmids were obtained as bacterial LB-agar stubs and used according to the supplier's instructions. Briefly, each stub was first plated on a 10 cm plate of agar LB (Bacto Agar; BD, USA). A single colony was then inoculated into a flask containing LB (BD Difco LB Broth, Lennox; BD, USA) and 100 μg / ml penicillin (BI, Israel). Transfection-grade plasmid DNA was isolated from each flask using the ZymoPURE II Plasmid Maxiprep Kit (Zymo Research, USA) according to the manufacturer's instructions.

[0274] HEK293T cells were transfected with GFP expression plasmid and packaging plasmid using TransIT-LT1 transfection reagent (Mirus Bio, USA) according to the supplier's instructions. Briefly, 6.65 μg of GFP lentivector plasmid, 3.3 μg of pVSV-G and 5 μg of psPAX2 were mixed with 45 μl of TransIT-LT1 in Opti-MEM reduced serum medium (Gibco, USA), kept at room temperature to allow complexation, and then added to each plate. After 18 hours of incubation, the transfection medium was replaced with 293T medium, and virus-rich supernatants were harvested after 48 and 96 hours. The supernatants were clarified by centrifugation (500×g, 5 min) and filtered (0.45 μm, Millex-HV, Merck Millipore). Packaging plasmids were kindly provided by the Nissim Benvenisti Lab (HUJI, Jerusalem, Israel).

[0275] Organoid culture hiPS-derived cardiomyocytes and microvascular cardiac endothelial cells (VEC Technologies, USA) were counted and 6.8 × 10 4The cells were mixed in a 2:1 ratio in growth factor-reduced Matrigel (BD Biosciences, San Jose, CA) at a cell density of 1.1 μl / μl. A volume of 1.1 μl of the gel-embedding mixture (7.48×10^4 cells) was injected into each microwell and allowed to spontaneously form in RPMI-1640 supplemented with B27 minus insulin and 5 ng / ml vascular endothelial growth factor (VEGF-A, Peprotech) until their spontaneous beating resumed after 6–10 days.

[0276] Immunostaining of cardiac organoids Cardiac organoids were fixed with 4% paraformaldehyde (PFA) for 1 h on ice and washed three times with Dulbecco's phosphate buffered saline (DPBS) containing calcium and magnesium (Sigma-Aldrich, USA). Samples were incubated with 100 mM glycine for 1 h at room temperature and washed with DPBS for 30 min. Permeabilization was performed with 0.5% TritonX-100 in DPBS at 4 °C overnight. Blocking buffer consisted of 3.8 g NaCl, 0.94 g NaHPO4, 0.2 g NaH2PO4, 5 g bovine serum albumin fraction V (MP Biomedicals, USA), 0.5% TritonX-100, and 0.25 ml Tween-20 in 50 ml distilled water. Samples were incubated in blocking buffer for 48 h at 4 °C, washed, and incubated with primary antibodies diluted in blocking buffer for another 48 h at 4 °C. After washing the samples for 24 h at room temperature, secondary antibodies diluted in blocking buffer were added and incubated at 4°C for 48 h. Nuclei were counterstained with Hoechst33258 (Sigma-Aldrich, USA) at a concentration of 1:1,000. After washing the samples for 24 h, microscopy was performed. Confocal microscopy was performed on an LSM-700 Zeiss microscope.

[0277] antibody Rabbit anti-cTNT Abcam: ab45932 1:100;mouse anti-α-actinin thermofisher: A7811 1:100;rabbit anti-MLC2v Abcam: ab79935 1:100;rabbit anti-WT1 Abcam: ab89901 1:100;rat anti-HCN4 Abcam: ab32675 1:100;mouse anti-SHOX2 Abcam: ab55740 1:100;rabbit anti-cardiac troponin I Abcam: ab52862 1:100;rabbit anti-periostin Abcam: ab215199 1:100;mouse anti-CD31 Abcam: ab215199 1:100;rabbit anti-TBX18 Abcam: ab115262 1:100;donkey anti-rabbit Alexa Fluor594 Jackson ImmunoResearch: 711-585-152 at 1:100; donkey anti-mouse Alexa Fluor488 Jackson ImmunoResearch: 715-546-150 at 1:100; donkey anti-rat Alexa Fluor647 Jackson ImmunoResearch: 712-605-150 at 1:100; and donkey anti-mouse Alexa Fluor647 Abcam: ab150111 at 1:100.

[0278] RNA-Seq analysis RNA-seq data of adult and fetal human cardiomyocytes were downloaded from GSEA series GSE126573 with accompanying metadata. RNA extraction from cell cultures was performed using RNeasy Micro kit (Qiagen, USA) according to the manufacturer's instructions. Library preparation and RNA sequencing were performed by the Hebrew University Center for Genomic Technologies. Briefly, library construction was performed using Illumina TruSeq RNA Library Prep V2 Kit (Illumina, USA) and sequenced on an Illumina NextSeq 500 with single-end 86bp reads using the High Output V2 Kit. Sequencing reads were mapped to the UCSC human transcriptome (genome build hg19) using Bowtie2. Expression levels of all genes were quantified using RSEM to obtain an expression matrix of inferred gene counts. Differential expression analysis was performed using the R package DESeq2 using default parameters. p-values ​​from negative binomial Wald tests are reported.

[0279] Visual contraction analysis High-speed phase-contrast imaging (17 frames / sec) was performed using an LSM-700Zeiss microscope at appropriate magnification. Time-lapse photomicrographs were analyzed using custom MATLAB® code. Briefly, multiple regions of interest (ROIs) were selected on the cardiac tissue borders to generate distinct regions with changes representative of cardiac contraction. Dynamic changes in ROI intensity were measured over time and converted to beating rate using a fast Fourier transform (FFT). Contractility was calculated based on the magnitude of frequency amplification.

[0280] Fabrication of optically tolerant confined microelectrode arrays (MEAs) The fabrication procedures were carried out in a class 100 cleanroom environment at the Harvey M. Krueger Family Center of Nanoscience and Nanotechnology at the Hebrew University of Jerusalem. Microelectrode array and disposable PDMS microwell insert design was performed using CleWin 5® and SolidWorks® (SolidWorks, USA). Standard commercial 75 × 50 × 1 mm microscope slides (Corning®) were cleaned sequentially with isopropanol, piranha solution (a mixture of sulfuric acid, water, and hydrogen peroxide), and deionized water. Electrode patterns were etched in dual positive photoresist (AZ1505 and LOR 5b) using direct laser writing (Microtech®, LE405-A), followed by development in AZ726MIF for 2 min. Patterns were sputtered with a 10 nm titanium layer and 150 nm gold using a Thin Film Deposition Systems vacuum coater (TFDS-141®; VST). Electrodes were then formed using a 2 min lift-off process in 100% AR grade acetone. PDMS microwell inserts were fabricated using laser cutting. Briefly, a thin sheet of PDMS (Dow Corning) was cast to a height of 0.7 mm using a motorized film applicator (Erichsen) and cured at 70 °C for 1 h. Microwells were cut to 1.5 mm diameter and 3 mm center-to-center distance using a 355 nm pulsed Nd-YAG laser (3D-Micromac). PDMS inserts were washed with 70% (vol / vol) ethanol (EtOH), dried with nitrogen, and covalently bonded to clean 0.5 mm thick glass coverslips (Schott) or clean optically tolerant microelectrode arrays using oxygen plasma activation.

