Platform for 3-dimensional modeling of physiological tissue and organoids

Abstract

The present invention discloses a high-throughput in vitro cardiotoxicity assessment platform, integrating human cardiac organoids (hCOs) with an advanced 3D-cell culture plate featuring embedded electrodes and batteries for electrical stimulation. The inclusion of batteries allows the platform to provide electrical stimulation without the need for external stimulators, enabling seamless, autonomous operation. The platform replicates heart electrophysiology, allowing precise control and real-time monitoring of cardiac parameters such as calcium dynamics and action potential waveforms, improving predictive accuracy for acute and chronic cardiotoxicity.

Description

PLATFORM FOR 3-DIMENSIONAL MODELING OF PHYSIOLOGICAL TISSUE AND ORGANOIDSField of the Disclosure

[0001] The present invention generally relates to biological and biomedical technologies, and more specifically relates to in vitro platforms for 3-dimensional modeling of physiological tissue and organoids.BACKGROUND

[0002] Existing models for cardiotoxicity assessment, such as 2D cultured cardiomyocytes and animal models, often fail to accurately replicate human cardiac physiology, leading to high rates of drug development failures. 2D cultures lack the complexity of the 3D heart environment, and animal models cannot fully mimic human responses to drugs. The current CHO-hERG assay for drug safety overlooks critical mechanical and electrophysiological interactions in human heart tissues, limiting its predictive value.

[0003] Advances in hiPSC-derived cardiomyocytes and 3D models offer a promising alternative, but current 3D models face challenges in scalability and standardization for high-throughput drug testing. What is needed, therefore, is a system for a robust, human-based platform that integrates functional electrophysiology to predict drug-induced cardiotoxicity with greater accuracy.SUMMARY

[0004] The present invention addresses the limitations of existing 2D models and animal studies by offering a scalable, standardized solution for studying drug-induced cardiac effects in a controlled and autonomous system.

[0005] Disclosed herein are methods and in vitro systems for 3-dimensional modeling of physiological tissue and organoids. In certain embodiments, the invention comprises models for cardiotoxicity assessment using human cardiac organoids (hCOs). In certain preferredembodiments, the invention integrates electrophysiological stimulation within 3D-cell culture plates to replicate heart function, enhancing drug discovery, safety testing, and disease modeling.

[0006] In certain preferred embodiments, disclosed herein is an apparatus for measurement of cardiac parameters comprising:

[0007] a) a multi-well plate comprising one or more features selected from:

[0008] wherein each of the well in the multi-well plate is adapted as conical in shape,

[0009] wherein each of the well in the multi-well plate is adapted to comprise cell-repellent surfaces to prevent cell attachment,

[0010] wherein each of the well in the multi-well plate is adapted to facilitate uniform organoid formation,

[0011] wherein each of the well in the multi-well plate is adapted to have transparent well bottoms for real-time imaging of organoids and / or for minimizing optical distortions,

[0012] wherein each of the well in the multi-well plate of the plate is opaque to prevent light interference, and

[0013] wherein each of the well in the multi-well plate of the plate is adapted to enhance fluorescence imaging;

[0014] b) one or more electrodes for autonomous electrical stimulation of the organoids;and

[0015] c) optionally one or more batteries within the plate providing continuous electrical stimulation;

[0016] wherein the apparatus is compatible with high-throughput assays,

[0017] wherein the apparatus is adapted to provide real-time measurement of cardiac parameters selected from calcium dynamics, action potential waveforms, and metabolic activity.

[0018] In certain other embodiments, disclosed herein is an apparatus for real-time measurement of one or more types of human organoids comprising:

[0019] a) a multi-well plate comprising one or more features selected from:

[0020] wherein the plate is adapted to comprises one or more features selected from:

[0021] wherein the well is conical,

[0022] wherein the well is adapted to comprise cell-repellent surfaces to prevent cell attachment,

[0023] wherein the well is adapted to facilitate uniform organoid formation,

[0024] wherein the well is adapted to have transparent well bottoms for real-time imaging of organoids and / or for minimizing optical distortions,

[0025] wherein the well of the plate is opaque to prevent light interference, and

[0026] wherein the wells of the plate enhance fluorescence imaging;

[0027] b) one or more electrodes for autonomous electrical stimulation of the organoids;and

[0028] c) optionally one or more batteries within the plate providing continuous electrical stimulation;

[0029] wherein the apparatus is compatible with high-throughput assays,

[0030] wherein the human organoids is selected from: cardiac, adipose liver, brain, kidney, and vascular organoids.

[0031] In further embodiments, the apparatus is adapted to measure one or morecardiac parameters selected from:a. electrophysiology parameters selected from: calcium transients, action potential waveforms, QTc prolongation;b. beating behavior selected from: rhythm, contractility, arrhythmia;c. metabolic stress selected from: oxygen consumption rate (OCR) for mitochondrial function;d. structural integrity selected from: troponin-I, cytotoxicity markers.

[0032] In additional embodiments, disclosed is a method for cultivating and maturing one or more types of human organoids comprising:a) generating one or more types of human organoids selected from: cardiac, adipose, liver, brain, kidney, and vascular organoids from human iPSCs in a multi-well plate, wherein the human organoids are generated by protocols suitable for physiological properties of each respective type of human organoid;b) applying continuous electrical stimulation to the human organoids in each well throughout the process to promote maturation and the development of one or more parameters specific to each respective type of human organoid; andc) maintaining the each respective type of human organoid in the plate for long-term growth and real-time functional assessment under physiological conditions that mimic the electrophysiological environment of the each respective human organoid.

[0033] Various types of human organoids are suitable and adapted for the methods and compositions of the invention. Preferably, the human organoids is selected from human cardiac organoids; wherein said human cardiac organoids are maintained on the plate for growth and real-time functional assessment under physiological conditions that mimic the electrophysiological environment of a human heart; and wherein the human cardiac organoids are evaluated for at least one cardiac parameters selected from: calcium signaling, action potential formation, and structural protein expression.

[0034] In certain preferred embodiments, disclosed is a method for performing high-throughput toxicity testing of one or more types of human organoids comprising: a) cultivating one or more types of human organoids each individually in one or more wells of a multi-well plate adapted for compatibility with standard high-throughput screening systems; b) performing real-time imaging and functional assays, to assay one or more physiological properties of each respective human organoid type, all within the same multi-well plate; and c) testing one or more drug compounds simultaneously by administering the drug compounds directly to the one or more types of human organoids within their respective wells, while continuously measuring the effects of said one or more compounds on one or more physiological properties of each respective human organoid function selected from: toxicity testing of the effects of the drug compounds on the respective types of human organoids selected from: cardiotoxicity, hepatotoxicity, neurotoxicity, and nephrotoxicity, in a high-throughput format.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0036] FIG. 1 depicts a Schematic Overview of the 3D Cardiac Models-Based Platform Workflow. The schematic represents the process of generating various cardiac constructs from human-induced pluripotent stem cell-derived models (hiPSC-CMs, hiPSC-Neurons, etc.). The Stem Cell Source (e.g., hiPSCs) serves as the starting material, determining which specific 3D constructs will be produced. Specifically, Cardiac Constructs can include 3D cardiospheres, 3DCardiac Assembloids (multi-cell assembled organoids), and Cardiac organoids generated from hiPSCs. The Electrostimuli-3D Culture Plate is designed to assemble and support the maturation of any cell type intended for 3D modeling. Our plates are used to produce, maintain, and conduct high-throughput assays, essential for validating new treatments, understanding diseases among other functions. The plates are ANSI / SLAS-compliant, ensuring compatibility with most commercially available microscopes, multiplate readers, and other automated systems. The combination of our plate with the optimized 3D cardiac models that are produced, maintained, matured, and electrically stimulated throughout the process forms the 3D Cardiac Models-based Platform.

[0037] FIG. 2 depicts a Schematic of the Electrostimuli-3D Culture Plate. The autonomous version can be seen in FIG. 2A, and describes the electrostimuli-3D Culture Plate Design: The 96-Well Plate Layout schematic depicts the configuration, highlighting the distribution of electrodes, batteries (FIG. 2B), circuits (FIG. 2C), and the placement of 3D cardiac models within the wells. FIG. 2B represents the well design (lateral view): Electrode 1 (continuous line) and Trails are depicted on one side of the well for stimulation, electrode 2 (dashed line) and trails are on the opposite side. Well Placement: The positioning of the cardiac constructs within each well is shown, ensuring precise contact with the electrodes for effective stimulation.Additionally, the schematic depicts the black walls of the wells and their conical shape, which are integral to the plate's design for enhanced organoid formation, imaging and stimulation efficiency. FIG. 2C represents the well bottom cross-section, showing the cardiac construct as well as the position of the electrodes.

[0038] FIG. 3 represents a schematic of the Electrostimuli-3D Culture Plate - Non- Autonomous version with Standalone base for electrostimulation. FIG. 3A represents the electrostimuli-3D Culture Plate Design: The 96-Well Plate Layout schematic depicts the configuration, highlighting the distribution of electrodes and the placement of 3D cardiac models within the wells. FIG. 3B represents the well design (lateral view): Electrode 1 (continuous line) and Trails are depicted on one side of the well for stimulation, electrode 2 (dashed line) and trails are on the opposite side. Well Placement: The positioning of the cardiac constructs within each well is shown, ensuring precise contact with the electrodes for effective stimulation. Additionally, the schematic depicts the black walls of the wells and their conical shape, which are integral to the plate's design for enhanced organoid formation, imaging and stimulation efficiency. FIG. 3Crepresents the well bottom cross-section, showing the cardiac construct as well as the position of the electrodes. FIG. 3D represents the standalone base for the Electrostimuli-3D Culture Plates electrical stimulation control. In Example 2, plates can be maintained per base. FIG. 3E represents the standalone base lateral view (cross-section): Electrodes have direct contact with conductors installed in the base to transfer electricity pulses for 3D cardiac models' stimulation. No wires are required to connect the plate to the electro-stimulator (base). FIG. 3F shows the representative computer screen to exemplify the use of a dedicated software capable of controlling hardware (Base and plates).

[0039] FIG. 4 depicts the progressive complexity within different in vitro cardiac models, showcasing most models available on the market for cardiovascular research. Starting with 2D hiPSC-CMs, it features human induced pluripotent stem cell-derived cardiomyocytes cultured in a simple two-dimensional monolayer format, suitable for basic studies of cardiac cellular function. Moving to a higher level of complexity, 3D Cardiospheres of hiPSC-CMs which presents spherical clusters of cardiomyocytes that promote enhanced cell-cell interactions, better mimicking the natural 3D environment of heart tissue. Then, 3D Cardiac Assembloids (Multicells produced in 2D and assembled in 3D) 3D organoids generated by the assembly of different cell types produced using hiPSCs in 2D culture conditions. Cells like cardiomyocytes, endothelial cells and fibroblasts are co-cultivated in a plate that allow 3D formation. This model carry more complexity than cardiospheres and is considered a type of organoids. And the most sophisticated level, 3D Cardiac Organoids, which replicates essential aspects of cardiac tissue development, architecture and function, providing a comprehensive model for studying heart biology and disease mechanisms.

[0040] FIG. 5 depicts the 3D hiPSC-CM-derived Cardiospheres production Workflow. The workflow starts with the isolation of adult somatic cells from individuals, specifically erythroblasts or urine progenitor cells (UPCs). These cells undergo reprogramming via transcription factors (OCT4, SOX2, KLF4, and c-MYC) to generate hiPSCs. The hiPSCs differentiated into cardiomyocytes in 2D, and then they are self-assembled in a 3D microenvironment in multi-well plate formats (96-well plates), this is where the Electrostimuli-3D Culture Plate takes place.

[0041] FIG. 6 depicts the 3D Multi-cell type hiPSC— derived Cardiac Assembloids production Workflow. The workflow starts with the isolation of adult somatic cells from individuals,specifically erythroblasts or urine progenitor cells (UPCs). These cells undergo reprogramming via transcription factors (OCT4, SOX2, KLF4, and c-MYC) to generate hiPSCs. The hiPSCs differentiated into cardiomyocytes, endothelial cells, fibroblasts, among others, in 2D conditions, are mixed to form self-assembled 3D Cardiac Assembloids in a 3D microenvironment in multiwell plate formats (96-well plates), this is where the Electrostimuli-3D Culture Plate takes place.

[0042] FIG. 7 depicts the 3D hiPSC-derived Cardiac Organoids production Workflow. The workflow starts with the isolation of adult somatic cells from individuals, specifically erythroblasts or urine progenitor cells (UPCs). These cells undergo reprogramming via transcription factors (OCT4, SOX2, KLF4, and c-MYC) to generate hiPSCs. The hiPSCs differentiated into cardiac organoids in a 3D microenvironment in multi-well plate formats (96-well plates). From pluripotent stem cells to complex Cardiac Organoids, every step is conducted in 3D microenvironment, this is where the Electrostimuli-3D Culture Plate take place.

[0043] FIG. 8 depicts the 3D Cardiospheres produced with ventricular-like cardiomyocytes (from hiPSCs genetically modified to express GFP when these cells transform into ventricular cardiomyocytes that express myosin-light chain 2), on day 20. FIG. 8A and FIG. 8B represent the generation of 3D Cardiospheres, starting by using the microtissues technology. 1.5% Agarose gels were printed using microtissues molds (FIG. 8A), and they were pre-loaded with hiPSC-CMs maintenance medium for up to 2h before beginning cell seeding, as shown in FIG. 8B. FIG.8C represents agarose gel loaded with day 50 hiPSC- CMs after 15 minutes seeding. FIG.8D and FIG. 8E represents multiple 3D Cardiospheres 96h after seeding. FIG. 8F represents how a second hiPSC line (non-GFP tagged) was differentiated. On Day 20, non-GFP hiPSC-CMs were loaded with CFDA-SE, a short-term live-cell tracker. 3D Cardiospheres are very compact after 24h, suffering cell accommodation into the space, making them smaller in diameter.

[0044] FIG. 9 represents the comparison of Cardiosphere formation using two ultra-low-attachment plate geometries. FIG. 9A depicts representative bright-field images (40x) of human iPSC-derived Cardiospheres formed in conical, flat-bottom 96-well plates and tubular, U-bottom 96-well plates. Arrows indicate multiple micro-aggregates and fragmented spheroids observed in the tubular U-bottom format. FIG. 9B represents circularity analysis showing significantly higher morphological uniformity and compactness in spheroids generated using the conical-flat-bottom plates (p = 7 x 107). C) Diameter comparison indicating slightly larger but less uniform spheroids in the tubular U-bottom plates (p = 5 * IO3). Together, these results highlight thatconical, flat-bottom geometries promote improved spheroid integrity and reproducibility compared with conventional U-bottom wells.

[0045] FIG. 10 depicts the time-course analysis of Cardiospheres morphology and size using conical, flat-bottom ultra-low-attachment plates. FIG. 10A depicts representative bright-field images of Cardiospheres generated in conical, flat-bottom 96-well plates at different time points, illustrating consistent morphology and compaction over time. FIG. 10B represents quantitative analysis of the mean Cardiospheres diameter and circularity across the 48-192 h culture period, showing stable growth dynamics and structural integrity. FIG. IOC represents the violin plots of Cardiospheres diameter distributions at each time point, revealing reduced variability and gradual compaction during culture. FIG. 10D represents the violin plots of Cardiospheres circularity, indicating increasing shape uniformity and roundness over time. FIG. 10E represents the diameter evolution of Cardiospheres formed from different initial cell seeding densities (2.5K-20K cells per well) across independent experiments, showing size dependence on initial cell number. FIG. 10F represents the mean Cardiospheres diameter over time for each seeding condition, demonstrating reproducible growth kinetics and convergence toward stable dimensions after 144 h.

[0046] FIG. 11 represents the generation of self-assembled hiPSC-derived 3D cardiac organoids using agarose gel mold technology (Version 1 protocol) and cardiomesoderm phase characterization. FIG. 11 A-F represents the two-phase protocol, which involves an initial 2D differentiation step followed by 3D aggregation within agarose gel micro-molds. 2D phase: Human induced pluripotent stem cells (hiPSCs) were seeded onto Geltrex®-coated six-well plates at -60% confluence and maintained in mTeSR medium until reaching full confluence (-4 days). On day 0, the medium was replaced with RPMI + B27 minus insulin (RB(-)) supplemented with 6-9 pM CHIR99021 for 24 h to induce mesodermal differentiation. On day 1, cultures received RB(-) medium supplemented with CHIR99021 and 10 nM BMP4. 3D phase: On day 3, cells were dissociated with TrypLE Express, counted, and seeded into 1.5% agarose micro-mold wells containing RB(-) medium supplemented with 2 pM Wnt-C59 to promote cardiomesoderm aggregation and self-assembly. Spheroids were imaged daily for 4 days to assess growth dynamics and morphology. FIG. 11 A and FIG. 1 IB depict representative micrographs and diameter quantification of 150 pm cardiomesoderm spheroids showing a minor but statistically significant reduction in diameter over time. FIG. 11C and FIG. 1 ID represent therepresentative images and quantification of 300 pm spheroids exhibiting a slightly more pronounced yet consistent reduction in diameter during culture. FIG. 1 IE and FIG. 1 IF represent the metabolic viability analysis by cumulative lactate quantification in the culture medium over 4 days. Parallel 2D cultures were maintained at low (10,000 cells / well) and high (1,000,000 cells / well) confluence to simulate 3D cell density. No significant increase in lactate release or morphological evidence of cell death was observed, confirming that 3D aggregation within agarose moulds preserves viability in both 150 pm and 300 pm spheroids, (p < 0.05).

[0047] FIG. 12 represents the generation and maturation of self-assembled hiPSC-derived 3D Cardiac Organoid using a magnetic levitation system. FIG. 12A represents a schematic overview of heart organoid formation using the magnetic levitation platform (n3D system, Greiner Bio-One). FIG. 12B represents the experimental timeline and representative images. Human induced pluripotent stem cells (hiPSCs) were cultured as single cells in pluripotent stem cell medium until reaching full confluence (~4 days). On day 0, monolayers were induced toward mesodermal lineage using CHIR99021, a selective GSK3 inhibitor. On day 3, mesoderm-like cells were loaded with magnetic nanoparticles, detached under single-cell conditions, and levitated for 16-24 h in the presence of a Wnt pathway inhibitor to promote cardiac lineage specification. On day 5, the resulting cardiac spheroids were transferred to cardiomyocyte maturation medium and cultured long term. Beating behavior emerged between days 10 and 15, becoming progressively more synchronized by day 45. FIG. 12C represents the histological analysis of day 45 heart organoids (H&E, 2 pm serial sections, 25 x magnification) showing internal cavities resembling primitive cardiac chambers. FIG. 12D represents the immunofluorescence staining of a medial section from a day 45 mini-heart, showing expression of human adult cardiac troponin I (cTnl; gene symbol TNNI3) and nuclear counterstaining (40 magnification), confirming advanced cardiomyocyte maturation.

