Heart-on-a-Miniscope: A Miniaturized Solution for Electrophysiological Drug Screening in Cardiac Organoids

Abstract

Cardiovascular toxicity remains a primary concern in drug development, accounting for a significant portion of post-market drug withdrawals due to adverse reactions such as arrhythmias. Traditional preclinical models, predominantly based on animal cells, often fail to replicate human cardiac physiology accurately, complicating the prediction of drug-induced effects. Although human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a more genetically relevant system, their use in 2D, static cultures does not sufficiently mimic the dynamic, 3D environment of the human heart. 3D cardiac organoids made from human iPSC-CMs can potentially bridge this gap. However, most traditional electrophysiology assays, developed for single cells or 2D monolayers, are not readily adaptable to 3D organoids. This study uses optical calcium analysis of human organoids combined with miniaturized fluorescence microscopy (miniscope) and heart-on-a-chip technology. This simple, inexpensive, and efficient platform provides robust on-chip calcium imaging of human cardiac organoids. The versatility of the system is demonstrated through cardiotoxicity assay of drugs known to impact cardiac electrophysiology, including dofetilide, quinidine, and thapsigargin. The platform promises to advance drug testing by providing a more reliable and physiologically relevant assessment of cardiovascular toxicity, potentially reducing drug-related adverse effects in clinical settings.

Keywords: calcium imaging; cardiac organoids; cardiotoxicity; miniaturized fluorescence imaging; miniscope.

Figure 1

Schematic representation of the components and the working principle of heart‐on‐a‐miniscope device. A) Schematic of the organoid chip with the microwells for organoid positioning and the carbon electrodes for on‐chip stimulation. B) 3D printed housing with the slider to assemble the chip and the imaging module. C) The long working distance miniscope and its components connected to Data Acquisition module (DAQ). D) Exploded diagram of the imaging module.

Figure 2

Fabrication steps of the organoid chip. A bench‐top 3D printer was used to create the molds, which were then used for replica molding of PDMS slabs. The microwells were then punched using a biopsy punch. The chip was sealed and assembled using a layer of pressure sensitive adhesive followed by securing the carbon rod electrodes in the chip.

Figure 3

Optical characterization of the setup. A) schematic showing 1951 USAF Target and fluorescent reference slide placement for characterization experiments. B) Images collected of the illuminated mask with the original Miniscope and C) by the LWD Miniscope at working distances determined for different focusing slider CMOS sensor positions. D) Heatmap of intensities from fluorescent sub‐diffraction sized beads (n = 30) in xy with E) a magnified view of a singular bead. Scale bar is 5 µm. F) A blown‐up image of the smallest line pair where G) plots the intensity values demonstrating resolving capability for objects down to 4.4 µm. Scale bar is 50 µm unless otherwise specified.

Figure 4

Electrical and pharmacological control over organoid beating. COMSOL simulation results, A) Schematic representation of the geometries used in the simulation, B) heat map cross‐section analysis of the electric field distribution in the chip. C) line profile of normalized electric filed along a line in y‐direction crossing a single well (represented with a red line). Ivabradine knock‐out of spontaneous beating with D) Cal‐520 detected intracellular calcium activity within organoids before and E) after treatment with 1 µm ivabradine overnight. F) pharmacological increase of calcium transient peak height with isoproterenol captured by our heart‐on‐a‐miniscope. G) Observed beating frequency plotted against input pacing (n = 3 organoids, n ≥ 5 ROIs per organoid) experimentally demonstrates successful pacing. H) Example calcium traces from organoids paced at 0.5, 1, and 1.5 Hz with 5, 10, and 15 fluctuation cycles, respectively, over the course of a 10 s video demonstrating consistent and regular pacing. Error bars represent the standard deviation.

Figure 5

Calcium imaging data collection and processing. A) Representative video frames from a series demonstrating the progression of Cal‐520 fluorescent intensity in a cardiac organoid during a 1 Hz pacing cycle. The images show dynamic changes in intracellular calcium levels. Scale bar is 100 µm. B) Kymograph generated from a region of interest (ROI) within the organoid, showing the continuous visual progression of calcium signals over time. C) Schematic representation of a calcium transient with labeled temporal parameters, including Time to Peak (TTP), Calcium Duration 90 (CD90), and decay times (Decay 70, Decay 50, Decay 30), used to quantify the kinetics of calcium handling in the cardiomyocytes. D) The frequency dependence of temporal parameters extracted from videos of cardiac organoids paced at different frequencies (0.5 Hz to 2 Hz), showing the shortening of temporal parameters with increased pacing frequency.(n ≥ 5 ROIs per organoid, data shown as mean ± standard deviation.).

Figure 6

Effect of dofetilide, thapsigargin, and quinidine on the calcium transient in cardiac organoids. A) Changes in calcium transient shape after the addition of 100 nm dofetilide compared to control. B) Analysis of changes in Decay 50, Decay 30, and CD90 temporal parameters with increasing concentrations of dofetilide (0, 10, and 100 nm). C) Changes in calcium transient shape after the addition of 1 µm thapsigargin compared to control. D) Analysis of changes in Decay 50, Decay 30, and CD90 with increasing concentrations of thapsigargin (0, 0.1, and 1 µm). E) Changes in calcium transient shape after the addition of 30 µm quinidine compared to control. F) Analysis of changes in Decay 50, Decay 30, and CD90 with increasing concentrations of quinidine (0, 3, and 30 µm). Data were obtained from n = 2–4 biologically independent organoids, with n ≥ 5 ROIs per organoid. Statistical significance was determined using one‐way ANOVA followed by Tukey's test, with significant differences defined by ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. Data shown as mean ± standard deviation.