[0281] 3D Printing of Cardiac Microphysiological Platform The microelectrode connector and casing design was done using Solidworks®. SUP706®, VeroBlackPlus®, and VeroWhitePlus® were deposited according to the design pattern using a Connex3 Objet260® 3D printer. The 3D printed parts were cleaned from the support material (SUP706®) in 2% sodium hydroxide and 4% sodium metasilicate solution overnight. The connectors were attached to the printed parts and assembled into a microrheology platform, and the metal connectors were assembled. Before use, the entire platform was cleaned in 70% ethanol overnight and sterilized in UV for 3 hours.

[0282] Electrophysiological recordings The MEA design and printed connections allowed intermittent access to 45 different electrodes in the platform via compatible sensor circuits (Figures 9A-9B). Spontaneous cardiac field potentials (FPs) were recorded from cardiac organoids using an integrated signal conditioning circuit (AD8232®) with a 2-pole adjustable high-pass filter, a 3-pole adjustable low-pass filter, adjustable gain, and a medical device amplifier to remotely extract, amplify, and filter the extracellular field potentials of organoids within the confined wells. The custom-designed circuit was connected to an Arduino MEGA2560® microcontroller that served as an analog-to-digital converter (ADC) to the integrated control system. Spontaneous cardiac field potentials were measured at a sampling rate of 100 Hz in each microwell. Field potential rhythmic frequencies were calculated in real time using custom-built MATLAB® software imposing a Fourier transform (FFT) on the generated kinetic data (Figures 9C-9D).

[0283] Real-time oxygen and contraction rate measurement Real-time oxygen measurements were performed based on the aforementioned elements. Briefly, a phosphorescent ruthenium microprobe (CPOx-50-RuP) exhibits a decrease in phosphorescence decay time as a function of oxygen concentration, allowing the measurement of oxygen content. A controlled sinusoidally modulated 532 nm LED signal excites an embedded oxygen sensor, which emits a sinusoidally amplified modulated light at 605 nm with a phase shift due to oxygen quenching. The phase shift is measured between the emission hardware filtered first detector (PMT) and the second excitation detector (cPMT). The 2-PMT system allows a 40-fold increase in measurement resolution and real-time measurement of tissue contraction (Figure 8). A two-frequency phase modulation of 53.5 kHz and 31.3 kHz was used to filter background interference. Measurements were performed at a sampling rate of 10 Hz. Contraction rate and oxygen rhythm frequency were calculated in real time using custom-made MATLAB software that imposes a Fourier transform (FFT) on the kinetic data generated. Using custom MATLAB code, we calculated cardiac organoid contraction velocity directly from the oxygen reader based on the emission intensity measured in peak-to-peak voltage from the oxygen bead and its displacement using a second PMT system. Cardiac displacement was measured by oxygen beads embedded within the cardiac organoid during contraction. As the beads moved different distances from the focus, changes in peak-to-peak voltage occurred and contractile behavior was detected. These data were converted to frequency data using a fast Fourier transform (FFT) with the same MATLAB® code.

[0284] Mitochondrial membrane potential assay (MMP) Mitochondrial membrane potential (MMP / Δψm) was estimated using JC-1 and TMRE dyes according to the manufacturer's instructions (Invitrogen, USA). Cells were loaded with 2 μM JC-1 or 2 nM TMRE dye in RPMI supplemented with B27-I for 30 min at 37 °C, washed with PBS, and measured in RPMI supplemented with B27-I using a LSM-700 Zeiss microscope with continuous incubation at 5% CO2 and 37 °C. JC1 measurements were performed by sequential excitation at 488 nm and 570 nm with a sampling rate of 5 Hz. Mitochondrial membrane potential was calculated from the ratio of the mean emission intensity of green (530 nm) and red (590 nm) in each measurement. TMRE measurements were performed by excitation at 549 nm with a sampling rate of 10 Hz. Mitochondrial membrane potential was calculated from the mean emission intensity at 574 nm. Kinetic analysis was performed using multiple regions of interest (ROIs) representing the mitochondrial network. Changes in fluorescence intensity were used to determine oscillation velocity and amplitude. Spatial analysis of mitochondrial membrane potential oscillations was performed using a custom MATLAB code. Briefly, each successive mitochondrial membrane potential (MMP) fluorescence time series micrograph was split into 10 μm 2 The mitochondrial membrane potential oscillations were generated using ImageJ software (National Institutes of Health, USA).

[0285] MCU Inhibition Test For mitochondrial calcium live imaging, different hiPSC-derived cardiomyocytes were cultured in RPMI supplemented with B27 minus insulin and 0.1% DMSO (control) or 10 μM KB-R7943 mesylate (Tocris Bioscience, USA). Mean mitochondrial calcium was measured using live imaging of Rhod-2AM dye after 0, 30 and 90 s exposure to DMSO (control) or 10 μM MCU inhibitor KB-R7943 mesylate.

[0286] For simultaneous real-time oxygen, contraction velocity, and field potential measurements, cardiac organoids were cultured in RPMI supplemented with B27 minus insulin, 5 ng / ml VEGF, and 0.1% DMSO (control) or 10 μM KB-R7943 mesylate. Real-time oxygen, contraction velocity, and field potential were measured after 0, 15, and 25 min exposure to DMSO (control) or 10 μM KB-R7943 mesylate.

[0287] Generation of MCU-KO and control (NT) CRISPR / Cas9 plasmids For MCU-KO, two different sgRNA oligos-MCU HGLibA_28660 and HGLibB_28619- from the GeCKO v.2 human CRISPR knockout pool library (Addgene#1000000048) were cloned into the lentiCRISPRv2 plasmid (Addgene#52961). For the control (NT), NT1 and NT2 non-targeting / control sgRNA oligos were cloned into the lentiCRISPRv2 plasmid.

[0288] sgRNA cloning was performed according to the instructions for the human GeCKO v.2 system. Briefly, two oligos containing each sgRNA insert were synthesized with BsmBI compatible ends, then phosphorylated and annealed in a single session: phosphorylation with T4 PNK (NEB-M0201S), followed by heating to 95°C for 5 min and controlled cooling to allow annealing. Vector plasmids were digested with BsmBI (FastDigest Esp3I, FD0454, Thermo), dephosphorylated (FastAP thermosensitive alkaline phosphatase, EF0651, Thermo), and gel extracted (QiaQuick gel extraction, Qiagen). Vector and insert fragments were ligated (T4 DNA ligase, EL0011) and transformed into chemically competent Stbl3 cells (Mix&Go! E. coli transformation kit, T3001, Zymo). Proper insertion was verified by Sanger sequencing using the LKO.1 primer.

