Protocol to study electrophysiological properties of hPSC-derived 3D cardiac organoids using MEA and sharp electrode techniques

Figures

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  Figure 1. Organoid replating onto 35 mm glass bottom dishes (A) Wide bore pipette preparation using 200 μL pipette and clean scissors. (B) Tissue culture hood with a microscope for organoid plating. (B) 35 mm dishes coated with matrix, scissors, and 200 μL pipette for transferring the organoids. (D) Re-plated organoids in a 35 mm dish.

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  Figure 2. MEA plate with organoids under a stereo microscope (A) 48-well MEA plate under a stereo microscope. (B) Organoid on top of MEA recording electrodes.

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  Figure 3. Electrophysiology rig setup 1. Microscope, 2. Head stage, 3. Micromanipulator controller unit, 4. Temperature controller unit, 5. Display monitor, 6. HEKA patch clamp amplifier, 7. Light source, 8. Antivibration table, and 9. Faraday cage.

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  Figure 4. Sharp electrode preparation (A) P-1000 micropipette puller. (B) Backfilling of the sharp electrode. (C) Filling the tip of the sharp electrode.

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  Figure 5. Close-up view of the 35 mm dish containing organoids placed on an electrophysiology rig heating platform (A) 1. Ground, 2. Bath temperature monitor, 3. Heating pad, and 4. Heating platform. (B) Close-up view of the replated organoid.

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  Figure 6. Action potential recording steps

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  Figure 7. Axion Maestro Pro System

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  Figure 8. Electrical activity in a 3D organoid measured using a 48-well MEA plate This image shows electrical signals from a 3D organoid, captured to assess its electrophysiological properties.

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  Figure 11. Analyzed action potential parameters (A) Amplitude. (B) Maximum rise slope. (C) Maximum diastolic potential. (D) Action potential durations (APD); APD30, APD50, APD90. (E) Triangulation (APD30-APD90). (F) APD Poincare plot. Error bars indicate standrad error,

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  Figure 9. Examples of good and bad intracellular action potential recordings (A) This figure illustrates a good intracellular action potential recording characterized by a stable maximum diastolic potential (MDP) and a stable amplitude. (B) This figure displays a poor intracellular recording where the action potential exhibits a reduced amplitude and a baseline that progressively decays, indicating declining recording quality.

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  Figure 10. Steps involved in importing and extracting AP parameters using Axon Clampfit software (A) Import the ASC file. (B–D) Identify peaks using threshold search and generate event files. (E and F) Extract AP properties.

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  Figure 14. Analyzed FP parameters (A) Beat period. (B) Rate corrected FP duration (FPDc). (C) Spike amplitude. (D) Coefficient of variance of beat period. (E) Beat period Poincare plot. Error bars indicate standrad error,