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Atrial and Ventricular Cardiomyocytes on the Axion Maestro MEA User Guide

Product Information

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Catalog. No. Product Name Format Stock Conc. Storage on Arrival Thawing Instructions Storage Once Thawed
ax2508 Human iPSC-Derived Ventricular Cardiomyocytes (Male) 1 million cells/vial N/A Liquid Nitrogen Follow protocol N/A
ax2518 Human iPSC-Derived Atrial Cardiomyocytes (Male) 1 million cells/vial N/A Liquid Nitrogen Follow protocol N/A
ax2530-500 Cardiomyocyte Maintenance Medium 1 x 500 mL Basal Medium
1 x 10 mL Supplement		 1x
1x		 Store the Basal Medium at 4ºC and the Supplement at -80ºC Thaw the Supplement overnight at 4ºC Once thawed store at 4ºC. If required, the medium can be aliquoted and stored at -80ºC for later use.
ax0050 Human Fibronectin 1 vial N/A Aliquot and store at -80ºC Thaw at 4ºC Once diluted, use immediately
ax2500 Human iPSC-Derived Ventricular Cardiomyocyte Kit – Male Kit Components:

  • 1 million cells
  • Cardiomyocyte Maintenance Medium
  • Fibronectin Coating Solution
 See above for component details See above for component details See above for component details See above for component details
ax2510 Human iPSC-Derived Atrial Cardiomyocyte Kit – Male Kit Components:

  • 1 million cells
  • Cardiomyocyte Maintenance Medium
  • Fibronectin Coating Solution
 See above for component details See above for component details See above for component details See above for component details

Additional Reagents

Product Name Supplier Product Code
Y-27632 dihydrochloride (ROCK inhibitor) Focus Biomolecules 10-2301
Fetal bovine serum (FBS)-EU Approved heat inactivated Sigma Aldrich F9665-500ML

Individual experimental results may vary depending on the supplier and batch of FBS used. For Japan, we recommend the use of heat-inactivated FBS (Netherland origin) #S-FBS-NL-025 from Serana.

Lot specific information such as specifications and quality control details are stated in the Certificate of Analysis

Introduction

axoCells Cardiomyocytes exhibit typical biochemical, electrophysiological, mechanical, and pathophysiological characteristics of native human cardiac myocytes. Due to their human origin, high-purity, functional relevance, and ease of use, axoCells Cardiomyocytes represent an optimal test system for investigating cardiomyocyte biology for basic research and drug development.

The Maestro Edge and Pro multielectrode array (MEA) systems from Axion BioSystems are a non-invasive, label-free platforms that measures local field potentials of electrically active cells, representing the summed activity of the underlying ion channels and the contractility of the syncytium.

Together, axoCells Cardiomyocytes and Axion’s MEA technology form an excellent, non-invasive platform for in vitro screening of compound effects on human cardiomyocyte physiology.

This user guide describes how to handle axoCells Atrial and Ventricular Cardiomyocytes for use on the Maestro MEA system and provides basic instructions for compound treatments, data acquisition, and analysis.

Recommendations

  • Recommended culture vessel: Axion CytoView MEA 48 #M768-tMEA-48B
  • Recommended culture vessel coating: Fibronectin
  • Recommended cell culture medium: Cardiomyocyte Maturation Medium
  • Recommended seeding density for assay: 100,000 – 200,000 cells/cm2
  • Recommended centrifugation speed: 200 x g for 5 minutes
  • Recommended days in culture before assay: 7 to 10 days

Important! Cardiomyocyte Maintenance Medium = Basal medium + Supplement DOES NOT contain antibiotics or antifungal agents. Axol Bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted.