[0289] The plasmids were named MCU-KO.A (MCU HGLibA_28660), MCU-KO.B (HGLibB_28619), control NT1 and control NT2 lentiCRISPR v2. Transfection-grade plasmid DNA was isolated using ZymoPURE Plasmid Miniprep Kit (D4209, Zymo Research) according to the manufacturer's instructions. The lentiCRISPR v2 plasmid was kindly donated by Nissim Benvenisti lab (HUJI, Jerusalem, Israel).

[0290] Generation of MCUKO hiPSC-derived cardiomyocytes MCU knockouts (KO) were generated by double lentiviral transduction of MCU-KO.A and MCU-KO.B lentiCRISPR v2 plasmids. Control transduction was performed similarly by double lentiviral transduction of control NT1 and control NT2 non-targeting lentiCRISPR v2 plasmids. Lentiviral stocks were generated as previously described.

[0291] Transduction stocks were prepared by mixing the corresponding lentiviral stocks (MCU-KO.A / B and NT1 / 2) at a 1:1 ratio. hiPSC-CMs were infected with 50% viral stock dilution for two consecutive sessions of 12 h each. Cell viability was not altered by transduction.

[0292] Mitochondrial calcium imaging Mitochondrial calcium uptake in human iPSC-derived cardiomyocytes was measured using Rhod-2AM dye according to the manufacturer's instructions (Abcam, USA). Cells were loaded with 2 μM Rhod-2AM in RPMI supplemented with B27-I for 30 min at 37°C, washed with PBS and incubated with 10 μM KB-R7943 or RPMI supplemented with 0.1% DMSO and B27-I. Measurements were performed continuously using an LSM-700 Zeiss microscope incubated at 5% CO2 and 37°C. Rhod-2AM measurements were performed by exciting at 552 nm with a sampling rate of 10 Hz. Mitochondrial calcium uptake was calculated by the change in mean emission intensity at 581 nm. Time-lapse micrographs were analyzed using multiple regions of interest (ROIs) selected based on Mitotracker staining generating distinct areas with changes representative of the mitochondrial network. Changes in fluorescence intensity were used to determine oscillation velocity and amplitude.

[0293] Ex vivo pig studies Tissues were procured post-mortem from a 70 kg domestic female pig that underwent laparoscopic liver surgery (ethical approval number MD-21-16533-3) and was euthanized using a lethal dose of potassium chloride. The pig was pronounced dead when the ECG tracing showed a flat line and ETCO2 was 0, and the left thorax was opened outward on the sternum. The lungs were deflated and the pericardium incised. The ascending aorta, inferior vena cava and superior vena cava were divided, and the pulmonary veins and heart were dissected out from the chest and placed on ice.

[0294] The anterior wall of the left ventricle was incised from base to apex, and several sections of 2-3 mm length and 1-2 mm thickness were obtained using a #10 scalpel blade. The cardiac tissue was further cut into 500±200 μm thick sections and washed extensively with 37°C pre-warmed ex vivo medium consisting of M199 medium supplemented with 1× insulin-transferrin-selenium (ITS), 10% fetal bovine serum (FBS), and 5 ng / mL vascular endothelial growth factor (VEGF) and 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

[0295] Cardiac explants were then embedded with oxygen sensors, placed on the MEA chip, and transferred to a 37°C temperature-controlled incubation chamber containing M199 medium supplemented with 1x ITS, 10% FBS, 5ng / mL VEGF, and 10mM HEPES to support long-term function as previously described. The explants were continuously point stimulated with a cycle length of 2Hz. Contraction, field potential, and oxygen were assessed using the integrated metabolic electromechanical sensor chip.

[0296] Cardiac explants were paced at 2 Hz using a custom designed Arduino control point stimulator. The custom designed circuit consisted of an Arduino MEGA2560 microcontroller, a pulse width modulation (PWM) for voltage, and a digital-to-analog module. The custom code is provided in the relevant availability section.

[0297] In validation assays, simultaneous real-time oxygen, contraction velocity, and field potential were measured in cardiac explants as above additionally supplemented with 0.1% DMSO (control), 10 μM blebbistatin, 10 μM mitoxantrone, or 10 μM mitoxantrone and 100 μM metformin (mitoxantrone + metformin).

[0298] Quantification of metabolic function Mitochondrial function was measured using Seahorse XF Cell Mito Stress Test Kit according to the manufacturer's instructions (Agilent, Santa Clara, CA). Briefly, cardiac organoids or human iPSC-derived cardiomyocytes were seeded on Seahorse XFp miniplates coated with 1% Matrigel at a density of 1 organoid or 3,000 cardiomyocytes per well. Cells were allowed to acclimate for 24 hours. Cultures were then incubated in unbuffered XF basal medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate and 10 mM glucose (pH 7.4) at 37°C in a non-CO2 incubator for 1 hour. We measured the basal oxygen consumption rate (OCR) for 30 minutes and then injected 1 μM oligomycin, a mitochondrial complex V inhibitor that blocks oxidative phosphorylation. The decrease in OCR due to oligomycin treatment was defined as the oxidative phosphorylation rate. Maximal mitochondrial activity was measured by adding 0.5 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler, at 60 min, and complete inhibition was induced at 90 min using a mixture of 0.5 μM antimycin A and rotenone, mitochondrial complex III and mitochondrial complex I inhibitors. Results were normalized to total cell number per well. 2D hiPSC-CMs remained at approximately seeding number by the time of assay, as determined by Hoechst staining after Mito stress assay. Briefly, two separate fields per well were imaged on an Olympus X81 microscope, raw images were exported, and cell nuclei were counted in Cell Profiler as primary objects.

[0299] For cell counting of cardiac organoids, genomic DNA (gDNA) was extracted from single organoids or known amounts of 2D hiPSC-CM cells using the Quick-DNA miniprep plus kit (D3024, Zymo Research) according to the manufacturer's instructions. The gDNA concentration and total gDNA extracted per sample were measured using Nanodrop 1000. To find the number of cardiomyocytes per organoid, we utilized qPCR with human specific primers to amplify a 156bp region of the gene EDEM1. qPCR was performed as previously described. Multiple single organoids and 10 5 Templates from iPSC-CM 2D cells were diluted 2-fold from 10 ng to 0.625 ng per reaction and all samples / dilutions were evaluated in quadruplicate. The average Ct values ​​per template amount were plotted and samples with comparable slopes / efficiencies were further evaluated. Given the similar qPCR efficiency of the latter, the ΔCt between organoids and 2D cardiac cells was used to calculate the percentage of human cardiac cells in each organoid. 5 The number of cardiac cells per organoid was estimated by comparing the amount of total gDNA extracted from iPSC-CM 2D cells to the total gDNA extracted from each organoid and applying the qPCR estimated percentage of cardiac cells. Human cardiomyocytes in each organoid were significantly more oxidative than rat endothelial cells and therefore, they are considered the only contributing factor to the assay results.

