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Robust Scale-Up of hPSC-Derived Cardiomyocytes in 2D Monolayer Culture

10 Parts 19 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 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.

Scale-up cultureware diagram showing the estimated cardiomyocyte yield from dishes to multilayer flasks.

Figure 1. Scale-Up Cultureware

This simplified reference diagram illustrates the four stages of the cardiomyocyte expansion protocol. In the first two stages, hPSCs are cultured with TeSR™ medium and then differentiated into cardiomyocytes using STEMdiff™ Ventricular/Atrial Cardiomyocyte Differentiation Kit. These early-stage cardiomyocytes are then expanded with STEMdiff™ Cardiomyocyte Expansion Kit and maintained throughout the rest of the protocol with STEMdiff™ Cardiomyocyte Maintenance Kit.

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:

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)
    • See More
    • 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)
    See Less

Part I. Preparation of Media

The four stages of the cardiomyocyte expansion protocol

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.

A. Preparation of STEMdiff™ Ventricular Cardiomyocyte Differentiation Media (A, B & C)

Prepare STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium (Differentiation Basal Medium + Differentiation Supplement A, B, or C). The following example is for preparing 100 mL of STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium A. If preparing other volumes, adjust reagent quantities accordingly. For Medium B and Medium C, follow the instructions below, replacing Differentiation Supplement A with Differentiation Supplement B or Differentiation Supplement C, respectively.

  1. Thaw Differentiation Supplement A at room temperature (15 - 25°C). Mix thoroughly.
    Note: If not used immediately, aliquot supplement and store at -20°C. Do not exceed the shelf life of the supplement. Once aliquots are thawed, do not re-freeze.
  2. Add 10 mL of Differentiation Supplement A to 90 mL of Differentiation Basal Medium. Mix thoroughly.
    Note: If not used immediately, store STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium A, B, or C at 2 - 8°C for up to 2 weeks. Warm medium to room temperature before use.

B. Preparation of STEMdiff™ Cardiomyocyte Maintenance Medium

Prepare STEMdiff™ Cardiomyocyte Maintenance Medium (Maintenance Basal Medium + Maintenance Supplement). The following example is for preparing 500 mL of complete medium. If preparing other volumes, adjust reagent quantities accordingly.

  1. Thaw Maintenance Supplement at room temperature (15 - 25°C). Mix thoroughly.
    Note: If not used immediately, aliquot supplement and store at -20°C. Do not exceed the shelf life of the supplement. Once aliquots are thawed, do not re-freeze.
  2. Add 10 mL of Maintenance Supplement to 490 mL of Maintenance Basal Medium. Mix thoroughly.
    Note: If not used immediately, store STEMdiff™ Cardiomyocyte Maintenance Medium at 2 - 8°C for up to 4 weeks. Warm medium to room temperature before use.

C. Preparation of STEMdiff™ Cardiomyocyte Passaging Medium

Prepare complete STEMdiff™ Cardiomyocyte Passaging Medium (STEMdiff™ Cardiomyocyte Support Medium + STEMdiff™ Cardiomyocyte Passaging Supplement [100X]). The following example is for preparing 100 mL of complete medium. If preparing other volumes, adjust reagent quantities accordingly.

  1. Thaw STEMdiff™ Cardiomyocyte Support Medium at room temperature (15 - 25°C) or overnight at 2 - 8°C. Mix thoroughly.
  2. Thaw STEMdiff™ Cardiomyocyte Passaging Supplement (100X) at room temperature. Mix thoroughly.
    Note: Once thawed, use immediately or aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not re-freeze.
  3. Add 1 mL of STEMdiff™ Cardiomyocyte Passaging Supplement (100X) to 99 mL of STEMdiff™ Cardiomyocyte Support Medium. Mix thoroughly.
    Note: If not used immediately, store the complete STEMdiff™ Cardiomyocyte Passaging Medium at 2 - 8°C for up to 2 weeks. Warm medium to room temperature before use.

