Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

1. Preparation of Medium Components

1. Prepare Wash Medium. Per 100 ml of DMEM/F12, add 1 ml of 100x Penicillin/Streptomycin (Pen/Strep), 1 ml of 100x L-glutamine, and 5 ml of serum replacement.
2. Prepare the chemically defined Basal Cardiac Induction Medium according to the manufacturer's instructions.
3. Prepare the following stock reagents for media that will be used in this protocol.
   1. L-Ascorbic Acid: Prepare a stock solution of 5 mg per ml in 4 °C, sterile ultra-pure distilled water in a conical tube.
      1. Leave this solution on ice and vortex the tube periodically until the solute is completely dissolved. Filter-sterilize the Ascorbic Acid solution using a 0.22 μm syringe filter.
      2. Prepare 1 ml aliquots of the ascorbic acid solution and store these aliquots at -20 °C. Use a freshly thawed aliquot each time medium is prepared.
   2. On the day of medium preparation, dilute 13 μl of monothioglycerol (MTG) in 1 ml of Basal Cardiac Induction Medium. Discard unused, diluted MTG.
   3. Prepare 1 ml aliquots of Transferrin (30 mg/ml) to store at -20 °C. Store thawed aliquots at 4 °C for up to 3 months.
   4. Prepare 4 mM hydrochloric acid (HCl) containing 0.1% Bovine Serum Albumin (BSA). In a fume hood, add 30 μl of 6.0 N HCl solution to 50 ml of ultra-pure distilled water. Filter-sterilize the solution using a 0.22 μm syringe filter. Add 2 ml of 25% BSA solution to the 50 ml HCl solution.
   5. Prepare Phosphate Buffered Saline (PBS) containing 0.1% BSA. Add 20 μl of 25% BSA solution per ml PBS.
   6. Prepare Human Bone Morphogenetic Protein 4 (BMP-4) stock solution (10 ng/µl). Dissolve 10 µg lyophilized BMP-4 in 1 ml of 4 mM HCl buffer solution containing 0.1% BSA. Prepare 50 µl aliquots to store at -20 °C.
   7. Prepare Human Fibroblast Growth Factor 2 (bFGF) stock solution (10 ng/µl). Dissolve 10 µg lyophilized bFGF in 1 ml of Phosphate Buffered Saline (PBS) containing 0.1% BSA. Prepare 50 µl aliquots to store at -20 °C.
   8. Prepare Human Vascular Endothelial Growth Factor (VEGF) stock solution (5 ng/µl). Dissolve 5 µg lyophilized VEGF in 1 ml of PBS containing 0.1% BSA. Prepare 50 µl aliquots to store at -20 °C.
   9. Prepare Activin A stock solution (10 ng/µl). Dissolve 10 µg lyophilized Activin A in 1 ml of PBS containing 0.1% Bovine Serum Albumin (BSA). Prepare 50 µl aliquots to store at -20 °C.
   10. Prepare Inhibitor of Wnt Production-2 (IWP-2) stock solution (10 mM). Dissolve 2 mg IWP-2 in 429 µl Dimethyl Sulfoxide (DMSO). Prepare 10 µl aliquots to store at -80 °C.
4. Prepare Complete Cardiac Induction Medium. To 100 ml of Basal Cardiac Induction Medium, add 1 ml of 100x Pen/Strep, 1 ml of 100x L-glutamine, 500 µl of Transferrin, 1 ml of freshly thawed ascorbic acid, and 300 µl of MTG. Discard unused medium.

2. Preparation of the Microwell Plate

NOTE: All steps should be performed in a biological safety cabinet.

1. Add 0.5 ml Rinsing Solution (kit component) to each well that will be used on the plate. To ensure that the solution contacts the entire surface inside each microwell, centrifuge the plate at 840 x g for 2 min.
2. Incubate the plate for 30 to 60 min at RT.
3. Aspirate the Rinsing Solution from the wells. Wash each well twice as follows: Add 1 ml of PBS to each well, centrifuge the plate at 840 x g for 2 min, and then aspirate the PBS.

