How to Dissociate and Plate Human Pluripotent Stem Cell-Derived Cardiomyocytes for Microelectrode Array (MEA) Assay

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer an attractive model for studying heritable and drug-acquired arrhythmias. One of the common assays used for these types of studies is the microelectrode array (MEA). In the MEA assay, arrays of microscopic electrodes are distributed over a small surface area at the bottom of a glass or plastic multi-well plate or a single-well (chip). Electroactive cells, such as cardiomyocytes, can be cultured over the electrodes forming cohesive networks over time, and their signal output (excitability) recorded in the form of a voltage map. The protocol described here outlines the key steps of preparing the cells for plating on an MEA chip. For a more in-depth discussion on how hPSC-derived cardiomyocytes can be used to model arrhythmias watch this webinar by Drs. Vincenzo Macri and Stacie Chvatal.


Materials

  • STEMdiff™ Cardiomyocyte Differentiation Kit (Catalog #05010)
  • STEMdiff™ Cardiomyocyte Dissociation Kit (Catalog #05025)
  • STEMdiff™ Cardiomyocyte Maintenance Kit (Catalog #05020)
  • Corning® Matrigel® hESC-Qualified Matrix (Corning 354277)
  • Trypan Blue (Catalog #07050)
  • Hemocytometer (ex. Neubauer hemocytometer) and coverslip
  • MEA plates (Supplier of MEA systems, ex. Axion BioSystems)

Protocol

  1. Coat MEA plates with Corning® Matrigel® hESC-Qualified Matrix (Corning Catalog #354277). The volume of Matrigel® used depends on the size of the MEA plates used, as follows:
    • For 24-well: 500 µL matrix/well
    • For 48-well: 250 µL matrix/well
    • For 96-well: 100 µL matrix/well

    Note: For coating plates with Matrigel®, refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1 (Document #10000005505) or TeSR™-E8™ (Document #10000005516).
  2. Dissociate and harvest monolayer of beating hPSC-CMs using the STEMdiff™ Cardiomyocyte Dissociation Kit as described in the corresponding Product Information Sheet. Resuspend the cell pellet from the dissociated hPSC-CMs in STEMdiff™ Cardiomyocyte Support Medium (provided as a component in the Dissociation Kit) to generate a single-cell suspension.

    Note: When using the STEMdiff™ Cardiomyocyte Differentiation and Maintenance Kits for the generation of hPSC-CMs, the beating monolayer cultures are often dissociated and harvested on Day 15 after initiation of differentiation (Day 0).
  3. Count cells using Trypan Blue and a hemocytometer.
  4. Plate cells onto MEA plates. The number of cells seeded and the volume per well depends on the size of the well. Refer to the table shown below.

    MEA Plate Format
    Cells per Well
    Total Volume of STEMdiff™ Cardiomyocyte Support Medium per Well
    24-well plate
    600,000
    1000 μL
    48-well plate
    300,000
    500 μL
    96-well plate
    150,000
    250 μL

  5. Incubate cells at 37°C for 24 hours.
  6. Remove medium using a pipette (no aspiration) and add the same volume of fresh STEMdiff™ Cardiomyocyte Maintenance Medium to each well. Incubate at 37°C for 24 hours.
  7. Perform a full-medium change as described in step 6. Incubate at 37°C. Perform a full-medium change every 2 days.
  8. Cardiomyocytes will re-form monolayers and start beating between 2 - 5 days after re-plating, and can be assessed after 7 days in culture. MEA recordings can be analyzed using the AxIS software (Axion BioSystems) following the supplier’s instructions.
  9. Electrophysiology experiments are usually performed 7 - 12 days after replating the hPSC-CMs. Metrics analyzed include beat period, spike amplitude, and field potential duration (FPD).
  • Document #PR00025
  • Version 1.0.0
  • September 2020


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