D. Preparation of STEMdiff™ Cardiomyocyte Expansion Medium

To prepare complete STEMdiff™ Cardiomyocyte Expansion Medium (STEMdiff™ Cardiomyocyte Maintenance Basal Medium + STEMdiff™ Cardiomyocyte Expansion Supplement [50X]). The following example is for preparing 500 mL of complete medium. If preparing other volumes, adjust reagent quantities accordingly.

  1. Thaw STEMdiff™ Cardiomyocyte Expansion Supplement (50X) at room temperature (15 - 25°C). Mix thoroughly.
    Note: Once thawed, use immediately or aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not re-freeze.
  2. Add 10 mL of STEMdiff™ Cardiomyocyte Expansion Supplement (50X) to 490 mL of STEMdiff™ Cardiomyocyte Maintenance Basal Medium. Mix thoroughly.
    Note: If not used immediately, store complete STEMdiff™ Cardiomyocyte Expansion Medium at 2 - 8°C for up to 2 weeks. Warm medium to room temperature before use.

E. Preparation of STEMdiff™ Cardiomyocyte Plating Medium

Prepare STEMdiff™ Cardiomyocyte Plating Medium (Support Medium + Plating Supplement). The following example is for preparing 500mL of complete medium. If preparing other volumes, adjust reagent quantities accordingly.

  1. Thaw STEMdiff™ Cardiomyocyte Plating Supplement (100X) at room temperature (15 - 25°C). Mix thoroughly.
    Note: Once thawed, use immediately or aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not re-freeze.
  2. Add 5 mL of STEMdiff™ Cardiomyocyte Plating Supplement (100X) to 495 mL of STEMdiff™ Cardiomyocyte Support Medium. Mix thoroughly.
    Note: If not used immediately, store complete STEMdiff™ Cardiomyocyte Plating Medium at 2 - 8°C for up to 2 weeks. Warm medium to room temperature before use.

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.

Table 1. Recommended Volumes of Matrigel® for Various Cultureware

CULTUREWARE
VOLUME OF MATRIGEL® REQUIRED FOR COATING
6-well plate
1 mL/well
10 cm2 dish
6 mL/dish
T-875 cm2 flask
90 mL/flask
5-chambered CellSTACK®
330 mL/CellSTACK®
  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.

    4. 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.

    6. 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.
    Phase image of pluripotent stem cells at > 95% confluence ready for differentiation into cardiomyocytes

    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.

    3. 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.
Three microscopy images of hPSC-derived ventricular cardiomyocytes during day 11, day 18, and day 25 of the scale up protocol.

Figure 4. Scaling Up SCTi003-A Ventricular hPSC-CMs

SCTi003-A hPSC-CMs were differentiated using STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. The first microscopy image shows SCTi003-A hPSC-CMs on Day 11, prior to using STEMdiff™ Cardiomyocyte Expansion Kit. The middle image shows expanding SCTi003-A hPSC-CMs on Day 18 (end of passage 1), after 1 week of using STEMdiff™ Cardiomyocyte Expansion Kit. The image on the right shows expanding SCTi003-A hPSC-CMs on Day 25 (end of passage 2).

Three microscopy images of pluripotent stem cells differentiating into atrial cardiomyocytes following the scale-up protocol.

Figure 5. Scaling Up F016 Atrial hPSC-CMs

F016 hPSC-CMs were differentiated using STEMdiff™ Atrial Cardiomyocyte Differentiation Kit. The first microscopy image shows F016 hPSC-CMs on Day 11, prior to using STEMdiff™ Cardiomyocyte Expansion Kit. The middle image shows expanding F016 hPSC-CMs on Day 18 (end of passage 1), after 1 week of using STEMdiff™ Cardiomyocyte Expansion Kit. The image on the right shows expanding F016 hPSC-CMs on Day 25 (end of passage 2).

