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Differentiation of hPSCs to Ventricular Cardiomyocytes in 3D Culture

4 Parts 14 Materials Print

Cardiovascular disease is the leading cause of death globally, and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a powerful platform that can be used for modeling heart disease, drug discovery, and regenerative medicine. Researchers working with hPSC-CMs need to efficiently generate large numbers of highly pure functional cardiomyocytes. To meet this demand, many in the field are moving toward 3D hPSC expansion and differentiation.

Here, we describe a scalable protocol that enables reproducible production of high-quality ventricular cardiomyocytes in 3D suspension culture using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. In just 14 days, you can generate cultures of beating aggregates containing over 80% cardiac troponin T-positive (cTnT+) ventricular cardiomyocytes, with yields of up to 5 × 107 cells per PBS-MINI 0.1 MAG Single-Use Vessel.

This 3D protocol is a valuable option for customers already expanding their hPSCs in 3D suspension culture, as it can be seamlessly incorporated into their workflow. Furthermore, this 3D protocol improves efficiency over the monolayer protocol by reducing the length of the protocol by 2 days and reduces cost per cardiomyocyte by decreasing media usage. Finally, scaling up differentiation in 3D generates a homogeneous culture, ensuring consistent, reproducible results and providing another option to customers looking to generate large numbers of high-quality ventricular cardiomyocytes from hPSCs.

Cells respond differently in 3D suspension culture compared to 2D adherent culture. This 3D method builds on the 2D monolayer protocol to deliver a more scalable approach for differentiating hPSCs into cardiomyocytes. Table 1 highlights the key differences between the two formats.

Table 1. Recommended Protocol Modifications When Using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit in 3D Suspension Culture

Process Parameter 2D Monolayer Protocol Recommendation for 3D Suspension Culture
Seeding Density (viable cells / mL) 3.5 – 8 × 105 6-Well Plate and Nalgene™ Bottle: 4 – 6 × 104
PBS-MINI: 6 – 8 × 104
Seeding Method Single Cell Clump
Media Change Strategy 100% change 100% change when switching media;
75% media change otherwise
Feeding Volume (mL / 6wp) 4 2
Days in TeSR™ Medium before Differentiation Initiation 2 1
Days in Medium A 2 1
Matrigel® Addition in Medium A? Yes No
Agitation N/A Reference
Table 1. Suspension Culture Vessels

Materials

Reagents:

3D Suspension Culture Vessels:

Cardiomyocyte Differentiation in 3D Suspension Culture Protocol Diagram

Figure 1. Cardiomyocyte Differentiation in 3D Suspension Culture Protocol Diagram

hPSCs are maintained in TeSR™-AOF 3D suspension culture for at least two passages. One day before beginning the differentiation protocol, hPSC aggregates are harvested and seeded as clumps in TeSR™-AOF 3D using one of the recommended 3D suspension culture vessels. After one day (Day 0), the TeSR™-AOF 3D medium is replaced with Medium A to initiate differentiation toward a cardiomyocyte fate. On Day 1, perform a 100% medium change with Medium B. On Day 3, perform a 100% medium change with Medium C, followed by a 75% medium change with Medium C on Day 5. On Day 7, perform a 100% medium change to switch to Maintenance Medium. On Days 9, 11, and 13, perform 75% medium changes with Maintenance Medium to promote further differentiation into cardiomyocytes. Beating cardiomyocytes are typically observed as early as day 7, with more than 80% of aggregates beating by the Day 14 harvest.

Part 1. Preparation of Media

For complete instructions on preparation of media and storage conditions, refer to the following Product Information Sheets (PIS):

Part 2. hPSC Seeding, Day -1

Start with a 3D hPSC suspension culture maintained in TeSR™-AOF 3D for at least 2 passages. It is critical to start with high-quality hPSC cultures that express markers of the undifferentiated state (e.g. OCT4 and TRA-1-60) to ensure efficient cardiomyocyte differentiation.

