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

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
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