Table 2. Recommended Volumes of STEMdiff™ Ventricular Cardiomyocyte Differentiation Medium for Various Cultureware

CULTUREWARE
VOLUME OF STEMdiff™ CARDIOMYOCYTE DIFFERENTIATION & MAINTENANCE MEDIUM
6-well plate
4 mL/well
T-175 cm2 flask
100 mL/flask
T-875 cm2 flask
500 mL/flask
5-chambered CellSTACK®
1800 mL/CellSTACK®

Part V. Dissociating and Plating Day 11 hPSC-CMs

The following instructions are for dissociating a 10 cm2 dish of Day 11 early-stage ventricular or atrial hPSC-CMs generated using STEMdiff™ Ventricular or Atrial Cardiomyocyte Differentiation Kit and plating into a 5-layered T-875 cm2 flask.

Note: For other cultureware, adjust volumes according to the tables referenced below. For the preparation of complete STEMdiff™ Cardiomyocyte Passaging Medium, refer to Part I. Preparation of Media.

Day 11

  1. Coat the desired culture vessel for expansion (e.g. 5-layered T-875 flask) with Corning® Matrigel® hESC-Qualified Matrix and bring to room temperature (15 - 25°C) for at least 1 hour prior to use. Refer to Table 1 for the volume used.
    Note: For complete instructions on coating plates with Corning® Matrigel®, refer to the Technical Manual for mTeSR™1 or contact us to request a physical copy.
  2. Warm STEMdiff™ Cardiomyocyte Passaging Medium to room temperature. Warm STEMdiff™ Cardiomyocyte Dissociation Medium to 37°C.
    Note: For complete instructions on preparing STEMdiff™ Cardiomyocyte Dissociation Medium, refer to the Product Information Sheet for STEMdiff™ Cardiomyocyte Dissociation Kit.

    Harvest the Day 11 hPSC-CMs as follows:

    1. Wash each 10 cm2 dish with 10 mL of D-PBS (Without Ca++ and Mg++).
    2. Gently remove the wash and add 10 mL/well of warm (37°C) STEMdiff™ Cardiomyocyte Dissociation Medium.
    3. Incubate at 37°C and 5% CO2 for 10 - 12 minutes.
    4. Add 20 mL of STEMdiff™ Cardiomyocyte Passaging Medium per well. Dislodge cells by pipetting up and down 3 - 5 times using a 25 mL serological pipette.
    5. Immediately transfer and pool cells from each 10 cm2 dish to a 225 mL conical tube containing 30 mL of STEMdiff™ Cardiomyocyte Passaging Medium.
    6. Centrifuge at 600 x g for 5 minutes. Remove and discard supernatant, by pouring into a waste bottle.
    7. Gently resuspend the cell pellet with 16 mL of STEMdiff™ Cardiomyocyte Passaging Medium for each 10 cm2 dish harvested.
  3. Perform a cell count using an automated cell counter (e.g. NucleoCounter® NC-250™) or with Trypan Blue and a hemocytometer.
    Note: It is recommended to perform flow cytometry on the harvested cells to assess for cardiac troponin T (cTnT) expression. The cTnT expression should reach ≥ 80% before proceeding with expansion. Proceeding with atrial or ventricular hPSC-CMs that are < 80% cTnT+ may result in decreased expansion and purity.
  4. Remove Corning® Matrigel® from the coated culture vessel (prepared in step 1). Add 90 mL of STEMdiff™ Cardiomyocyte Passaging Medium per T-875 flask. For other cultureware, refer to Table 3 for recommended volumes.

    5. Table 3. Recommended Volume of Medium for Plating Day 11 hPSC-CMs in Various Cultureware

    CULTUREWARE
    VOLUME OF STEMdiff™ CARDIOMYOCYTE PASSAGING MEDIUM
    6-well plate
    1 mL/well
    T-175 cm2 flask
    18 mL/flask
    T-875 cm2 flask
    90 mL/flask
    5-chambered CellSTACK®
    330 mL/CellSTACK®
  5. Plate cells at a density of 4.6 x 107 cells per 5-layered T-875 flask. Refer to Table 4 for recommended plating densities for other cultureware.
    Note: Adjust the volume of STEMdiff™ Cardiomyocyte Passaging Medium added to the culture vessel relative to the cell suspension volume, such that the total volume added does not exceed the recommended volume listed in Table 3.
    For example, to plate a cell solution containing 4.6 x 107 cells in 90 mL, first add 74 mL of STEMdiff™ Cardiomyocyte Passaging Medium to a conical tube containing 16 mL of cell suspension, for a final plating volume of 90 mL.