  1. Prepare sufficient TeSR™-AOF 3D supplemented with 10 μM Y-27632 to resuspend and seed all conditions. Warm the medium to 37°C in an incubator with the cap slightly loosened to allow for CO₂ equilibration.
  2. Passage hPSCs as small clumps using Gentle Cell Dissociation Reagent (GCDR) and a 37 µm Reversible Strainer as detailed in the . Resuspend the clumps in TeSR™-AOF 3D + 10 μM Y-27632.
    Note: Use a Small 37 µm Reversible Strainer if passaging a 6-Well Plate, and a Large 37 µm Reversible Strainer if passaging a Nalgene™ Bottle or PBS-MINI.
  3. Gently resuspend the clumps by flicking the tube. Remove a sample for viable cell counts and calculate the volume of cell suspension required for seeding at a density of:
    1. 6-Well Plate or Nalgene™ Bottle: 4 – 6 × 104 viable cells/mL
    2. PBS-MINI: 6 – 8 × 10⁴ viable cells/mL
    Note: It is recommended to use the A100-B Assay on the ChemoMetec NucleoCounter® NC-250™ for clumpy suspension cell counts.
    Note: When seeding based on clump counts, assume approximately 100 viable cells per clump. Automated viable cell counting is recommended to reduce seeding variability.
  4. Add required volume of clump suspension to the culture vessel, then top up with warmed TeSR™-AOF 3D + 10 μM Y-27632 to desired total volume.
  5. Incubate for 24 hours at 37°C at the optimized agitation rate (RPM) as detailed in Table 2.
    Note: As the Nalgene™ Rapid-Flow™ Bottles do not have vented caps, open the caps slightly when placing them in the incubator to allow gas exchange.

Table 2. Agitation Rate for Recommended Culture Vessels

Culture Vessel Culture Volume Recommended Agitation Rate
6-Well Flat-Bottom Plate, Non-Treated 2 mL 70 RPM (2.5 cm orbital diameter)
Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles 15 mL
30 mL
60 mL		 40 RPM
55 RPM
65 RPM (2.5 cm orbital diameter)
PBS-MINI 0.1 MAG Single-Use Vessel 100 mL 40 RPM
PBS-MINI 0.5 MAG Single-Use Vessel 500 mL 45 RPM

For complete instructions on maintaining hPSCs in TeSR™-AOF 3D, refer to the Technical Manual.

Part 3. Differentiation – Media Changes, Days 0–13

For preparation of STEMdiff™ Ventricular Cardiomyocyte Differentiation and Maintenance media, refer to Part 1 - Preparation of Media.

For Day 0 to 13, perform the medium changes shown in Table 3.

Table 3. Medium Change Workflow

Day Medium % Medium Change
0 STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium A 100
1 STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium B 100
3 STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium C 100
5 STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium C 75
7 STEMdiff™ Cardiomyocyte Maintenance Medium 100
9 STEMdiff™ Cardiomyocyte Maintenance Medium 75
11 STEMdiff™ Cardiomyocyte Maintenance Medium 75
13 STEMdiff™ Cardiomyocyte Maintenance Medium 75

The following are instructions for performing medium changes in different culture vessels.


6-Well Plate

  1. Aliquot the required volume of fresh medium for the medium change into a tube or bottle, and warm to 37°C in an incubator with the cap slightly opened to allow for CO₂ equilibration. Once warmed, proceed to step 2.
  2. Swirl plate to center the aggregates, then place the plate at a 30 - 45° angle and allow aggregates to settle for ~ 1 minute.
  3. Remove spent medium carefully (2 mL for 100% change; 1.5 mL for 75%).
    Note: It may be challenging to remove 2 mL per well without aspirating any aggregates. If needed, reduce to a 95% medium change instead of a 100% medium change in the 6-well plate.
  4. Replace with an equal volume of warmed fresh medium.

Nalgene™ Bottle

  1. Aliquot the required volume of fresh medium for the medium change into a tube or bottle, and warm to 37°C in an incubator with the cap slightly opened to allow for CO₂ equilibration. Once warmed, proceed to step 2.
  2. Remove required volume of spent media with a serological pipette and pass over a large 37 µm Reversible Strainer with the arrow pointing up to collect removed aggregates (75% or 100% of the seed volume).
  3. Flip strainer back over the Nalgene™ Bottle such that the arrow is pointing down, and rinse aggregates back in with an equal volume of warmed fresh medium.

