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 generate large numbers of highly pure functional cardiomyocytes for high throughput in vitro assays.

Described below is a 2D protocol for differentiating hPSCs into hPSC-CMs using STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit and their subsequent large-scale expansion using STEMdiff™ Cardiomyocyte Expansion Kit. A large expansion of hPSC-CMs is achievable using this method; for example, if starting from 1 x 10⁸ hPSC-CMs, approximately 8.9 x 10⁸ hPSC-CMs can be generated within 32 days. In addition to generating large numbers of hPSC-CMs, it has also been shown that cardiomyocyte purity steadily increases with each passage.

Important Notes

• This protocol describes the steps required for expanding hPSC-CMs in 2 x 5-layered T-875 cell culture flasks and a 5-chambered CellSTACK®; if using different cultureware formats, refer to the tables below to find the alternative volumes of reagents required.
• We recommend the direct pouring method for seeding, exchanging medium, and harvesting cells. To maintain sterility, it is critical to use a sterile technique and avoid making contact between open cell culture flasks or CellSTACK® ports and media bottles when pouring.
• Multi-layered cell culture flasks, such as a 5-layered T-875 or 5-chambered CellSTACK®, cannot be viewed under a standard bright field microscope. To monitor cell health and confluence, it is recommended to seed a “companion plate”, such as a 6-well plate, at the same cell density per cm², and feed it at the same frequency and with the same volume of STEMdiff™ Cardiomyocyte Expansion Medium per cm² as the 5-layer cell culture flask and CellSTACK®.
• A seeding density of approximately 5.2 x 10⁴ cells/cm² is recommended, however, the inherent biological variability of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines may mean that seeding density optimization is required.
• If you are following along with the volumes suggested in this protocol, you will need multiple kits:
  • 1 x STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit
  • 6 x STEMdiff™ Cardiomyocyte Expansion Kit
  • 8 x STEMdiff™ Cardiomyocyte Maintenance Kit

Materials

• STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Catalog #05010)
• STEMdiff™ Cardiomyocyte Dissociation Kit (Catalog #05025)
• STEMdiff™ Cardiomyocyte Expansion Kit (Catalog #100-1109)
• STEMdiff™ Cardiomyocyte Maintenance Kit (Catalog #05020)
• hESC-Qualified Corning® Matrigel® (Corning® Catalog #354277)
• mTeSR™1 (Catalog #85850)
• D-PBS (Without Ca++ and Mg++) (Catalog #37354)
• 5/10/25/50 mL Falcon® Serological pipettes (e.g. Catalog #38003)
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• Falcon® 6-Well Flat-Bottom Plate, Tissue Culture-Treated (Catalog #38016)
• Falcon™ Cell Culture Multi Flask, 875 cm2 (e.g. Fisher Scientific, Catalog #353144)
• CellSTACK®, 5 Chambers (Catalog #38076)
• Falcon® Conical Tubes, 15 mL (e.g. Catalog #100-0092)
• Falcon™ Polypropylene Centrifuge Tubes, 225mL (e.g. Fisher Scientific, Catalog #05-538-61)
• 1L Bottle (e.g. Nalgene, Sigma-Aldrich Catalog #Z364525)
• Gentle Cell Dissociation Reagent (Catalog #100-0485)
• AO-DAPI and automated cell counter, or Trypan Blue (Catalog #07050) and hemocytometer (e.g. Hausser Scientific™ Bright-Line Hemocytometer Catalog #100-1181)

For Optional Downstream Assays:

• STEMdiff™ Cardiomyocyte Cardiomyocyte Freezing Medium (Catalog #05030)
• STEMdiff™ Cardiomyocyte Plating Supplement (Catalog #100-1121)
• STEMdiff™ Cardiomyocyte Support Medium (Catalog #05027)
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Part I. Preparation of Media

Figure 2. Scale-Up of hPSC-Derived Cardiomyocytes Protocol

As you scale up your cardiomyocyte culture following this protocol, you will transition from culture dishes to layered cell culture flasks like the CellSTACK®. Estimates of cell yield at each step are shown.

