Atrial and Ventricular Cardiomyocyte User Guide
| Catalog. No. | Product Name | Format | Stock Conc. | Storage on Arrival | Thawing Instructions | Storage Once Thawed |
| ax2508 | Human iPSC-Derived Ventricular Cardiomyocytes (Male) | 1.0 million cells/vial | N/A | Liquid Nitrogen | Follow protocol | N/A |
| ax2518 | Human iPSC-Derived Atrial Cardiomyocytes (Female) | 1.0 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:
|
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:
|
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
Recommendations
- Recommended culture vessel: Greiner 96 well, Part no. 655090
- 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.
Coating the Cultureware
Fibronectin Coating
- Cell cultureware can be coated on the day of use, or overnight prior to use.
- Calculate the total surface area that requires coating.
- Dilute the stock Human Fibronectin in 20 mL D-PBS (without magnesium and calcium) to make a 1x working solution.
- Coat the surface of your culture vessel with the Fibronectin 1x working solution. We recommend coating at a minimum volume of 100 μl per well of a 96 well plate. However, please optimize for your experiments.
- Incubate the culture vessel for a minimum of 4 hours or overnight at 37°C in a humidified incubator.
- Coated plates can be stored at 4°C for up to 2 weeks. Any excess coating cannot be stored and should be discarded.
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 dates 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 & Plating 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 the required plating density.
- Remove the Fibronectin solution from the plate to be seeded, take care to work quickly so that the plate does not dry out.
- Plate the cells at a density of 100,000 – 200,000 cells/cm2. Depending on application, this density may require optimization.
Please note that this is a recommended seeding density, and that density may need to be optimized but the user to suit their culture dish size, culture conditions and the final assay
- Ensure that there is enough medium in the culture vessel to prevent drying and improper attachment. For example: include 2 mL total in a 6-well plate, 1 mL total in a 12-well plate, 500 μL in a 24-well plate and 200 μL in a 96-well plate.
- To ensure an even plating of cardiomyocytes, gently rock the culture vessel back and forth and side to side several times.
- Incubate the cells at 37°C, 5% CO2.
- The day after plating, replace the culture medium with fresh, pre-warmed (37ºC) Cardiomyocyte Maintenance Medium (without 10% FBS or Y-27632 2HCl (ROCK inhibitor) to remove any dead cells/debris.
Maintenance of axoCells Atrial & Ventricular Cardiomyocytes
- Every 2 days remove the medium and replace with the same volume of fresh, pre-warmed (37ºC) Cardiomyocyte Maintenance Medium.
- After 7 days in culture, the axoCells Atrial or Ventricular Cardiomyocytes should beat spontaneously (this can occur within 72 hours).
- After 7-10 days in culture, axoCells Atrial or Ventricular Cardiomyocytes will be ready for experimental assays. Atrial or Ventricular Cardiomyocytes can be cultured for longer depending on assay requirements.