Cardiomyocyte Differentiation.
Cardiomyocytes (CMs) were differentiated from human induced pluripotent stem cells (hiPSCs; Gibco human female episomal iPSCs) in high-density monolayer cultures using CDM3 medium83 and Wnt signal activation and inhibition84 (Fig. 1A). Briefly, hiPSCs were treated with 6 µM Chiron 99021 (Tocris), a glycogen synthase kinase 3 (GSK3) inhibitor at day 1, followed by 5 µM IWP2 (Tocris), a chemical Wnt inhibitor at day 3. Cardiac phenotype, expressed by beating cells, was visible between days 8 and 12 and upon beating, cardiomyocytes were cultured in RPMI 1640 medium with B27 supplement (RPMI + B27; Gibco). Cardiomyocytes differentiated from hiPSCs were used for the production of microtissues between days 14 and 18 of differentiation or were further purified with a lactate protocol. Cardiomyocytes designated for lactate purification were harvested and replated to new culture vessels coated with Matrigel® (Corning) in RPMI + B27. These cells were deprived of media change for 4 days and were fed with lactate media (DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate (D-5030, Sigma) + 4 mM L-glutamine, 1X Non-Essential Amino Acids, 1x Glutamax and 4 mM lactate, pH 7.4). Lactate purified cells were fed with RPMI + B27 + 1% penicillin/streptomycin (P/S). Cardiac purity was measured by flow cytometry analysis as previously described.85
Human Cardiac Fibroblast Culture.
Commercially available human cardiac fibroblasts (hCFs, Sigma) were maintained and passaged in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 4 ng/ml basic fibroblast growth factor (Reprocell). hCFs were incorporated into cardiac microtissues at cell passages P2-P4.
Fabrication of Hydrogels and 3D Culture.
Scaffold-free 3D spherical microtissues were generated using non-adhesive agarose gels with cylindrical microwells with hemispherical bottoms to guide self-assembly (Fig. 1B). Sterilized 2% (wt/vol) agarose was pipetted into molds designed for 24-well plates with 800-µm-diameter rounded pegs (3D Petri Dish®, MicroTissues). After being cooled to room temperature (~ 5 min), the agarose gels were separated from the molds and transferred to single wells of 24-well plates. For equilibration, 1 mL RPMI + B27 + 1% P/S medium was added to each well. Hydrogels were equilibrated for at least 1 hr at 37 °C in a humidified incubator with 5% CO2. Molds were transferred to 6-well plates for electrical stimulation, and hiPSC-CMs with or without additional 5% hCFs in suspension were added to the center of the hydrogel seeding chamber (420-840K cells/mold in 35 recesses) and allowed to settle into the recesses for 30 min. Medium (RPMI + B27 with 1% P/S and 10% FBS with 5 µM rock inhibitor (Y27632)) was then added to each well (5 mL). Medium was changed to RPMI + B27 with 1% P/S and 10% FBS at 24–48 hr, and cells were cultured for 6–8 days with media changes every other day. During the 3D culture period, the self-assembled microtissues were field stimulated with a 1 Hz, 10.0 V, 4.0 ms duration bipolar pulse train.
Microtissue Size Analysis.
Stitched 4X phase-contrast images of whole 24-well microtissue hydrogels were captured with a Nikon TE2000-U and a black and white/color digital camera (MicroVideo Instruments, Avon, MA). NIS Elements software was used for image acquisition and analysis. Image thresholding and particle size analysis was performed to determine the top view cross-sectional area of individual microtissues across each mold.
3D Tissue Sections and Immunohistochemistry.
Microtissues in 24-well hydrogels were fixed with 4% (vol/vol) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and 8% (wt/vol) sucrose in PBS overnight at room temperature. Molds were then rinsed twice with PBS and fully equilibrated (as indicated by their sinking, usually over 12 hrs) with 15% and then 30% (wt/vol) sucrose in PBS. Whole agarose gels containing microtissues were removed from sucrose solution, blotted dry, and embedded in Tissue-Tek CRYO-OCT Compound (Sakura). Blocks were stored at − 80 °C, sectioned on a Leica CM3050 cryostat microtome (Leica Biosystems, Buffalo Grove, IL) into 10-µm-thick sections, and placed on Superfrost Plus slides. After being air dried for 15 min, sections were post-fixed in 4% paraformaldehyde in PBS. For immunofluorescent staining at room temperature, frozen sections were rinsed 3 times for 5 min with 1X PBS wash buffer. Non-specific binding was blocked with 1.5% goat serum for 1 hr, followed by 24 hr incubation in primary and followed by a 1 hr incubation in secondary antibodies diluted in 1.5% goat serum. Primary antibodies were directed against cardiac troponin I (cTnI, 1:100, Abcam ab47003) and vimentin (1:100, Sigma V6630), and secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:200, Invitrogen). Coverslips were mounted with Vectashield mounting medium with DAPI. Images were taken with an Olympus FV3000 Confocal Microscope and processed using ImageJ.
Optical Mapping and Automated Action Potential Analysis.
Microtissues were loaded with voltage-sensitive di-4-ANEPPS (5 µM for 10 min at 35 °C) for measurements of membrane potential (Vm). Fluorescence images were acquired at 979 frames/s using a Photometrics Evolve + 128 EMCCD camera (2 × 2 binning to 64 × 64 pixels, 18.7 × 18.7-µm2 resolution, 1.2 × 1.2-mm2 field of view) and an Olympus MXV10 macroview optical system.86 Typically, four microtissues were recorded simultaneously per scan at this magnification. A step-by-step illustration of automated data analysis is available in Supplemental Fig. 1. Briefly, the pixels with APs were identified from Fast Fourier transformation (FFT) of fluorescence signals. After appropriate thresholding and image segmentation, the region of each microtissue was grouped and the fluorescence signals from the pixels in the same microtissue were average and used for AP analysis (Supplemental Fig. 1).
Validation and Screening of Toxicants for Arrhythmogenic Risk.
A single mold of microtissues was mounted on a temperature-controlled chamber (Dual Automatic Temperature Controller TC-344B, Warner Instrument) to maintain 35 ± 1 °C and bathed with Tyrode’s solution containing (in mM) 140 NaCl, 5.1 KCl, 1 MgCl2, 1 CaCl2, 0.33 NaH2PO4, 5 HEPES, and 7.5 glucose. Microtissues were stimulated with a platinum field stimulation electrode (Myopacer EP field stimulator, IonOptix, Milton, MA). The test compounds including E4031, 4-AP, BayK8644, ISO were purchased from Sigma Aldrich and dissolved in 100% DMSO to prepare 0.01–0.5 mM stock solutions. BPA was dissolved in 10% ethanol stock solution and diluted in Tyrode solution to the final concentration. Microtissues were exposed to vehicle (DMSO or ethanol) and the indicated concentrations of test compounds for 20 min, and action potentials were measured as described above.
Statistical Analyses.
The data from optical mapping are expressed as mean ± SD for n microtissues unless otherwise indicated. Statistical analyses were performed using Student’s two tailed paired and unpaired t-test. P values of 0.05 were considered statistically significant. Normality test was done using Kolmogorov-Smirnov test. The test for the equality of regression coefficients was done using Z statistics of two slopes and SE as described.87,88