RPMI (10x)

Dissolve 104 mg/mL of RPMI powder in ddH₂O under constant rotation at 37°C for 1 h. Sterilize by filtration. Store at -20°C for up to 12 months.

Optional: Add antibiotics such as 100 U/mL penicillin and 100 μg/mL streptomycin. Store at 4°C for up to 14 days.

IGF-1 (100 μg/mL)

Dissolve IGF-1 in PBS containing 0.1% recombinant albumin to obtain a 100 μg/mL stock solution. Aliquot and store at -20°C for up to 12 months. Once thawed, keep at 4°C for up to one week.

VEGF₁₆₅ (5 μg/mL)

Dissolve VEGF₁₆₅ in PBS containing 0.1% recombinant albumin to obtain a 5 μg/mL stock solution. Aliquot and store at -20°C for up to 12 months. Once thawed, keep at 4°C for up to one week.

FGF-2 (10 μg/mL)

Dissolve FGF-2 in PBS containing 0.1% recombinant albumin to obtain a 10 μg/mL stock solution. Aliquot and store at -20°C for up to 12 months. Once thawed, keep at 4°C for up to one week.

TGF-β1 (5 μg/mL)

Dissolve TGF-β1 in PBS containing 0.1% recombinant albumin to obtain a 5 μg/mL stock solution. Aliquot and store at -20°C for up to 12 months. Once thawed, keep at 4°C for up to one week.

L-Ascorbic Acid 2-Phosphate (300 mM)

Dissolve 4.3 g of L-ascorbic acid 2-phosphate in 50 mL ddH₂O to obtain a 300 mmol/L stock solution. Mix until dissolved and sterilize by filtration. Aliquot and store at -20°C for up to 12 months.

Enzymatic Dispersion of Human PSC-Derived Cardiomyocytes

Timing: 30–45 min

This step establishes a single cell suspension of PSC-derived cardiomyocytes for EHM generation.

CRITICAL: This protocol has been optimized for cardiomyocyte monolayer cultures. We typically use cardiomyocytes between days 18–25 of differentiation. For other cardiomyocyte differentiation protocols, e.g. 3D embryoid bodies, alternative digestion protocols have been published (Chen et al., 2015, Kattman et al., 2011, Tiburcy et al., 2017). For optimal standardization we recommend to plate cardiomyocytes in monolayer culture first (steps 1–3) and then use this digestion protocol when making EHM.

Note: Volumes are given for a T75 cell culture flask. For different culture formats (e.g., 6-well plates), volumes need to be adjusted according to culture plate surface area.

• 5.

  Warm Cardio Recovery Medium, PBS, and Accutase to 20–25°C. Warm Versene to 37°C.

• 6.

  Aspirate medium from flask with human PSC-derived cardiomyocytes.

• 7.

  Add 6 mL of Accutase to the cells and incubate for 15–45 min at 20–25°C until cells start to visibly detach (Figure 1).

  

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  Figure 1

  Enzymatic Dispersion of Monolayer Cardiomyocytes

  Brightfield images of monolayer cardiomyocytes (cultured according to steps 19–23) at indicated stages of enzymatic dispersion. Note, that Versene should be added once larger aggregates of cardiomyocytes lift off the culture plate surface. Bar graph: 50 μm.

Note: Depending on cell density, culture conditions, and time point of differentiation, this may occur after 15 min, but may take up to 45 min. Check detachment repeatedly under the microscope (Figure 1).

Note: During this incubation time, preparation of fibroblasts (see steps 22–26) can be commenced.

• 8.

  Add 6 mL of Versene to the Accutase and incubate at 20–25°C for a further 5 min.

• 9.

  Stop the digestion by adding 12 mL of Cardio Recovery Medium to the Accutase/Versene mix. Pipette up and down gently with a 10 mL serological pipette to ensure a single cell suspension, and transfer cells to a fresh collection tube.

• 10.

  Wash flask once more with 6 mL of Cardio Recovery Medium and add to the collection tube. Centrifuge the cells at 200 x g for 7 min at 4°C.

CRITICAL: At this stage, the cells are very sensitive; handle them carefully and avoid extensive pipetting. Use a serological pipette with a wide tip to minimize mechanical disruption of cells.

• 11.

  Carefully aspirate the supernatant and flick the tube to dislodge the pellet. Resuspend the cells in 10 mL of Cardio Recovery Medium.

• 12.

  Strain the suspension through a 70 μm mesh cell strainer.

• 13.

  Count the cells using an automated cell counter or hemocytometer. Leave the cells on ice.

