With this 8-days protocol, cardiomyocytes can be generated from hPSCs with high efficiency. Depending on the cellular line, first contracting cardiomyocytes can be observed already after 6 days of culture. 
After directed differentiation using StemMACS CardioDiff Kit XF (# 130-125-289), PSC-derived cardiomyocytes can be maintained in culture in StemMACS Cardiac Cultivation Medium XF for more than 30 days.

▲Note: To achieve an efficient differentiation, it is important to start with high quality PSCs. Monitor regularly the pluripotency status of the stem cell cultures by observing their morphology and staining for pluripotency markers. The PSCs culture should show a confluency of 75–85% before starting.

▲Note: Perform all steps under sterile conditions.

Day –1: Coat plates

1. Coat 12-well plates with Matrigel® or Biolaminin 521 LN (LN521) according to the manufacturer’s recommendation.
2. Thaw supplements at 2–8 °C overnight.

Day 0: Prepare media and seed cells

▲Note: The starting cell number is strongly line-dependent. Therefore, when using a stem cell clone for the first time it is recommended to perform a titration experiment in order to determine the best starting PSC concentration for differentiation. Suggested cell numbers are 125,000 cells/cm², 250,000 cells/cm², 300,000 cells/cm², and 400,000 cells/cm².

1. Prepare complete media as indicated in table 4. 

▲Note: Volumes given below are for hPSCs originally cultivated in a 6-well plate and transferred to a 12-well plate for induction. If using other culture ware adjust the volumes accordingly.

▲Note: (Optional) Antibiotic solutions might be added to prevent contamination, e.g. 100 U/mL penicillin and 100 μg/ mL streptomycin.

2. Aspirate cell culture medium and wash each well of the 6-well plate with 2 mL of D-PBS without Ca²⁺ and Mg²⁺.

3. Add 1 mL/well TrypLE™ Select Enzyme. Gently rock the plate to ensure even distribution of the solution.

4. Incubate for 5 minutes in the dark at 37 °C.

5. Stop enzymatic reaction by adding 1 mL/well of Soybean Trypsin Inhibitor (0.5 mg/mL).

6. Using a 5 mL serological pipette, dissociate to a single-cell suspension by carefully pipetting up and down.

7. Determine the cell number and viability. Viability should be >95%.

8. Transfer the desired cell number into a new tube. Centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.

9. Resuspend the cells in a sufficient amount of Mesoderm Induction Medium. Per well, 2 mL of Mesoderm Induction Medium are needed.

10. Transfer cells into a freshly-coated 12-well plate with 2 mL/well. Place the plate into the incubator (37 °C, 5% CO₂).

Day 1: Replace medium with Cardiac Cultivation Medium

1. 24 hours after seeding, aspirate Mesoderm Induction Medium from the culture plate.
2. Wash each well with 2 mL D-PBS to remove dead cells.
3. Replace with 2 mL/well of Cardiac Cultivation Medium.
    

Day 2: Replace medium with Cardiac Induction Medium

1. 24 hours after previous feeding, aspirate Cardiac Cultivation Medium from the culture plate.
2. Wash each well with 2 mL D-PBS to remove dead cells.
3. Replace with 2 mL/well of Cardiac Induction Medium.
    

Day 3: Replace medium with Cardiac Cultivation Medium

1. 24 hours after previous feeding, aspirate Cardiac Induction Medium from the culture plate.
2. Wash each well with 2 mL D-PBS to remove dead cells.
3. Replace with 2 mL/well of Cardiac Cultivation Medium.

Day 4–8: Replace medium with Cardiac Cultivation Medium daily

1. Replace old Cardiac Cultivation Medium with 2 mL/well fresh medium every 24 hours.
2. (Optional) Wash each well with 2 mL D-PBS to remove dead cells.

▲Note: Depending on the PSC line, first contractions can be observed already on day 6 of differentiation.

