• Cellartis Cardiomyocytes (from ChiPSC22) Kit (Takara Bio, Cat. # Y10075)
  • Cellartis Cardiomyocytes (from ChiPSC22)
  • Cellartis CM Thawing Base
  • Cellartis CM Culture Base
• Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. # 16140)
• Y-27632
• Electronic 48-well microtiter plate (E-Plate CardioECR 48; Cat. #300600940, ACEA Biosciences)
• Fibronectin (Sigma-Aldrich, Cat. No. F0895)
• PBS Dulbecco's with Ca²⁺ & Mg²⁺ (D-PBS +/+)
• xCELLigence RTCA CardioECR instrument (ACEA Biosciences)
• General cell culture equipment used in cell culture laboratory

A. Coating of the E-Plate CardioECR 48

1. Dilute the required volume of Fibronectin in D-PBS +/+ to a final concentration of 10 µg/ml.
2. Add the diluted Fibronectin solution into each well to be used. Use 50 µl/well.
3. Incubate at 37°C for 3 hr.
4. Aspirate the Fibronectin solution from the cell culture plate just before use.

B. Medium preparation

Preparing Cellartis CM Thawing Medium

1. Thaw Cellartis CM Thawing Base.
2. Decontaminate the external surface of all bottles with an appropriate disinfectant and place into the biological safety cabinet.
3. Add 8 ml FBS per 32 ml Cellartis CM Thawing Base to achieve Cellartis CM Thawing Medium.
   • Cellartis CM Thawing Medium should be stored at 4°C and expires one month after the date of preparation.
   • Always discard any leftover warmed Cellartis CM Thawing Medium.

Preparing Cellartis CM Thawing Medium with Y-27632

1. On the day of use, prepare Cellartis CM Thawing Medium with Y-27632 by adding Y-27632 to a final concentration of 10 μM to Cellartis CM Thawing Medium.
   • Cellartis CM Thawing Medium with Y-27632 should be used on the day of preparation.

Preparing Cellartis CM Culture Medium

1. Thaw Cellartis CM Culture Base.
2. Decontaminate the external surface of supplement and medium bottle with appropriate disinfectant and place into the biological safety cabinet.
3. Add 10 ml FBS per 90 ml Cellartis CM Culture Base to make the Cellartis CM Culture Medium.
   • Cellartis CM Culture Medium should be stored at 4°C and expires one month after the date of preparation.
   • Always discard any leftover warmed Cellartis CM Culture Medium.

Preparing Cellartis CM Culture Medium with Y-27632

1. On the day of use, prepare Cellartis CM Culture Medium with Y-27632 by adding Y-27632 to a final concentration of 10 μM to Cellartis CM Culture Medium.
   • Cellartis CM Culture Medium with Y-27632 should be used on the day of preparation.

C. Thawing and plating of Cellartis cardiomyocytes

1. Prepare the appropriate volume of Cellartis CM Thawing Medium with Y-27632 (see Section B above) and warm to room temperature (RT, 15–25°C).
2. As quickly as possible, transfer the frozen vial from liquid nitrogen to a 37°C ± 1°C water bath using forceps.
3. Thaw the cells by gently pushing the vial under the surface of the water, without swirling the vial. Do not submerge the cap of the vial in the water bath as this could contaminate the cells.
4. Take the vial out of the water bath as soon as the thawing is completed (approximately 3 min; the vial should still be cold on the outside).
5. Wipe the vial with an appropriate disinfectant and place into the biological safety cabinet.
6. As soon as possible, gently transfer the cell suspension into a sterile 50-ml tube by using a pipette.
7. Rinse the vial with 1 ml of Cellartis CM Thawing Medium with Y-27632 and carefully add it to the cell suspension dropwise.
8. Add 8 ml of Cellartis CM Thawing Medium with Y-27632 dropwise. Gently swirl the tube a few times in between.
9. Centrifuge the tube at 200g for 5 min at RT and remove the supernatant.
10. Carefully resuspend the cell pellet with Cellartis CM Thawing Medium with Y-27632, using 4 ml of medium per thawed vial.
11. Count the cells and measure viability.
12. Adjust the number of viable cells to 5 x 10⁵ cells/ml with Cellartis CM Thawing Medium with Y-27632.
    
    

    NOTE: Aspirate the Fibronectin solution just before adding the cell suspension. Prepare one column at a time since drying of the surface might result in crystallization of the Fibronectin and subsequent damaging of the cells.

13. Aspirate the Fibronectin solution just before adding the cell suspension, making sure the wells do not run dry.
14. Carefully mix your cell suspension and pipet 50 μl into each well (corresponding to 2.5 x 10⁴ cells/well).
15. Proceed rapidly with the remaining columns.
16. Place the plate in the incubator (37°C ± 1°C, 5% CO2, and >90% humidity).
17. After 3 hr, carefully add 130 µl Cellartis CM Thawing Medium with Y-27632 to each well to reach a final volume of 180 μl.

D. Medium change

It is recommended to do the first medium change 48 hours after thawing and plating, and further on every 2–3 days (e.g. Monday, Wednesday, Friday) until analysis.

Medium preparation

1. Prepare the appropriate volume of Cellartis CM Culture Medium with 10 µM Y-27632 as described in Section B above and warm to 37°C ± 1°C before use.

Medium change

1. Replace the medium with 180 μl of fresh Cellartis CM Culture Medium with Y-27632.
2. Place the plate in the incubator (37°C ± 1°C, 5% CO₂, and >90% humidity).