[0048] FIG. 13 depicts the iron-gold nanoparticle (NP) coating of self-assembled 3D cardiac organoids and assessment of post-loading viability. FIG. 13A-E depict how day 30 cardiac organoids were subjected to a nanoparticle surface-coating protocol using iron-gold (Fe-Au) nanoparticles designed for future conjugation with drugs, dyes, or antibodies. FIG. 13 A represents magnetic separation of iron-gold nanoparticles from suspension using a magnetic rack, demonstrating the strong magnetic responsiveness of the particles. FIG. 13B represents how organoids were incubated for 2 h with different NP concentrations to evaluate dose-dependent tolerance. FIG. 13C depicts how, following incubation, nanoparticle-coated organoids were magnetically separated from the medium, confirming successful NP adherence. FIG. 13D represents the global viability assessment of 300 pm 3D cardiac organoids 24 h after NP loading using the MTT assay, indicating preserved metabolic activity across conditions. FIG. 13E reveals the morphological evaluation at day 45 of cardiac organoids pre-loaded with nanoparticles at day 30. Organoids (300 pm diameter) exposed to a 1:500 NP dilution displayed morphology comparable to untreated controls, whereas smaller (150 pm) organoids exhibited slightly more heterogeneous borders after NP loading (right panel, high magnification).

[0049] FIG. 14 depicts the functional characterization of 3D Cardiospheres at day 30. FIG. 14A-D represent the confocal microscopy and functional analyses demonstrating calcium and membrane potential dynamics in spontaneously beating 3D Cardiospheres. FIG. 14A depicts representative confocal images (Z-stack planes and corresponding maximum intensity projection) of a Cardiosphere loaded with a fluorescent calcium indicator for quantification of intracellular calcium transients. FIG. 14B depicts representative confocal images (Z-stack planes and maximum intensity projection) of a Cardiosphere loaded with a voltage-sensitive fluorescent dye for quantification of action potential-associated membrane potential changes. FIG. 14C depicts the representative calcium transient trace obtained from time-lapse fluorescence recordings of a Cardiosphere loaded with the calcium-sensitive dye shown in panel A. FIG. 14D depicts representative membrane potential trace obtained from time-lapse fluorescence recordings of a Cardiosphere loaded with the voltage-sensitive dye shown in panel B. All fluorescence data were acquired using linear temporal acquisition settings, and quantitative analyses — including transient detection and parameter extraction — were performed using custom MATLAB scripts developed in-house.

[0050] FIG. 15 represents 3D Cardiospheres generated in conical, flat-bottom ultra-low-attachment (ULA) 96-well plates exhibit enhanced expression of MLC2v, a marker of ventricular cardiomyocyte maturation. FIG. 15A reveals gene expression data for MLC2a (MYL7) and MLC2v (MYL2) obtained from hiPSCs during 2D cardiac differentiation (data derived from our transcriptomic dataset; the original dataset is part of a study recently accepted for publication in Stem Cell Research & Therapy, BMC, IF: 8.098). FIG. 15B depicts representative fluorescence and bright-field images of hiPSC-eGFP-MLC2v cardiomyocytes at day 30, showing that not all cells express MLC2v at this stage. FIG. 15C depicts representativeconfocal fluorescence and bright-field merged images comparing 2D and 3D cultures of hiPSC-eGFP-MLC2v cardiomyocytes. A substantially higher proportion of cells within the 3D Cardiospheres express eGFP at day 30 compared with their 2D counterparts. Confocal images are shown as maximum intensity projections derived from 10-15 Z-stack planes (~5 pm spacing). FIG. 15D depicts the percentage of eGFP-positive cells over time in 2D cultures of hiPSC-eGFP-MLC2v cardiomyocytes. FIG. 15E depicts the normalized eGFP intensity over time in 3D cultures of Cardiospheres.

[0051] FIG. 16 depicts the acute Dofetilide treatment protocol and functional response of spontaneously beating 3D Cardiospheres generated in conical, flat-bottom ultra-low-attachment (ULA) 96-well plates. FIG. 16A reveals the experimental design illustrating the stepwise Dofetilide exposure protocol. Dose selection was based on current literature and regulatory recommendations from the FDA and NIH for preclinical cardiac safety assessment. FIG. 16B depicts the representative calcium transient traces recorded from the same 3D Cardiosphere exposed sequentially to increasing concentrations of Dofetilide, showing a progressive dosedependent prolongation of calcium transient duration and reduced beating frequency. FIG. 16C depicts the detection of Dofetilide-induced arrhythmic events. Representative fluorescence traces from a single Cardiosphere exposed to escalating Dofetilide concentrations display irregular beating patterns and waveform abnormalities consistent with drug-induced arrhythmia.

[0052] FIG. 17 depicts the comparative validation of 3D Cardiospheres and 2D monolayer cultures for cardiac safety assessment using CiPA reference compounds. FIG. 17A depicts the representative fluorescence traces obtained from high-throughput voltage imaging of 3D Cardiospheres generated in conical, flat-bottom ultra-low-attachment (ULA) 96-well plates and exposed to increasing concentrations of reference drugs. FIG. 17B depicts the CiPA compound test set and calibration drugs classified by risk level (high, intermediate, and low) used to benchmark the assay’s predictive performance. FIG. 17C depicts the representative schematic of electrophysiological readouts extracted from fluorescence recordings, showing the measurement of action potential duration (APD20, APD50, and APD90). FIG. 17D-G represents the sensitivity and specificity analysis comparing 3D Cardiospheres with 2D monolayer hiPSC-cardiomyocyte cultures under acute and chronic exposure conditions. The 3D Cardiospheres exhibited superior sensitivity for detecting both high-risk (100%) and intermediate-risk (70-100%) compounds, and improved specificity for non-cardiotoxic (low-risk) drugs (1 h: 100%; 24 h: 62.5%) comparedwith 2D cultures (1 h: 62.5%; 24 h: 37.5%). These results demonstrate the enhanced predictive accuracy and translational reliability of the 3D Cardiospheres model for preclinical cardiotoxicity screening.

[0053] FIG. 18 represents the electrostimulation plate configurations and well layouts. FIG. 18A represents the autonomous 96-well plate (60 active wells) — Columns 11-12 house internal batteries (FIG. 18B) and a controller (FIG. 18C) that delivers programmed biphasic electrical pulses to embedded electrodes in columns 1-10. Enables wireless operation without external wiring. FIG. 18B represents the standalone-base 96-well plate (96 active wells) — All wells functional. Electrical stimulation supplied through contact pads on the plate bottom that interface with an external base station for continuous powered operation and programmable control. FIG.18C represents the autonomous 384-well plate (-320 active wells) — Conceptual high-density version maintaining on-board batteries and controller occupying two columns (23-24). Designed for compact micro-battery integration while preserving independent stimulation across remaining wells. FIG. 18D depicts the standalone-base 384-well plate (384 active wells) — High-throughput embodiment powered via external base contacts; all wells active for synchronized or independent stimulation. Suitable for automated or parallel assays. Each configuration includes paired micro-electrodes per well spaced <1 mm apart around the central spheroid / organoid, with black well walls for optical isolation and clear bottoms for imaging.DEFINITIONS

[0054] As used herein, a “totipotent” cell refers to a cell capable of differentiating into any cell type of the body, including both somatic and germ line cells, and of forming extraembryonic tissues such as the trophoblast. A totipotent cell can therefore give rise to an entire organism under suitable developmental conditions. Totipotency is characteristic of the zygote and early blastomere stages in mammalian development. The cells used in the methods according to the present invention are preferably not totipotent, but rather are restricted to pluripotent or lineage-committed states. In certain embodiments, the inventive methods utilize cells that have reduced developmental potential relative to totipotent cells, thereby ensuring formation of structured organoid models that recapitulate organ-specific morphogenesis without generating whole-organismal potential.

[0055] As used herein, a “pluripotent” stem cell refers to a cell that, while not capable of developing into an entire organism, possesses the ability to give rise to derivatives of all three primary germ layers: ectoderm, mesoderm, and endoderm, and can thereby generate all somatic cell types of the body. Pluripotency may occur naturally, as in embryonic stem cells, or may be induced artificially through reprogramming of somatic, multipotent, unipotent, or progenitor cells. In certain preferred embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPSC) generated from patient-derived somatic cells. Such pluripotent cells can be directed to differentiate in vitro under defined conditions to yield organoid cultures that reproduce structural and functional characteristics of human tissues. The resulting organoids may be used to study disease phenotypes, model developmental abnormalities, or assess therapeutic responses in a patient-specific manner.

[0056] As used herein, the term “multipotent cell” refers to a cell that is capable of differentiating into two or more distinct, lineage-specific cell types within a particular germ layer or tissue lineage. In certain embodiments, a multipotent cell may give rise to multiple specialized cell types found within a defined organ or tissue (for example, hepatic progenitor cells capable of producing hepatocytes and cholangiocytes). Multipotent cells retain a capacity for self-renewal for at least a limited number of divisions and may be derived from pluripotent stem cells, primary tissues, or organoid cultures. In the context of human organoids, multipotent cells serve as intermediate progenitors that drive the development and maintenance of multiple cell lineages representative of the in vivo organ architecture.

[0057] As used herein, the term “unipotent cell” refers to a cell that is restricted to differentiation into a single mature cell type. A unipotent cell may possess proliferative capacity but is committed to a single lineage fate. In certain embodiments, unipotent cells may arise during in vitro organoid differentiation as terminally committed precursors or as tissue-specific progenitors that sustain renewal of a single functional cell population (for example, basal cells of airway epithelium or absorptive enterocytes in intestinal organoids).

[0058] As used herein, the term “human induced pluripotent stem cell” or “hiPSC” refers to a pluripotent stem cell of human origin that has been reprogrammed from a somatic cell type, such as a fibroblast, peripheral blood cell, keratinocyte, or other differentiated human cell, through the introduction or induction of specific genetic, epigenetic, or chemical factors that restore pluripotency. hiPSCs exhibit characteristics substantially similar to those of human embryonicstem cells, including self-renewal and the capacity to differentiate into derivatives of all three germ layers — ectoderm, mesoderm, and endoderm. In certain embodiments, hiPSCs are derived using reprogramming factors such as OCT4, SOX2, KLF4, and c-MYC, or functional equivalents thereof. In the context of the present invention, hiPSCs may serve as the cellular source for generating organoids that recapitulate the cellular complexity, architecture, and function of human tissues, allowing modeling of normal physiology, disease pathogenesis, or therapeutic response in a patient-specific manner.

[0059] As used herein, the term “human cerebral organoid” or “hCO” collectively referred to as “hCOs”, refers to a three-dimensional, multicellular in vitro structure derived from human pluripotent stem cells, including human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), that recapitulates key cellular, structural, and functional features of the developing or mature human brain. hCOs comprise organized regions containing neural progenitor cells, neurons, glial cells, and other brain-associated cell types arranged in layered or compartmentalized architectures that mimic aspects of human cortical development. hCOs may be generated through self-organization under defined culture conditions that promote neuroectodermal differentiation, or through guided differentiation protocols employing specific morphogens, signaling molecules, or extracellular matrix components to direct regional identity (e.g., forebrain, midbrain, or hindbrain). In certain embodiments, hCOs produced according to the present invention are used to model neurodevelopmental or neurodegenerative disorders, assess neuronal connectivity or electrophysiological function, or evaluate the efficacy and safety of therapeutic agents in a human-specific, physiologically relevant context.

[0060] As used herein, the term “organoid” refers to a three-dimensional, multicellular structure grown in vitro from pluripotent stem cells, multipotent progenitor cells, or tissue-derived stem cells, that self-organize and differentiate to recapitulate certain structural, molecular, and functional characteristics of a corresponding in vivo organ. Organoids comprise multiple interacting cell types arranged in a spatially organized manner that reflects the native tissue architecture and can exhibit organ-specific functionality, such as electrical activity, contractility, or metabolic activity. In the context of the present invention, organoids include, but are not limited to, cardiac organoids, brain organoids, kidney organoids, and liver organoids. A cardiac organoid refers to a three-dimensional tissue construct comprising cardiomyocytes, cardiac fibroblasts, endothelial cells, and / or other cardiac-resident cell types, which together modelaspects of human heart development, contractile physiology, or disease pathology. Cardiac organoids may exhibit spontaneous beating, calcium transients, or electrophysiological properties consistent with native myocardium and may be used to study cardiac morphogenesis, cardiotoxicity, ischemic injury, or regenerative responses.

[0061] A brain organoid, including a human cerebral organoid (hCO), refers to a three-dimensional neural tissue model containing differentiated neuronal and glial cell populations organized into cortical- or region-specific domains that reproduce features of human brain development. Brain organoids may display radial organization, neuronal layering, and spontaneous electrical activity, and can be employed to model neurodevelopmental or neurodegenerative disorders, neuronal connectivity, or drug responses.

[0062] A kidney organoid refers to a three-dimensional cell culture model comprising nephron progenitor cells, endothelial cells, and stromal components that self-organize to form structures resembling nephron-like units, including glomeruli, proximal and distal tubules, and collecting ducts. Kidney organoids exhibit filtration and transport properties reflective of renal function and can be employed to study nephrogenesis, renal pathophysiology, nephrotoxicity, or regenerative mechanisms.

[0063] A liver organoid refers to a multicellular construct composed of hepatocytes, cholangiocytes, hepatic stellate cells, and / or endothelial cells that recapitulates key aspects of liver tissue organization and function. Liver organoids may demonstrate bile canaliculi formation, albumin secretion, cytochrome P450 activity, and metabolic processing characteristic of mature hepatic tissue. Such organoids can be used to model liver development, fibrosis, steatosis, viral infection, or drug-induced liver injury.

[0064] An adipose organoid refers to a three-dimensional multicellular construct composed of adipocytes, preadipocytes, stromal vascular fraction cells, and / or endothelial cells that recapitulates key aspects of adipose tissue organization and function. Adipose organoids may exhibit lipid droplet accumulation, adipokine secretion (such as leptin and adiponectin), insulin responsiveness, and extracellular matrix remodeling characteristic of mature adipose tissue. Such organoids can be used to model adipogenesis, obesity, metabolic dysfunction, insulin resistance, or adipose tissue inflammation.

[0065] In certain embodiments, organoids of the present invention are derived from patientspecific hiPSCs to enable precision modeling of cardiac or neurological diseases, or for use inpharmacological screening, regenerative medicine, or mechanistic studies of human organogenesis.

[0066] As used herein, the term “progenitor cell” refers to a proliferative, lineage-restricted cell that originates from a stem or multipotent cell and gives rise to one or more differentiated progeny cells. Progenitor cells generally exhibit limited self-renewal capacity relative to stem cells and function as transient amplifying intermediates during tissue development or regeneration. In the context of human organoids, progenitor cells populate expanding cellular compartments that recapitulate developmental hierarchies and contribute to the morphogenesis, maturation, and maintenance of organoid structure and function.

[0067] As used herein, the term “differentiate” or “differentiation” refers to the biological process by which a less specialized cell, such as a stem cell or progenitor cell, undergoes molecular and phenotypic changes resulting in the acquisition of specialized structural and functional characteristics of a specific cell type. Differentiation may occur spontaneously or may be induced under defined in vitro conditions by exposure to one or more stimuli, such as growth factors, cytokines, extracellular matrix components, or small molecules. In the context of organoid generation, differentiation encompasses the stepwise progression from pluripotent or multipotent states toward lineage-restricted progenitors and ultimately to mature, functionally specialized cells that collectively form a three-dimensional tissue-like structure. Differentiation may be partial or complete, reversible or irreversible, depending on the stage of development and the applied culture conditions.

[0068] As used herein, the term “metabolic analysis” refers to the assessment, quantification, and characterization of cellular bioenergetic activity, including but not limited to measurements of mitochondrial respiration, glycolysis, oxidative phosphorylation, and related metabolic pathways. Metabolic analysis may be conducted using biochemical assays, imaging techniques, or real-time metabolic flux measurements. In certain embodiments, metabolic analysis is performed using extracellular flux analysis technology, such as Seahorse XF analyzers (Agilent Technologies), which measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as indicators of mitochondrial and glycolytic function, respectively. In the context of organoids, metabolic analysis enables evaluation of cellular energy metabolism, substrate utilization, and mitochondrial integrity within three-dimensional tissue constructs, and may beused to assess developmental maturity, disease-associated metabolic remodeling, or responses to pharmacological agents.

[0069] As used herein, the term “electrophysiology” refers to the study and measurement of the electrical properties and activity of biological cells and tissues. Electrophysiological analysis encompasses techniques for recording electrical signals such as action potentials, field potentials, ion channel currents, and transmembrane voltage changes. In certain embodiments, electrophysiological characterization of organoids is performed using microelectrode array (MEA) platforms, patch-clamp recordings, voltage-sensitive dyes, or optical mapping systems. In the context of cardiac organoids, electrophysiological analysis may be used to assess conduction velocity, excitability, and arrhythmic behavior, whereas in brain organoids it may be used to evaluate neuronal firing, synaptic activity, or network connectivity. Electrophysiology thus provides functional readouts of cellular and tissue-level excitability, enabling the investigation of physiological maturation, disease phenotypes, and drug-induced effects in organoid models.

[0070] As used herein, the term “cardiotoxicity assessment” refers to the evaluation of adverse structural, functional, or electrophysiological effects of a compound, biologic agent, or environmental factor on cardiac cells, tissues, or organoid models. Cardiotoxicity assessment may include the measurement of parameters indicative of cardiac cell viability, contractility, mitochondrial function, electrophysiological activity, or calcium handling. In certain embodiments, cardiotoxicity is evaluated using cardiac organoids, engineered heart tissues, or cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), which provide physiologically relevant human-specific test systems. Cardiotoxicity assessment may employ analytical methods such as metabolic flux analysis (e.g., Seahorse technology), electrophysiological recordings (e.g., microelectrode arrays), imaging-based contractility assays, or biomarker profiling. In the context of the present invention, the cardiotoxicity assessment is preferably performed using the 3D BioSol™ Heart Cardiotox Platform. In the context of the present invention, cardiotoxicity assessment may be used to predict or quantify compound-induced effects such as arrhythmia, impaired contraction, mitochondrial dysfunction, or structural cytotoxicity, thereby enabling preclinical evaluation of cardiac safety and therapeutic response.

[0071] As used herein, the term “No-Float™” refers to a hydrogel-based matrix system designed to stabilize 3D adipose tissue organoids, addressing buoyancy and enabling long-term culture. This system is unique in its ability to maintain organoid integrity during media changes and experimental protocols.

[0072] As used herein, the term “PDOO” refers to patient-derived obese organoids.

[0073] As used herein, the term “PDOC” refers to patient-derived cardiac organoids for personalized cardiotoxicity risk.

[0074] As used herein, the term “PDOL” refers to patient-derived liver organoids for NAFLD and fibrosis.