PBS-MINI MAG Single-Use Vessel

  1. Aliquot the required volume of fresh medium for the medium change into a bottle, and warm to 37°C in an incubator with the cap slightly opened to allow for CO₂ equilibration. Once warmed, proceed to step 2.
  2. Remove required volume of spent media with a serological pipette and pass over a large 37 µm Reversible Strainer with the arrow pointing up to collect removed aggregates (75% or 100% of the seed volume).
  3. Add 80% of the required volume of warmed fresh media into the PBS-MINI.
  4. Flip strainer back over the PBS-MINI such that the arrow is pointing down and rinse aggregates back in with the remaining 20% of the required volume of fresh medium.
    1. This eliminates the “death drop” of the aggregates being rinsed back into an empty vessel and colliding with the wheel.

Part 4. Harvesting Differentiated Cells (Day 14)

The following are instructions for harvesting the cTnT+ aggregates from different culture vessels and dissociating them to single cells.

Table 4. Required Reagent Volumes for the Harvest Protocol

Culture Vessel Culture Volume Volume STEMdiff™ Cardiomyocyte Dissociation Medium + DNase I Solution Volume STEMdiff™ Cardiomyocyte Plating Medium
6-Well Flat-Bottom Plate, Non-Treated 2 mL 1 mL 3 - 4 mL
Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles 15 mL
30 mL
60 mL		 10 mL
15 mL
25 mL		 7.5 mL
15 mL
30 mL
PBS-MINI 0.1 MAG Single-Use Vessel 100 mL 40 mL 45 mL
PBS-MINI 0.5 MAG Single-Use Vessel 500 mL 150 mL 250 mL

6-Well Plate:

  1. Prepare required volume of STEMdiff™ Cardiomyocyte Dissociation Medium with a final concentration of 0.1 mg/mL DNase I Solution. Warm to 37°C.
  2. Warm STEMdiff™ Cardiomyocyte Plating Medium to room temperature.
  3. Prepare one 15 mL conical tube per harvested well with 2 mL STEMdiff™ Cardiomyocyte Plating Medium.
  4. Swirl plate to center the aggregates, then place the plate at a 30 - 45° angle and allow aggregates to settle for ~ 1 minute.
  5. Remove as much spent media as possible per well without aspirating aggregates.
  6. Add 1 mL D-PBS to each well.
  7. Allow the aggregates to settle for ~ 1 minute then remove as much of the D-PBS as possible per well without aspirating aggregates.
  8. Add 1 mL Dissociation Medium + DNase to each well.
  9. Incubate at 37° on an orbital shaker at 70 RPM for 15 minutes.
  10. Use a P1000 to triturate each well to single cells.
  11. Transfer the cell suspension in each well to a tube with 2 mL Plating Medium (prepared in Step 3).
  12. Rinse each well with 1 mL Plating Medium to collect any remaining single cells and transfer to the corresponding tube.
  13. Centrifuge at 300 x g for 5 minutes. Remove and discard supernatant.
  14. Resuspend in 1 - 2 mL Plating Medium or other medium for downstream applications / analysis.
    Note: Viability of dissociated cardiomyocytes is expected to be > 80%.

Nalgene™ Bottle / PBS-MINI MAG Single-Use Vessel:

  1. Prepare required volume of STEMdiff™ Cardiomyocyte Dissociation Medium with a final concentration of 0.1 mg/mL DNase I Solution. Warm to 37°C.
  2. Warm STEMdiff™ Cardiomyocyte Plating Medium to room temperature.
  3. Filter out non-aggregated single cells by passing the entire volume of the culture through a Large 37 µm Reversible Strainer with the arrow pointing up into a 50 mL conical tube or sterile bottle.
    Note: Multiple strainers may be required for the PBS-MINI cultures.
    Note: Aggregates can also be settled and spent media aspirated out of the PBS-MINI to reduce volume filtered.
  4. Rinse the strainer with 5 mL D-PBS to wash away residual spent media.
  5. Flip strainer back over the Nalgene™ Bottle / PBS-MINI such that the arrow is pointing down, and rinse aggregates back in with required volume of Dissociation Medium + DNase I.
  6. Place the Nalgene™ Bottle / PBS-MINI back in the incubator and follow the RPM protocol detailed in Table 5.
    Note: Optimal incubation time and RPM may vary depending on the cell line.
  7. Transfer Nalgene™ Bottle / PBS-MINI contents to a 50 mL conical tube.
    Note: Multiple conical tubes will be required for the PBS-MINI 0.5 harvest. Combine cell suspensions at the end into a sterile bottle.
  8. Centrifuge at 300 x g for 5 minutes.
  9. Aspirate the supernatant, leaving ~ 1 mL in the conical tube.
  10. Use a P1000 to triturate to single cells.
  11. Add required volume of Plating Medium (as detailed in Table 4) or other medium for downstream applications / analysis.
    Note: Viability of dissociated cardiomyocytes is expected to be > 80%.

