• Cellartis Cardiomyocytes (from ChiPSC22) Kit 
  • Cellartis Cardiomyocytes (from ChiPSC22)
  • Cellartis CM Thawing Base
  • Cellartis CM Culture Base
• Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. # 16140)
• Y-27632
• Fibronectin (Sigma-Aldrich, Cat. # F0895)
• CardioExcyte 96 Sensor Plate (Cat. # 20 1001, Nanion Technologies)
• PBS Dulbecco's with Ca2+ & Mg2+ (D-PBS +/+)
• CardioExcyte 96 instrument (Nanion Technologies)
• General cell culture equipment used in a cell culture laboratory

A. Coating of the CardioExcyte 96 Sensor Plate

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 a minimum of 1.5 hours.
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 make 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

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 the 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 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.

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. Transfer the frozen vial from liquid nitrogen to a 37°C ± 1°C water bath as quickly as possible, 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 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 drops.
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 6 ml medium per thawed vial.
11. Count the cells and measure viability.
12. Adjust the number of viable cells to 1.5–2.0 x 10⁵ cells/ml with Cellartis CM Thawing Medium with Y-27632.
    
    

    NOTE: It is recommended to remove the coating solution and add the cell suspension column by column. Preparing more wells at a time might result in a drying of the surface, resulting in crystallization of the Fibronectin and damaging of the cells afterward.

13. Aspirate the Fibronectin solution from the first column.
14. Carefully mix your cell suspension and pipet 200 μl into each well (corresponding to 3–4 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).

D. Medium change

It is recommended to do the first medium change 24 ± 2 hrs. after thawing and plating, and further on day 2, 3 and 4. From day 4 and onwards, it is sufficient to do a medium change every other day.

Medium preparation

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

Medium change

1. Aspirate 100 µl medium per well column-by-column and add 100 µl of fresh medium. Be careful not to touch the electrodes on the bottom of the well.
2. Repeat one time for each well (column-by-column).
3. Place the plate in the incubator (37°C ± 1°C, 5% CO2, and >90% humidity).