    6. Table 4. Recommended Cell Plating Density of hPSC-CMs for Various Cultureware

    CULTUREWARE
    PLATING DENSITY OF hPSC-CMs
    6-well plate
    5.0 x 105 cells/well
    T-175 cm2 flask
    9.1 x 106 cells/flask
    5-layered T-875 cm2 flask
    4.6 x 107 cells/flask
    5-chambered CellSTACK®
    1.7 x 108 cells/CellSTACK®
  6. Incubate at 37°C and 5% CO2 for 24 hours. Do not disturb the cells. Proceed to Part VI.

Part VI. Expansion of hPSC-CMs

For the preparation of STEMdiff™ Cardiomyocyte Expansion Medium, refer to Part I. Preparation of Media. The following instructions are for the expansion of Day 12 hPSC-CMs in 1 x 5-layered T-875 flask. Refer to Table 5 for recommended volumes for various cultureware. It is recommended to seed a companion plate (e.g. 6-well tissue culture-treated plate), as cells in the 5-layered T-875 flask cannot be directly viewed under a microscope.

    Day 12 - 18

  1. Warm prepared STEMdiff™ Cardiomyocyte Expansion Medium to room temperature (15 - 25°C).
  2. Remove STEMdiff™ Cardiomyocyte Passaging Medium from the well and add 180 mL of STEMdiff™ Cardiomyocyte Expansion Medium. For other cultureware, refer to Table 5 for recommended volumes.

    3. Table 5. Recommended Volumes of Medium for Various Cultureware

    CULTUREWARE
    VOLUME OF STEMdiff™ CARDIOMYOCYTE EXPANSION MEDIUM FOR FEEDING DURING hPSC-CM EXPANSION
    6-well plate
    2 mL/well
    T-175 cm2 flask
    36 mL/flask
    T-875 cm2 flask
    180 mL/flask
    5-chambered CellSTACK®
    660 mL/CellSTACK®
  3. Incubate at 37°C and 5% CO2 for 48 hours.
  4. Day 14 and 16: Perform a full-medium change by repeating steps 1 - 3.
  5. Day 18: Expanded hPSC-CMs are ready for harvesting. To continue expanding hPSC-CMs into passage 2 (P2), proceed to Part VII. Passaging Expanding hPSC-Derived Cardiomyocytes. To stop expanding for downstream assays, proceed to Part VIII. Stopping Expansion of hPSC-Derived Cardiomyocytes.
    Note: Atrial or ventricular hPSC-CMs may continue to be expanded and passaged up to 5 times by repeating Part VII & Part VIII, respectively, from Day 18 - 46.

Part VII. Passaging Expanding hPSC-Derived Cardiomyocytes

The following instructions are for the passaging of expanding hPSC-CMs in 1 x T-875 flask. By Day 18, hPSC-CM cultures should typically reach 80 - 100% confluency before passaging. For other cultureware, adjust volumes according to Tables 1, 3 - 6. It is recommended to seed a companion plate (e.g. 6-well tissue culture-treated plate), as cells in the 5-chambered CellSTACK® cannot be directly viewed under a microscope.