3. Formation of hPSC Aggregates in the Microwell Plate

NOTE: All steps should be performed in a biological safety cabinet.

1. Place Wash Medium in a 37 °C water bath.
   NOTE: The volume required = 1 ml x number of wells of hPSC that will be dissociated.
2. Dissociate hPSCs to single cells
   NOTE: These steps describe the dissociation of cells cultured on 6 well plates — the reagent volumes can be scaled proportionally for different culture formats.
   1. Aspirate the culture medium from each hPSC culture well for dissociation. Rinse each well with 1 ml of dissociation enzyme, and then immediately aspirate the dissociation enzyme from each well.
      NOTE: Residual dissociation enzyme should be sufficient to dissociate the cells.
   2. Incubate the plate at 37 °C for 3 min.
   3. Add 1 ml of Wash Medium to each well and mechanically dissociate the cells from the tissue culture surface by pipetting the Wash Medium with a P1000 micropipettor over the tissue culture surface. If the dissociation appears to be incomplete and cell clumps remain, pass the suspension through a strainer at this point.
   4. Transfer the cell suspension to a 15 ml conical tube and store the tube in the incubator while the cell count is performed in the next step.
   5. Perform a cell count:
      1. Take a 10 µl sample of the cell suspension collected in the previous step. Add 30 µl of Trypan Blue and mix the suspension well by pipetting.
      2. Transfer 10 µl of Trypan Blue-stained cells to each chamber of a hemocytometer and visualize under the inverted microscope with the 10X objective. Count the cells.
   6. From the cell count results, calculate how many wells can be seeded at 1.2 x 10⁶ cells per well of the microwell plate. Calculate Aggregation Medium required to seed the cells at 1 ml per well.
   7. Prepare aggregation medium. Per 10 ml of Complete Cardiac Induction Medium, add 0.5 µl of BMP4 stock solution (final concentration = 0.5 ng/ml) and Y-27632 ROCK Inhibitor (final concentration = 10 µM).
   8. Remove the conical tube containing the hPSCs from the incubator and centrifuge the tube at 200 x g for 5 min. Aspirate the wash medium and resuspend the cells in aggregation medium at a density of 1.2 x 10⁶ cells per ml.
   9. Aspirate the PBS wash solution from each well of the microwell plate. Using a P1000 micropipettor, evenly distribute and seed 1 ml of the cell suspension to each well on the microwell plate. To seed a large number of wells, periodically vortex the cell suspension to prevent settling.
   10. Centrifuge the plate at 200 x g for 5 min. Observe the plate under the microscope to confirm cells have spun to the bottom of each microwell.
   11. Incubate the plate for 24 hr at 37 °C in a 5% CO₂, 5% O₂ (hypoxic) incubator.

4. Cardiac Induction Stage 1

1. One day following aggregation (day 1), prepare the required volume of Stage 1 Induction medium (for a 24 well plate, volume = 1 ml x number of wells). For 1 ml of complete cardiac induction medium, add 1 µl of BMP4 stock solution (final concentration = 10 ng/ml), 0.5 µl of bFGF stock solution (final concentration = 5 ng/ml), and 0.6 µl of Activin A (final concentration = 6 ng/ml). Place the medium in a 37 °C water bath for at least 15 min.
2. Remove the microwell plate from the incubator. Inspect the aggregates under the microscope. Compared to immediately post-centrifuge aggregation, they should appear intact with smooth edges (more round and less square than the previous day).
3. Remove the supernatant without disturbing the aggregates inside the microwells:
   1. Hold the microwell plate horizontally level (i.e., do not tilt it). For each well on the microwell plate, place the tip of a P1000 micropipettor at the surface of the culture medium and against the edge of the well.
   2. Slowly remove the medium, being careful not to disturb the aggregates at the bottom of the microwells. After reducing the medium level to about 1 to 2 mm from the textured microwell surface, slowly tilt the plate to collect medium at one side of the well (once the volume is sufficiently low, fluid motion is greatly reduced and it is easier to avoid aggregates getting lifted out of their individual microwells). Slowly pipette out the remaining medium in the well.
4. To add fresh Stage 1 Induction Medium while ensuring the aggregates remain in their individual microwells: Draw 1 ml of Stage 1 induction medium with a P1000 micropipettor. Hold the pipette tip against the inside edge of the well and very slowly dispense the medium against the inside wall of the well. Repeat for the remaining wells.
5. Return the plate to the incubator under hypoxic conditions for 3 days.