[0075] As used herein, the term “CardioToxScreen™” or “CardioTox” refers to an An integrated 3D cardiac safety platform offering high-throughput assessment of acute and chronic cardiotoxicity. This platform provides multiparametric readouts including calcium dynamics, action potential waveforms, and beating behavior.DESCRIPTIONS OF THE EMBODIMENTS

[0076] The present invention provides significant improvements that address in whole or in part challenges in drug development, such as the high failure rate of drugs due to unreliable preclinical models, high costs, and extended timelines. Current non-human models often lack predictive accuracy, resulting in expensive late-stage failures. The present invention provides improved models, such as a human-based cardiotoxicity platform, that significantly mitigates these risks by enhancing accuracy and reducing development time and costs.

[0077] The present invention provides apparatus and methods for improved 3-dimensional modeling of physiological tissue, such as human adipose and cardiac tissue, and further provides organoids which model the 3-dimensional environment of organs comprising such tissue, such as adipose and cardiac or heart. Thus, in certain embodiments, the invention provides for improved advanced cardiotoxicity assessment, combining the processes of manufacturing, cultivating, and testing human cardiac organoids (hCOs) within the same 3D-cell culture plate. This unique platform maintains continuous electrical stimulation of the organoids throughout the entire process, closely mimicking the natural electrophysiological environment of a human heart. By integrating all aspects of organoid creation and testing in one system, the platform significantlyreduces the need for transfers between vessels and minimizes experimental variability, offering a seamless workflow.CELLULAR MODEL

[0078] 3D cell culture systems have revolutionized how researchers study cellular behavior by creating more physiologically relevant environments compared to traditional 2D cultures. These systems better mimic in vivo tissue organization and function, providing more accurate models for drug discovery, disease modeling, and metabolic studies.

[0079] The technology is flexible, adaptable to a wide range of human cell types, and applicable to various adipose depots, including visceral and subcutaneous fat. The platform allows for the differentiation of progenitor cells or stromal vascular fractions (SVF) into adipocytes, thereby generating organoids from distinct fat depots for specific metabolic research needs.

[0080] In addition to adipose tissue, the system is also applicable to other disease models and organoid types, such as those used for liver disease research. For example, it can be adapted to generate organoids for non-alcoholic fatty liver disease (NAFLD) and liver fibrosis models, offering valuable insights into metabolic dysfunctions across tissues. Furthermore, the platform is compatible with adipose tissue organoids derived from induced pluripotent stem cells (iPSCs), enabling personalized disease modeling and drug testing for a broader range of therapeutic applications.

[0081] Extending this versatility, the BioSol™ platform also supports the generation of cardiac constructs (human cardiac organoids and cardiospheres derived from hiPSCs). These constructs replicate functional aspects of the human heart, including spontaneous beating, response to pacing, and electrophysiological readouts. By integrating adipose, liver, and cardiac models, the platform enables multi-tissue disease modeling and cross-validation of metabolic and safety outcomes.

[0082] The 3D BioSol™ platform technology enables continuous, high-content analysis of organoid morphology across tissues. Subtle changes in volume, area, and structure can be tracked in real time throughout treatments. For adipose organoids, the technology can assess adipocyte size, lipid accumulation, and tissue remodeling. For cardiac organoids, the technology can assess measurements that extend to beating amplitude, rhythm regularity, and contractility dynamics, directly linked to calcium transients and action potentials.

[0083] As demonstrated further herein, the present invention provides advantages in overcoming, in whole or in part, the challenges discussed above with respect to 3 -dimensional modeling of adipose tissue and cardiac tissue. As further demonstrated herein, the present invention can be readily adapted to provide for 3-dimensional modeling of other tissue and organoids, including hepatic tissue / liver organoids (e.g., NAFLD, fibrosis), renal tissue / kidney organoids (e.g., diabetes), neural tissue / brain organoids (e.g., neurodegenerative disease).Having read the instant application, the skilled artisan is able to practice the invention with respect to such tissue / organoid models. Accordingly, such modifications and adaptations form a part of the present invention as described and claimed herein.

[0084] In some embodiments, human cardiac organoids (hCOs) are derived from hiPSCs.

[0085] In some embodiments, the hCOs are cultured and matured in the plate, exhibiting key features of human heart tissue, including the ability to spontaneously contract and respond to electrical stimuli.

[0086] In some embodiments, the organoids can be maintained and cultivated long-term in the plate under conditions that promote long-term growth and maturation, allowing for extended experimentation and drug testing.

[0087] In some embodiments, cardiac organoids are cultured through the use of electrostimuli in 3D culture plates. ANSI / SLAS-compliant 96-well plates with embedded electrodes and batteries providing autonomous electrical stimulation to cardiac organoids. These plates enable maturation, long-term pacing, and high-throughput cardiotoxicity testing.

[0088] In some embodiments, the organoids can be functionally assessed in real-time during culturing.

[0089] In some embodiments, the platform allows for real-time measurement of cardiac parameters such as calcium dynamics, action potential waveforms, and other physiological responses.

[0090] In some embodiments, electrical stimulation is applied from the moment the organoids are generated and continues throughout their development, ensuring that the organoids mimic real heart conditions more accurately than traditional models.

[0091] In some embodiments, the platform is employed to enhance the maturation of cardiac organoids through chronic electrical stimulation. Using embedded electrodes, electrical pulses are delivered at controlled intervals to the organoids throughout their development, promotingthe expression of key cardiac proteins such as troponin T and ion channels critical for cardiac function. Over time, the platform reduces spontaneous beating rates to resemble adult heart rhythms, while continuously monitoring calcium transients and action potentials. The stimulation protocols are optimized to mimic real-life heart conditions, ensuring that the organoids mature into functionally relevant models for long-term drug testing and research.

[0092] In some embodiments, the platform integrates autonomous, battery-powered electrical stimulation directly into the 96-well plates, allowing continuous stimulation throughout organoid cultivation. This feature mimics the natural electrophysiological environment of the heart, promoting the maturation of hCOs to a degree unmatched by traditional models. By maintaining electrical activity, the organoids develop into physiologically relevant cardiac tissues, providing more accurate predictions of how drugs will interact with the human heart. By maintaining electrical activity, the organoids develop into physiologically relevant cardiac tissues, providing more accurate predictions of how drugs will interact with the human heart.

[0093] In certain embodiments, the present invention provides a battery-operated electrical stimulation system designed to deliver continuous electrical pulses to organoids within each well, without the need for external power sources or wires, and operate autonomously for extended periods, ensuring that electrical stimulation continues without interruption during longterm organoid culturing and testing.

[0094] In some embodiments, the platform offers a human-relevant alternative to animal testing, addressing growing industry and regulatory demands for more ethical and sustainable research practices. By reducing reliance on animal models, the platform not only aligns with ethical research goals but also provides a more accurate prediction of human responses to drug treatments, further decreasing the risk of clinical trial failures.

[0095] In some embodiments, the BioSol™ Technology prioritizes the use of human cells, enabling organoids that replicate in vivo physiology.

[0096] In some embodiments, 3D adipose tissue modeling and / or the modeling of adipose organoids are relevant for obesity, diabetes, and metabolic dysfunction. Culturing adipocytes in 3D presents unique challenges, particularly due to the buoyant nature of adipocytes in liquid media. This buoyancy complicates handling and makes it difficult to generate stable, high-throughput adipose models. These challenges are further amplified in the formation of adiposeorganoids or spheres, limiting the ability to accurately reproduce the in vivo environment of adipose tissue.

[0097] In some embodiments, 3D cardiac models and / or cardiac organoid modeling are relevant for drug safety, arrhythmia prediction, and cardiometabolic cross-talk. In parallel, cardiotoxicity is a leading cause of drug attrition. Current 2D monolayers and animal models fail to capture human heart electrophysiology, leading to poor translation. Generating functional 3D cardiac organoids requires overcoming limitations in cell immaturity and ensuring long-term maturation and stimulation. This creates a strong demand for integrated cardiac platforms capable of predictive safety testing.

[0098] Cardiac organoids of the present invention are produced using optimized differentiation protocols from hiPSCs. A key innovation is the Electrostimuli-3D Culture Plate, designed with embedded electrodes and batteries to provide autonomous electrical stimulation. This continuous pacing drives maturation, stabilizes functional properties, and enables real-time monitoring of calcium dynamics, action potential waveforms, and contractility. The system is ANSI / SLAS-compliant, ensuring compatibility with HTS imaging and automated liquid handling.

[0099] In some embodiments, cardiac organoids are derived from human iPSCs using small molecules and media such as CHIR-99021, Wnt-C59, RPMI-B27 (± insulin), and antioxidant supplements.

[0100] In some embodiments, adipose organoids are generated from primary stromal vascular fraction (SVF) cells, using proprietary in-house protocols that combine hormones, recombinant proteins, and lipid precursors.

[0101] In some embodiments, vascular organoids are also derived from hiPSCs, using BMP4, bFGF, and VEGF in media such as StemPro-34, EGM-2.

[0102] In some embodiments, liver and kidney models can originate either from hiPSCs or primary cells, depending on the application.

[0103] In some embodiments, described herein is a hydrogel-based matrix system designed to stabilize 3D adipose tissue organoids, addressing buoyancy and enabling long-term culture.

[0104] In certain embodiments, the No-Float™ component is implemented as a hydrogel-based organoid retention system designed to counteract buoyancy of lipid-laden adipocytes and maintain stable 3D positioning during culture, stimulation, and imaging. Suitable materials include low-percentage alginate (e.g., 0.25-1.0% w / v), agarose (0.25-0.75% w / v), gelatin-methacrylate (GelMA 3-7% w / v), or synthetic PEG-based hydrogels with tunable mesh size (50-300 nm) to permit nutrient diffusion while restraining vertical drift. In some embodiments, the retention matrix is patterned as a thin dome or ring at the well bottom (thickness -150-500 pm) to avoid optical obstruction. For long-term adipocyte maintenance, the matrix may be combined with ECM additives (collagen I, laminin) or a micro-mesh insert to preserve unilocular morphology and endocrine function. This configuration addresses known challenges in mature adipocyte / 3D adipose culture where buoyancy and fragility confound handling and imaging. The system is advantageous in its ability a to maintain organoid integrity during media changes and experimental protocols (Dufau J, et al., (2021). Am J Physiol Cell Physiol, 320(5):C822-C841; ComperaN, et al., (2022). RSC Publishing, 22:4332-4347; Yon H, et al., (2023). Organoid, 3:el).

[0105] In certain embodiments, organoid imaging is performed using high-content widefield and confocal systems configured for multiwell plates. Suitable modalities include brightfield, live / dead fluorescence, calcium imaging (e.g., Calbryte™ 630-AM), optical action potential mapping (e.g., FluoVolt™), and time-lapse high-content acquisition for morphology and contractility analysis. For example, an inverted imaging platform (e.g., EVOS™ M7000) supports multi-channel fluorescence and automated stage control for ANSI / SLAS-compliant plates. Calcium imaging dyes (e.g., Calbryte™ 630-AM) provide deep-red emission with high signal-to-background and cytosolic localization advantageous for thick 3D constructs, while membrane-potential dyes (e.g., FluoVolt™) enable rapid optical recording of action potentials in hiPSC-derived cardiomyocytes and organoids for higher-throughput assays. These configurations permit quantitative measurements of beating, calcium transients, and voltage dynamics within the same plate used for culture and stimulation. Evidence of EVOS-based organoid calcium imaging and high-throughput optical AP recording is well documented.DEVICE

[0106] In some embodiments, the Electrostimuli-3D culture plate features conical wells with cell-repellent surfaces to promote the formation of uniform organoids. The 1mm transparent bottoms ensure proper z-positioning for imaging and reduce optical distortion, while the black walls eliminate light interference, providing clearer fluorescence imaging.

[0107] In some embodiments, the plate is equipped with electrodes embedded in each well, providing autonomous electrical stimulation to the organoids without the need for external wiresor stimulators. The integration of batteries ensures continuous electrical stimulation throughout the experiment, closely mimicking real heart conditions.

[0108] In some embodiments, the embedded batteries allow the plate to operate independently, simplifying the setup and ensuring consistent stimulation across all wells, thus eliminating the complexities associated with external stimulation systems.

[0109] In some embodiments, the design adheres to ANSI / SLAS standards, ensuring compatibility with commercial imaging and analysis systems. The platform supports multiple assays with its 96-well plate format, including protein imaging, calcium dynamics, and metabolic analysis, all within the same plate.

[0110] In some embodiments, the integration of batteries into the plates for electrical stimulation makes the platform fully autonomous, eliminating the need for external power sources or wiring. This innovation simplifies experimental setup, enhances portability, and allows for use in diverse laboratory environments, including those with limited access to complex electrical systems.

[0111] In some embodiments, the integration of organoid generation, stimulation, and testing combined in one platform significantly reduces the need for multiple pieces of equipment, consumables, and labor. The minimized handling, reduced plastic waste, and automation of many experimental steps translate to lower operational costs and faster turnaround times for drug testing and research projects.

[0112] In some embodiments, the integrated design reduces the need for handling and transferring organoids between different systems for imaging, testing, and culturing. This reduction in manual intervention minimizes variability between experiments, enhancing the reproducibility and reliability of results. It also reduces the risk of contamination and damage to the organoids, ensuring consistent data across multiple trials.

[0113] The invention thus offers a method for reducing experimental variability and contamination risk, comprising conducting all steps of organoid generation, cultivation, and testing within the same 96-well plate, eliminating the need for transferring organoids between different vessels, and minimizing manual intervention, thereby reducing contamination risk, experimental variability, and damage to organoids during handling.

[0114] In some embodiments, by offering a fully integrated solution for the production, cultivation, and testing of cardiac organoids, this invention streamlines the cardiotoxicity assessment process while improving predictive accuracy and scalability for drug discovery.

[0115] In some embodiments, described herein is a customized 3D cell culture plates specifically designed for high-throughput screening of adipose organoids. These plates facilitate advanced drug discovery and metabolic assays in a scaffold-free environment.

[0116] In certain embodiments, embedded microelectrodes (e.g., Au or Pt, -100-300 pm feature size) deliver biphasic square pulses for synchronized pacing of cardiac organoids. Representative parameters include frequency -1 Hz, pulse width -2 ms, and peak-to-peak amplitude -1.0-1.5 Vpp, selected to achieve capture without electrolysis or thermal loading. Protocols may be chronic (e.g., continuous pacing >7 days) to promote maturation or intermittent (e.g., duty cycles such as 2 h on / 4 h off) to mitigate fatigue. During optical assays, simultaneous readout with voltage dyes (FluoVolt™) and calcium indicators (e.g., Calbryte™ 630-AM) allows confirmation of capture, APD metrics, and excitation-contraction coupling in a high-throughput format. Published methods demonstrate feasibility of optical AP mapping in hiPSC-CMs (including mini-tissue / 3D contexts) and dual optical mapping of voltage and calcium using these dyes, supporting the disclosed ranges.

[0117] In some embodiments, the disclosed system enables electrical pacing of three-dimensional cardiac organoids within standard 96-well ANSI / SLAS-compatible microplates, using embedded miniature electrodes and either an autonomous self-powered mode or a standalone base-powered mode.

[0118] In preferred embodiments, the system provides electrical stimulation to three-dimensional cardiac organoids cultured in standard 96-well microplates. In one embodiment, the plate is autonomous, containing embedded microelectrodes and a power circuit housed within two peripheral columns of the plate. The electrodes are spaced 0.3-1.0 mm apart, generating electric fields of 1-100 V cm1(preferably 3-10 V cm ') using biphasic charge-balanced pulses of 0.1-20 ms duration delivered at frequencies of 0.1-20 Hz. The circuitry operates from one or more coin-cell lithium batteries (3.0-4.2 V, 50-250 mAh each) with boost regulation to 1-20 V per channel, enabling 24-120 hours of continuous stimulation. In another embodiment, the system comprises a standalone base unit capable of powering multiple plates (e.g., four plates simultaneously) through contact pads on the plate bottom, allowing full 96-well use and programmable independent stimulation profiles. Both embodiments incorporate per-well current limiting (0.1-50 mA), thermal protection, and galvanic isolation, and may optionally include wireless connectivity for remote parameter configuration or synchronization. The dual-modedesign permits autonomous operation or base-connected operation using the same plate architecture.

[0119] In some embodiments, the electrode spacing ranges between 0.1-1.5 mm between opposing poles within each well.

[0120] In preferred embodiments, the electrode spacing ranges between 0.3-1.0 mm between opposing poles within each well.

[0121] In some embodiments, the wall geometry of conical wells has a bottom diameter ranging between about 0.3-1.5 mm.

[0122] In preferred embodiments, the wall geometry of conical wells has a bottom diameter ranging between about 0.8-1.0 mm.

[0123] In some embodiments, the wall geometry of organoids ranges between 100-900 pm in diameter, positioned centrally between electrodes.

[0124] In preferred embodiments, the wall geometry of organoids ranges between 300-600 pm in diameter, positioned centrally between electrodes.

[0125] In some embodiments, the effective electric-field ranges between .05-200 V cm ', equivalent to 0.05-2.0 V potential difference across a 1 mm gap.

[0126] In preferred embodiments, the effective electric-field ranges between .1-100 V cm ', equivalent to 0.1-1.0 V potential difference across a 1 mm gap.

[0127] In some embodiments, the typical operating range is between 1-15 V cm1(0.1-1.5 V across electrodes) for consistent pacing and minimal electrolysis.

[0128] In preferred embodiments, the typical operating range is between 3-10 V cm1(0.3-1.0 V across electrodes) for consistent pacing and minimal electrolysis.

[0129] In preferred embodiments, the electrode material is selected from carbon, platinum, platinum-iridium, or gold, optionally coated with PEDOT or conductive carbon ink.

[0130] In some embodiments, the waveform is biphasic, charge-balanced square pulse with optional interphase delay (0-1 ms).

[0131] In some embodiments, the pulse width per phase ranges between 0.1- 20 ms.

[0132] In preferred embodiments, the pulse width per phase ranges between 0.5-5 ms.

[0133] In some embodiments, the frequency ranges between 0.1-20 Hz (6-1200 bpm), with a typical cardiac stimulation of 0.5-6 Hz.

[0134] In some embodiments, the amplitude ranges from about 0.1-2 V per well (corresponding to 1-100 V cm1for <1 mm spacing).

[0135] In some embodiments, the output current limit ranges from about 0.1-50 mA per pulse (user- or circuit-limited).

[0136] In some embodiments, the waveform options are selected from square, biphasic, monophasic, sinusoidal, or custom-programmable.

[0137] In some embodiments, the duty cycle is <10 % to minimize heating and electrolysis.

[0138] In some embodiments, each autonomous plate incorporates a battery and micro-driver circuit occupying the physical footprint of two columns of wells (columns 11 and 12).

[0139] In some embodiments, the battery chemistry is lithium coin-cell or equivalent (Li-MnCL or Li-ion).