Preparing the MEA plate

  • Calculate the total volume of fibronectin that is required for coating.
  • Dilute the stock Human Fibronectin in 20 mL D-PBS (without magnesium and calcium) to make a 1x working solution.
  • At least 4 hours before plating cardiomyocytes, place an 8 μL droplet of the 1x fibronectin working solution over the recording electrode area of each well in the MEA plate. Do not let the tip touch the electrodes. See Figure 1 and 2 for appropriate drop placement.
  • Add 1 mL of SDW to the area surrounding the wells (MEA reservoirs) of the MEA plate to prevent substrate evaporation. Do not allow the water into the wells of the MEA plate.
  • Incubate the fibronectin-coated MEA plate in a cell culture incubator at 37°C, for at least 4 hours.
  • Any excess coating cannot be stored and should be discarded.

    Care should be taken when using higher volumes of water in the MEA reservoirs to prevent sloshing and spilling.

Preparation of reagents

Cardiomyocyte Maintenance Medium

  • Upon receipt store Cardiomyocyte Maintenance Medium at or below 4°C and Supplement at -80°C.
  • Remove and discard 10 mL of the basal medium and then add 10 mL Supplement to the remaining 490 mL Cardiomyocyte Maintenance Basal Medium.
  • For long-term storage, prepare aliquots of Cardiomyocyte Maintenance Medium and store at -80°C. The Cardiomyocyte Maintenance Medium is then stable from the first expiry date of media/supplement (please refer to the Certificate of Analysis).

Plating Medium

  • When ready to use, thaw an aliquot of Cardiomyocyte Maintenance Medium overnight in the dark at 4°C.
  • Take an aliquot of Cardiomyocyte Maintenance Medium and add 10% fetal bovine serum (FBS) and Y-27632 2HCl (ROCK inhibitor) to a final concentration of 10 μM to make the Plating Medium. Complete plating media must be used fresh and cannot be stored once supplemented.
  • Before use, pre-warm an aliquot of Plating Medium at 37°C
Supplement Stock Concentration Final Concentration Final Volume in 45 ml of Medium
Y-27632 dihydrochloride (ROCK inhibitor) 10 mM 10 μM 50 μl
Fetal bovine serum (FBS) NA 10% 5 ml

Thawing axoCells Atrial & Ventricular Cardiomyocytes

  • On the day of thawing axoCells Atrial or Ventricular Cardiomyocytes, prepare the Cardiomyocyte Maintenance Medium and Plating Medium and pre warm before use.
  • To thaw the cells, transfer the cells from liquid nitrogen storage by carrying cells buried in dry ice. Remove the cells from dry ice and transfer them immediately to a 37°C water bath.
  • Quickly thaw the vial of cells in a 37°C water bath, taking care not to completely submerge the vial (only up to two thirds should be placed in the water). Remove the vial before the last bit of ice has melted, ~ 2 minutes.
  • Do not shake the vial whilst thawing.
  • Take the vial of cells to a biological cabinet, spraying it thoroughly with 70% ethanol and wipe with an autoclaved paper towel before placing in the culture hood.
  • Once thawed, use a P1000 to immediately transfer the cells to a 15 ml sterile conical tube.
  • Using a P1000, wash the now empty cryovial with 1 ml of room temperature Plating Medium. Add the 1 mL of Plating Medium to the conical tube containing the Cardiomyocytes.
  • Important – the Plating Medium should be added to the cells drop-wise whilst gently swirling the conical tube. This should take ~ 60 seconds to dispense the 1 ml of medium. This is a necessary step to prevent osmotic shock to the cells and improve post thaw viability.
  • Using a 10 ml stripette, slowly add 8 ml of room temperature Plating Medium to conical tube containing the cells. This should take ~ 60 seconds to dispense the 8 ml of medium.
  • Centrifuge the cells at 200 g for 5 minutes at room temperature.
  • Aspirate and discard the supernatant, taking care not to disturb the cell pellet.
  • Using a P1000, resuspend the pellet in 1 ml of warm (37°C) Plating Medium. Gently resuspend the cell pellet until a single cell suspension is obtained.
  • Perform a cell count to ensure optimal seeding density. Remove 10 μl of cell suspension and mix it with 10 μl of trypan blue solution. Count the cells.
  • Using warm, 37°C Plating Medium, resuspend the cells to give to 6,250,000 plated cardiomyocytes/mL. Alternative seeding densities and volumes can be calculated at https://www.axionbiosystems.com/cell-plating-calculator. Refer to the Axion guide at https://www.axionbiosystems.com/resources/product-videos/cell-plating-calculator-how-guide.