5. Cardiac Induction Stage 2

1. On day 4, place wash medium (volume = number of wells x 2 ml) in a 37 °C water bath for a minimum of 15 min.
2. Prepare the necessary volume of Stage 2 Induction Medium (volume = number of wells x 1 ml): Per ml of Complete Cardiac Induction Medium, add 2 µl of VEGF stock solution (final concentration = 10 ng/ml) and 0.5 µl IWP-2 stock solution (final concentration = 5 µM). Place the prepared Stage 2 Induction medium in a 37 °C water bath for a minimum of 15 min.
3. Using a 5 ml serological pipette, harvest the aggregates from each well of the microwell plate and collect the aggregate suspension in a 15 ml conical tube (collect up to 10 wells per 15 ml tube).
4. Allow the aggregates to settle for 15 min in a hypoxic incubator.
   NOTE: This step is important to separate single cells and cellular debris from the intact aggregates.
5. Aspirate the supernatant carefully and resuspend the aggregates in 10 ml of the pre-warmed Wash Medium to remove residual inductive cytokines (e.g., Activin A is a potent signaling molecule even at very low concentrations).
6. Centrifuge the aggregates at 50 x g for 2 min. Aspirate the supernatant. Resuspend the pelleted aggregates in the pre-warmed Induction 2 Medium.
7. Transfer the aggregate suspension to a 24-well ultra-low attachment (ULA) plate at 1 ml per well. Observe the aggregates under the microscope as uniform, tight cell clusters. Incubate under hypoxic conditions until day 6.

6. Cardiac Induction Stage 3

1. On day 6, prepare the necessary volume of Stage 3 Induction Medium (volume = number of wells x 1 ml). For 1 ml of Complete Cardiac Induction Medium, add 2 µl VEGF stock solution (final concentration = 10 ng/ml), and 0.5 µl of bFGF stock solution (final concentration = 5 ng/ml). Place prepared Stage 3 induction medium in a 37 °C water bath for a minimum of 15 min.
2. Use a 5 ml serological pipette to transfer the aggregates to 15 ml conical tubes, pooling up to 10 ml of aggregates per tube.
3. Allow 10 min for the aggregates to settle. Aspirate the supernatant and resuspend the aggregates in the pre-warmed Stage 3 Induction Medium. Using a 5 ml serological pipette, redistribute the aggregates into a 24-well ULA plate at 1 ml per well.
4. On day 10, prepare the necessary volume of Stage 3 Induction Medium (volume = number of wells x 1 ml) as per Step 6.1.
5. Use a 5 ml serological pipette to transfer the aggregates to 15 ml conical tubes, pooling up to 10 ml of aggregates per tube.
6. Allow 10 min for the aggregates to settle. Aspirate the supernatant and resuspend the aggregates in Stage 3 Induction Medium. Using a 5 ml serological pipette, redistribute the aggregates into a 24-well ULA plate at 1 ml per well.
7. Incubate under hypoxic conditions for two days. On day 12, begin incubating the cells at normoxic oxygen levels for the remainder of the culture period (37 °C, 20% O₂, 5% CO₂).
   NOTE: After this time point, cells are no longer cultured under hypoxic conditions.
8. Repeat this complete medium exchange (Steps 6.2 and 6.3) every 4 days, beginning day 14 until cell harvest (typically, peak cardiomyocyte concentrations are observed after day 14 of differentiation).