    Day 18

  1. Coat the desired culture vessel(s) for expansion (e.g. 5-chamber CellSTACK®) with Corning® Matrigel® hESC-Qualified Matrix and bring to room temperature (15 - 25°C) for at least 1 hour prior to use.
  2. Warm STEMdiff™ Cardiomyocyte Passaging Medium (see Part I. Preparation of Media) to room temperature. Warm thawed STEMdiff™ Cardiomyocyte Dissociation Medium to 37°C.
  3. Harvest the expanding hPSC-CMs as follows:
    1. Rinse the T-875 flask with 90 mL of D-PBS (Without Ca++ and Mg++).
    2. Gently remove the wash and add 90 mL of warm (37°C) STEMdiff™ Cardiomyocyte Dissociation Medium to the well. Refer to Table 4 for recommended volumes.
    3. Incubate at 37°C and 5% CO2 for 3 - 10 minutes.
      Note: For later passages, a longer incubation time of up to 10 minutes may be required.
    4. Add 180 mL of STEMdiff™ Cardiomyocyte Passaging Medium directly to the T-875 flask. Dislodge cells by tapping the T-875 on each side.
    5. Immediately transfer cells to a sterile 1 L bottle containing 270 mL of Passaging Medium. Refer to Table 4 for recommended volumes.
    6. Aliquot the contents of the sterile 1 L bottle into equal volumes for 225 conical tubes.

      7. Table 6. Recommended Volumes of Media for Harvesting Expanding hPSC-CMs in Various Cultureware

      CULTUREWARE
      VOLUME OF STEMdiff™ CARDIOMYOCYTE DISSOCIATION MEDIUM
      VOLUME OF STEMdiff™ CARDIOMYOCYTE PASSAGING MEDIUM*
      6-well plate
      1 mL/well
      5 mL/well
      T-75 cm2 flask
      8 mL/flask
      40 mL/flask
      T-175 cm2 flask
      18 mL/flask
      90 mL/flask
      T-875 cm2 flask
      90 mL/flask
      450 mL/flask
      *Total volume needed to perform steps 3d and 3e.
    7. Centrifuge at 600 x g for 5 minutes. Remove and discard supernatant.
    8. Gently resuspend the cell pellet with 90 mL of STEMdiff™ Cardiomyocyte Passaging Medium.
    9. Perform a cell count using an automated cell counter (e.g. NucleoCounter® NC-250™) or with Trypan Blue and a hemocytometer.
    10. Remove Corning® Matrigel® from the coated vessel(s) prepared in step 1. Add 330 mL of STEMdiff™ Cardiomyocyte Passaging Medium per 5-chamber CellSTACK®. Refer to Table 6 for recommended volumes for various cultureware.
    11. Plate cells at a density of 2.3 x 108 cells/CellSTACK® (7 x 104 cells/cm2). Refer to Table 4 for recommended plating densities for various cultureware.
      Note: Adjust the volume of STEMdiff™ Cardiomyocyte Passaging Medium added to the culture vessel relative to the cell suspension volume, such that the total volume added does not exceed the recommended volumes listed in Table 1.
  4. Incubate at 37°C and 5% CO2 for 24 hours. Do not disturb the cells.

Part VIII. Stopping Expansion of hPSC-Derived Cardiomyocytes

The greatest expansion potential is observed during passages 1 and 2. However, atrial or ventricular hPSC-CMs may continue to be expanded and passaged up to 5 times by repeating sections B and C in Part I, respectively, from Day 18 - 46 if desired. Stop expansion for downstream assays on Day 25 (or after additional expansion and passaging) using STEMdiff™ Cardiomyocyte Maintenance Kit (Catalog #05020) as follows:

    Day 25

  1. Prepare STEMdiff™ Cardiomyocyte Maintenance Medium (as described in Part I. Preparation of Media) and warm to room temperature (15 - 25°C).
  2. Remove medium and add 360 mL of STEMdiff™ Cardiomyocyte Maintenance Medium per 5-chambered CellSTACK®. For other cultureware, refer to Table 7 for recommended volumes.

