How to Dissociate and Plate Human Pluripotent Stem Cell-Derived Cardiomyocytes for Microelectrode Array (MEA) Assay
Step by step guide for dissociating and plating human pluripotent cell-derived cardiomyocytes for performing multiple electrode assays for studying arrhythmias
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. Whether you are planning to evaluate key indicators of cardiomyocyte function, monitor long-term electrophysiological maturation, or record real-time responses to experimental stimuli, the Maestro MEAâ„¢ system is a versatile, user-friendly platform that is ideal for a wide variety of MEA experiments. 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
- STEMdiffâ„¢ Ventricular Cardiomyocyte Differentiation Kit (Catalog #05010)
- STEMdiffâ„¢ Atrial Cardiomyocyte Differentiation Kit (Catalog #100-0215)
- STEMdiffâ„¢ Cardiomyocyte Dissociation Kit (Catalog #05025)
- STEMdiffâ„¢ Cardiomyocyte Plating Supplement (Catalog #100-1120)
- STEMdiffâ„¢ Cardiomyocyte Maintenance Kit (Catalog #05020)
- Corning® Matrigel® hESC-Qualified Matrix (Corning 354277)
- Trypan Blue (Catalog #07050)
- Hemocytometer and cover slip (e.g. Hausser Scientificâ„¢ Bright-Line Hemocytometer, Catalog #100-1181)
- MEA Instrument and Software — e.g. Maestro MEA™ by Axion BioSystems®
- Maestro Proâ„¢ (Catalog #200-0887)
- Cardiac Software Module for Maestro Proâ„¢ (Catalog #200-0908)
- MEA Plates — e.g. CytoView MEA™ Plate
- 24-well (Catalog #200-0874)
- 48-well (Catalog #200-0872 or #200-0873)
- 96-well (Catalog #200-0870 or #200-0871)
Add Protocol Flexibility with Cryopreserved Cells
- For researchers looking for highly characterized human iPSC lines for their workflows, genotypically diverse, cryopreserved human iPSC control cell lines provide a reliable alternative to in-house reprogramming and quality characterization.
Explore available cell lines > - Alternatively, for researchers looking to streamline their workflows further, cryopreserved cells differentiated (using STEMdiffâ„¢ kits) from the Healthy Control Human iPSC Line, SCTi003-A (Catalog #200-0511), offer a time-saving alternative to in-house differentiation.
Explore available cell types >
Protocol
- 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 () or TeSR™-E8™ (). - 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 Plating Medium (STEMdiff™ Cardiomyocyte Support Medium + STEMdiff™ Cardiomyocyte Plating Supplement [100X]) 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). - Count cells using Trypan Blue and a hemocytometer. You can follow the steps found in this protocol: How to Count Cells with a Hemocytometer.
- 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 FormatCells per WellTotal Volume of STEMdiff™ Cardiomyocyte Plating Medium per Well24-well plate300,000600 μL48-well plate150,000300 μL96-well plate75,000150 μL - Incubate cells at 37°C for 24 hours.
- 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.
- Perform a full-medium change as described in step 6. Incubate at 37°C. Perform a full-medium change every 2 days.
- MEA data can be recorded and analyzed on the Maestro Proâ„¢ using the Cardiac Software Module. Refer to the Maestro MEAâ„¢ and the for detailed guidance on data acquisition and analysis.
- 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).
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