Enzymatic Dispersion of Human Fibroblasts

Timing: 20 min

The culture of human foreskin fibroblasts has been described in detail (Schlick et al., 2019). This step establishes a single cell suspension of fibroblasts for EHM generation.

Alternatives: Fibroblasts from other sources including PSC-derived fibroblasts can be used.

Note: Volumes are given for a T75 cell culture flask. For different culture formats (e.g., 6-well plates), volumes need to be adjusted by scaling to surface area.

• 14.

  Warm Cardio Culture Medium and TrypLE Express to 37°C.

• 15.

  Wash cells with 6 mL TrypLE Express, aspirate and add 6 mL of TrypLE Express again. Incubate at 37°C for 5–10 min.

• 16.

  Stop the digestion by adding 12 mL of Cardio Culture Medium.

• 17.

  Pipette up and down, and pass the cell suspension through a 70 μm mesh cell strainer into a fresh 50 mL centrifuge tube to remove excess extracellular matrix.

• 18.

  Count the cells using an automated cell counter or hemocytometer. Leave the cells on ice.

Cryo-Preserved Human Fibroblasts (Alternative)

Timing: 15 min

EHM can be made directly from cryo-preserved fibroblasts. The advantage of this approach is that fibroblast properties are very standardized over experimental series if the same pool of fibroblasts is used. The following steps describe the preparation of cryo-preserved fibroblasts for EHM generation.

• 1.

  Warm Cardio Culture Medium to 15–25°C.

• 2.

  Remove cryo-vials containing fibroblasts from liquid nitrogen and thaw at 37°C for 3 min.

• 3.

  Disinfect cryo-vial with 70% EtOH.

• 4.

  Remove cell suspension from cryo-vial with a 2 mL serological pipette and transfer to a tube. Wash cryo-vial with 1 mL of Cardio Culture Medium, add dropwise to the cells.

• 5.

  Slowly add another 8 mL of Cardio Culture Medium.

• 6.

  Centrifuge the tube at 300 x g for 5 min at 4°C.

• 7.

  Carefully aspirate the supernatant and resuspend the cells in 10 mL of Cardio Culture Medium.

• 8.

  Strain the suspension through a 70 μm mesh cell strainer.

• 9.

  Count the cells using an automated cell counter or hemocytometer. Leave the cells on ice.

Generation of EHM

Timing: 1.5 h

In this step cardiomyocytes (from step 13) and fibroblasts (from step 18 or 27) are combined into a cell mix and then reconstituted in a collagen hydrogel. The cell-hydrogel mixture is cast into molds and left to solidify (condense) for 1 hr in an incubator. This step is completed by adding medium to the condensed EHM hydrogels.

• 19.

  Determine the number of EHM to be prepared based on the number of cardiomyocytes and fibroblasts prepared on ice (live cells):

• 20.

  Mix the appropriate number of cardiomyocytes and fibroblasts into a new tube according to Table 1 and centrifuge tube at 200 x g for 7 min at 4°C.

  Table 1

  Preparation of Cell Mix (Including a 10% Surplus to Account for Pipetting Error)

  EHM Number:	1	6	48
  Cardiomyocytes	0.385 million	2.31 million	18.48 million
  Fibroblasts	0.165 million	0.99 million	7.92 million

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• 21.

  Aspirate the supernatant and resuspend the cell mix in 4°C cold EHMM (volume according to cell mix in Table 2). Leave the cell mix on ice.

  Table 2

  Preparation of EHM Hydrogel (Including a 10% Surplus Accounting for Pipetting Errors)

  	Volume (μl)
  EHM number:	1	6	48
  Collagen (6.84 mg/mL)	44	264	2,112
  2x RPMI	44	264	2,112
  0.1 N NaOH	6	36	288
  Cell mix in EHMM	104	624	4,992
  Total volume	198	1,188	9,504

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Note: A collagen concentration of 0.3 mg/EHM is recommended. A collagen stock solution containing 6-7 mg/mL is recommended. Volumes of the other EHM components have to be adapted according to the volume of the collagen stock solution needed to obtain on optimal EHM collagen content. The NaOH amount may have to be adapted individually for optimal pH neutralization (see Critical point below).

• 22.

  Prepare the EHM hydrogel on ice in the exact order listed in Table 2. To a 50 mL centrifuge tube on ice:

• a.

  Add the acid soluble collagen.

• b.

  Add the 2x RPMI and mix by swirling the tube.

• c.

  Add 0.1 N NaOH and mix by swirling the tube.

• d.

  Add the cell mix.

Mix the entire suspension well by triturating >10 times with a serological pipette.