    3. Table 7. Recommended Volumes of Medium for Maintenance of hPSC-CMs After Expansion in Various Cultureware

    CULTUREWARE
    VOLUME OF STEMdiff™ CARDIOMYOCYTE MAINTENANCE MEDIUM
    6-well plate
    4 mL/well
    T-175 cm2 flask
    72 mL/flask
    T-875 cm2 flask
    360 mL/flask
    5-chambered CellSTACK®
    1300 mL/CellSTACK®
  3. Incubate at 37°C and 5% CO2 for 48 hours.

    4. Day 27

  4. Perform a full-medium change with STEMdiff™ Cardiomyocyte Maintenance Medium every 2 days until Day 32.

    5. Day 32

  5. Atrial or ventricular hPSC-derived cardiomyocytes are ready to be harvested with STEMdiff™ Cardiomyocyte Dissociation Kit and STEMdiff™ Cardiomyocyte Plating Kit for cryopreservation or downstream assays, such as electrophysiology, flow cytometry, or immunocytochemistry.
    6. Microscopy image of cardiomyocytes that have been expanded using the scale up protocol

    Figure 6. Post-expansion hPSC-CMs on Day 32

    Replacing STEMdiff™ Cardiomyocyte Expansion Media with STEMdiff™ Cardiomyocyte Maintenance Media for 7 days will stop hPSC-CMs from expanding. The resulting hPSC-CMs are functional and will maintain the high purity observed during expansion. The scaled-up hPSC-CMs can now be used for downstream applications.

    Day 32+

  6. To continue maintaining hPSC-CMs for 1 month or longer, perform a full-medium change every 2 days with STEMdiff™ Cardiomyocyte Maintenance Medium.

    7. Part IX. Cryopreservation of Post-Expansion hPSC-Derived Cardiomyocytes (Optional)

    Post-expansion hPSC-derived cardiomyocytes can be cryopreserved after at least 7 days of maintenance in STEMdiff™ Cardiomyocyte Maintenance Medium (Catalog # 05020) on Day 32+ as follows:

    Day 32+

  7. Prepare STEMdiff™ Cardiomyocyte Dissociation Kit, STEMdiff™ Cardiomyocyte Plating Media, and STEMdiff™ Cardiomyocyte Freezing Medium as described in Part I. Preparation of Media and warm to room temperature (15 - 25°C).
  8. Cryopreserving hPSC-derived cardiomyocytes:
    1. Rinse the 5-chambered CellSTACK® with 330 mL of D-PBS (Without Ca++ and Mg++).
    2. Pour out the wash into a waste bottle and add 330 mL of warm (37°C) STEMdiff™ Cardiomyocyte Dissociation Medium to the well. Refer to Table 8 for recommended volumes.
    3. Incubate at 37°C and 5% CO2 for 3 - 10 minutes.
    4. Add 660 mL of STEMdiff™ Cardiomyocyte Plating Medium directly to the 5-chambered CellSTACK®. Dislodge cells by tapping the 5-chambered CellSTACK® on each side.
    5. Immediately transfer cells and distribute evenly to 2 x sterile 1 L bottles, each containing 495 mL of Plating Medium. Refer to Table 8 for recommended volumes.
    6. Aliquot the contents of the 1 L bottles into conical tubes of equal volumes for centrifugation (e.g. 165 mL cell suspension / 225 mL conical tube, for a total of 6 x 225 mL conical tubes).
    7. Centrifuge at 600 x g for 5 minutes. Remove and discard supernatant.
    8. Gently resuspend the cell pellet in each 225 mL conical tube with 30 mL of Plating Media. Pool cell suspensions from each 225 mL conical tube as desired.
    9. Perform a cell count using an automated cell counter (e.g. NucleoCounter® NC-250™) or with Trypan Blue and a hemocytometer.
    10. Centrifuge cell suspensions in 225 mL conical tubes at 600 x g for 5 minutes. Gently resuspend the cells in STEMdiff™ Cardiomyocyte Freezing Medium to a final concentration of 5 x 105 cells/mL.
    11. Transfer 1 mL of cell suspension per labeled cryovial.
    12. Freeze cell suspension using a standard slow rate-controlled cooling protocol that reduces temperatures at approximately -1°C/minute, followed by long-term storage at -135°C (liquid nitrogen) or colder. Long-term storage at -80°C is not recommended.