[0300] Metabolic measurements and ATP production Amperometric 4-analyte sensors (B.LV5) were purchased from Innovative Sensor Technology (IST, Switzerland). Measurements were performed and calibrated for sensitivity loss by an on-chip potentiostat (IST, Switzerland). Oxygen, glucose, lactate and glutamine fluxes were measured by calculating the change in metabolite concentration over time. Glutamine measurements allowed the calculation of lipid metabolic flux. Oxidative phosphorylation flux was calculated by dividing the oxygen uptake rate by 6. We estimated 32 ATP molecules generated by the complete oxidation of glucose. Glycolytic flux was calculated by dividing the lactate production rate by 2, with the maximum rate defined as the glucose uptake rate minus the oxidative phosphorylation flux. ATP production in glycolysis was estimated to be 2 molecules per glucose molecule. Glutaminolysis was calculated directly by glutamine uptake. ATP production in glutaminolysis was estimated to be three molecules per molecule of lactate produced.

[0301] Quantitative RT-PCR Total RNA isolation from hiPS-derived CMs or cardiac organoids was performed using the NucleoSpin RNA II kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. RNA concentration and purity were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA synthesis was performed using qScript™ cDNA Synthesis (Quanta BioSciences) according to the manufacturer's instructions. 1 μg of purified RNA was used for each reaction.

[0302] qRT-PCR was performed on an Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System using KAPA SYBR FAST (Kapa Biosystems). Data analysis was performed by normalizing the Ct values ​​of the target genes to the Ct value of the RPL32 gene, and data were presented as relative quantification (RQ) values. Primers used were (5' to 3'): EDEM1-F-GGCCCCCGCGCTTTAAAATA (SEQ ID NO: 1); EDEM1-R-GGAAAGCGCTGGTAGAAGCC (SEQ ID NO: 2); MCU-F-CACACAGTTTGGCATTTTGG (SEQ ID NO: 3); and MCU-R-CGTGACTTTTTGGCTCCTTT (SEQ ID NO: 4).

[0303] Genomic data processing, analysis and graphic display Principal component analysis (PCA; prcomp package), scatter plots and volcano plots (ggplot package) were performed using R studio (www.rstudio.com / ).

[0304] Hierarchical clustering, heat maps, correlation plots, and similarity matrices were generated in Morpheus. Gene ontology enrichment analysis and clustering were performed using DAVID Informatics Resources 6.7 and the PANTHER classification system. Metabolic network maps were generated using McGill's Network Analyst Tool using the KEGG database.

[0305] Scanning electron microscopy Organoid samples were precoated with a Au-Pd nanolayer using an SC7640 Sputter. SEM imaging was performed using an FEI Sirion High-Resolution Scanning Electron Microscope (HR SEM, Holland). Images were taken using high-resolution mode with secondary electron (SE) detection at an accelerating voltage of 5 kV, spot size of 4.0, and working distance of 5.3 mm. A TSL-EDAX (EDAX, USA) system was installed for electron backscatter diffraction (EBSD).

[0306] Quantitative and statistical analysis All experiments were performed with at least three biological replicates. Measurements were performed in either technical triplicates or quadruplicates, and images were analyzed in ≥5 fields of view. Graphs show mean ± SEM. Pairwise comparisons were performed using Student's t-test. Mann-Whitney U test was used when distributions could not be determined to be normal. False discovery rate (FDR) correction was used to adjust for multiple comparisons and RNA-seq comparisons. Unless otherwise stated, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0307] Software Resources Custom analysis software is available at github.com / mohammadghosheh95 / Heart-on-a-Chip.

[0308] statistical analysis Experiments were repeated three times with triplicate samples for each experimental condition unless otherwise stated. Data from a representative experiment are presented, and similar trends were seen across multiple tests. Parametric two-tailed Student's t-tests were used to calculate significant differences between groups. All error bars represent ± SE unless otherwise stated. EXAMPLES

[0309] Generation of vascularized multi-chamber cardiac organoids by anisotropic stress Developmental studies have shown that complex cardiac partitioning occurs only after the development of the cardiovascular system. To mimic this developmental stage, we seeded a mixture of hiPSC-derived cardiomyocytes (hiPSC-CMs) and cardiac microvascular endothelial cells into microwells in a basement membrane matrix (Figure 1A-1B). The tissue contracted into a single mass in 4 days and started beating after 10 days. Cardiac organoids were cultured over the following week, obtaining a smooth appearance and synchronized behavior after 25 days of culture (Figure 1B). GFP-expressing endothelial cells reveal that a network is formed after 10 days, followed by the development of a complex circumferentially aligned vascular network after 25 days (Figure 1B, 1L). Confocal microscopy of cardiac organoids showed a complex structure supporting two to three chambers in both UN-1- and ACS-1021-derived organoids (Figure 1M-1N). Immunostaining for TUNEL and carbonic anhydrase IX (CAIX), an endogenous marker of hypoxia, indicates that the formation of chambers is not the result of hypoxia or apoptosis during organoid formation (Figure 1O).

[0310] To better understand this complex organization, we modeled the von Mises stress distribution and Gaussian displacement of cardiac organoids undergoing contraction in different conditions (Figures 1C-1D and Materials and Methods). Unstressed cardiac organoids without geographic confinement or vasculature resulted in a uniform stress distribution associated with the formation of a solid spheroid. Geometric confinement in the absence of vasculature (i.e., without the addition of endothelial cells) resulted in an isotropic stress gradient, which resulted in a single chamber (Figures 1C-1D, 7A-7B). However, the incorporation of vessel-like structures within the mass by including endothelial cells produced an anisotropic stress gradient, resulting in multiple low-stress regions where the tissue separated into multiple chambers (Figures 1C-1D, 1M-1N, and 7A-7B).

[0311] To validate the results of the finite element model, we used confocal microscopy to analyze the distribution of mechanotransduction markers Lamin A / C (LMNA) and YAP1 in different conditions (Figure 1E). Open organoids showed uniform expression of LMNA and YAP1, indicating a uniform stress distribution. Geometric confinement resulted in a circumferential ring of high stress characterized by a gradient of LMNA, YAP1 surrounding a central cavity. Incorporation of vessel-like structures within the mass resulted in an anisotropic stress gradient, resulting in high stress regions characterized by LMNA, separating multiple chambers. Mechanosensor expression patterns correspond to the modeled stress patterns (Figure 1D-E, Figure 7C).