[0140] In some embodiments, the nominal battery voltage ranges from about 3.0-3.7 V, with a maximum of 4.2 V.

[0141] In some embodiments, the configurable boost converter output ranges from 1-20 V per channel.

[0142] In some embodiments, the battery capacity ranges from 50-250 nAh per cell, as multiple cells may be paralleled.

[0143] In some embodiments, the average power draw ranges from 2-10 mW per plate during continuous 2 Hz pacing (0.5-1 V pulses, 2 ms width).

[0144] In some embodiments, the autonomy capabilities range from 24-120 h (1-5 days) of continuous stimulation depending on frequency and duty cycle, with replaceable batteries.

[0145] In some embodiments, the batteries are rechargeable with optional wireless or padcontact charging through integrated contacts.

[0146] In some embodiments, the power safety is per-well current limiting, watchdog cutoff to prevent DC bias, and thermal shutdown.

[0147] In some embodiments, the preset mode is a factory-programmed pulse pattern (fixed amplitude and frequency) for simplified use.

[0148] In some embodiments, the advanced mode contains an optional wireless (Wi-Fi / Bluetooth Low Energy) communication allowing adjustment of frequency (0.1-20 Hz), pulse width (0.1-20 ms), and amplitude (0.1-2 V).

[0149] In some embodiments, the synchronization embodies an optional phase-locking across plates for simultaneous pacing of multiple constructs.

[0150] In some embodiments, the battery and circuit are located in columns 11-12 of the 96-well array, leaving 60 functional wells (columns 1-10) fully available for organoid culture and stimulation.

[0151] In some embodiments, the operation of each well pair (or group) is driven by the embedded circuit, with no external wiring required.

[0152] In some embodiments, the replaceable coin cells are accessible through a sealed rear or side compartment, the replacement interval 2-5 days under continuous pacing.

[0153] In some embodiments, the dual-mode compatibility plates include contact pads enabling optional use with the standalone base to bypass the internal battery (preserving charge during non-autonomous runs).

[0154] In some embodiments, the contactless interface standalone base station includes conductive pads aligned with mating pads on the plate bottom; mechanical pressure or magnetic alignment ensures contact.

[0155] In some embodiments, the standalone base station capacity is up to four plates (384 wells total) operated simultaneously, each under independent stimulation parameters.

[0156] In some embodiments, the standalone base station power source is externally regulated DC (5-24 V), supplying individual driver channels for each plate.

[0157] In some embodiments, the standalone base station is a wired or wireless computer interface for programming pulse sequences, logging duty cycles, and coordinating multiple plates.

[0158] In some embodiments, the standalone base station advantages include full 96-well functionality (no wells lost to batteries / circuits) and unlimited runtime.

[0159] In some embodiments, the wells range from 60-384 wells.

[0160] In some embodiments, the transparent well bottom ranges from 0.1-lmm.

[0161] In some embodiments, the transparent well bottom ranges from l-2mm.

[0162] In some embodiments, the transparent well bottom ranges from 2-5mm.

[0163] Across all embodiments, wells are manufactured with black walls to minimize optical crosstalk and clear bottoms to enable microscopy and optical readouts. The embedded microelectrodes are fabricated from carbon, platinum, platinum-iridium, or gold and may becoated with conductive polymers such as PEDOT to reduce impedance. The spacing between electrodes (typically 0.3-1.0 mm) ensures homogeneous field exposure to organoids of 300-600 pm in diameter positioned at the well center.

[0164] Collectively, the embodiments shown in Figure 18 provide a scalable electrostimulation platform capable of operating from 60 to 384 active wells per plate, depending on configuration, while preserving identical electrode geometry and electrical field characteristics. The dual-mode design allows seamless transition between wireless autonomous operation and externally powered base operation, supporting a broad range of laboratory and industrial applications.

[0165] In some embodiments, the total configurable wells can be seen in Table 1, and FIG. 18.

[0166] Table 1.Plate Type Total Active Power Source Wells WellsAutonomous 96-well 96 60 On-board batteries & circuits Standalone-base 96-well 96 96 External powered base Standalone-base 384-well (future 384 384 External powered base embodiment)(Optional) Autonomous 384-well 384 240-320 On-board micro-batteries(feasibility dependent)DISEASE MODELING

[0167] In some embodiments, the platform is used for disease modeling in cardiovascular research. Patient-derived hCOs, generated from hiPSCs or CRISPR-edited cell lines, can be cultured in the plate to simulate specific cardiac conditions, such as inherited heart diseases or drug-induced cardiomyopathies. Continuous electrical stimulation during culturing promotes the maturation of the organoids, ensuring that they replicate key physiological characteristics of the human heart. Researchers can use the platform to study disease progression, cardiac regeneration, or test new therapeutic compounds, offering a precise model for precision medicine applications.

[0168] In addition to cardiac organoids, the platform can be adapted for the culturing and testing of other human organoids, such as liver, brain, or kidney organoids. By utilizing the same 96-well plate design with embedded electrodes, researchers can simulate electrophysiological conditions relevant to these tissues. For example, brain organoids can be electrostimulated to study neurotoxicity or neurodegenerative diseases, while liver organoids can be used forhepatotoxicity screening. This flexibility makes the platform a valuable tool for multi-organ drug testing and toxicity assessment, providing a scalable solution for comprehensive preclinical testing.

[0169] In some embodiments, the platform is applied to adipose disease models. The organoids recapitulate obesity-related phenotypes, enabling Patient-Derived Obese Organoids (PDOO) for precision metabolic testing.

[0170] In some embodiments, the platform is applied to cardiac disease models. Patient-derived cardiac organoids can be generated to model inherited arrhythmias, drug-induced cardiomyopathies, and chronic drug exposure. Continuous pacing protocols improve maturation, reducing spontaneous immature beating rates and enhancing predictive accuracy for safety testing.

[0171] In certain embodiments, hepatic organoids are used to evaluate hepatocellular function and drug-induced liver injury. Quantitative readouts include albumin secretion (ELISA), urea synthesis, and cytochrome P450 activity (e.g., CYP3A4 induction / inhibition assays), along with bile canaliculi integrity by live imaging. Liver organoids and liver-on-chip systems have been shown to maintain improved hepatic phenotype and metabolic competence in vitro, enabling predictive toxicity and metabolism studies (Liu S, et al., (2024). PMC, Review; Telles-Silva KA, et al., (2022). PMC, Review).

[0172] In certain embodiments, brain organoids are profiled by electrophysiological activity (e g., multi-electrode array burst rate / frequency, local field potentials) and calcium imaging, providing quantitative drug response metrics for excitability and network synchronization.Mature organoids exhibit coherent oscillatory dynamics and network-level field potentials that are modulated by pharmacology (Passaro AP, et al., (2021). PMC; Sharf T, et al., (2022). Nat Commun).

[0173] In certain embodiments, kidney organoids are evaluated by functional transporter activity, albumin reabsorption / handling, urea production / clearance surrogates, and barrier metrics (e.g., TEER when integrated on chips). Kidney organoids recapitulate nephron-like segments and have been advanced toward translational modeling and chip integration for function assessment (Tabibzadeh N, et al., (2023). PMC; Miyoshi T, et al., (2019). PMC, Review).

[0174] In certain embodiments, vascular function is quantified by barrier integrity (e g., permeability assays to macromolecular tracers) and transendothelial electrical resistance (TEER) where available. Microvessel arrays and 3D endothelialized channels provide reproducible permeability measurements and compound-induced barrier modulation (Lee H, et al., (2014). PubMed; Pauty J, et al., (2017). Pubmed; Nguyen HT, et al., (2024). Taylor & Francis Online).

[0175] In some embodiments, while the platform is designed for cardiac organoids, the platform's versatile architecture allows it to be adapted for other organoid types, including liver, brain, adipose, and kidney organoids. This flexibility expands its applications beyond cardiotoxicity testing, making it suitable for multi-organ toxicity assessments and complex disease modeling. Researchers can simulate various organ systems within the same platform, making it a powerful tool for studying drug effects across different tissues.

[0176] In some embodiments, the platform can also be employed to test the long-term effects of chronic drug exposure on human cardiac organoids. Using its embedded electrical stimulation system, the platform simulates continuous heart function while organoids are exposed to varying doses of drugs over extended periods. Researchers can monitor changes in organoid function, such as alterations in contraction rates, calcium signaling, and structural protein integrity. This embodiment is particularly useful for evaluating drugs with cumulative toxicity, such as cancer therapies, allowing early detection of adverse cardiac effects before clinical trials.

[0177] In some embodiments, the platform adheres to ANSI / SLAS standards, making it fully compatible with high-throughput screening systems. Its 96-well plate format allows simultaneous testing of multiple compounds, making it ideal for pharmaceutical companies and research institutions engaged in drug discovery. This scalability significantly reduces the time and cost associated with traditional testing methods, particularly for early-stage cardiotoxicity assessment, enabling the rapid screening of large compound libraries.

[0178] In certain embodiments, patient-derived organoids (PDOs) or patient-specific iPSC-derived organoids are employed to capture an individual’s genetic background for functional precision medicine. Such PDO / iPSC systems enable (i) ex -vivo drug response profiling on patient-matched tissues, (ii) modeling of inherited electrophysiology phenotypes (e.g., long-QT variants) in 2D and 3D cardiac constructs, and (iii) establishment of biobanked panels for responder / non-responder stratification. The platform thereby supports prospective selection of efficacious compounds and early detection of patient-specific safety liabilities (Simons E, et al.,(2023). Biomedicines, 11 (2):334; Tong L, et al., (2024). Med (Cell Press), 5(7): 1000-1019; Qu S, et al., (2024). Signal Transduct Target Ther, 9:199).ASSESSMENTCardiac Assessment

[0179] Some embodiments of the invention involve the use of the 3D BioSol™ Heart Cardiotox Platform to perform high-throughput cardiotoxicity testing. Human cardiac organoids (hCOs) are generated from hiPSCs within the 96-well plate, and electrical stimulation is applied continuously throughout the culturing process. The hCOs are maintained under conditions that mimic human heart electrophysiology, allowing real-time monitoring of critical parameters such as calcium dynamics and action potential waveforms. During drug testing, the platform supports assays to evaluate acute and chronic cardiotoxicity. The system enables simultaneous testing of multiple compounds, measuring their effects on cardiac function with over 95% predictive accuracy.

[0180] In certain embodiments, the BioSol™ platform enables metabolic-cardiac cross-talk analyses, allowing simultaneous assessment of compound efficacy in adipose organoids and potential cardiotoxicity in cardiac organoids. For example, glucagon-like peptide- 1 (GLP-1) receptor agonists such as semaglutide or tirzepatide induce lipolysis and mitochondrial biogenesis in adipose organoids, improving metabolic efficiency, while parallel cardiac organoid assays confirm preserved contractility and only modest chronotropic shifts. Clinical and mechanistic studies report modest increases in resting heart rate (~3-5 bpm) with GLP-1 RAs, consistent with a physiological chronotropic effect rather than overt cardiotoxicity (Mansoor et al., 2025). This integrated workflow helps identify compounds that achieve metabolic benefits without inducing adverse cardiac changes, combining efficacy and safety screening in a unified platform.

[0181] In some embodiments, the BioSol™ platform improves on drug testing by integrating hCOs and spheroids — advanced human cardiac models — with microelectronics to provide an electrophysiological microenvironment in a high-throughput, compact system. The platform features a unique 96-well plate design that embeds circuits and batteries for real-time monitoring and precise control. This setup enables mid-to-large scale production and long-term cultivation of hCOs and human cardiac spheroids (hCSs) under electrophysiological standards, allowing for reliable and accurate measurement of cardiac parameters, such as calcium dynamics and actionpotential waveforms, with an estimate of over 95% prediction accuracy (based on proprietary tests with 3D models) for acute and chronic cardiotoxicity.

[0182] By automating and scaling cardiac safety assessments, the 3D BioSol™ Heart Cardiotox Platform overcomes key challenges in drug development, providing a more reliable method to predict drug-induced cardiac effects. This advancement enhances the accuracy of preclinical testing, potentially reducing late-stage drug failures and improving patient safety. Based on preliminary data in 2D (Shim, 2023), the platform could improve cardiotoxicity prediction accuracy by >95%, reducing drug development costs by up to 30%.

[0183] The platform is further useful for in vitro cardiotoxicity assessment. The platform features a robust protocol for generating, maintaining, and testing hCOs within a versatile 96-well plate designed for 3D culture with embedded electrodes for electrical stimulation. This setup promotes hCO maturation and accurately mimics human heart electrophysiology. The platform is useful to predict drug-induced cardiotoxicity, enable early detection of cardiotoxic effects, reduce clinical trial failures, and minimize reliance on animal testing, thereby saving time, reducing costs, and de-risking the drug development process.

[0184] The invention described herein is further useful for disease modeling in cardiovascular treatments, enabling precision medicine. Generating, maintaining, and testing hCOs using the methods and apparatus of the invention that mimic heart electrophysiology, enabling modeling of specific cardiovascular conditions and inherited cardiac diseases using patient cells or CRISPR-gen erated models. This enables comprehensive testing, including cardiotoxicity, electrophysiology, metabolism, and regeneration, to develop targeted therapies. Replacing 2D technologies with the 3D platform of the present invention enhances predictive accuracy, offering a powerful tool for developing new treatments, improving patient outcomes, and reducing costs. The present invention supports neurotoxicity assessments and disease modeling for brain diseases. By generating, maintaining, and testing human brain organoids using the innovative plates of the present invention, researchers can model neurological conditions and inherited diseases with patient cells or CRISPR-generated models in a 3D electrostimulated environment. This setup allows for assessing neurotoxicity, studying neural electrophysiology, and investigating aspects such as metabolic dysregulation and electrochemical neural networking, thereby speeding up therapy discovery. Moving from 2D to 3D technologies boostsassay accuracy, providing a strong toolkit for targeted treatment development and better patient outcomes.

[0185] In certain embodiments, validation studies comparing the BioSol™ Heart Cardiotox Platform with clinical and preclinical reference data demonstrate predictive accuracy values ranging from -93-96 % for acute and chronic cardiotoxicity classification. Statistical correlation analyses between organoid-derived QTc-like parameters, troponin I release, and published human outcomes confirm that 3D organoid models substantially outperform conventional 2D assays. These results indicate that, when operated under standardized stimulation and imaging protocols, the platform achieves up to 95 % concordance with clinically observed drug-induced QTc prolongation and contractility changes.

[0186] The invention described herein enables the seamless production, cultivation, maturation, and testing of cardiac organoids in a single platform, eliminating the need for transfers between different vessels. This all-in-one solution simplifies workflows, reduces the potential for human error, and minimizes time and resource consumption. Researchers can carry out drug testing, electrophysiological assessments, and imaging all within the same plate, streamlining the experimental process and enhancing reproducibility.

[0187] In some embodiments, by utilizing advanced 3D human cardiac organoids, coupled with continuous electrical stimulation, the platform offers over 95% accuracy in predicting acute and chronic cardiotoxic effects of drugs. This level of precision is critical in reducing late-stage drug failures, which often arise due to the poor predictability of current 2D models and animal studies. The platform's ability to mimic human heart physiology leads to more reliable and clinically relevant results.

[0188] In some embodiments, the platform offers about 80% or greater accuracy in predicting acute and chronic cardiotoxic effects of drugs.

[0189] In some embodiments, the platform offers about 85% or greater accuracy in predicting acute and chronic cardiotoxic effects of drugs.

[0190] In some embodiments, the platform offers about 90% or greater accuracy in predicting acute and chronic cardiotoxic effects of drugs.

[0191] In some embodiments, the platform offers about 95% or greater accuracy in predicting acute and chronic cardiotoxic effects of drugs.

[0192] In some embodiments, the platform offers about 99% or greater accuracy in predicting acute and chronic cardiotoxic effects of drugs.

[0193] In some embodiments, the platform also allows for detailed electrophysiological testing of cardiac parameters under various experimental conditions. During experiments, calcium imaging and action potential measurements are performed directly in the plate using commercial high-throughput imaging systems. By maintaining the organoids in conical wells with transparent bottoms, the platform minimizes optical distortions and ensures that fluorescence signals are accurately detected. Researchers can assess the effects of different compounds on heart function, such as QTc-prolongation, and use the data to predict drug-induced arrhythmias or other cardiac adverse effects.

[0194] In some embodiments, the 3D BioSol™ Heart Cardiotox Platform is integrated with automated pipetting, mixing, and imaging systems. The plate’s design, compliant with ANSI / SLAS standards, allows it to be used with commercial laboratory automation equipment. This setup supports high-throughput workflows, enabling the simultaneous generation, cultivation, and testing of hundreds of organoids in a streamlined process. Automated liquid handling systems can be used for media exchanges, drug administration, and sample collection, further enhancing the platform’s efficiency in large-scale drug discovery programs.

[0195] Thus, the present invention enables early integration with functional human iPSC-derived cardiac models, such as cardiospheres and cardiac organoids, in order to detect potential cardiotoxicity risks of compounds while also testing for positive effects on metabolic function, before reaching animal studies. In certain embodiments, an initial assessment can be made of mitochondrial respiration (OCR) to ensure that compounds enhancing adipose metabolism are not dangerously increasing cardiac metabolic rates. This can be followed by viability analysis of cardiac organoids, observation of beating behavior, and electrophysiological profiling. These functional readouts — based on calcium handling, action potential waveform analysis, and structural integrity — allow early identification of potential cardiotoxicity in a human-relevant system.

[0196] In some embodiments, the invention described herein contains applications in drug discovery and pre-clinical studies. For cardiac applications, assessments include, but are not limited to, high-throughput cardiotoxicity screening with greater than 95% predictive accuracy for acute and chronic toxicity, IND-enabling cardiac safety packages, cardio-metabolic cross-validation to ensure metabolic therapies do not overload cardiac mitochondrial function, personalized medicine via PDOCs.

[0197] In some embodiments, further measurements include, but are not limited to, OCR / ECAR profding, including electrophysiology (calcium transients, action potential waveforms, and QTc prolongation), beating behavior (rhythm, contractility, and arrhythmia detection), metabolic stress (oxygen consumption rate (OCR) for mitochondrial function), and structural integrity (troponin-I and cytotoxicity markers).

[0198] In certain embodiments, cardiac organoids are assayed by (i) optical action potential (APD metrics using FluoVolt™ or equivalent), (ii) calcium transients (AF / Fo amplitude, rise / decay kinetics with Calbryte™ 630-AM or equivalent), (iii) contractility by video-based displacement, and (iv) troponin release and viability markers for structural integrity. Neural organoids are profded by MEA (spike / burst rate, synchrony), calcium imaging of network oscillations, and pharmacologic modulation of excitability. Hepatic organoids are evaluated by albumin, urea, CYP450 activity and bile canaliculi imaging; kidney organoids by transporter function, albumin handling, and TEER on chip; vascular constructs by permeability and TEER. These assays are compatible with ANSVSLAS-compliant plates and high-content imaging workflows, enabling multi-tissue screening within a unified platform.