    9. Table 8. Recommended Volumes of Media for Harvesting Non-Expanding hPSC-CMs in Various Cultureware

    CULTUREWARE
    VOLUME OF STEMdiff™ CARDIOMYOCYTE DISSOCIATION MEDIUM
    VOLUME OF STEMdiff™ CARDIOMYOCYTE PLATING MEDIUM*
    6-well plate
    1 mL/well
    5 mL/well
    T-75 cm2 flask
    8 mL/flask
    40 mL/flask
    T-175 cm2 flask
    18 mL/flask
    90 mL/flask
    T-875 cm2 flask
    90 mL/flask
    450 mL/flask
    5-chambered CellSTACK®
    330 mL/CellSTACK®
    1650 mL/CellSTACK®

Part X. Thawing Expanded hPSC-Derived Cardiomyocytes (Optional)

Cryopreserved hPSC-derived cardiomyocytes should be thawed and plated onto Corning® Matrigel®-coated cultureware. For coating plates with Corning® Matrigel®, refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1 or Technical Manual: TeSR™-E8™.

For storage, stability, and preparation instructions for STEMdiff™ Cardiomyocyte Plating Kit, and STEMdiff™ Cardiomyocyte Maintenance Kit, refer to the linked Product Information Sheets.

The following instructions are for a 12-well tissue culture plate. For other cultureware, adjust reagent amounts accordingly.

  1. Coat a 12-well tissue culture plate with Corning® Matrigel® hESC-Qualified Matrix and bring to room temperature (15 - 25°C) for at least 1 hour prior to use.
  2. Prepare STEMdiff™ Cardiomyocyte Plating Supplement and STEMdiff™ Cardiomyocyte Support Medium as per Part I. Preparation of Media and warm to room temperature (15 - 25°C).
  3. Thaw hPSC-derived cardiomyocytes in a 37°C water bath by gently shaking the cryovial continuously until only a small frozen cell pellet remains.
  4. Add 5 - 7 mL of STEMdiff™ Cardiomyocyte Plating Medium to a 15 mL conical tube (e.g. Catalog #38009).
  5. Using a 2 mL pipette, gently transfer the contents of the cryovial to the tube from step 4.
  6. Centrifuge the cells at 300 x g for 5 minutes at room temperature (15 - 25°C).
  7. Aspirate the supernatant and gently add 1 - 2 mL of STEMdiff™ Cardiomyocyte Support Medium to resuspend cells.
  8. 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.
  9. Aspirate Corning® Matrigel® from the tissue culture plate prepared in step 1. Add 2 mL of STEMdiff™ Cardiomyocyte Plating Medium per well.
  10. Add cells at a density appropriate for downstream assays or other applications. Incubate at 37°C for 24 hours.
  11. Remove the medium and add 2 mL of STEMdiff™ Cardiomyocyte Maintenance Medium per well. Incubate at 37°C.
  12. Every 2 days, perform a full medium change with 2 mL of STEMdiff™ Cardiomyocyte Maintenance Medium per well.

Graphs showing cardiomyocyte yield, purity, and total cell yield following expansion

Figure 7. Cardiomyocyte Purity, Total Cell Yield, and Fold Expansion Observed When Scaling Up hPSC-CMs

(A) Cardiomyocyte purity increases from 81% at Day 11 to 93% at Day 18 and remains at 92% at the end of the scale-up protocol (Day 32). (B) Total cardiomyocyte yield increases from 6.3 x 10⁷ live cells on Day 11 to 3.2 x 10⁸ live cells on Day 18 (D18) and to 5.9 x 10⁸ live cells on Day 32 (D32). (C) The expansion of hPSC-CMs shows high growth rates when early-stage (D18) and continues with some expansion even when late-stage (D32).

  • Document #PR00088
  • Version 1.0.0
  • May 2024
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