•  Cellartis Cardiomyocytes (from ChiPSC22) Kit
  
  •  Cellartis Cardiomyocytes (from ChiPSC22)
  • Cellartis CM Thawing Base
  • Cellartis CM Culture Base
• Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Cat. # 16140063) 
• Y-27632
• Cell culture T25 flask(s)
• Fibronectin (Sigma-Aldrich, Cat. # F0895)
• Accumax
• Stopping medium (i.e. any medium w serum, w/o antibiotics)
• External solution (140 mM NaCl, 10 mM HEPES, 5 mM Glucose, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 298 mOsm, pH 7.4)
• Blebbistatin
• PBS-EDTA
• Sterile H₂O

A. Coating the T25 flask(s) with fibronectin

1. Dilute the required volume of fibronectin in D-PBS +/+ (final concentration 10 µg/ml).
2. Add the fibronectin solution to each T25 flask to be used. Use 2 ml per T25 flask. Make sure the entire surface is covered.
3. Incubate at 37°C ± 1°C and 5% CO₂ for at least 3 hours.
4. Remove the fibronectin solution from the cell culture vessels just before use.

B. Preparing medium

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 the Cellartis CM Thawing Medium.
   Cellartis CM Thawing Medium should be stored at 4°C and expires one month after the date of preparation.
4. 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 Cellartis CM Thawing Medium to a final concentration of 10 μM.

• 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.

C. Thawing and plating 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. 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 additions.
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 3 ml medium per thawed vial.
11. Count the cells and measure viability.
12. Adjust the number of viable cells to 2 x 10⁵ cells/ml with Cellartis CM Thawing Medium with Y-27632.
    
    

    NOTE: Aspirate the fibronectin solution just before adding the cell suspension. Drying of the surface might result in crystallization of the fibronectin that could damage the cells.

13. Aspirate the fibronectin solution from the T25 flask.
14. Carefully mix your cell suspension and add 7.5 ml into each flask (corresponding to 1.5 x 10⁶ cells/flask).
15. Transfer the cell culture unit to an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity and leave untouched for 24 ± 2 hrs.

D. Changing medium

At day one after thawing, wash away the non-attached cells and change the medium. After day one, change the medium every second to third day.

Medium Preparation

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

Medium Change Day 1

1. Wash the cells carefully by aspirating the Cellartis CM Thawing Medium and gently flush the cells with the medium two to three times in order to remove non-attached cells.
2. Aspirate the old medium and add warm Cellartis CM Culture Medium (0.4–0.6 ml/cm²).
3. Return the cell culture unit to an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity.

Medium Change Day 3 Onwards

1. Gently aspirate the entire volume of medium from the culture vessel and discard.
2. Add warm Cellartis CM Culture Medium, 0.4–0.6 ml/cm².
3. Return the cell culture vessel to the incubator with 37°C ± 1°C, 5% CO₂, and >90% humidity.

E. Detaching Cellartis Cardiomyocytes for automatic patch clamp recordings

On day 7–9, for one fibronectin coated T25 flask:

1. Rinse 2x with PBS-EDTA.
2. Add 1 ml 1X Accumax and incubate up to 20 min at 37°C (no pipetting during this step).
3. Add 1 ml stopping medium to block Accumax dissociation (no pipetting during this step) and place at 4°C for 15 min (cell membranes become more stable at low temperatures, for trituration/pipetting up and down).
4. Add 2 ml external solution (+ 1 μM Blebbistatin).
5. Use the cells for downstream automatic patch clamp applications.