[0199] In certain embodiments, cardiac function and toxicity are quantified through a combination of electrophysiological, structural, and biochemical readouts. Typical functional parameter ranges (in certain embodiments) include the beating rate (~0.5 to 1.2 Hz under spontaneous contraction), contraction amplitude (-50 to 150 pm displacement (via high-speed video analysis), calcium transient amplitude (AF / Fo) (1.5 to 2.0, e.g. with Calbryte™ 630-AM dye), action potential duration (APD90) (-280 to 380 ms from FluoVolt™ or equivalent optical potential probes), QTc prolongation threshold (an increase greater than 10 ms relative to baseline in matched control organoids), and troponin-I release (-0.2 ng / mL under non-damaging conditions). These parameters are illustrative of sensitivity to both acute and chronic cardiotoxic insults (e.g. hERG block, mitochondrial impairment). Use of organoid-based screening platforms has been demonstrated (Mills RJ, et al., (2017). Proc Natl Acad Sci USA, 114: E8372-E8381) in 96-well functional devices, and 3D arrhythmia phenotypes have been modeled under drug perturbation conditions (Kawatou M, et al., (2017). Nature Communications, 8:1078).

[0200] In certain embodiments, cardiac function and toxicity are quantified through a combination of electrophysiological, structural, and biochemical readouts.

[0201] In some embodiments, the beating rate of the cardiac organoids ranges from -0.5-1.2 Hz under spontaneous contraction.

[0202] In some embodiments, the contraction amplitude ranges from -50 to 150 pm displacement (via high-speed video analysis).

[0203] In some embodiments, the calcium transient amplitude (AF / Fo) ranged from -1.5 to 2.0, e.g. with Calbryte™ 630-AM dye.

[0204] In some embodiments, the action potential duration (APD90) ranges from -280 to 380 ms from FluoVolt™ or equivalent optical potential probes.

[0205] In some embodiments, the QTc prolongation threshold ranges from an increase greater than 10ms relative to baseline in matched control organoids.

[0206] In some embodiments, the troponin-I release is less than -0.2 ng / mL under nondamaging conditions.Tissue-specific measurements

[0207] In some embodiments, tissue-specific measurements pertain to cells selected from cardiac, adipose, liver, brain, kidney, and vascular cells.

[0208] In some embodiments, the functional and biochemical endpoints for adipose, hepatic, renal, and vascular organoids differ from those of cardiac or neuronal tissues, except for general viability markers (e.g., ATP production, cytotoxicity).

[0209] In some embodiments, liver-specific measurements include, but are not limited to, albumin secretion, urea synthesis, and CYP450 enzyme activity (e.g., CYP3A4).

[0210] In some embodiments, kidney-specific measurements include, but are not limited to, barrier integrity (TEER), albumin reabsorption, and injury markers such as KIM-1 or NGAL.

[0211] In some embodiments, vascular-specific measurements include, but are not limited to, permeability, nitric oxide production, and VE-cadherin integrity.

[0212] In some embodiments, multiparametric profiling of human adipose organoids can simultaneously measure mitochondrial respiration (OCR), lipid mobilization (glycerol / FFA), and morphological changes (organoid imaging); and multiparametric profiling of human cardiac organoids can simultaneously measure mitochondrial respiration (OCR), beating behavior, and calcium / AP waveform.

[0213] In other embodiments, the present invention comprises a human-relevant, multiparametric screening platform designed to accelerate the discovery of compounds that modulate adipose tissue metabolism and morphology. Using 3D human adipose organoids, the metabolic screening embodiment captures key functional endpoints, such as energy expenditure, lipid mobilization, and mitochondrial activity, enabling scientists to identify compounds with true therapeutic potential for treatment of patients with metabolic disorders and / or dysfunction.

[0214] Obesity is a complex and urgent health crisis that demands therapeutic solutions targeting metabolism without compromising safety. Unfortunately, metabolic modulation can be a doubleedged sword: compounds that shift energy balance may inadvertently harm the heart. This underscores the need for integrated, human-relevant models capable of identifying both efficacy and toxicity early in development.

[0215] 3D Human adipose organoids are derived from human tissue, and provide models that better mimic in vivo tissue architecture and metabolic complexity. The present invention allows simultaneous measurement of multiple parameters, including mitochondrial respiration (OCR), lipid mobilization (Glycerol / FFA), and morphological changes (Organoid imaging). The present invention further allows non-destructive, scalable readouts ideal for compound libraries of 5,000-10,000 molecules. Screening of 3D adipose organoids not only identifies active compounds, but also provides early mechanism-oriented insight into critical processes as thermogenesis, ATP- wasting pathways, and non-canonical energy expenditure, giving researchers additional data that can be utilized to prioritize compounds for further testing and translational development.

[0216] In adipose metabolic embodiments, the invention comprises methods that allow discovery of energy expenditure-enhancing compounds, validation of lipid-depleting agents, early screening for UCP1 -independent thermogenic pathways, early-stage profiling of mitochondrial modulators, functional cardiotoxicity screening of metabolic hits, and integrated safety and efficacy screening in other human-relevant systems.

[0217] In some embodiments, adipose-specific measurements include, but are not limited to, lipid droplet morphology, triglyceride turnover, and OCR / ECAR metabolic activity.

[0218] In some embodiments, adipose organoid tissue assessments include both morphological analysis, such as organoid size, adipocyte size, and lipid accumulation, and metabolic assays, including mitochondrial activity, glucose metabolism, and Seahorse analysis.

[0219] In some embodiments, high-content imaging solutions for adipose organoids include a suite of optical and confocal microscopy solutions specifically designed for the real-time analysis of organoid morphology. This product enhances the ability to monitor changes in organoid and adipocyte size during drug treatments.

[0220] In some embodiments, combined applications, such as cross-talk analysis (e.g. metabolic improvement vs cardiac safety) can be applied. Together, these readouts provide multiparametric insights into both efficacy (adipose models) and safety (cardiac models). Future expansion will allow other organoids, such as liver organoids, to be analyzed in various ways, such as steatosis progression, fibrosis markers, and structural hepatotoxicity changes.

[0221] In some embodiments, described herein is a Seahorse XF Analyzer Integration for BioSol™ Technology. The integration and optimization of 3D adipose tissue organoids with Seahorse XF technology enabling real-time metabolic analysis, including mitochondrial function and cellular respiration.

[0222] In certain embodiments, tissue-specific function is quantified by validated biochemical and imaging endpoints: (a) Liver — albumin secretion (ELISA), urea synthesis, and CYP450 activity (e.g., CYP3A4 induction / inhibition) with bile-canalicular structure imaging; (b) Kidney — segment-specific transporter function, albumin handling / uptake, and barrier integrity (TEER when on-chip), with urea product! on / clearance surrogates under flow; (c) Brain — network electrophysiology via MEA burst frequency, local field potentials, and calcium-imaging synchrony; (d) Vascular — microvessel permeability to fluorescent tracers and TEER where applicable. These metrics are widely used in organoid and organ-on-chip studies to benchmark maturation, stability, and pharmacological response, and are compatible with multiwell plate workflows.Preclinical Phase Integration

[0223] The present invention represents a major advancement in humanized 3D culture systems. By integrating both adipose and cardiac models, it provides stable, physiologically relevant organoids; allows dual endpoints for efficacy and safety and compatibility with automation and HTS workflows. This dual capability reduces late-stage failures, improves translational predictivity, and strengthens commercialization opportunities across obesity, cardiometabolic, and cardiovascular markets.

[0224] The platform improves translatability by offering dual-function testing (e.g., adipose efficacy + cardiac safety) early in the pipeline. By mimicking the in vivo environment more accurately than 2D systems, 3D BioSol™ Technology enhances the ability to predict drug efficacy and toxicity before moving into clinical trials. This technology can be used in various stages of the pre-clinical phase, particularly in decision-making processes for compound selection.

[0225] In some embodiments, the invention described herein contains applications in drug discovery and pre-clinical studies. For adipose applications, assessments include, but are not limited to, compound screening for anti-obesity and insulin-sensitizing agents, depot-specific efficacy, and personalized medicine via PDOOs.

[0226] In certain embodiments, the platform enables parallel multi-organ screening to evaluate efficacy and safety within a unified workflow. By way of example, adipose organoids (efficacy arm) are exposed to a candidate peptide and quantified for lipid droplet area, adipokine secretion, and metabolic flux (OCR / ECAR), while matched cardiac organoids (safety arm) are assessed for metabolic flux (OCR / ECAR), beating metrics, calcium transients, and optical action potentials / QTc surrogates under identical dosing regimens. Data are integrated at the well and batch levels to flag compounds that achieve metabolic benefit without electrophysiologic or structural cardiotoxicity. Multi-organ integration is consistent with microphy si ologi cal systems (MPS) approaches, combining tissues for systems-level pharmacology and toxicity profiling (Zommiti M, et al., (2022). Bioengineering, 9(11):646).EXAMPLES

[0227] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice.

[0228] However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0229] EXAMPLE 1- ADVANCED 3D BTOSOL™ HEART CARDIOTOX PLATFORM— ORGANOIDS AND ELECTROSTIMULI-3D CULTURE PLATES.

[0230] Developing safe cardiovascular drugs remains a significant challenge due to the inability of current animal and cellular models to accurately replicate human cardiac physiology. Despite advancements such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-cardiomyocytes) and 3D cultures, existing models fall short due to the complexity and variability of human heart tissue. This gap leads to high rates of late-stage drug failures and safety concerns (Sun, 2022), underscoring the urgent need for more predictive and standardized in vitro testing methods (Dariolli, 2021).

[0231] The key technical challenge preventing this problem from being solved is that hiPSC-cardiomyocytes exhibit biological and technical limitations including molecular and functional immaturity, fetal resemblance, and cultivation primarily in 2D, which does not match the organ’s reality (Van de Sande, 2023). The few existing 3D models are incompatible with high throughput (Nakar, 2022), limiting scalability and making them difficult to manipulate (Dariolli, 2021). Solving these issues requires an integrated solution that utilizes human cells to create a 3D model replicating heart physiology and electrophysiology, which current models cannot achieve. The current CHO-hERG assay for drug safety has limitations in representing the human heart, failing to recapitulate mechanical forces, complex interactions between different cell types (D'Costa, 2020), and the effect of well-established ion channels involved in cardiac homeostasis, such as sodium and calcium channels (Bers, 2002).

[0232] 3D models for cardiovascular research is challenging due to the complexity of replicating heart structure and function (FIG. 4). Unlike 2D cultures, 3D models require precise spatial organization of cells and matrix, often needing advanced scaffolding or self-organization.Scaling for high-throughput use adds challenges in managing nutrient distribution, waste removal, and oxygen supply. Ensuring reproducibility is also difficult, as small changes in microenvironment conditions can lead to inconsistent results.

[0233] The present invention provides a platform for in vitro cardiotoxicity assessment composed of an innovative 3D cell culture plate and human cardiac organoids (hCOs) (FIG. 1). The 96-well plate will have embedded electrodes that provide electrical stimulation to promote maturation, mimicking real-life heart electrophysiology. It will also enable convenient large-scale manufacturing, cultivation, and manipulation of hCOs.

[0234] Plate design: The Electrostimuli-3D culture plates are a groundbreaking innovation that integrates several unique features like conical wells with cell-repellent surfaces and black walls (FIG. 2). The cell-repellent surfaces prevent cell attachment, optimizing organoid formation, and the conical wells avert sample loss during aspiration by creating a physical barrier. The 1mm bottom means organoids are kept in the correct z-position, reducing optical distortions, while the black walls eliminate light interference, ensuring clear fluorescence imaging. Autonomous operation: A first-of-its-kind embedded electrodes and batteries within plate provide autonomous electrical stimulation to the organoids, eliminating need for external wires / stimulators. Every plate is a standalone device, which simplifies experiment configuration and improves high-throughput capabilities. Organoid culturing and maintenance: hCOs are generated from hiPSCs within the culture plate. Organoids are cultured under conditions that allow for long-term growth and maturation, aided by proprietary media and chronic electrophysiological stimulation protocols.

[0235] Electrical stimulation is applied concurrently with organoid culturing, enabling better maturation of hCOs and allowing for assessment of cardiac function and responses to stimuli. This facilitates the measurement of parameters such as calcium dynamics and action potential waveforms, with hCOs behaving as expected for a real human heart under pacing. High throughput testing: Based on ANSI / SLAS standards for dimensions (ANSI / SLAS, 2021), the plates support simultaneous morphologic and protein imaging, calcium, action potential, DNA / RNA extraction, and metabolic assays, reducing the need for transferring organoids to other vessels by 86% (transfer only in 1 out of 7 assays). This efficiency minimizes time, costs, and plastic waste, enhancing overall experimental throughput and reliability (FIG. 1, FIG. 2).

[0236] EXAMPLE 2- 3D CARDIOSPHERES.

[0237] Experimental overview:

[0238] Differentiators and main workflow features:Systematic identification of time-regulated genes and miRNAs during hiPSC- cardiomyocyte differentiation and maturation.Combined bioinformatic and experimental approach: multi-dataset screening followed by in-house time-course validation (7 time points, 30-day protocol).Inverse miRNA-gene pairing strategy (R < -0.5) used to pinpoint regulators of cardiomyocyte maturation.- Functional testing of top candidate miRNAs in hiPSC-CMs from multiple clones, assessing structure, calcium handling, and contractility.

[0239] Main results of the experiment:- Validated 16 specific miRNA-gene pairs in experimental hiPSC-CM differentiation.Demonstrated that miR-124 and others modulate calcium handling and sarcomere, maturation, influencing functional maturation of hiPSC-CMs.- Provides a time-based regulatory map of transcripts that can modulate cardiomyocyte differentiation and maturation (FIG. 3, FIG. 18).

[0240] These results are described in the following references: Munoz JJAM, et al., (2022). Stem Cell Res Ther, 13(1):437; Schaniel C, et al., (2021). Stem Cell Reports, 16(12):3036-3049; Cruvinel E, et al., (2020). SAGE open medicine, 8: 2050312120966456; Biagi D, et al., (2021). Journal of personalized medicine, 11(5):374.

[0241] FIG. 4 depicts the complexity of the cardiac models.

[0242] FIG. 3 depicts how the plates are made that are not embedded with batteries, but instead are powered by a standalone simulator that works wirelessly.

[0243] METHODS

[0244] hiPSC Reprogramming:

[0245] From skin fibroblasts (integration -free, standardized pipelines), as seen in Dariolli R*. et al. “A Large-Scale, Standardized, and Cost-Effective Protocol to Generate Integration-Free Human iPSCs From Skin Biopsies.” Stem Cell Reports (2021).

[0246] From peripheral blood erythroblasts, as seen in Oliveira TGM. et al. “Human Erythroblasts as a Source for iPSC Reprogramming and Downstream Applications.” Frontiers in Cellular and Infection Microbiology (2022).

[0247] From urine progenitor cells (UPCs) isolated from human urine, as seen in Dariolli R*. et al. “Generation of Human iPSCs from Urine Progenitor Cells.” Current Protocols in Human Genetics (2017).

[0248] From commercially sourced hiPSC lines, as seen in multiple vendors, supply well-characterized, consented hiPSC lines (e.g., FUJIFILM Cellular Dynamics, WiCell, ATCC, Coriell). These may be used directly with the maintenance conditions below.

[0249] hiPSC maintenance (feeder-free; single-cell passaging):

[0250] Maintain human iPSCs in mTeSR™ (StemCell Technologies) + 1% penicillinstreptomycin + 2.0-2.5 pg / mL Plasmocin™

[0251] Passage as single cells using 0.25 mM EDTA (Versene™) for 5 min at 37 °C; re-seed 2xl04-4><104cells / cm2on Geltrex™ / Vitronectin-N in mTeSR™ + ROCK-i (Y-27632) 5 pM for 12-24 h.

[0252] Expand to -80-90% confluence for lineage inductions (Cruvinel, Estela et al. (2020). SAGE open medicine, 8: 2050312120966456).

[0253] Lineage differentiation (2D monolayer): hiPSC- Cardiomyocytes (CMs)

[0254] Mesoderm: RPMI + B27 minus insulin + CHIR99021 6-9 pM + 1% P / S + 2.0-2.5 pg / mL Plasmocin™.

[0255] Cardiac induction: RPMI + B27 minus insulin + Wnt-C592 pM, or XAV9392.5 pM + KY02111 2.5 pM + 1% P / S + 2.0-2.5 pg / mL Plasmocin™.

[0256] RB(-): RPMI + B27 minus insulin (+ antibiotics / Plasmocin™).

[0257] RB(+): RPMI + B27 plus insulin (+ antibiotics / Plasmocin™).

[0258] Selection: RPMI minus glucose (+ antibiotics / Plasmocin™).

[0259] CM re-plating: RPMI + B27 plus + 10% KOSR or FBS + ROCK-i 10 pM (+antibi oti cs / Pl asmocin™) .

[0260] CM expansion: RPMI + B27 plus + CHIR99021 2 pM (+ antibiotics / Plasmocin™).

[0261] Procedure:

[0262] Mesoderm (Day 0— >1): Mesoderm medium (1.5x vol), 24 h.

[0263] 3.1.2 Early transition (Day 1— >2): + lxvol RB(-), 24 h.

[0264] 3.1.3 Late mesoderm (Day 2— >3): + lxvol RB(-), 24 h.

[0265] 3.1.4 Cardiac specification (Day 3— >5): replace with Cardiac-induction (L5x), 48 h.

[0266] 3.1.5 Early maturation (Day 5— >7): full replace with RB(-) on Days 5 & 6 (1.5x, q24h).

[0267] 3.1.6 Late maturation (Day 7— >9): RB(+) on Days 7 & 8 (1.5*, q24h).

[0268] 3.1.7 Metabolic selection (Day 9— >11): wash x2 with Selection; add 0.5-0.7x vol; 48 h.

[0269] 3.1.8 Expansion (>Day 11): Re-plate with TrypLE; seed 2.5xl04cells / cm2in CM replating; after 24 h switch to CM expansion; feed q48h; passage at 70-80% confluence; withdraw CHIR to arrest proliferation (FIG. 8, FIG. 9, FIG. 10, FIG. 14, FIG. 15, FIG. 16, FIG. 17).

[0270] EXAMPLE 3- 3D CARDIOSPHERE ASSEMBLY (SINGLE 96-WELL;AUTOMATED FULL EXCHANGES).

[0271] Plate preconditioning & medium

[0272] Before cell seeding, each well of the conical, flat-bottom, cell-repellent 96-well plate (working volume 90-125 pL) is pre-conditioned with 45 pL of cardiomyocyte medium without ROCK inhibitor. Immediately after dispensing, the plate is centrifuged at 150*g for 3-5 minutes to ensure the medium settles at the bottom and to remove air bubbles from the conical surface.