[0312] Confocal microscopy revealed the robust formation of multi-chamber cardiac organoids (Figure 7C), composed of circumferentially aligned cardiomyocytes surrounding multiple hollow chambers (Figure 1E, 1M). Immunostaining for α-actinin and cardiac troponin revealed that the organoids were composed of elongated cardiomyocytes with an organized sarcoid pattern (Figure 1G). Confocal and scanning electron microscopy showed patent endothelial capillaries penetrating throughout the chamber walls (Figure 1F). Structural analysis shows that POSTN+ cardiac fibroblast-like cells are woven into a cardiomyocyte layer decorated by pacemaker-like cell clusters positive for HCN4 and SHOX2 (Figure 1H). Confocal microscopy revealed an outer shell of epicardial-like cells positive for WT1 and TBX18 (Figure 1I). The cardiac chambers appear partially layered with PECAM-1 positive endocardial-like cells (Figure 1J). These structural findings were further validated by RNA sequencing of cardiac organoids compared to human tissues and hiPSC-CMs derived from 2D cultures.Cardiac organoids displayed expression signatures associated with pacemaker cells (sinoatrial and atrioventricular nodes), endocardial and epicardial cells, and cardiac fibroblasts (Figure 1K). EXAMPLES

[0313] Functional characterization of multi-chamber cardiac organoids To assess cardiac maturity of the model, RNA-Seq analysis was performed on multi-chamber cardiac organoids compared to hiPSC-CMs grown in two dimensions as well as adult and fetal cardiac tissues (Figure 2A-B). Transcriptomic analysis of multi-chamber cardiac organoids showed functional gene expression that correlated with adult but not fetal myocardium (Figure 2A). Genes involved in cardiac execution (KCNJ2, KCNJ8), ultrastructure (TMNI3, MYH7, AKAP6), energetics (PPKAA2, PGC1A), and calcium handling (RAR2, CASQ2, CAV3) were differentially upregulated in cardiac organoids compared to both hiPSC-CMs (2D culture) and fetal myocardium (Figure 2A). Distinguishing between adult and fetal cardiac expression Principal component analysis (PCA) of 513 genes differentially expressed between cardiac organoids and hiPSC-CMs (2D) clusters cardiac organoids with adult tissues (Figure 2B).

[0314] Vascularized cardiac organoids exhibited spontaneous synchronous beating at 66±5 beats per minute (bpm) as well as physiological responses to drugs. Epinephrine stimulation increased beating rate to 88±7 bpm from baseline values ​​(n=6, p<0.001) and contractile amplitude by 18% (n=6, p<0.01). In contrast, the antiarrhythmic drug amiodarone decreased contractile rate to 52±4 bpm from baseline values ​​(n=6, p<0.01) and contractile amplitude by 28% (n=6, p<0.01; Fig. 2C). Importantly, cardiac organoids disclosed herein exhibited 35% and 85% higher basal respiration and oxidative phosphorylation, respectively, than hiPSC-CMs (n=3, p<0.05; Fig. 2D). Mitochondrial maximum capacity, an indicator of metabolic maturation, was two-fold higher than basal respiration (Fig. 2D).

[0315] The metabolic dynamics of cardiac organoid contraction and relaxation were tracked using oxygen-sensing beads embedded in the organoids (Figure 2E-F and Figure 3A) as well as electrochemical sensors to monitor glucose, lactate and glutamine. Metabolic flux balance analysis (see Materials and Methods) demonstrates the dominance of fatty acid oxidation over other metabolic pathways characteristic of mature myocardium (Figure 2F). This metabolic analysis showed that changes in interstitial oxygen occur with subsecond resolution during the cardiac cycle (Figure 2F). EXAMPLES

[0316] Implanted sensors link breathing to the heart's electromechanical rhythm Cardiac rhythm occurs with sub-second resolution, too fast for most metabolic sensors to record changes. To address this issue, we designed a dual photomultiplier tube (PMT) sensing platform that allows for real-time active background noise reduction, enabling phosphorescence measurements from oxygen sensors embedded in cardiac organoids with sub-100 ms resolution (Figures 3A-3E; and Figure 8A). Phase-shift measurements were used to track oxygen consumption, while sensor reflection of the excitation wavelength allowed precise measurements of tissue displacement (Figure 3B; and Figures 8A-8B). Integration of this new system with a microelectrode array (MEA, Figures 3C-3E and Figures 8C-8D) surrounded by nanofabricated optically transparent microwells uniquely enables simultaneous measurements of cardiac contraction (Figure 3F), field potential (Figure 3G), and oxygen consumption (Figure 3H) in real time. Tracking of the implanted optical sensors showed that the organoids contracted 9.3 ± 1.1% along their axis during the pulsatile cycle (Figure 3F and Figure 9A). Organoid contractions were analyzed by fast Fourier transform (FFT) showing a dominant frequency of 0.94 ± 0.03 Hz, indicating 58 ± 2 bpm (n = 9; Figure 3F and Figure 9B). Recorded field potentials of each organoid were filtered and amplified in real time, showing a similar dominant frequency at 0.99 ± 0.03 Hz (n = 9; Figure 3G and Figure 9A-D).

[0317] Although metabolic flux is thought to vary on timescales ranging from minutes to hours, the real-time optical measurements disclosed herein of interstitial oxygen concentration in cardiac organoids showed rapid oscillations with a dominant frequency of 0.92 ± 0.03 Hz (n = 9; Figure 3H and Figures 9A-D), indicating a link between respiration and the organoid electromechanical cycle. Similar oscillations were also recorded in ACS-1021 and ACS-1028 cardiac organoids (Figure 9A). EXAMPLES

[0318] Elucidation of electrometabolic coupling in human cardiac tissue Epinephrine, released during the physiological fight-or-flight response, rapidly increases contraction rate and contractility and, on longer time scales, increases cardiac glucose metabolism. To study the dynamics of this metabolic response in sudden stress, we exposed cardiac organoids to 100 μM epinephrine and tracked their mechanometabolic response in real time. As expected, organoid contraction frequency doubled from 0.95 Hz to 1.95 Hz (n=3, p<0.001; Fig. 3I), while its contraction increased by 33% within 15 min of stimulation (n=3, p<0.001). Interstitial oxygen concentration also showed a corresponding oscillation, with the frequency similarly doubling from 1.11 Hz to 1.83 Hz within 15 min of stimulation (n=3, p<0.001; Fig. 3J). Interestingly, increased organoid contraction was associated with amplification of interstitial oxygen oscillations, suggesting coordination between cardiac function and oxygen consumption (Figure 10A-F).

[0319] To determine whether the respiratory cycle is driven by mechanical force or depolarization, we used blebbistatin, an excitation-contraction decoupling compound, and Na vCardiac organoids were treated with tetrodotoxin (TTX), a channel inhibitor. Blebbistatin inhibits myosin II, thereby preventing myosin contraction without affecting the action potential of cardiac cells. Blebbistatin treatment blocked cardiac contractions and sensor movement but did not affect the frequency and intensity of field potential or oxygen oscillations (Figure 3K and Figure 10G). Tetrodotoxin treatment eliminated field potential generation, mechanical contractions, and oxygen oscillations in cardiac organoids within seconds of effect onset (Figure 3L). These results demonstrate that oxygen oscillations are related to electrical activity rather than mechanical activity of cells. EXAMPLES

[0320] Dynamics of mitochondrial function in beating hiPSC-derived cardiomyocytes Rapid metabolic changes on the millisecond scale are more likely to be controlled by ion fluxes than by enzyme cascades. Mitochondrial membrane potential (MMP / ΔΨm) is a critical regulator of respiratory function, as is mitochondrial calcium ([Ca 2+ Mitochondrial membrane potential is a mitochondrial membrane potential (MMP)-dependent indicator. Mitochondrial membrane potential was measured in beating hiPSC-derived cardiomyocytes and non-beating cells in 2D culture by live cell imaging using TMRE (Figure 4A). Beating cells showed MMP oscillations that correlated precisely with cardiomyocyte contraction (Figure 4A and Figures 11A-H). Non-beating cells in the same field did not show an oscillatory mitochondrial membrane potential and, on average, had 3-fold lower MMP than spontaneously contracting cells (n=5, p<0.001; Figure 4A and Figures 11A-H). Rhod-2AM is a Ca fluorescence-dependent indicator. +2 Live imaging of hiPSC-derived cardiomyocytes in 2D culture (see Materials and Methods) measures mitochondrial calcium [Ca] in beating cells. 2+ ] m The results showed rapid oscillations in [Ca] during the contraction cycle, which correlated precisely with the contraction frequency of the cells (Fig. 4B and Fig. S11I). Together, these data indicate that the [Ca 2+ This strongly suggests that changes in ]m lead to rapid oscillations in oxygen consumption.