Possible next steps

• If required, cardiomyocytes can be maintained in culture for more than 30 days if fed with StemMACS™ Cardiac Cultivation Medium XF (# 130-125-287) every two days. For details refer to 3. Long-term culturing.
• After 10 days, cells can be harvested. For details refer to 3.1 Cell harvest.
• Cells can be frozen for long-term storage. For details refer to 3.3 Cell freezing and thawing.
• Cardiomyocyte differentiation can be qualitatively and quantitatively evaluated by immunocytochemistry or flow cytometry.  For details refer to 5.1 Flow cytometry or immunofluorescence microscopy analysis of cardiomyocytes.
• In some cases further selection of correctly differentiated cardiomyocytes might be needed. For details refer to 4. Isolation of cardiomyocytes.

After directed differentiation using StemMACS CardioDiff Kit XF, PSC-derived cardiomyocytes can be maintained in culture in StemMACS Cardiac Cultivation Medium XF (# 130-125-287) for more than 30 days. – Media change needs to be performed every second day.

Prepare complete media

1. Thaw supplement at 2–8 °C overnight.
2. Mix StemMACS CardioDiff Basal Medium XF with StemMACS CardioDiff Cardiac Cultivation Supplement XF.
   ▲Note: (Optional) Antibiotic solutions might be added to prevent contamination, e.g. 100 U/mL penicillin and 100 μg/ mL streptomycin.
3. Equilibrate complete StemMACS Cardiac Cultivation Medium XF at room temperature (20–25 °C) prior to use.
4. Replace old StemMACS Cardiac Cultivation Medium XF with fresh medium every 48 hours: 2 mL per well of a 12-well plate.
   ▲Note: In case dead cells are visible in the supernatant, washing with D-PBS (2 mL per well) is recommended before adding fresh StemMACS Cardiac Cultivation Medium XF.

After day 10 cells can be harvested for:
- Re-plating for long-term culturing.
 ▲Note: Cells should be transferred into a freshly coated culture ware just if necessary, e.g. when cells show signs of detachment from the plate.

- Freezing
- Cell isolation
- Flow cytometry

Cell harvest with Multi Tissue Dissociation Kit 3

To achieve best performance, dissociate the monolayer cultures into a single-cell suspension using the Multi Tissue Dissociation Kit 3 (# 130-110-204). The Multi Tissue Dissociation Kit 3 has been developed for the gentle, rapid, and effective generation of single-cell suspensions and results in high yield of viable cells.

▲Note: Volumes below are given for 12-well plates.

1. Remove cell culture supernatant from the cultured cells.
2. Wash 3 times with 1 mL PBS per well.
3. Prepare enzyme mix by adding Enzyme T to Buffer X of the Multi Tissue Dissociation Kit 3 in a ratio of 1:10, for example, add 50 μL of Enzyme T to 450 μL of Buffer X.
4. Add to each well containing cells 400 μL enzyme mix.
5. Incubate cells for 10 minutes at 37 °C.
6. Add to each well containing cells 600 μL of DMEM/F12 supplemented with 20% FBS.
7. Gently detach cells from the dish by pipetting 3 times up and down using a 1 mL pipette.
   ▲Note: For late stage differentiation it may be necessary to pipette more often.
8. Transfer the cells to a MACS® SmartStrainer (70 μm) placed on a 50 mL tube.
9. Wash each well with 1 mL DMEM/F12 supplemented with 20% FBS and transfer also to the MACS SmartStrainer (70 μm).
10. Wash MACS SmartStrainer (70 μm) with 1 mL DMEM/F12 supplemented with 20% FBS.
11. Determine the cell number and proceed to subsequent application (3.2 Cell seeding, 3.3 Cell freezing and thawing, 4. Isolation of cardiomyocytes, 5.1 Flow cytometry).

Cell harvest with TrypLE™ Select Enzyme

1. Aspirate cell culture medium and wash each well of the 6-well plate with 2 mL of D-PBS without Ca²⁺ and Mg²⁺.
2. Add 1 mL/well TrypLE™ Select Enzyme. Gently rock the plate to ensure even distribution of the solution.
3. Incubate for 10 minutes in the dark at 37 °C.
4. Stop enzymatic reaction by adding 1 mL/well of Soybean Trypsin Inhibitor (0.5 mg/mL).
5. Using a 5 mL serological pipette, dissociate to a single-cell suspension by carefully pipetting up and down.
   ▲Note: It is important to obtain a single-cell suspension.
6. Determine the cell number and proceed to subsequent application (3.2 Cell seeding, 3.3 Cell freezing and thawing, 4. Isolation of cardiomyocytes, 5.1 Flow cytometry).