Table 5. RPM Protocol for Recommended Culture Vessels and Dissociation Volumes

Culture Vessel Dissociation Volume RPM Protocol
Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles 10 mL 1. 40 RPM for 10 minutes
2. 50 RPM for 5 minutes
15 mL 1. 45 RPM for 10 minutes
2. 55 RPM for 5 minutes
25 mL 1. 55 RPM for 10 minutes
2. 65 RPM for 5 minutes
PBS-MINI 0.1 MAG Single-Use Vessel 40 mL 1. 30 RPM for 5 minutes
2. 40 RPM for 9 minutes
3. 60 RPM for 1 minute
PBS-MINI 0.5 MAG Single-Use Vessel 150 mL
Aggregate Morphology Throughout 3D Cardiomyocyte Differentiation

Figure 2. Aggregate Morphology Throughout 3D Cardiomyocyte Differentiation

Representative images of the aggregates on (A) Day 0, (B) Day 1, (C) Day 7, and (D) Day 14. Aggregates grow in size and remain compact over the course of the differentiation to ventricular cardiomyocytes.

hPSC Aggregate Size Distributions on Day 0

Figure 3. hPSC Aggregate Size Distributions on Day 0

hPSC aggregate size distributions for 6-Well Plate and PBS-MINI 0.1 MAG Single-USe Vessel cultures on Day 0, with the color denoting the seeding density (orange = 4 × 104 and grey = 6 × 104 viable cells/mL). Each point represents the diameter of one aggregate, and the bars represent the median aggregate diameter for each condition. hPSC aggregate size distributions vary slightly across seeding densities and culture vessels, with median diameters of ~ 130 - 140 µm on Day 0.

Aggregates on the 3D Cardiomyocyte Harvest Day

Figure 4. Aggregates on the 3D Cardiomyocyte Harvest Day

Representative image of cardiomyocytes cultured in a PBS-MINI 0.1 MAG Single-Use Vessel on the 3D ventricular cardiomyocyte harvest day. Cardiomyocyte aggregates are distributed throughout the vessel and are visible by eye.

Efficient and Robust Generation of cTnT-Positive Cardiomyocytes in 3D Suspension Culture

Figure 5. Efficient and Robust Generation of cTnT-Positive Cardiomyocytes in 3D Suspension Culture

hPSCs were cultured for 14 days in one of the recommended 3D suspension culture vessels using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. Representative results for the 6-well plate and PBS-MINI 0.1 MAG Single-Use Vessel are shown. At the end of the culture period, cells were harvested, counted, and analyzed by flow cytometry for expression of cardiac troponin T (cTnT). Data shown are (A) histogram analysis for cardiomyocyte cell marker cTnT for a culture of SCTi003-A cells (filled = sample; blank = unstained control); (B) percentage of cells expressing cTnT; (C) number of viable cTnT+ cells per mL of differentiation culture volume; and (D) total number of viable cTnT+ cells per well / vessel. Points for the 6-well plate culture represent the mean of three technical replicates for each cell line, and bars represent the mean. Shape denotes the seeding density: ▲ = 4 ×104, ⏺ = 5 × 104, ▼ = 6 × 104 viable cells / mL.

3D-Generated Ventricular Cardiomyocytes Exhibit a Robust and Stable Excitability Profile

Figure 6. 3D-Generated Ventricular Cardiomyocytes Exhibit a Robust and Stable Excitability Profile

Microelectrode array (MEA) recordings of hPSC-derived cardiomyocytes cultured in 2D and 3D using the STEMdiff™ Ventricular Cardiomyocyte Kit. The cardiomyocytes differentiated in 2D were dissociated to single cells, plated down on the MEA plate, and recorded 7 days later. The cardiomyocytes differentiated in 3D were transferred to the MEA plate as whole aggregates and recorded 2 days later. Cardiomyocytes had expected (A) Field Potential Duration (FPD), (B) Beat Period, and (C) Beats Per Minute (BPM), with slight differences between 2D and 3D culture formats. MEA recordings were performed using the Maestro Pro™ MEA System.

  • Document #PR00110
  • Version 1.0.0
  • December 2025
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