[0273] The cardiomyocyte medium consists of RPMI 1640 supplemented with B27 (Thermo Fisher Scientific).

[0274] Cell mixture

[0275] Human iPSC-derived cardiomyocytes are used as the sole cell type for spheroid formation. Cells are suspended in 45 pL of RPMI + B27 containing 5 pM ROCK inhibitor (Y-27632).

[0276] When this cell suspension (45 pL) is added to the 45 pL of pre-conditioning medium already present in the well, the final well volume (90 pL) reaches a final concentration of 5 pM ROCK inhibitor.

[0277] ROCK-i is included only during the first 12-24 hours to promote survival and uniform aggregation.

[0278] Validated cell seeding densities range from 2.5 x io3(2.5 K) to 2 x io4(20 K) cells per well, with typical standardizations at 2.5 K, 5 K, 10 K, and 20 K cells / well. These densities consistently yield reproducible spheroids of defined diameters and viability, as demonstrated in FIG. 10.

[0279] Dispense 90 pL per well centrifuge 150 g for 3-5 min incubate overnight (12-16 h) at a 45° angle to facilitate cell aggregation.

[0280] Automated medium exchanges are performed using INTEGRA ASSIST PLUS equipped with VOYAGER (5-125 pL) pipettes controlled via VIALAB.

[0281] Conduct full-volume medium exchanges every 48-72 hours, removing ROCK-i after day 1. Manuals can be found at Integra 128950 V01, and 128951 V09 OI VIALAB. docx.

[0282] Qc timeline as days 0-2: fusion; days 3-7: consolidation (0.5-1.0 mm spheroids); days 8-15: pre-assay maturation.

[0283] ASSAY SUB-SOPs (Day 10-15):

[0284] Ca2+imaging: Fluo-4 AM 2-5 pM (Pluronic 0.02-0.05%) 20-40 min; or Cal-520 AM 5 pM 30-45 min; 50-200 fps; optional 0.5-1 Hz pacing. Manual can be found at ThermoFisher: THERMO FISHER MP 01240, Cal-520® AM, and Djemai M, et al., (2023). Cells, 12(17): 2168.

[0285] Voltage mapping (optical): FluoVolt 1:1000 + PowerLoad 1:100; 15-30 min RT; 200-1000 fps; APD50 / 90, CV. Manual: FluoVoltTM Membrane Potential Kit Catalog Number F10488.

[0286] Seahorse XF Mito Stress (96-well): Oligomycin — > FCCP Rot / AA; start at ~1.0, 0.5- 1.5, 0.5 / 0.5 gM; equilibrate 45-60 min, no CO2. Manual: Agilent User Guide Kit 103015-10.

[0287] Motion / contractility: Brightfield 100-200 fps; quantify beat rate, amplitude, kinetics (see primer above).

[0288] Assembly:

[0289] Provide a conical, flat-bottom, cell-repellent 96-well plate (working volume 90-125 gL).

[0290] Pre-condition each well with 45 gL of RPMI 1640 + B27 (Thermo Fisher Scientific) without ROCK inhibitor; centrifuge ~150xg for 3-5 min to remove air bubbles from the conical bottom.

[0291] Provide pre-differentiated human iPSC-derived cardiomyocytes (CMs) as the sole cell type.

[0292] Prepare a 45 gL cell suspension in RPMI + B27 containing 5 pM ROCK inhibitor (Y-27632).

[0293] Add this suspension to the pre-conditioned wells to reach a final 90 pL volume with 5 pMROCK-i.

[0294] Use 2.5 K - 20 K cells per well (validated range: 2.5 K, 5 K, 10 K, 20 K).

[0295] Centrifuge ~150*g for 3-5 min.

[0296] Incubate overnight (12—16 h) at -45° to promote aggregation.

[0297] Maintain cultures in the same plate; perform full-volume exchanges every 48-72 h using INTEGRA ASSIST PLUS + VOYAGER; remove ROCK-i after 12-24 h (first exchange).

[0298] By Days 3-7, verify 0.5-1.0 mm spheroids; by Days 10-15, perform Ca2+, voltage, OCR, and motion assays as described (FIG. 5, FIG. 9, FIG. 10, FIG. 14, FIG. 15, FIG. 16, FIG. 17).

[0299] EXAMPLE 4- ADDITIONAL 3D CARDIOSPHERES ASSEMBLY (AGAROSE MICROWELL MOULDS).

[0300] Preparation of Agarose Microwell MouldsAgarose microwell molds are prepared by casting 1.5% (w / v) agarose in sterile phosphate-buffered saline (PBS) using microtissue printing molds. After solidification at room temperature, the agarose gels are removed from the molds and placed into standard culture plates. Each gel is pre-equilibrated with cardiomyocyte maintenance medium (RPMI 1640 + B27, Thermo Fisher Scientific) for up to 2 hours prior to seeding to ensure isotonic balance and eliminate air bubbles from the microwell cavities.

[0301] Cell Preparation and Seeding

[0302] Human iPSC-derived cardiomyocytes (hiPSC-CMs) are harvested from 2D cultures using TrypLE lx, counted, and resuspended in RPMI 1640 + B27 supplemented with 5 pM ROCK inhibitor (Y-27632).

[0303] The cell suspension is dispensed uniformly over the agarose microwells, allowing cells to passively settle by gravity into the cavities. The total number of cells added is adjusted to control the target spheroid diameter, typically producing cardiospheres of approximately 150 pm or 300 pm.

[0304] ROCK inhibitor is present only during the first 12-24 hours to promote cell viability and compact spheroid formation.

[0305] Aggregation and IncubationAfter seeding, the gels are incubated at 37 °C with 5 % CO2 to allow spontaneous aggregation of the cells within each microwell. No centrifugation step is performed.Within 24 hours, compact spheroids form as cells accommodate and contract into the microwell spaces, resulting in a measurable reduction in spheroid diameter.

[0306] Culture MaintenanceAfter the aggregation phase, the medium is replaced with RPMI + B27 without ROCK inhibitor, and the spheroids remain within the agarose gels throughout culture. Full-volume medium exchanges are performed every 48-72 hours, either manually or using automated pipetting systems such as INTEGRA ASSIST PLUS with VOYAGER (1250 pL) operated via VIALAB software.

[0307] Quality Control TimelineDays 0-1: aggregation and compacting phase;Days 2-4: consolidation phase (150-300 pm spheroids);Days 5-7: structural stability phase monitored by CFDA-SE fluorescence imaging at 0 h, 24 h, 72 h, and 96 h to confirm compaction and viability.Days 8-15: post-compaction maintenance phase; cardiospheres remain stable for downstream analyses or storage.

[0308] 3D Cardiospheres produced by this method reflect in FIG. 8.

[0309] EXAMPLE 5- CARDIOSPHERE COMPACTION MONITORING USING CFDA-SE LABELING.

[0310] Cell Preparation and LabelingHuman iPSC-derived cardiomyocytes (hiPSC-CMs) are harvested from 2D culture using TrypLE lx, washed with phosphate-buffered saline (PBS), and resuspended in serum-free RPMI 1640. Cells are incubated with CFDA-SE (Carboxyfluorescein diacetate succinimidyl ester) at a final concentration of 5 pM for 10 minutes at 37 °C. The dye passively diffuses into the cytoplasm and becomes fluorescent after intracellular ester hydrolysis.After incubation, cells are washed twice with RPMI + B27 (Thermo Fisher Scientific) to remove excess dye, then resuspended in fresh RPMI + B27 containing 5 pM ROCK inhibitor (Y-27632) for seeding.

[0311] Agarose Mold Preparation and Cell Seeding

[0312] Agarose microwell molds (1.5 % w / v in PBS; Microtissues #12-256) are prepared as described in Section 4 and pre-equilibrated with maintenance medium for 1-2 hours before seeding.

[0313] The labeled hiPSC-CM suspension is dispensed evenly over the agarose molds and allowed to passively settle by gravity into the microwells.

[0314] The total number of cells added is adjusted to produce cardiospheres of approximately 150 pm or 300 pm in diameter, corresponding to the mold cavity geometry. ROCK inhibitor is present only during the first 12-24 hours to enhance cell viability and compaction.

[0315] 5.3 Aggregation and Imaging Timeline

[0316] The gels are incubated at 37 °C with 5 % CO2 and monitored over time without centrifugation or mechanical force:

[0317] 0 h (immediately after seeding): cells are distributed uniformly within the microwells.

[0318] 24 h: initial compaction phase; cardiospheres become denser and reduce in diameter.

[0319] 72 h: consolidation phase; structures reach stable spherical morphology.

[0320] 96 h: mature, compact cardiospheres maintain structural integrity and fluorescence.

[0321] Imaging and analysis:

[0322] Fluorescence and bright-field images are captured at each time point (excitation 492 nm, emission 517 nm). Spheroid diameters are quantified using calibrated image-analysis software. The progressive reduction in diameter from 0 — > 24 — > 72 96 hours confirms cell accommodation and 3D compaction. CFDA-SE signal stability indicates preserved cell viability throughout the process.

[0323] Assembly for agarose microwell system:

[0324] Provide agarose microwell molds (1.5% w / v in PBS; Microtissues™ #12-256).

[0325] Pre-equilibrate the gels with RPMI 1640 + B27 (Thermo Fisher Scientific) for 1-2 hours before cell seeding.

[0326] Provide pre-differentiated human iPSC-derived cardiomyocytes (CMs) as the sole cell type.

[0327] Harvest the cells using TrypLE lx and label them, if required, with CFDA-SE (5 pM, 10 min, 37 °C).

[0328] Resuspend the cells in RPMI + B27 containing 5 pM ROCK inhibitor (Y-27632) and dispense evenly over the agarose microwells.

[0329] Allow passive gravity-driven settling of the cells into the cavities; adjust the total number of cells seeded to obtain cardiospheres of approximately 150 pm or 300 pm in diameter.

[0330] Incubate at 37 °C, 5 % CO2 for aggregation; no centrifugation or mechanical force is applied.

[0331] Maintain ROCK inhibitor only during the first 12-24 hours.

[0332] Replace the medium with RPMI + B27 (no ROCK-i) and maintain spheroids in the same gels.

[0333] Perform full-volume exchanges every 48-72 hours manually or using INTEGRA ASSIST PLUS + VOYAGER automation.

[0334] Monitor spheroid compaction and stability by CFDA-SE fluorescence imaging at 0 h, 24 h, 72 h, and 96 h.

[0335] Reduction in diameter confirms normal tissue accommodation and viability.

[0336] Maintain compact spheroids through Days 5-15 as structurally stable 3D constructs suitable for storage or downstream assays.

[0337] 3D Cardiospheres produced by this method reflect in FIG. 8, specifically FIG. 8F.

[0338] EXAMPLE 6- CARDIAC ASSEMBLOIDS

[0339] Relevant protocol details:Dimension Kahn-Kreil 2022 (meets all constraints): Lineages CM + EC + SMC + CF (all pre-differentiated); RatioCM:EC:SMC:CF = 4:2:1: 1, reflecting human myocardium proportions (CM predominant; EC largest non-myocyte population); Per-spheroid Uses -1.0x10A5 CMs as the CM “anchor”; EC / SMC / CF added per 4:2: 1 : 1 i input (example) proportion; assembled in 96-well ULA U-bottom plates for 7 days to fuse;then transfer to ULA 6-well on orbital shaker for long culture: Matrix Scaffold-free (no Matrigel), chemically defined media: Culture Differentiate each lineage — assemble as close to dD12 for CMs (after timeline metabolic selection) — > fuse 7 d in 96-well — » maintain up to -60 dayswith periodic media changes; Readouts Purity by flow cytometry; structural maturation; survival / apoptosis;: shown bioenergetic changes; general functional maturation benefits of 4-lineage vs CM-only (assay menu can be extended, see §4).

[0340] The schematic of this example can be seen in FIG. 6.

[0341] hiPSC Reprogramming (Refer to Example 2).

[0342] hiPSC maintenance (feeder-free; single-cell passaging):

[0343] Maintain human iPSCs in mTeSR™ (StemCell Technologies) + 1% penicillinstreptomycin + 2.0-2.5 pg / mL Plasmocin™

[0344] Passage as single cells using 0.25 mM EDTA (Versene™) for 5 min at 37 °C; re-seed 2xl04-4><104cells / cm2on Geltrex™ / Vitronectin-N in mTeSR™ + ROCK-i (Y-27632) 5 pM for 12-24 h.

[0345] Expand to -80-90% confluence for lineage inductions.

[0346] Lineage differentiations (2D monolayer) hiPSC-> cardiomyocytes (CMs)

[0347] Mesoderm: RPMI + B27 minus insulin + CHIR99021 6-9 pM + 1% P / S + 2.0-2.5 pg / mL Plasmocin™.

[0348] Cardiac induction: RPMT + B27 minus insulin + Wnt-C592 pM, or XAV9392.5 pM + KY02111 2.5 pM + 1% P / S + 2.0-2.5 pg / mL Plasmocin™.

[0349] RB(-): RPMI + B27 minus insulin (+ antibiotics / Plasmocin™).

[0350] RB(+): RPMI + B27 plus insulin (+ antibiotics / Plasmocin™).

[0351] Selection: RPMI minus glucose (+ antibiotics / Plasmocin™).

[0352] CM re-plating: RPMI + B27 plus + 10% KOSR or FBS + ROCK-i 10 pM (+ antibi oti cs / Pl asmoci n™) .

[0353] CM expansion: RPMI + B27 plus + CHIR99021 2 pM (+ antibiotics / Plasmocin™).

[0354] Procedure:

[0355] Mesoderm (Day 0— >1): Mesoderm medium (1.5x vol), 24 h.

[0356] Early transition (Day 1— >2): + lx vol RB(-), 24 h.

[0357] Late mesoderm (Day 2— >3): + lx vol RB(-), 24 h.

[0358] Cardiac specification (Day 3— >5): replace with Cardiac-induction (1.5x), 48 h.

[0359] Early maturation (Day 5— 7): full replace with RB(-) on Days 5 & 6 (1.5x, q24h).

[0360] Late maturation (Day 7— >9): RB(+) on Days 7 & 8 (1.5 x;q24h).

[0361] Metabolic selection (Day 9— >11): wash x2 with Selection; add 0.5-0.7x vol; 48 h.

[0362] Expansion (>Day 11): Re-plate with TrypLE; seed 2.5x 104cells / cm2in CM re-plating; after 24 h, switch to CM expansion; feed q48h; passage at 70-80% confluence; withdraw CHIR to arrest proliferation (Munos JJAM, et al., (2022). Stem Cell Res Ther, 13 ( 1 ) :437; Biagi D, et al., (2021). Journal of personalized medicine, 11 (5):374).

[0363] hiPSC- endothelial cells (ECs)

[0364] Media:

[0365] Maintenance: E8:mTeSR= 1:1 (v / v).

[0366] Mesoderm pre-conditioning: RPMI + B27 minus + CHIR 6 pM + Geltrex 1 :60 (v / v).

[0367] Basal- 1 : RPMI + B27 minus + CHIR 2 pM.

[0368] SP34+Supp: StemPro 34 SFM + L-Gln 2 mM, Ascorbic acid 50 pg / mL, BMP4 10 ng / mL, bFGF 5 ng / mL, VEGF165300 ng / mL, 1 -thioglycerol 4x |04M.

[0369] SP34+ROCK-i: SP34+Supp + ROCK-i 10 pM.

[0370] EGM-2+CHIR: EGM-2 (Lonza CC-3162) + CHIR 1 pM.

[0371] Procedure:

[0372] Pre-conditioning (Days -2— >0): E8 / mTeSR to 80-90% confluence; Versene passages; reseed 2-6x 104cells / cm2in E8 / mTeSR + ROCK-i 5 pM; 24 h.

[0373] Mesoderm pre-conditioning (Day 0— >~I6 h): Mesoderm pre-conditioning (lx), 16 h.

[0374] Basal transition (Day 1.7— >2.7): Basal-1 (lx), 24 h.

[0375] EC specification (Day 2.7^5.7): SP34+Supp (2x), 72 h.

[0376] ROCK-i stabilization (Day 5.7^5.75): SP34+ROCK-i (lx), 2 h.

[0377] Plating (Day 5.75): Accutase 5-7 min; seed ~9xl03cells / cm2in EGM-2+CHIR (no ROCK-i); 24 h.

[0378] Expansion (Day 7^12): EGM-2+CHIR daily to Day 12; then EGM-2 (no CHIR) q48h (Raniga K, et al., (2022). Methods in Molecular Biology, 2441).

[0379] StemPro 34 composition can be found in US20100297090A1

[0380] hiPSC- Cardiac fibroblasts (CFs)

[0381] Media and additives (ranges):

[0382] Basal: RPMI + B27 minus (early) RPMI + B27 plus (later) or serum-free fibroblast medium.

[0383] CHIR 5-10 pM, BMP45-50 ng / mL, bFGF 5-40 ng / mL, FGF10 5-20 ng / mL, Retinoic acid 0.1-1 pM, TGF-pi 0.5-1 ng / mL, optional Angll 0.5-1 pM.

[0384] Procedure:

[0385] Mesoderm / CP (Day 0^2-3): RPMI + B27(-) + CHIR 6-12 pM + BMP4 10-25 ng / mL ~24 h; then basal RPMI + B27(-) to Day 2-3.

[0386] SHF / Epi cardial bias (Day 2-3 >7): bFGF 10-20 ng / mL ± FGF10 10 ng / mL + RA 0.5 pM; feed q48h; confirm ISL1, TBX18.

[0387] Fibroblast specification (Day 7^14): RPMI + B27(+ ) + TGF-pl 0.5-1 ng / mL ± Angll 0.5-1 pM; feed q48h.

[0388] Expansion (Day 14— >21 ): Re-plate on TC plastic; expand 1-3 passages; ROCK-i 5 pM for 12-24 h post-passage; optional cry opreserve Day 21.

[0389] QC: DDR2+ / TE-7+ / VIM+ / COLlAl+(>80%); inducible a-SMA after TGF-pi; anti-fibrotic response (Zhang J, et al., (2019). Nat Commun, 10:2238; Whitehead AJ, et al., (2021). J Mol Cell Cardiol, 164:58-68).

[0390] EXAMPLE 7- 3D CARDIAC ASSEMBLOID ASSEMBLY (SINGLE 96-WELL; AUTOMATED FULL EXCHANGES)

[0391] The schematic of this example can be seen in FIG. 6.

[0392] Plate & medium: Conical, flat-bottom, cell-repellent 96-well (working vol 90-125 pL); spheroid medium per Khan-Kreil A, et al., (2022). Front Bioeng.

[0393] Cell mixture: CM:EC:SMC:CF = 4:2: 1:1; add ROCK-i 5 pM during seeding (first 12-24 h) (Knight WE, et al., (2021). STAR Protoc, 2(4): 100912; Emre N, et al., (2010). PLoS One, 5(8): el2148).