1. Follow the preferred protocol for cell harvesting “3.1 Cell harvest”.
2. Centrifuge at 200×g for 5 minutes.
3. Aspirate supernatant completely.
4. Resuspend in an appropriate amount of StemMACS Cardiac Cultivation Medium XF (2 mL per well of a 12-well plate. If using different culture ware adjust volumes accordingly).
   ▲Note: It is recommended to seed 300,000 cells/cm² up to 600,000 cells/cm².
5. Transfer the cells in a freshly coated plate.
6. Swirl the plate to ensure even distribution of cells and transfer to an incubator 37 °C, 5% CO₂. Leave the cells undisturbed for 24 hours.
7. The day after seeding it is recommended to wash once with D-PBS to remove cellular debris and dead cells and to feed with fresh StemMACS Cardiac Cultivation Medium XF.

Freezing

1. Follow the preferred protocol for cell harvesting 3.1 Cell harvest.
2. Transfer desired cell number into a 15 mL conical tube.
   ▲Note: It is recommended to freeze cardiomyocytes in aliquots of 250 µL containing 5×10⁶ cells.
3. Centrifuge for 5 minutes at 200×g.
4. Resuspend the cell pellet in StemMACS Cryo-Brew to 2×10⁷ cells per mL.
5. Quickly transfer the cell suspension into suitable cryogenic vials (250 µL per vial).
6. Place the vials into an isopropanol freezing container and immediately store at –80 °C.
7. After 24 hours transfer cells into a liquid nitrogen tank for long-term storage.
    

Thawing

• Work quickly to avoid loss of cells.

1. Take a vial with cells out of the liquid nitrogen container.
2. Incubate the vial in a 37 °C water bath until only a little lump of ice is left.
3. Quickly transfer cell suspension into a 15 mL conical tube and dropwise add 5 mL of used cell culture medium.
4. Centrifuge for 5 minutes at 200×g.
5. Aspirate supernatant.
6. Resuspend the cell pellet in the culture medium supplemented with StemMACS™ Y27632 10 µM or StemMACS™ Thiazovivin 0.5–10 μM.
7. Seed 300,000 cells/cm² in freshly coated plates.

After differentiation with StemMACS Cardio Diff Kit it is possible to further enrich the cardiomyocyte culture by isolating cardiomyocytes with the  PSC-Derived Cardiomyocyte Isolation Kit, human (# 130-110-188). Moreover, prolonged culturing can increase the purity of the cardiomyocyte population. 

It is recommended to perform the isolation step in case:

• End-experiment requires highly homogeneous cardiomyocytes populations. 
• Some genetically modified PSC lines might not respond well to induction protocols and show low efficiency rates.

The isolation is performed either as a single-step or two-step protocol, depending on the initial differentiation efficiency (see isolation strategy below). As a general guideline, we recommend performing the second (positive selection) step if the culture contains <50 % of cardiomyocytes.

The standard protocol enables enrichment of cardiomyocytes from up to 5×10⁶ total cells, and upscaling is possible for up to 1×10⁷ total cells.

Download kit data sheet

PSC-Derived Cardiomyocyte Isolation Kit, human

Different cardiomyocyte subpopulations can be identified and analyzed using techniques such as patch clamp, immunocytochemistry or flow cytometry. Compared to other techniques, flow cytometry is a fast and efficient method to analyze and quantify different cell types within a cell population, without difficult techniques or genetic engineering of the starting material.

• See also the application note "Characterization by flow cytometry PSC-derived subtypes".