Part II. Dissociation of hPSCs into a Single-Cell Suspension

Start with a clump culture of hPSCs maintained in a TeSR™ media on Corning® Matrigel®-coated 6-well plates. It is critical to start with high-quality hPSC cultures for efficient and effective cardiomyocyte differentiation. hPSCs must have high expression of pluripotency markers, e.g. OCT4 and TRA-1-60. For complete instructions on maintaining hPSCs in TeSR™ media or coating plates with Corning® Matrigel®, refer to their respective Technical Manuals.

1. Coat a 10 cm² dish with Corning® Matrigel® and bring to room temperature (15 - 25°C) for at least 1 hour prior to use.
2. Wash each well to be passaged with 6 mL of D-PBS (Without Ca++ and Mg++).
3. Aspirate the wash and add 1 mL/well of Gentle Cell Dissociation Reagent.
4. Incubate at 37°C and 5% CO₂ for 8 - 10 minutes.
5. In each well, dislodge cells by pipetting up and down 3 - 4 times using a pipette with a 1000 μL tip.
6. Immediately transfer cells to a tube containing 6 mL of TeSR™ media per well harvested.
7. Centrifuge at 300 x g for 5 minutes. Remove and discard supernatant.
8. Gently resuspend the cell pellet with 1 - 2 mL of TeSR™ media supplemented with 10 μM Y-27632.
9. Perform a cell count using an automated cell counter (e.g. NucleoCounter® NC-250™) or with Trypan Blue and a Hausser Scientific™ Bright-Line Hemocytometer.

Part III. Culture of Single-Cell hPSCs

Day -2

1. Aspirate Matrigel® from a coated 10 cm2 dish (prepared in Part II, step 1). Add 10 mL of TeSR™ media supplemented with 10 μM Y-27632 per well.
2. Add hPSCs (from Part II) at a density of 4.0 x 106 - 1.13 x 107 cells/10 cm2 dish. Gently rotate the dish to ensure uniform distribution of cells.
   Note: A range of seeding densities is provided in this step, to account for differences in hPSC lines and variations in their rate of proliferation during maintenance culture.
3. Incubate at 37°C for 24 hours.

Day -1

4. Remove the medium and replace with 30 mL of fresh TeSR™ media (without Y-27632). Incubate at 37°C for 24 hours.
5. Assess cells for confluency.


Critical: Cells must reach > 95% confluency before starting the differentiation protocol (Part IV. Ventricular hPSC-Cardiomyocyte Differentiation and Maintenance [Day 0 - 11]). Figure 3 is a representative example of this level of confluency. If cells are < 95% confluent, do not continue incubation. Instead, repeat steps 1 - 5, seeding cells at a higher density than previously used.


Figure 3. SCTi003-A hPSCs at > 95% Confluency (Day 0 of hPSC-CM Differentiation)

This figure shows an example phase microscopy image of how your hPSCs should appear when they are ready to begin the cardiomyocyte differentiation step.

6. Once > 95% confluency is achieved, proceed to Part IV for ventricular cardiomyocyte differentiation and maintenance.

Part IV. Ventricular hPSC-Cardiomyocyte Differentiation and Maintenance (Day 0 - 11)

Day 0

1. Thaw Matrigel® on ice. Add 300 μL of Matrigel® to 30 mL of STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium A (1 in 100 dilution).
2. Remove the medium from the 10 cm2 dish from Part III. Add 30 mL of STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium A supplemented with Matrigel® (prepared in step 1) per dish. Incubate at 37°C for 2 days.

Days 2 - 11

3. Perform a full-medium change on Day 2 and every 2 days until Day 11, as follows:
   1. Using a pipettor, gently remove the medium from the wells (do not aspirate).
   2. Gently add 30 mL of medium per well. For different volumes required for other culture vessels see Table 2. Incubate at 37°C.
4. On Day 11, hPSC-derived ventricular cardiomyocytes are ready to be harvested for scale-up.