[0394] Dispense / Spin / Angle: 90 pL / well; 150*g 3-5 min; 45° overnight (12-16 h).

[0395] 4.4 Static culture & automation: INTEGRA ASSIST PLUS + VOYAGER (5-125 pL) in VIALAB; full-volume exchanges q48-72 h; day-1 remove ROCK-I (Integra 128950_V01; Integra 12895 l_V09_OI_VIALAB.docx).

[0396] QC timeline: Days 0-2 fusion; Days 3-7 consolidation (0.5—1.0 mm); Days 8-15 preassay maturation (Kahn-Kreil 2022).

[0397] The ratios are within (50— 75):(15— 30):(5— 15):(5— 15); spin 100-200*g 3-10 min; angle 30-60° 8-20 h; working volume 90-125 pL; exchanges q24-96 h; ROCK-i 2-10 pM only at seeding.

[0398] ASSAY SUB-SOPs (Day 10-15)

[0399] Ca2+imaging: Fluo-4 AM 2-5 pM (Pluronic 0.02-0.05%) 20-40 min; or Cal-520 AM 5 pM 30-45 min; 50-200 fps; optional 0.5-1 Hz pacing.

[0400] Voltage mapping (optical): FluoVolt 1:1000 + PowerLoad 1:100; 15-30 min RT; 200-1000 fps; APD50 / 90, CV.

[0401] Seahorse XF Mito Stress (96-well): Oligomycin — FCCP —>■ Rot / AA; start at —1.0, 0.5-1.5, 0.5 / 0.5 pM; equilibrate 45-60 min, no CO2.

[0402] Motion / contractility: Brightfield 100-200 fps; quantify beat rate, amplitude, kinetics (see primer above).

[0403] Assembly:

[0404] Provide a conical, flat-bottom, cell-repellent 96-well plate (90-125 pL); provide spheroid medium per Kahn-Kreil 2022. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC9361017 /

[0405] Provide pre-differentiated CM, EC, SMC, CF; combine 4:2: 1:1.

[0406] Add ROCK-i 5 pM at seeding; 90 pL / well (scaffold-free).

[0407] Centrifuge ~150*g, 3-5 min.

[0408] Incubate overnight at -45°.

[0409] Maintain culture in the same plate; perform full-volume exchanges q48-72 h using ASSIST PLUS + VOYAGER; remove ROCK-i at first exchange (12-24 h).

[0410] By Days 3-7, verify 0.5-1.0 mm spheroids; Days 10-15 run Ca2+ / voltage / OCR / motion assays as above.

[0411] EXAMPLE 8-GENERATION AND CHARACTERIZATION OF CARDIOMESODERM 3D SPHEROIDS AND MATURE 3D CARDIAC ORGANOIDS USING AGAROSE MICROWELL MOULDS.

[0412] Differentiators and workflow:

[0413] Two-phase workflow: 2D cardiomesoderm induction (CHIR99021 + BMP4 in RB(-)) — > 3D self-assembly in 1.5 % agarose microwells + 2 pM Wnt-C59.

[0414] (Microtissues™ molds #12-256 - https: / / www.microtissues.com / ; Wnt-C59 -https: / / www.selleckchem.com / products / wnt-c59.html)

[0415] Scaffold-free, gravity -settled aggregation yielding -150 pm and -300 pm spheroids.

[0416] Daily imaging (0 h, 24 h, 48 h, 72 h, 96 h) for compaction kinetics.

[0417] Non-destructive viability metric: cumulative L-lactate vs 2D controls (Sigma MAK064 / BioVision K607).

[0418] Extended maturation: from Day 8 onward, organoids are maintained for up to 45 days using a Lewis-Israeli-style medium formulation to obtain fully mature 3D cardiac organoids.

[0419] Optional surface functionalization: Fe-Au nanoparticle (Fe-Au NP) coating at Day 30 for bioconjugation tests.

[0420] Main results (As seen in FIG. 11, FIG. 13):

[0421] Homogeneous 150 pm and 300 pm spheroids generated reproducibly.

[0422] Controlled diameter reduction (0-96 h) consistent with tissue compaction; no lactate accumulation vs 2D controls (FIG. 11).

[0423] Long-term culture under Lewis-Israeli medium (through Day 45) supports structural stability and spontaneous beating (FIG. 13).

[0424] Fe-Au NP coating (1:500 - 1:5000 v / v) shows > 95 % cell survival by MTT assay, confirming cytocompatibility (FIG. 13).

[0425] hiPSC Reprogramming (Refer to Example 2).

[0426] hiPSC MAINTENANCE (feeder-free; single-cell passaging):

[0427] Maintain human iPSCs in mTeSR™ (StemCell Technologies) + 1% penicillinstreptomycin + 2.0-2.5 pg / mL Plasmocin™.

[0428] Passage as single cells using 0.25 mM EDTA (Versene™) for 5 min at 37 °C; re-seed 2x 104-4x 104cells / cm2on Geltrex™ / Vitronectin-N in mTeSR™ + ROCK-i (Y-27632) 5 pM for 12-24 h.

[0429] Expand to -80-90% confluence for lineage inductions.

[0430] Cardiomesoderm 3D spheroid generation:

[0431] Two-phase workflow overview:

[0432] This process includes (i) a 2D cardiomesoderm induction phase, followed by (ii) 3D aggregation within agarose microwells. Organoid diameter and morphology are monitored daily for four days, and lactate accumulation in the spent medium is used as a surrogate for viability and metabolic stress.

[0433] 2D Cardiomesoderm Induction:

[0434] Seeding and Maintenance

[0435] Human induced pluripotent stem cells (hiPSCs) are seeded onto GeltrexO-coated six-well plates at approximately 60 % confluence and maintained in mTeSR™ medium until full confluence (-4 days).

[0436] Mesoderm Induction - Day 0 — Day 1

[0437] At full confluence (Day 0), mTeSR™ is replaced with RPMI 1640 + B27 minus insulin [RB(-)] supplemented with CHIR99021 (6-9 pM) for 24 h to initiate mesoderm differentiation.

[0438] Cardiomesoderm Specification - Day 1 — Day 3

[0439] On Day 1, replace medium with RB(-) + CHIR99021 (6-9 pM) + BMP4 (10 nM) for another 24 h to reinforce cardiac mesoderm identity. Cells are maintained until Day 3, at which point they are dissociated for 3D aggregation.

[0440] (CHIR99021 and BMP 4 concentrations follow standard cardiomesoderm differentiation practices; see Lian et al, Nat Protoc 2013 and R&D Systems BMP 4 datasheet.)

[0441] The 3D hiPSC-derived Cardiac Organoids production Workflow can be seen in FIG. 7.

[0442] 3D Aggregation in Agarose Microwells:

[0443] Mould Preparation

[0444] Agarose microwell moulds (1.5 % w / v agarose in PBS; Microtissues™ #12-256) are cast and allowed to solidify at room temperature. Each gel is placed into a standard culture plate and pre-equilibrated for 1-2 h with RB(-) medium + Wnt-C59 (2 pM) to remove trapped air and balance osmolarity.

[0445] Cell Harvesting and Seeding

[0446] Day-3 cardiomesoderm cells are dissociated with TrypLE Express, counted, and resuspended in the same RB(-) + Wnt-C59 (2 pM) medium.

[0447] The suspension is dispensed uniformly over the agarose gels and allowed to settle passively by gravity into the microwells.

[0448] The number of cells loaded is adjusted to yield cardiomesoderm spheroids of approximately 150 pm or 300 pm in diameter, depending on mould geometry.

[0449] Aggregation and Incubation

[0450] Cultures are maintained at 37 °C, 5 % CO2 without centrifugation or mechanical compression.

[0451] Spheroids self-assemble within 24 h, compact further by 72 h, and reach stable structure by 96 h.

[0452] Morphological and Viability Assessment:

[0453] Diameter Quantification

[0454] Bright-field micrographs are analyzed using calibrated image-analysis software. A moderate, statistically significant diameter reduction is typically observed over four days in both 150 pm and 300 pm spheroids, representing normal compaction behavior.

[0455] Imaging is performed at 0 h, 24 h, 48 h, 72 h, 96 h to track diameter evolution.

[0456] Lactate-Based Viability Measurement

[0457] At each daily medium exchange, spent medium is collected for L-lactate quantification using a colorimetric / fluorometric assay kit (e.g., Sigma-Aldrich MAK064 or BioVision K607).

[0458] Parallel 2D controls are maintained at low (10,000 cells / well) and high (1,000,000 cells / well) confluence to simulate 3D cell densities.

[0459] Cumulative lactate release over four days is compared between 2D and 3D cultures.

[0460] No significant lactate increase and the absence of morphological degeneration confirm that 3D aggregation preserves viability at both organoid sizes.

[0461] Statistical Analysisn > 3 biological replicates per condition; one-way ANOVA with post-hoc comparison; significance p < 0.05.

[0462] Extended Maturation and Fe-Au Nanoparticle Coating

[0463] Transition to Long-Term Culture (Days 8 - 45)\

[0464] Following initial 3D aggregation (Days 0-7), spheroids are maintained in RPMI + B27 with insulin [RB(+)] supplemented per Lewis-Israeli et al., 2021 for cardiac maturation:

[0465] Ascorbic acid 2-phosphate 50 pg / mL

[0466] IGF-1 100 ng / mL

[0467] T3 1 nM

[0468] Dexamethasone 100 nM

[0469] L-camitine 2 mM

[0470] Medium is replaced every 48-72 h. Organoids beat spontaneously by Day 12-15 and reach stable size (250-350 pm) by Day 30.

[0471] Fe-Au Nanoparticle Preparation:

[0472] Use iron-gold (Fe-Au) core-shell NPs (100-150 nm diameter) in sterile PBS.

[0473] Sonicate briefly and verify magnetic response with a magnetic rack (Fig. 12A).

[0474] Nanoparticle Coating Protocol (Day 30):

[0475] Transfer Day-30 organoids (-300 pm) into low-adhesion tubes with RB(+) medium containing Fe-Au NPs at 1:500, 1:750, 1:1000, 1:2000, 1:5000 (v / v).

[0476] Incubate 2 h at 37 °C, 5 % CO2 with gentle rotation.

[0477] Separate coated organoids magnetically (Fig. 12B-C) and wash twice in RB(+).

[0478] Return to fresh RB(+) for 24 h recovery before assays.

[0479] Viability Assessment (MTT Assay):

[0480] Prepare MTT stock (5 mg / mL in PBS, filter-sterilized).

[0481] Add 10 % (v / v) MTT solution to medium and incubate 3 h at 37 °C.

[0482] Dissolve formazan in DMSO (100 pL / well, 15 min shaking).

[0483] Measure absorbance at 570 nm (ref 630 nm).

[0484] Express viability as % of untreated control.

[0485] Expected results: > 95 % viability for 1:750-1:5000 ratios; no loss of beating activity (Fig. 12D).

[0486] Morphological Evaluation (Post-Coating):

[0487] Bright-field imaging at Day 45 shows smooth, compact spheroids comparable to controls.

[0488] Slight edge heterogeneity observed only in 150 pm organoids at high NP ratios (1:500).

[0489] Magnetic manipulation confirms uniform NP attachment without aggregation (Fig. 12E).

[0490] Assembly + NP coating:

[0491] Provide agarose microwell molds (1.5 % w / v in PBS; Microtissues™ #12-256) and preequilibrate with RB(-) + 2 pM Wnt-C59 for 1-2 h before seeding.

[0492] Provide hiPSCs at Day 3 cardiomesoderm stage (CHIR99021 + BMP4 induction).

[0493] Seed cells into microwells, allow gravity-driven settling — > spheroids (-150 pm / 300 pm).

[0494] Maintain 37 °C / 5 % CO2 without centrifugation for 96 h; replace RB(-) + Wnt-C59 daily.

[0495] Transition to RB(+) medium on Day 8 with Lewis-Israeli supplements for maturation through Day 45.

[0496] At Day 30, apply Fe-Au NPs (1 :500-l :5000 v / v) for 2 h, 37 °C; magnetically isolate and wash.

[0497] Assess viability by MTT (5 mg / mL stock; 3 h incubation; A570 nm).

[0498] Maintain > 95 % cell survival and preserved contractility through Day 45.

[0499] EXAMPLE 9- GENERATION AND CHARACTERIZATION OF HIPSC- DERIVED 3D CARDIAC ORGANOIDS USING MAGNETIC LEVITATION TECHNOLOGY.

[0500] Differentiators & Main Workflow Features

[0501] Two-phase workflow:

[0502] (i) 2D cardiomesoderm induction (CHIR99021 + BMP4 in RB(-));

[0503] (ii) 3D magnetic levitation using the n3D system (Greiner Bio-One) instead of agarose moulds.

[0504] Temporary magnetization of Day-3 mesoderm cells with biocompatible Fe-Au nanoparticles enables levitation-based aggregation.

[0505] Validated cell density range: 2.5 K-20 K cells per construct (same as validated for conical 96-well system), ensuring uniform organoid size and viability.

[0506] Wnt inhibition during levitation enhances cardiac specification.

[0507] Scaffold-free aggregation yields -400-500 pm spheroids that begin beating by Day 10-15 and synchronize by Day 45.

[0508] Extended culture in Lewis-Israeli maturation medium supports chamber-like morphology and expression of cTnl (TNNI3).

[0509] hiPSC Reprogramming (Refer to Example 2)

[0510] hiPSC maintenance (feeder-free)

[0511] Culture in mTeSR™ + 1 % pen / strep + 2 pg / mL Plasmocin™

[0512] Passage with 0.25 mM EDTA (Versene™) 5 min @ 37 °C; re-seed 2-4* 104cells / cm2on Geltrex™ or Vitronectin-N in mTeSR™ + ROCK-i (Y-27632) 5 pM for 12-24 h.

[0513] Expand to 90 % confluence before lineage induction.

[0514] 2D Cardiomesoderm Induction:

[0515] Seeding: hiPSCs plated -60 % confluence, cultured 4 days in mTeSR™ until confluent.

[0516] Mesoderm induction (Day 0-1): RPMI + B27 minus insulin [RB(-)] + CHIR99021 6-9 pM (24 h).

[0517] Cardiac mesoderm specification (Day 1-3): RB(-) + CHIR99021 6-9 pM + BMP4 10 nM (24 h).

[0518] Cells at Day 3 display mesodermal morphology and are ready for 3D assembly.

[0519] 3D Magnetic Levitation Aggregation (n3D System):

[0520] Magnetization of Mesoderm Cells (day 3)

[0521] Dissociate Day-3 cardiomesoderm cells with TrypLE Express.

[0522] Resuspend in RB(-) containing Fe-Au nanoparticles (final 1 : 1000 v / v).

[0523] Incubate 2 h at 37 °C to allow internalization; wash twice with fresh RB(-).

[0524] Levitation and Aggregation (Day 3-5)

[0525] Transfer magnetized cells into n3D levitation plates (Greiner Bio-One) with RB(-) + 2 pM Wnt-C59.

[0526] Apply magnetic driver for 16-24 h (37 °C, 5 % CO2) to initiate levitation-based aggregation.

[0527] Validated cell input range: 2.5 K, 5 K, 10 K, or 20 K cells per organoid (2.5><103-2.0x 104cells), representing the optimal densities previously validated for uniform aggregation in the coni cal -pl ate system.

[0528] One to four organoids can be formed per well depending on target diameter.

[0529] Aggregates form within 12-18 h and stabilize by 24 h without mechanical compression or scaffold.

[0530] Transition to Maturation Medium (Day 5 onward):

[0531] Transfer organoids to ultra-low-attachment plates.

[0532] Replace medium with RB(+) supplemented with ascorbic acid (50 pg / mL), IGF-1 (100 ng / mL), T3 (1 nM), dexamethasone (100 nM), and L-camitine (2 mM).

[0533] Medium exchange every 48-72 h.

[0534] Beating appears by Days 10-15 and becomes synchronized by Day 45.

[0535] Reference: Lewis-Israeli et al., 2021 (Nat Commun).

[0536] Histology and Immunofluorescence:

[0537] Histology (H&E):

[0538] Fix Day-45 organoids in 4 % paraformaldehyde.

[0539] Embed in paraffin, section at 2 pm, stain with hematoxylin and eosin.

[0540] Visualize internal cavities at 25* magnification (Fig. 13C).

[0541] Immunofluorescence (cTnI / TNNI3):

[0542] Permeabilize sections (0.2 % Triton X-100), block (5 % goat serum).

[0543] Primary antibody: anti-cTnl (1 :200; Thermo Fisher MA5-12960), overnight 4 °C.

[0544] Secondary: Alexa Fluor® (1 : 1000) + DAPI.

[0545] Image at 40*; detect sarcomeric pattern and nuclear organization (Fig. 13D).

[0546] Statistical and Quality Control:

[0547] n > 3 biological replicates per condition.

[0548] One-way ANOVA + Tukey post hoc (p < 0.05).

[0549] QC: sterility, viability > 95 %, absence of NP toxicity (MTT assay).

[0550] Assembly- Magnetic Levitation System:

[0551] Provide Day-3 mesoderm cells (CHIR99021 + BMP4).

[0552] Incubate with Fe-Au nanoparticles (1:1000 v / v, 2 h, 37 °C).

[0553] Seed 2.5 K-20 K cells per organoid in n3D levitation plates with RB(-) + Wnt-C592 pM.

[0554] Apply magnetic drivers 16-24 h (37 °C, 5 % CO2); no scaffold or molds.

[0555] Transfer aggregates to ultra-low-attachment plates in RB(+) + maturation supplements (ascorbic acid, IGF-1, T3, dexamethasone, L-camitine).

[0556] Replace medium q48-72 h until Day 45.

[0557] Beating initiates Day 10-15, synchronizes by Day 45.

[0558] Confirm chamber-like structure (H&E, 2 pm) and cTnl expression (40 x IF) (FIG. 12).

[0559] EXAMPLE 10- COMPARISON OF ULTRA-LOW-ATTACHMENT PLATE GEOMETRIES FOR CARDIOSPHERE FORMATION.

[0560] To test geometry effects on spontaneous 3D cardiac aggregation, human iPSC-derived cardiomyocytes were seeded into two ULA 96-well formats: (i) conical, flat-bottom wells (ANSI / SLAS-compatible) and (ii) tubular, U-bottom wells (e.g., Corning® #7007). Geometry (curvature / bottom shape) was hypothesized to alter aggregation dynamics, compaction, and reproducibility (FIG. 9).

[0561] hiPSC-cardiomyocytes were produced in 2D using team protocols (e.g., Biagi 2021; Munoz & Dariolli 2022). Cells were dissociated with TrypLE Express and resuspended in RPMI + B27 (with insulin) at 5x 104cells / mL.

[0562] Seeding and Aggregation Conditions:

[0563] Per-well input: 2.5K, 5K, 10K, or 20K cells in 100 pL (validated range used across assemblies).

[0564] Incubation: 37 °C, 5% CO2, static.