1. Harvest cultured purified cardiomyocytes. For details refer to 2.2 Cell harvest.
2. Count isolated cardiomyocytes by flow cytometry using the MACSQuant® Analyzer 10.
3. Transfer 5×10⁵ cells per staining into a 1.5 mL microtube and centrifuged at 300×g for 5 minutes.
4. Remove the supernatant and resuspend the pellet in InsideFix (a component of the Inside Stain Kit) 1:1 diluted in DPBS.
5. Incubate cells for 10 minutes at room temperature.
6. Wash the cells with 1 mL autoMACS® Running Buffer – MACS® Separation Buffer and centrifuge at 300×g for 5 minutes.
7. Remove the supernatant.
8. Resuspend cells in 80 μL Inside Perm (a component of the Inside Stain Kit). Add 10 μL each of the following antibodies:
   Anti-Cardiac Troponin T-FITC and Anti-α-Actinin (Sarcomeric)-VioBlue®
   Anti-Cardiac Troponin T-FITC and Anti-Myosin Heavy Chain-APC
   Anti-Cardiac Troponin T-TnT-FITC and Anti-MLC2a-APC
   Anti-Cardiac Troponin T-TnT-FITC and Anti-MLC2v-PE
   Anti-MLC2a-APC and Anti-MLC2v-PE
9. Incubate cells for 10 minutes at room temperature and in the dark.
10. Wash cells with 1 mL Inside Perm and centrifuge at 300×g for 5 minutes.
11. Remove the supernatant and resuspend the cell pellet in 250 μL autoMACS Running Buffer – MACS Separation Buffer.
12. Analyze the stained cells on the MACSQuant® Analyzer 10.

Besides analysis by flow cytometry, cardiomyocytes are often examined by microscopy using immunofluorescence staining. Recombinantly engineered REAfinity™ Antibodies enable unambiguous analysis of cardiomyocytes.

1. Wash cells cultures twice with PBS.
2. Dilute Inside Fix (from the Inside Stain Kit) 1:1 with PBS.
3. Fix cells with Inside Fix for 10 minutes in the dark at room temperature.
4. Wash cells twice with PBS.
   ▲Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
5. Add the Anti-α-Actinin (Sarcomeric) pure or Anti-Cardiac Troponin T pure antibody 1:100 to Inside Perm, e.g., 1 μL antibodies to 99 μL Inside Perm.
6. Add the antibody solution from step 5 to cells and incubate for 10 minutes in the dark at room temperature.
7. Wash cells twice with PBS.
8. Add an appropriate secondary antibody, e.g., Anti-IgG (H+L)-Vio® 515, 1:100 to Inside Perm. Add the solution to the cells.
   ▲Note: If using a cell nucleus marker, e.g., DAPI, add it at this point as well.
9. Incubate for 10 minutes in the dark at room temperature.
10. Wash cells twice with PBS.
11. Cells are now ready for immunofluorescence microscopy.
    ▲Note: Samples can be stored at 2–8 °C in the dark for up to one week.

1. Lian, X. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109: E1848–E1857.
2. Lian, X. et al. (2013) Nat. Protoc. 8: 162–175.
3. Burridge, P.W. et al. (2014) Nat. Methods 11: 855–860.
4. Zhang, J. et al. (2012) Circ. Res. 111: 1125–1136.

The following is a list of relevant resources that might be of interest:

• Cardiovascular research solutions – Conveniently groundbreaking
• PSC-derived Cardiomyocyte Isolation Kit, human – Gentle and fast purification of hPSC-derived cardiomyocytes
• REAfinity™ Recombinant Antibodies – Flow cytometry is in their genes
• REAfinity Recombinant Antibodies – Frequently Asked Questions
• Application: Cardiomyocyte purification from pluripotent stem cells
• Application: Characterization by flow cytometry of PSC-derived cardiomyocyte subtypes
• Immunofluorescence staining with Anti-α-Actinin (Sarcomeric) or Anti-Cardiac Troponin T antibodies for microscopy (includes protocols for cultivated cells, acetone-fixed and formalin-fixed tissue sections).
   
• Noack, K., et al. (2016) Highly efficient immunomagnetic purification of cardiomyocytes derived from human pluripotent stem cells. ISSCR. San Francisco, USA.

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