CRITICAL: pH neutralization of the acid soluble collagen must be confirmed by inspection of the phenol red indicator transition from yellow to pink. At this point, the condensation (solidification) of collagen will commence and must not be disrupted so the following steps should be performed swiftly.

Note: As collagen is very viscous, pipette slowly to avoid air bubble formation.

• 23.

  Distribute 180 μl of EHM hydrogel with a 1 mL pipette tip evenly into each well of a 48-well EHM plate (Figure 2A). Ensure an even distribution of hydrogel in each well by lightly tapping the plate (Figure 2B).

  

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  Figure 2

  Generation and Parallel Culture of 48 EHM in a Multi-well Format

  (A) 48-well EHM plate used for the construction of EHM.

  (B) EHM forming between two poles over time. The prepared hydrogel becomes opaque after 1 h (dashed boxes indicate inner and outer boundary of the hydrogel) and compacts notably by 24 h. After four weeks in culture, EHM are typically analyzed.

  (C) Breaks in the hydrogel (arrow) will prevent correct EHM formation. Bar graph: 5 mm.

Note: If breaks in the hydrogel occur (Figure 2C arrow), an intact EHM loop will not form.

• 24.

  Carefully transfer the 48-well plate to the incubator. Let the reconstitution mix settle for 1 h, after which it will appear opaque (Figure 2B).

• 25.

  Carefully add 600 μl of 37°C EHMM+T per well and incubate for 24 h.

• 26.

  Exchange 500 μl of EHMM+T per well daily for the first three days of EHM formation. Afterwards, feed with EHMM (without TGF-β1) until analysis.

Note: After the initial phase of cell independent hydrogel condensation, fibroblasts will start to compact the cell-hydrogel mixture. Within 24 h, EHM should have almost completely formed a loop structure (Figure 2B). Spontaneous contractions of the EHM loops can typically be observed after 3 days of EHM formation and will noticeably increase over time in culture.

Potential Solution

At this stage care must be taken to not physically disrupt the hydrogel. Do not pipette culture medium directly onto the condensing EHM. Add culture medium slowly via the well wall.

Problem

EHM do not condense within 1 h after casting.

Potential Solution

Hydrogel condensation is a material intrinsic, i.e., largely cell independent, property of the collagen type I hydrogel (refer for details to Schlick et al. 2019). Care must be taken to not disrupt condensation by quickly casting the pH neutralized reconstitution mixture. In addition, the collagen hydrogel should be tested as to its condensation properties before use in EHM generation. Testing different suppliers and batches may be required for optimal results. Acetic acid solubilized collagen with a collagen concentration of >4 mg/mL gives best results. Care must be taken to store and handling of collagen at 4±2°C; collagen must never be frozen or handled at room temperature.

Problem

EHM do not compact within 3 days after casting.

Potential Solution

The compaction process is a function of the fibroblast component. Ensure fibroblasts viability and sufficient quantity preferably 30% of input cells. Optimal fibroblast content may have to be adapted depending on the source of fibroblasts. A too high fibroblast content will result in massive EHM compaction with a steep increase in EHM stiffness (Young’s Modulus) and inhibit EHM contractility; a too low fibroblast content will result in a lack of EHM compaction with negligible or no contractile force development (refer to Tiburcy et al., 2017 for details).

Problem

EHM do not contract visibly within 2 weeks after casting.

Potential Solution

EHM contractility is a function of the cardiomyocyte component. Ensure cardiomyocyte viability after enzymatic dispersion and purity at preferably 70% of input cells. Optimal cardiomyocyte content may have to be adapted depending on the source of cardiomyocytes. Too low or too high cardiomyocyte purity will result in suboptimal or no contractile performance (refer to Tiburcy et al., 2017 for details).

Problem

EHM do not contract uniformly in the 48-well plate.

Potential Solution

Casting procedure may have to be trained to ensure completion of the casting from the prepared reconstitution mixture within ideally 5 min. Note that the reconstitution mixture will start to condense after pH neutralization and that this process must not be inappropriately disrupted. However, ensure proper mixing of cells in the prepared reconstitution mixture by trituration (10x on ice) before casting. EHM may stop to contract spontaneously after extended culture, but will remain contractile upon external electrical stimulation.

Problem

EHM do not form a homogeneous loop.

This work was supported by the German Research Foundation (DFG TI 956/1-1 to M.T., ZI 708/10-1 to W.H.Z.; CRC 1002 C04 to M.T. and W.H.Z. and S01 to W.H.Z.), by the German Center for Cardiovascular Research (DZHK), and the Fondation Leducq (14CVD04).