[0321] The effect of [Ca] on rhythmic function of cardiomyocytes 2+ To examine the role of [Ca]m, we exposed cells to KB-R7943 (MCU; Figures 4B and 11J), a pharmacological inhibitor of the mitochondrial calcium uniporter. Short-term exposure to the MCU inhibitor did not significantly affect cardiomyocyte contraction frequency (Figure 4B), but dramatically reduced the magnitude of contraction by 45% and 70% at 30 and 90 s, respectively (n=4, p<0.01; Figures 4B and 11J-L). Parallel measurements of mitochondrial calcium after KB-R7943 exposure showed that [Ca 2+ ]m concentration, but [Ca 2+ The magnitude of mitochondrial calcium oscillations was reduced by 60% and 78% at 30 and 90 s, respectively (n=4, p<0.001; FIG. S11L). These data suggest that the magnitude of cardiomyocyte contraction depends on efficient oscillations of mitochondrial calcium. EXAMPLES

[0322] Real-time effects of MCU inhibition in vascularized cardiac organoids The current results suggest that mitochondrial activity oscillates in spontaneously beating cardiomyocytes due to mitochondrial calcium rhythms. To study this behavior in a physiological model, we exposed cardiac organoids cultured on the sensor-integrated platform disclosed herein to the MCU inhibitor, KB-R7943 (Figure 4C). MCU inhibition induced a progressive decrease in oxygen uptake in cardiac organoids (Figure 4D). Metabolic flux balance analysis showed a progressive decrease in respiration and fatty acid oxidation, leading to a decrease in ATP generation within 20 minutes of exposure to the MCU inhibitor (Figure 4D).

[0323] Analysis of cardiac organoid metabolic activity showed that the magnitude of oxygen oscillations significantly and progressively decreased from the moment of KB-R7943 exposure (n=9; Figure 4E). At the same time, the magnitude of cardiac organoid contraction and its depolarization showed a comparable and parallel decrease after MCU inhibition (Figure 4E). These data indicate a loss of function driven by the observed mitochondrial disruption. However, while cardiac organoid metabolic activity was decreased, cardiac contraction frequency, oxygen oscillation rate, and frequency of field potential oscillations significantly increased after MCU inhibition (Figure 4F). All three rates showed a 2.5-fold increase within 35 min of exposure, indicating arrhythmic behavior (n=9; Figure 4F). This arrhythmogenic behavior was associated with a loss of synchronization that shifted oxygen oscillations away from the contractile cycle (Figure 4G). Taken together, the data disclosed herein suggest that MCU inhibition severs the electrical mitochondrial coupling, resulting in arrhythmogenic behavior. EXAMPLES

[0324] CRISPR / Cas9 knockout of MCU disrupts electrical mitochondrial coupling and induces arrhythmic behavior. To further validate the proposed mechanism, CRISPR / Cas9 knockout of MCU was generated in hiPSC-CMs in 2D culture (see Materials and Methods). Because antibiotic selection prevents cardiac maturation and survival, the analysis relies on mixed culture. Live imaging of hiPSC-CMs (2D) showed mitochondrial calcium [Ca 2+ ] m In contrast, MCU knockout (MCU KO ) cells are [Ca 2+ ] m The results showed a 50% decrease in vibration frequency, but the vibration rate increased to 1.3 Hz (n=7, p<0.01; Fig. 5A and Fig. 12A).

[0325] Next, non-uniform MCU KOCardiomyocytes were used to form chimeric cardiac organoids (Figure 5B). RT-qPCR and confocal microscopy demonstrated a marked reduction in MCU expression at the mRNA and protein levels (Figure 5B and Figure 12B-C). Contraction, field potential, and interstitial oxygen were measured using an integrated metabolic electromechanical sensor chip in these cardiac organoids. MCU KO Cardiac organoids composed of MCU cells exhibited less contractility and lower magnitude of oxygen oscillations (n=9, p<0.001; Figure 5D). KO The organoids displayed increased beating frequency and irregular field potentials indicative of arrhythmogenic behavior (Figure 5C). Collectively, these findings suggest that electrical mitochondrial coupling mediates [Ca] in cardiomyocytes. 2+ ] m These results suggest that the regulation of muscle contraction is driven by vibration (Figure 5D). This regulation allows the muscle to prepare for the sudden increase in energy demand required for mechanical contraction (Figure 5D). EXAMPLES

[0326] Metformin reverses mitoxantrone-induced arrhythmias Mitoxantrone is a human topoisomerase II inhibitor used in the treatment of prostate cancer, non-Hodgkin's lymphoma, and multiple sclerosis. Mitoxantrone is one of several chemotherapeutics thought to be involved in cancer therapy-induced arrhythmias (CTIA). Similar cardiovascular risks also limit its use in multiple sclerosis. Recent studies have identified mitoxantrone as a selective inhibitor of MCU (Figure 6C-6D).

[0327] Metformin has been reported to increase mitochondrial calcium and MCU activity, suggesting that metformin may be able to reverse this effect. Indeed, exposure to metformin increased mitochondrial calcium concentrations 3.4-fold in mitoxantrone-treated cells (n=4, p<0.001; Figures 6A-6D).

[0328] To demonstrate this behavior in a physiological model, we exposed cardiac organoids cultured on the sensor-integrated platform disclosed herein to mitoxantrone or a combination of mitoxantrone and metformin (Figure 6E-6F). Mechanical contractions, electrical depolarization, and oxygen concentration were significantly reduced by mitoxantrone exposure (Figure 6E). Irregular contractions and abnormal field potentials were associated with disruption of electrical mitochondrial rhythms (Figure 6F). Contraction rates increased from 53 to 79 BPM (Figure 6E-6F), collectively indicating arrhythmogenic behavior. Combination of mitoxantrone and metformin reversed this effect (Figure 6E-6F). Organoid contractions became regular and the dominant field potential frequency was restored. Furthermore, oxygen consumption increased and the correlation between electrical and mitochondrial rhythms was restored (Figure 6E-6F). The beating rate decreased from 82 to 61 BPM, restoring normal cardiac organoid activity (Figure 6E). EXAMPLES

[0329] Validation of electrical mitochondrial coupling in an ex vivo pig model Previous studies have demonstrated that the use of cardiac tissue slices from an adult porcine model is a physiologically relevant model for human ex vivo validation, as it shares physiological and electrical properties with the human heart. Porcine myocardial tissue, 500 ± 200 µm thick, was surgically prepared from the left ventricle of a 70 kg sow pig heart, embedded with oxygen sensors, and placed on the MEA chip (Figure 13A-C). Contraction, field potential, and interstitial oxygen of the myocardial slices were measured using the integrated metabolic electromechanical sensor chip during 2 Hz point stimulation (see Materials and Methods).