7. Flow Cytometry Analysis of Cardiac Troponin T (cTnT) Expression Frequency of Microwell Culture Output

1. Dissociate the aggregates as follows:
   1. Use a 5 ml serological pipette to transfer 1 well of aggregates to a 15 ml conical tube. Centrifuge the aggregates at 50 x g for 2 min and carefully aspirate the supernatant.
   2. Add 1 ml of freshly dissolved 1 mg/ml Collagenase Type II solution to the aggregates. Transfer the aggregate suspension to a 1.5 ml microcentrifuge tube. Incubate the aggregates in Collagenase Type II overnight at room temperature.
   3. The following day, use a P1000 micropipettor to gently dissociate the aggregates into a homogenous single cell suspension. If the aggregates do not readily dissociate, settle the aggregates, aspirate the supernatant, and incubate the aggregates in 700 μl of Dissociation enzyme for 1 to 2 min at RT. Gently pipette the aggregates 1 to 2 times with a P1000 micropipettor to dissociate.
   4. Dilute the dissociation enzyme: Add 700 μl of Wash Medium containing 14 μl of 1 mg/ml DNAse stock solution. Take a 10 μl sample to perform a cell count, and centrifuge the remaining suspension using a bench-top microcentrifuge at 300 x g for 2 min.
2. Perform a cell count: Stain the 10 μl counting sample with an equal volume of Trypan Blue and count using a hemocytometer.
3. Remove the 1.5 ml microcentrifuge tube containing the remaining cells from the microcentrifuge. Aspirate the supernatant and resuspend the cells, at a concentration of 200,000 to 500,000 cells per 100 μl, in Hanks Balanced Salt Solution containing 2% Fetal Bovine Serum (HF).
4. For each condition, transfer 100 μl of cell suspension per well to 2 wells of a 96-well plate (one well will be stained with the cTnT antibody and the other will be the secondary antibody control).
5. Centrifuge the plate at 300 x g for 2 min. Remove the supernatant with a multichannel micropipettor.
6. Fix the cells: Add 200 μl of Fixation Solution (kit component) per well and incubate the plate for 15 min at RT.
7. Centrifuge the plate at 300 x g for 2 min. Use a multichannel micropipettor to carefully withdraw the supernatant from the wells. Dispose the supernatant in a paraformaldehyde waste container.
8. Wash the fixed cells twice: Add 200 μl of HF to each well. Centrifuge the plate at 300 x g for 2 min. Aspirate the supernatant, and repeat the wash step once more. Fixed cells can be stored for up to one week in HF at 4 °C.
9. Permeabilize the cells:
   1. Centrifuge the plate at 300 x g for 2 min.
   2. Add 100 μl of Permeabilization Solution (kit component) to each well and incubate the plate at room temperature for 5 min.
   3. Centrifuge the plate at 300 x g for 2 min and aspirate the supernatant.
10. Prepare a master mix of anti-cTnT in HF at the optimal concentration for the given lot number (The optimal dilution must be determined by titration and typically ranges from 1:500 to 1:2,000).
11. For each condition (2 wells per condition), add 100 μl of master mix to one well (stained sample) and 100 μl of plain HF to the other well (secondary antibody control). Incubate the cells at 4 °C for 30 min.
12. Centrifuge the cells at 300 x g for 2 min. Aspirate the supernatant and add 200 μl of HF per well. Repeat this wash step one more time.
13. Prepare a master mix of the secondary antibody. Transfer a volume of HF that corresponds to 100 μl for every well (secondary antibody control and cTnT-stained) being treated. Add 1 μl of goat anti-mouse-APC secondary antibody per 200 μl of HF (1:200 dilution).
14. Stain the samples. Add 100 μl of staining solution to each well (both the secondary antibody control and anti-cTnT-stained wells). Incubate cells in the dark at 4 °C for 30 min (keep the plate covered or in the dark after adding fluorescent secondary antibody to avoid photobleaching).
15. Centrifuge the cells at 300 x g for 2 min. Aspirate the supernatant and add 200 μl of HF per well. Repeat this wash step one more time.
16. Transfer the samples to 5 ml round bottom flow cytometer analysis tubes and perform flow cytometry for signal in the APC channel using the standard protocols for the instrument¹⁹.