Please note that this is a recommended seeding density and that density may need to be optimized by the user to suit their culture dish size, culture conditions and the final assay

Plating Cardiomyocytes onto the MEA

  • Aspirate, from a single row or column, most of the fibronectin solution from the MEA surface.

  • Place an 8 µl droplet of cardiomyocyte suspension (approx. 50,000 cardiomyocytes) over the recording electrode area of each well. See Figure 1 and 2 for appropriate drop placement.

Timing is critical in this step. Cardiomyocyte attachment is compromised if the fibronectin is allowed to dry. Under typical conditions, the well will begin to dry within a few minutes after aspiration of the excess fibronectin solution. At this point the residual fibronectin in the well will begin to crystallize, turn white, and the well should then be ignored as cardiomyocyte attachment will be suboptimal.

  • Repeat the previous steps until all rows or columns have been plated.
  • Incubate the MEA plate with the seeded cardiomyocytes in a cell culture incubator at 37°C, 5% CO2for 1 hour.
  • Gently add 150 μL of Plating Medium to each well of the MEA. Dispense into alternating sides of the well as adding medium too quickly will dislodge the adhered cardiomyocytes.
  • Using a pipettor, add medium first in a semi-circle along the outer edge of the flat bottom area. Progressively add medium so it fills evenly towards the centre, stopping before contact is made with the droplet in the centre. Gently bridge the gap with additional medium. The goal is to prevent a rush of medium in either direction that might dislodge the cardiomyocytes.
  • Slowly add the remaining volume to reach the plate recommended media volume. Recommended well volumes for each plate type: 12-well = 500 µL, 48-well = 300 µL, 96 well = 200 µL. At this point, MEA reservoir water is no longer required.
  • Incubate in a cell culture incubator at 37°C, 5% CO2 for 24 hours.
  • Figures adapted from Axion Biosystems user guides available at  https://www.axionbiosystems.com/ 

 

Figure 1: Drop Placement Diagram.

The layouts above represent the bottom surfaces of wells in a 12-well MEA (A), a 48-well MEA (B) and a 96-well MEA (C). Diagram A represents a 12-well MEA and the inner 64 dots of the electrode array with the 4 ground electrodes located in the corners. Diagram B represents a 48-well MEA and the inner 16 dots of the electrode array with the 4 ground electrodes located in the corners. Diagram C represents a 96-well MEA and the inner 8 dots of the electrode array with the 4 ground electrodes located in the corners. The red circles indicate the approximate size and location for the drop placement.

 

      • Orient tip just above the recording electrodes.
      • Eject droplet without touching the surface.
      • If droplet does not release gently, touch it down to the surface.
      • Cover recording electrodes completely while avoiding ground electrodes.

Figure 2: Drop Placement guide for both fibronectin coating and cardiomyocyte seeding on the Axion 48-well MEA plate.

 

Maintenance of axoCells Cardiomyocytes on the MEA

  • On the day after plating, replace 100% of the Plating Medium with Cardiomyocyte Maintenance Medium.
  • Culture cardiomyocytes on the MEA plate replacing 50% of the spent medium with Maintenance Medium every 2 days (see Figure 3).
  • After 7 days in culture, the Human iPSC-Derived Atrial or Ventricular Cardiomyocytes should beat spontaneously (this can occur within 72 hours).
  • For optimal results, perform MEA recordings 7-18 days after plating.

 

      • Expel 50-75 µl against the well wall.
      • Break the surface tensions between the media and cell droplets with the tip.
      • Continue adding a half volume of media slowly against the well wall.
      • Add half volumes to each well to minimize time cells are exposed in droplets then add second half.

 

Figure 3: Media change for cardiomyocyte culture on the Axion 48-well MEA plate