[0330] Porcine ex vivo myocardial tissue showed the same oxygen oscillations and responses recorded in human cardiac organoids (Figure 13D). Exposure to the myosin II inhibitor blebbistatin blocked mechanical contractions without affecting the field potential or oxygen oscillations. Meanwhile, exposure to the MCU inhibitor mitoxantrone reduced oxygen consumption and doubled the cardiac rhythm from 1 Hz to 2 Hz (Figure 13D). Co-treatment with mitoxantrone and metformin restored the beating frequency to 1.4 Hz, the field potential and the oxygen oscillations (Figure 13D).

[0331] Furthermore, addition of metformin 50 min after exposure to mitoxantrone gradually restored oxygen consumption over the course of 1 h (Figure S13E). These findings validate our previous results in human cardiac organoids linking electrical mitochondrial desynchronization to arrhythmogenic behavior (Figures 6A-F).

[0332] Consideration Microphysiological systems emulate important aspects of human physiology, but their true potential lies in their ability to discover new physiological mechanisms in a controlled environment. In this study, we developed a sensor-based platform that can correlate mechanoelectrical and metabolic activity in human cardiac organoids. Using this platform, we demonstrated an electrical-mitochondrial axis associated with human cardiac rhythm. Disruption of this electrical-mitochondrial rhythm leads to arrhythmias, which can be partially corrected by the energy disrupting agent metformin.

[0333] Early studies on small animal models showed that oxygen consumption correlated with cardiac mechanical activity, while others showed that cardiac electrical and mechanical activity are related. Further improvements in electrophysiological methods have allowed some groups to link mitochondrial calcium oscillations to electromechanical rhythms, suggesting the existence of a cardiac metabolic-electromechanical axis in animals. The current understanding is that an actomyosin mechanism leads to rhythmic depletion of ATP and calcium to drive the metabolic cycle. Advances in imaging techniques have allowed the detection of waves of mitochondrial membrane potential in guinea pig ventricular cells, demonstrating slow oscillations in glycolysis and mitochondrial activity during the cardiac cycle. Others have shown similar oscillations in rat cardiomyocytes. These studies strongly suggest that human cardiomyocyte metabolism can be driven to oscillate in a similar way by cardiac contraction.

[0334] Unfortunately, ion channel kinetics, contraction velocity, and lipid metabolism differ fundamentally between small animal models and humans, resulting in species-specific responses to pathological events at the molecular and metabolic levels. In this context, cardiac organoid technology offers human-relevant metabolism with more mature functionality than hiPSC-derived cardiomyocytes.

[0335] We seeded hiPSC-derived cardiomyocytes into geometrically confined microwells that were previously shown to promote the formation of endocardial-coated microchambers via WNT / β-catenin signaling. In this study, we used cardiac endothelial cells to generate anisotropic stress that drives the formation of multi-chamber organoids. Pacemaker-like clusters, endocardial-like cells, and an epicardial-like shell add to the complexity of these multi-chamber cardiac organoids (Figure 1).

[0336] One advantage of confining organoids in individual microwells is the ability to minimize electrical interference (Figure 3C). Similarly, simultaneous measurement of background fluorescence allowed us to reduce ambient noise, enabling real-time detection of oxygen consumption with 10 Hz resolution (Figure 8A). Comparing the two signals also allowed us to precisely monitor the mechanical motion of the organoids, yielding a coordinated measurement of cardiac function. Using this approach, we demonstrated 1 Hz oscillations in mitochondrial function, the fastest switching of metabolic flux recorded to date. Metabolic flux balance analysis indicates that ATP is primarily generated during cardiac relaxation (Figure 2F).

[0337] However, in contrast to rodents, the actomyosin machinery of human cardiac organoids does not seem to drive this metabolic oscillation as an electrical and metabolic rhythmic cycle even in the absence of mechanical activity (Figure 3A-L). Instead, this electrical-metabolic coupling is driven by [Ca]m oscillations in human cardiomyocytes (Figure 4A-G), as muscle depolarization leads to increased mitochondrial activity in preparation for the surge in energy demand of mechanical contraction. This electrical-mitochondrial coupling is facilitated by the mitochondrial calcium uniporter (MCU), which has been shown to play a role in cardiac homeostasis in rodents. MCU - / - Knockout mice were unable to increase their heart rate during stress and were more susceptible to ischemic injury. Our findings support earlier observations showing that even mild inhibition of MCU activity in human cardiac organoids results in loss of electrical mitochondrial synchronization and arrhythmogenic behavior (Figures 5A-E and 6A-F). Indeed, loss of electrical metabolic synchronization may underlie some cases of fatal arrhythmias occurring after ischemic injury, viral infection, or cancer therapy-induced arrhythmias.

[0338] Mitoxantrone is one of several chemotherapeutics thought to play a role in cancer therapy-induced arrhythmias (CTIA). Indeed, many drugs beyond simply cancer treatments are known to induce arrhythmias and other cardiac side effects (see Table 1). Recent studies have identified mitoxantrone as a selective inhibitor of MCU. Exposure of human cardiac organoids to mitoxantrone caused disruption of electrical mitochondrial rhythm, resulting in irregular contractions, abnormal field potentials, and increased heart rate (Figures 6A-6F and 7A-7D). Metformin is an energy disrupting agent that has recently been shown to increase mitochondrial calcium and MCU activity. Addition of metformin partially restored electrical mitochondrial coupling and resolved arrhythmias in both human organoids and ex vivo models of porcine myocardium (Figures 6A-6F and 7A-7D).

[0339] This study reveals a new physiological mechanism that may provide a novel target for the development of antiarrhythmic therapies. Further studies are needed to elucidate the exact molecular mechanism of electrical mitochondrial coupling and to demonstrate its role in adult human tissues. Indeed, observational studies are needed to delineate the interactions between metformin and cancer drugs, and further placebo-controlled studies are needed to elucidate the exact role of such interventions in different conditions. The present findings highlight the utility of microphysiological systems in the study of human physiology and suggest that novel metamaterial cells and sensors may go beyond the utility of animal models.

[0340] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A multi-chamber cardiac organoid comprising cardiomyocytes and endothelial cells and at least two synchronously beating chambers.

2. 2. The organoid of claim 1, wherein all chambers beat synchronously, the organoid produces biphasic pulsation, the organoid produces a basal pulsation cycle of 50-90 beats per minute (bpm), or a combination thereof.

3. The organoid of claim 1 or 2, said organoid comprises pacemaker-like cell cluster, and optionally said pacemaker-like cell cluster is positive for potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and short stature homeobox 2 (SHOX2).

4. 3. The organoid of claim 1 or 2, comprising an outer epicardium, optionally wherein the epicardium comprises cells positive for Wilms' tumor-1 (WT1) and T-box transcription factor 18 (TBX18).