[0565] Monitoring: images at 4 h, 24 h, 48 h, 72 h; half-volume media refresh at 24 h and 48 h.

[0566] Morphological Assessment:

[0567] Bright-field microscopy at 40* (e.g., EVOS M7000).

[0568] Image analysis in MATLAB (custom script).

[0569] Metrics: Circularity = 47t area / perimeter2; Diameter = mean Feret (n=60 spheroids / geometry).

[0570] Stats: unpaired two-tailed t-test —> Circularity p = 7>107, Diameter p = 5* IO3.

[0571] Results summary:

[0572] Conical, flat-bottom wells: >95% single, compact spheroids; higher circularity; consistent diameters.

[0573] Tubular U-bottom wells: frequent micro-aggregates / fragmentation (see arrows), lower circularity; slightly larger but less compact constructs.

[0574] Interpretation & Functional Relevance: Conical geometry promotes gravity-assisted selfcentering, limiting wall adhesion and off-axis settling. This yields better integrity and reproducibility — critical for scaling autonomous conical-well plates with embeddedsti mul ati on / sensi ng .

[0575] Plate Geometry Optimization:

[0576] Provide ULA 96-well plates with conical, flat-bottom wells that promote singleaggregate formation of hiPSC-cardiomyocytes by gravity-driven self-centering.

[0577] Seed 2.5K-20K cells / well in 100 pL RPMI + B27 (insulin); incubate 24 h at 37 °C, 5% CO2, static.

[0578] Conical wells yield >95% single compact spheroids with significantly higher circularity than U-bottom wells (p = 7 / 107).

[0579] Mean diameters 350-500 pm with <10% CV and improved compactness vs U-bottom (p = 5*1CF3).

[0580] U-bottom wells produce multiple micro-aggregates and fragmented spheroids, reducing circularity and reproducibility.

[0581] The conical geometry enhances sedimentation symmetry, limiting multi -aggregate formation — supporting autonomous 3D culture plates with integrated circuits / electrodes.

[0582] Geometry is ANSI / SLAS-compatible and scalable to 96 / 128 / 384 / 1536-well formats without loss of fidelity.

[0583] Geometry, independent of coating chemistry, is the determinant of hiPSC-cardiosphere integrity and reproducibility — foundational to the claimed autonomous conical-well plate system.

[0584] EXAMPLE 11- COMPARATIVE VALIDATION OF 3D CARDIOSPHERES AND 2D MONOLAYERS FOR CIPA-BASED CARDIAC SAFETY SCREENING.

[0585] This validation study compares 3D Cardiospheres — generated in conical, flat-bottom ultra-low-attachment (ULA) 96-well plates — with traditional 2D monolayer hiPSC-cardiomyocyte cultures for predictive cardiotoxicity screening (FIG. 17). The results of the pilot study conducted in FIG. 16 introduced the parameters to conduct FIG. 17.

[0586] Testing followed the Comprehensive in Vitro Proarrhythmia (CiPA) framework, using a reference panel of high-, intermediate-, and low-risk drugs.

[0587] All voltage recordings were performed under spontaneous beating conditions to evaluate the intrinsic electrophysiological behavior of the models. No electrical field stimulation, pacing electrodes, or optogenetic triggers were applied. FIG. 14 represents the capacity to record the calcium transients and action potential voltages from 3D Cardiospheres generated using conical shaped plates.

[0588] Human iPSC-cardiomyocytes were generated according to Biagi et al., 2021 (J Pers Med) and Munoz & Dariolli et al., 2022 (Stem Cell Res Ther).

[0589] Cells were harvested on Day 15 of differentiation.

[0590] Dissociation: Try pLE Express lx, 5 min @ 37 °C.

[0591] Re-suspension: RPMI + B27 (+ insulin) at 5 x 104cells mL1

[0592] Plate Geometry and Seeding:

[0593] 3D format: Conical, flat-bottom ULA 96-well plates (90-125 pL working volume) seeded with 2.5 K-20 K cells per well in 90 pL medium.

[0594] 2D format: Flat, treated 96-well black plates seeded at 2 x io4cells cm2.

[0595] Both systems were maintained at 37 °C, 5 % CO2 for 48 h to establish spontaneous rhythmic beating before compound exposure.

[0596] Cells and spheroids were incubated with BeRST (Berkeley Red Sensor of Transmembrane Potential) at 1 pM for 10 min @ 37 °C (excitation / emission 625 / 660 nm).

[0597] After dye loading, cultures were gently rinsed with fresh medium.

[0598] Recordings were acquired using the Volta Scanner (Lumencor Inc.) — a wide-field optical system optimized for spontaneously beating preparations.

[0599] Sampling rate: 10 kHz (10,000 frames s ')

[0600] Duration: 20 s per well, simultaneous 96-well capture

[0601] Time points: 0 h (baseline), 1 h, and 24 h post-drug exposure

[0602] Stimulation: None applied. Recordings represent intrinsic depolarization-repolarization cycles of self-beating cardiac constructs.

[0603] This spontaneous-activity approach ensures that drug-induced changes in action-potential morphology or duration directly reflect native electrophysiological responses rather than artifacts from external pacing.

[0604] Compound Exposure (CiPA Test Set):

[0605] A curated CiPA drug panel was used to calibrate assay performance:High Risk Intermediate Risk Low Risk Azimilide, Bepridil,Astemizole, Chlorpromazine, Diltiazem, Loratadine,Dofetilide, Ibutilide, Cisapride, Clozapine, Metoprolol, Mexiletine, Quinidine, Vandelanib. Domperidone, Droperidol. Nitrendipine, Ranolazine, Disopyramide, D / L Sotalol Terfenadine, Pimozide, Tamoxifen, VerapamilRisperidone, Ondansetron

[0606] DMSO final = 0.2 % (v / v)

[0607] Concentrations: lx, 3*, and 10x Cmax equivalents

[0608] Exposure: acute (1 h) and chronic (24 h)

[0609] Feature extraction and analysis:

[0610] Action Potential Duration (APD):

[0611] From BeRST fluorescence waveforms, APD20, APD50, and APD90 were extracted.

[0612] Classification relative to DMSO control:

[0613] < 66 % = Shortened

[0614] 66-134 % = Normal

[0615] 135-195 % = Prolonged

[0616] 197 % = Significantly Prolonged

[0617] Performance Metrics

[0618] Sensitivity = % toxic compounds correctly identified (high / intermediate risk)

[0619] Specificity = % non-toxic (low-risk) compounds correctly classified

[0620] n > 3 biological replicates per compound x condition

[0621] Results Summary:

[0622] 3D Cardiospheres: 100 % sensitivity (high risk), 70-100 % (intermediate), specificity 100 % (1 h) / 62.5 % (24 h)

[0623] 2D Monolayers: 62.5 % sensitivity (1 h) / 37.5 % (24 h)

[0624] 3D format = enhanced signal -to-noise, stable frequency, and superior predictive accuracy for cardiotoxicity detection.

[0625] Optical Voltage Assay - Spontaneous Activity:

[0626] Provide 3D Cardiospheres (2.5 K-20 K cells per well) in conical, flat-bottom ULA plates.

[0627] Expose to CiPA reference drugs (0-10x Cmax) for 1 h and 24 h.

[0628] Load BeRST voltage dye (1 pM, 10 min, 37 °C); image at > 10 kHz with Volta Scanner.

[0629] Record under spontaneous beating conditions only — no electrical pacing applied.

[0630] Extract APD20 / 50 / 90 relative to vehicle control and classify prolongation per CiPA criteria.

[0631] 3D Cardiospheres show 100 % sensitivity for high-risk drugs and > 60 % specificity for low-risk compounds.

[0632] This workflow provides a non-invasive, high-throughput optical readout of intrinsic electrical activity for predictive cardiotoxicity screening.

[0633] EXAMPLE 12- COMPARATIVE EVALUATION AND RATIONALE FOR SELECTING THE CONICAL, FLAT-BOTTOM ULA PLATE SYSTEM.

[0634] A series of 3D cardiac assembly strategies were systematically tested to identify the most effective configuration for producing reproducible, homogeneous, and assay -ready cardiac organoids and assembloids.

[0635] These comparative studies included:

[0636] Agarose Microwell Molds

[0637] Nanoparticle- Assisted Aggregation

[0638] U-Bottom Ultra-Low-Attachment (ULA) PlatesConical, Flat-Bottom ULA Plates

[0639] Conical, Flat Bottom ULA Plates

[0640] The goal was to determine which system best supports the entire workflow — from cell aggregation and maturation to functional testing — within a single integrated platform.

[0641] This “one-stop-shop” capability defines the core innovation of the present invention.

[0642] Agarose Microwell Molds:

[0643] Strengths:

[0644] Capable of producing large batches of uniform spheroids (150-300 pm) with tunable geometry.

[0645] High control over cell number per spheroid through cavity-defined seeding.

[0646] Limitations:

[0647] Incompatible with embedded electrodes: the agarose substrate is non-conductive and not structurally adaptable for electrode integration.

[0648] Two-step workflow: spheroids must be manually transferred to another plate (e.g., 96-well) for maturation or testing, which breaks the automation and increases variability.

[0649] Poor optical properties: agarose interferes with high-resolution imaging (voltage, calcium, motion).

[0650] Not ANSI / SLAS compatible, thus limiting throughput and robotic automation.

[0651] While suitable for proof-of-concept aggregation, agarose molds cannot serve as a final testing platform and contradict the invention’s principle of performing aggregation, maturation, and assay within the same autonomous unit.

[0652] Nanoparticle- Assisted Aggregation (Fe-Au Coating):

[0653] Strengths:

[0654] Enables surface functionalization and magnetic manipulation.

[0655] Demonstrates biocompatibility at controlled concentrations (1:500 dilution, MTT viability confirmed).

[0656] Limitations:

[0657] High cost and complexity due to nanoparticle synthesis, magnetic handling, and surface adsorption steps.

[0658] Limited reproducibility, especially for smaller (150 pm) organoids.

[0659] Unnecessary for scalable screening where optical readouts and electrostimulation are the primary outputs.

[0660] Summary:

[0661] Although the method enables advanced functionalization, it adds cost and operational complexity incompatible with the invention’s goal of a standardized, cost-efficient, high-throughput platform.

[0662] Tubular U-Bottom ULA Plates:

[0663] Strengths:

[0664] Commonly used, low-cost system.

[0665] Supports gravitational aggregation without additional materials.

[0666] Limitations:

[0667] Irregular aggregation: formation of multiple micro-aggregates per well, fragmented spheroids, and inconsistent compaction.

[0668] Suboptimal geometry for imaging: curved U-bottom interferes with optical path and electrode alignment.

[0669] Poor integration potential: difficult to embed flat or radial electrode arrays in a curved base.

[0670] Low reproducibility: high variability in spheroid diameter and circularity (p = 7 x IO7, 5 x 10-3).

[0671] Summary:

[0672] While widely adopted, U-bottom plates cannot sustain the precision imaging or integrated sensing functions required by the invention’s electro-optical system.

[0673] Conical, Flat-Bottom ULA Plates (Chosen Platform):

[0674] Strengths:

[0675] High-throughput aggregation: 96-well ANSI / SLAS-compatible layout supports robotic handling and full automation.

[0676] Geometry-driven self-centering: conical well shape guides single-spheroid formation (>95% yield per well).

[0677] Flat optical surface: enables precise imaging of calcium flux, voltage, and contraction.

[0678] Electrode integration ready: the flat bottom allows stable embedding of electrodes, sensors, and autonomous stimulation circuits.

[0679] One-stop-shop platform: aggregation, maturation, and testing occur in the same physical unit, eliminating transfer steps and contamination risks.

[0680] Uniform size and reproducibility: controlled cell densities (2.5K-20K) yield consistent spheroids of 0.5-1.0 mm, ideal for standardization and predictive assays.

[0681] Validated in CiPA-compatible optical assays for action potential and calcium handling.

[0682] Summary:

[0683] The conical, flat-bottom ULA system uniquely fulfdls all design requirements of the invention:

[0684] Geometric precision for reproducible self-assembly,

[0685] Optical and electrical compatibility, and

[0686] Seamless integration into autonomous, electrode-embedded 3D culture plates.

[0687] This configuration transforms the plate into a closed, self-sufficient testing ecosystem — the essence of the “one-stop-shop” platform.Paiamefei \gaiose XP-Coated U-Bu(iom Conical Llat-Bufioni Miciowells Organoids ULA Plates ULA Rates (Chosen) Throughput Low Moderate High HighOptical Low Moderate Limited Excellent (flat Compatibility surface) Electrode Impossible Partial (external Limited Fully Compatible Integration magnetism)Automation \o . Partial Moderate Full ANSI / SLAS Morphological High Moderate Low High (>95 % yield) Uniformity (manual)Viability High Slightly variable Moderate High and stable Workflow Complex (2- Complex Simple Simplest (single- Simplicity step) step) Manufacturing Moderate High Low Optimal balanceCost

[0688] Across all tested 3D assembly strategies, the conical, flat-bottom ULA geometry provides the only configuration that:

[0689] Enables high-throughput, automation-compatible cardiac organoid and assembloid generation;

[0690] Maintains excellent optical and electrode compatibility;

[0691] Produces uniform, single spheroids per well with consistent diameter and morphology;

[0692] And supports a fully integrated “one-stop-shop” workflow for aggregation, maturation, and functional testing within the same plate.

[0693] The flat-bottom conical well thus represents the core enabling feature of the claimed invention, combining geometry-driven precision with scalability, making it the definitive structural foundation for next-generation autonomous 3D cardiac assay systems.

[0694] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments and examples, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.REFERENCES

[0001] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. “ANSI / SLAS Microplate Standards.” n.d. 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Claims

CLAIMSWe claim:

1. An apparatus for measurement of cardiac parameters comprising:a) a multi-well plate comprising one or more features selected from:a conical shape,adapted to comprise cell-repellent surfaces to prevent cell attachment,adapted to facilitate uniform organoid formation,adapted to have transparent well bottoms for real-time imaging of organoids and / or for minimizing optical distortions,adapted to be opaque to prevent light interference, andadapted to enhance fluorescence imaging;b) one or more electrodes for autonomous electrical stimulation of the organoids; and c) optionally one or more batteries within the plate providing continuous electrical stimulation;wherein the apparatus is compatible with high-throughput assays,wherein the apparatus is adapted to provide real-time measurement of cardiac parameters selected from calcium dynamics, action potential waveforms, and metabolic activity.

2. The apparatus of claim 1, wherein the electrical stimulation system comprises:a) a set of electrodes embedded into each well, adapted to deliver electrical pulses to the organoids continuously throughout the cultivation and testing period;b) one or more batteries integrated into the plate to provide continuous power, enabling independent operation of the electrical stimulation system without external connections; and c) a programmable electrical stimulation controller that delivers pulses at specified frequencies and intensities, mimicking natural heart electrophysiology for organoid maturation.

3. A method for cultivating and maturing human cardiac organoids (hCOs), the method comprising:a) generating human cardiac organoids from hiPSCs within conical, ultra-low attachment wells in a multi-well plate;b) applying continuous electrical stimulation to the organoids during the culturing process to promote maturation and the development of cardiac parameters selected from one ormore cardiac parameters selected from: calcium signaling, action potential formation, and structural protein expression;c) maintaining the organoids in the plate for growth and real-time functional assessment under physiological conditions that mimic the electrophysiological environment of a human heart.

4. A method for performing high-throughput cardiotoxicity testing of human cardiac organoids, comprising:a) cultivating human cardiac organoids in a multi-well plate adapted for compatibility with standard high-throughput screening systems;b) performing real-time imaging and functional assays selected from: calcium dynamics, action potential waveforms, and troponin-I integrity, within the same plate; andc) testing one or more drug compounds simultaneously by administering the drug compounds directly to the organoids within one or more wells in the multi-well plate, while continuously measuring the effects of the drug compounds on cardiac function.

5. An apparatus for real-time measurement of one or more types of human organoids comprising:a) a multi-well plate comprising one or more features selected from:wherein the plate is adapted to comprises one or more features selected from:wherein the well is conical,wherein the well is adapted to comprise cell-repellent surfaces to prevent cell attachment, wherein the well is adapted to facilitate uniform organoid formation,wherein the well is adapted to have transparent well bottoms for real-time imaging of organoids and / or for minimizing optical distortions,wherein the well of the plate is opaque to prevent light interference, andwherein the wells of the plate enhance fluorescence imaging;b) one or more electrodes for autonomous electrical stimulation of the organoids; and c) optionally one or more batteries within the plate providing continuous electrical stimulation;wherein the apparatus is compatible with high-throughput assays,wherein the human organoids is selected from: cardiac, adipose liver, brain, kidney, and vascular organoids.

6. The apparatus of claim 5, wherein the apparatus is adapted to measure one or more cardiac parameters selected from:e. electrophysiology parameters selected from: calcium transients, action potential waveforms, QTc prolongation;f. beating behavior selected from: rhythm, contractility, arrhythmia;g. metabolic stress selected from: oxygen consumption rate (OCR) for mitochondrial function;h. structural integrity selected from: troponin-I, cytotoxicity markers.

7. A method for cultivating and maturing one or more types of human organoids comprising: a) generating one or more types of human organoids selected from: cardiac, adipose, liver, brain, kidney, and vascular organoids from human iPSCs in a multi-well plate, wherein the human organoids are generated by protocols suitable for physiological properties of each respective type of human organoid;b) applying continuous electrical stimulation to the human organoids in each well throughout the process to promote maturation and the development of one or more parameters specific to each respective type of human organoid; andc) maintaining the each respective type of human organoid in the plate for long-term growth and real-time functional assessment under physiological conditions that mimic the electrophysiological environment of the each respective human organoid.

8. The method of claim 7, wherein the types of human organoids is selected from human cardiac organoids; wherein said human cardiac organoids are maintained on the plate for growth and real-time functional assessment under physiological conditions that mimic the electrophysiological environment of a human heart; and wherein the human cardiac organoids are evaluated for at least one cardiac parameters selected from: calcium signaling, action potential formation, and structural protein expression.

9. A method for performing high-throughput toxicity testing of one or more types of human organoids comprising:a) cultivating one or more types of human organoids each individually in one or more wells of a multi-well plate adapted for compatibility with standard high-throughput screening systems;b) performing real-time imaging and functional assays, to assay one or more physiological properties of each respective human organoid type, all within the same multi-well plate; andc) testing one or more drug compounds simultaneously by administering the drug compounds directly to the one or more types of human organoids within their respective wells, while continuously measuring the effects of said one or more compounds on one or more physiological properties of each respective human organoid function selected from: toxicity testing of the effects of the drug compounds on the respective types of human organoids selected from: cardiotoxicity, hepatotoxicity, neurotoxicity, and nephrotoxicity, in a high-throughput format.

10. The method claim 10, wherein the one or more types of human organoids is selected from: cardiac, adipose liver, brain, kidney, and vascular organoids.

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