5. 3. The organoid of claim 1 or 2, comprising an inner endocardium, optionally wherein the endocardium comprises cells positive for platelet endothelial cell adhesion molecule (PECAM-1).

6. 3. The organoid of claim 1 or 2, wherein the organoid comprises vascular structures, circumferentially aligned cardiomyocytes surrounding a hollow chamber, elongated cardiomyocytes organized in a sarcoid pattern, capillaries within the walls of the chamber, and cardiac fibroblast-like cells, optionally wherein the fibroblast-like cells are periostin (POSTN) and / or vimentin positive.

7. and at least one parameter that is increased compared to isolated cardiomyocytes in culture or fetal cardiac tissue in culture, said parameter being TNNT2, TNNI3, Cx43, MYH7, AKAP6, GJA5, JPH2, SLC8A1, ATP2A2, CACNA1C, RYR2, CASQ2, PLN, CAMK2B, TRDN, CAV3, BIN1, AMP2, SCN5A, KIR2.1, ITPR. The organoid of claim 1 or 2, wherein the organoid is selected from the group consisting of basal respiration, oxidative phosphorylation, mitochondrial maximum capacity and expression of at least one factor selected from the group consisting of: 3, HCN2, SCN1B, HCN1, KCNJ8, KCNH2, PRKAA1, CPT1A, TFAM, PPARGC1A, PPA1, PPP2R4, SLC2A4, MAPK1, PRKACA, α1A, α1B, SCN4B, KCNE1.

8. A method for producing a multi-chamber cardiac organoid comprising at least two chambers that beat synchronously, the method comprising co-culturing a mass of cardiomyocytes and endothelial cells in a geometrically confined culture space to generate an anisotropic stress gradient in the mass of cells, thereby producing a multi-chamber cardiac organoid.

9. 9. The method of claim 8, comprising culturing about 6.8 x 10^4 cells in microwells comprising a diameter of 1 to 1.2 mm, or wherein the co-culture comprises a ratio of cardiomyocytes to endothelial cells of 1.5:1 to 2.5:1, or both.

10. 10. A multi-chamber cardiac organoid comprising at least two synchronously beating chambers produced by the method of claim 8 or 9.

11. A method for assessing cardiac cell function, comprising exposing a multi-chamber cardiac organoid according to claim 1 or 2 to a condition and measuring at least one parameter of the multi-chamber cardiac organoid.

12. 12. The method of claim 11, wherein the condition is selected from the group consisting of drug or chemical application, hypoxia, circulatory conditions, changes in metabolite exposure, changes in hormone exposure, and genetic mutations of cells within the organoid.

13. 12. The method of claim 11, wherein the at least one parameter is electromitochondrial synchronization.

14. 1. A sensing system comprising: an illumination source; a first photomultiplier tube (PMT) sensor; a second PMT sensor; a controller, controlling the illumination source to illuminate microparticles embedded in tissue or cell aggregates with a photon beam having a first wavelength; detecting, by the first PMT sensor, a first signal indicative of photons reflected from the particulate at the first wavelength; detecting, with the second PMT sensor, a second signal indicative of emission from the microparticle comprising an excitable molecule quenchable by a cofactor at a second wavelength; measuring a shift between the frequency of the first signal and the frequency of the photon beam, determining background noise based on the measured shift, and reducing the background noise from the second signal; and calculating the temporal cofactor consumption of the tissue or cell aggregate based on the background noise-reduced second signal; a controller configured to: a sensing system including:

15. The system of claim 14 , wherein the temporal cofactor consumption indicates oxygen levels in the tissue or cell aggregate.

16. 15. The system of claim 14, wherein the controller is further configured to detect a change in intensity of the first signal and calculate a relative displacement of the microparticle based on the detected change.

17. 17. The system of claim 16, wherein the detected change in intensity of the signal indicates a relative displacement of the microparticle, optionally wherein the displacement is measured in an axis perpendicular to the photon beam.

18. 15. The system of claim 14, wherein the controller is further configured to sense a field potential of the tissue or cell aggregate from an array of microelectrodes to measure electrical activity of the tissue or cell aggregate simultaneously with detecting the first signal and the second signal.

19. 1. A method for assessing cellular function, comprising: a. Placing a tissue, organoid or cell aggregate in a sensing system according to any one of claims 14 to 18; b. applying a condition to the tissue, organoid, or cell aggregate; c. Measuring at least cofactor consumption in the tissue, organoid or cell aggregate; thereby assessing cellular function; and A method for assessing cell function, comprising:

20. 20. The method of claim 19, wherein the sensing system is the sensing system of claim 18, and wherein measuring comprises measuring cofactor consumption, displacement and field potential in the tissue, organoid or cell aggregate, wherein a significant deviation in displacement, cofactor consumption and field potential after applying the condition compared to the displacement, cofactor consumption and field potential before applying the condition or compared to a control untreated tissue, organoid or cell aggregate indicates electrical mitochondrial desynchronization.

21. 20. The method of claim 19, wherein a cardiac organoid or a cerebral organoid is placed within the sensing system.

22. 20. The method of claim 19, wherein applying the condition is selected from application of a drug or chemical, application of hypoxic conditions, application of circulatory conditions, altered metabolite exposure, altered hormone exposure, and genetic mutation of cells in the tissue, organoid, or aggregate.

23. a. regulating mitochondrial calcium concentration in diseased or disordered tissue in a subject; b. modulating mitochondrial calcium channel activity in said tissue; or c. A combination thereof 1. A method for selecting a subject suffering from a disease or disorder suitable for treatment with an agent capable of: The method comprises determining the presence of electrical mitochondrial desynchronization in the subject, wherein the presence of the desynchronization indicates that the subject is suitable for treatment.

24. The determining step comprises: a. determining a mitochondrial calcium concentration in a sample obtained from said subject, wherein a concentration above a predetermined threshold indicates desynchronization, optionally said predetermined threshold being the calcium concentration of a healthy subject or a subject suffering from said disease or disorder not characterized by electrical mitochondrial desynchronization; b. observing the patient for arrhythmic or proarrhythmic symptoms that are unresponsive to antiarrhythmic therapy that targets electrical activity via membrane channels, thereby indicating desynchronization, optionally wherein the antiarrhythmic therapy is selected from sodium channel blockers, beta blockers, potassium channel blockers, non-dihydropyridine calcium channel blockers, adenosine, and digoxin; c. an EKG, EEG, or EMG, wherein the abnormal readings indicate desynchronization, optionally the abnormal readings include at least one of late potentials, reduced R waves, and an increased R / R ratio; d. Identifying exposure to an agent known to cause electrical mitochondrial desynchronization, optionally wherein the agent is selected from those provided in Table 1 and toluene, trichloroethane, xylene, heptane, hexane, ethyl ether trichloroethylene, trichlorotrifluoroethane, carbon monoxide, carbon disulfide, pesticides, methane-derived halogenated hydrocarbons, organic nitrates, arsenic, cadmium, cobalt, organic solvents, and metals; e. Ascertaining a medical history including a diagnosis of conditions indicative of electrical mitochondrial desynchronization; 24. The method of claim 23, comprising at least one of:

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