Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Summary

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Abstract

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 10⁶/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Introduction

Cardiac side effects such as the drug-induced long QT syndrome have led to market withdrawals over the past years. Statistics indicate that about 45% of all withdrawals are due to unwanted effects on the cardiovascular system¹. This drug failure after the expensive developmental process and approval is the worst-case scenario for pharmaceutical companies. Research and development departments therefore focus on detection of such unwanted cardiovascular effects early on. For economic and ethical concerns, efforts to reduce animal experiments and replace them with new in vitro screening assays are ongoing.

A set of established assays are included in the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines for preclinical evaluation of proarrhythmic drug effects². The technology of reprogramming somatic cells followed by differentiation of human induced pluripotent stem cells (hiPSC) boosted this research field³. It now offers the possibility to screen new drug candidates on human cardiomyocytes in vitro and avoids issues with inter-species differences. Recent cardiac differentiation protocols^4,5 provide unlimited supply of cardiomyocytes without ethical concern. However, the measurement of contractile force, the most important and best characterized in vivo parameter of cardiomyocytes, is not well established. This is related to the relative immaturity⁶ of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as compared to the adult cardiomyocyte. A possible advancement is to engineer 3-dimensional heart tissue from single cells⁷ (engineered heart tissue, EHT). The EHT protocol is based on embedding single murine or human cardiomyocytes^8,9,10 in fibrin hydrogel between two flexible polydimethylsiloxane (PDMS) posts¹¹ in 24-well format. Within a few days the cardiomyocytes start to contract spontaneously as single cells and start to form cellular networks. After 7-10 days, macroscopic contractions of the entire tissue are visible. During this process the extracellular matrix is remodeled, which leads to a decrease of diameter and length. The shortening of the EHT results in bending of the PDMS post even during rest, subjecting cardiomyocytes in the developing EHT to continuous load. EHTs continue to perform auxotonic muscle contractions over several weeks. Human EHTs show responses to physiological and pharmacological stimulation indicating their suitability for drug screening and disease modeling⁷.

In this manuscript we present a robust and easy protocol for the generation of human EHT, and the automated contractility analysis of concentration dependent changes of the contraction pattern in the presence of hERG channel inhibitors.

Protocol

NOTE: The following steps describe a cell culture protocol. Please perform under sterile conditions and use pre-warmed media.

1. Cardiac Differentiation of hiPSC

1. Cultivate the hiPSC
   1. Coat 6-well plates (1 mL/well) or T75 flasks (7 mL/flask) with reduced growth factor basement membrane matrix (e.g. geltrex, 1:200; see table of materials) diluted in Dulbecco's modified Eagle medium (DMEM) for 30 min at 37 °C. Prepare FTDA medium¹² by mixing basic DMEM/F-12 medium with 2 mM L-Glutamine, 0.5% penicillin/streptomycin, 5 mg/L transferrin, 5 µg/L selenium, 0.1% human serum albumin, 1x lipid-mix, 5 mg/L insulin, 50 nM dorsomorphin, 2.5 ng/mL activin A, 0.5 ng/mL human TGFß1 and 30 ng/mL FGF2.
   2. Seed hiPSC (~3 x 10⁵/ 10 cm²) in FTDA medium and culture at 37 °C, 5% CO₂, 21% O₂ with daily medium change (Figure 1A). At 80% confluency, passage (1:2-1:6) with ethylene diamine tetraacetic acid (EDTA; 0.5 mM; incubation time ~4 min).
2. Formation of embryoid bodies (EB)
   1. Cultivate hiPSC in T75 flasks to high density.
      1. Wash flasks with confluent cells twice with phosphate buffered saline without calcium and magnesium (PBS) and incubate in 0.5 mM EDTA for ~10 min. Make sure the cells do not detach from the bottom of the flask.
      2. Remove EDTA with glass pipette, flush off cells with 10 mL PBS, tap flasks at the bench to detach cells.
         NOTE: Use cell scraper if necessary.
      3. Collect cells in 50 mL conical tube, add ¼ of volume (e.g. 10 mL RMPI to 40 mL cell suspension) RPMI 1640 medium (or any other standard medium containing 1.8 mM calcium) and centrifuge at 250 x g for 5 min.
   2. Resuspend cell pellet in FTDA medium supplemented with 4 mg/mL polyvinyl alcohol (PVA) and 10 µM Y27632 and count the cells in a Neubauer chamber.
   3. Fill the spinner flask with the cell suspension (30 x 10⁶ cells/100 mL FTDA medium supplemented with PVA and Y-27632; see step 1.2.2) and initiate EB formation in spinner flasks (adjust rotor speed of magnetic stirrer to 40 rpm) in the cell culture incubator (37 °C, 5% CO₂, 5% O₂) over night.
3. Mesodermal differentiation
   1. During EB formation, coat cell culture flasks suitable for suspension culture with non-ionic surfactant (14 mL/T175) over night at 37 °C.
   2. The next morning, wash surfactant-coated flasks twice with PBS (30 mL/T175). Add PBS again and keep in the incubator until needed.
   3. Remove the spinner flask from the incubator.
      NOTE: Cell suspension should be clear with small macroscopically visible EBs (Figure 1B).
   4. Transfer 50 mL suspension to a 50 mL conical tube and let EBs settle for 5-20 min.
   5. Remove the supernatant and wash the pellet with 7 mL of RPMI 1640 supplemented with 2% PVA and 10 µM Y-27632. Transfer suspension to a graduated 15 mL conical tube and estimate EB volume (~50-100 µL).
   6. Transfer contents of the spinner flasks to uncoated T175 flasks and leave this on V-shaped rack for sedimentation for up to 20 min. Remove supernatant and wash embryoid bodies as stated above. Do not fill the T175 flask with more than 200 mL as sedimentation would take too long. If initial spinner flask volume exceeds 200 mL, split EB suspension to several T175 flasks and combine EBs again after washing.
   7. Remove washing medium and resuspend in medium for mesodermal differentiation, that is RPMI supplemented with 4 mg/mL PVA, 0.5% penicillin/streptomycin, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.05% human serum albumin, 5 µg/mL transferrin, 5 ng/mL sodium selenite, 0.1% lipid mix, 250 µM phospho-ascorbate, 10 µM Y-27632, 10 ng/mL bone morphogenic protein-4 (BMP-4), and 3 ng/mL activin A, 5 ng/mL stable FGF2.
   8. Remove PBS from surfactant-coated T175 flasks and fill with cell suspension (200 µL EBs in 40 mL). Use smaller flasks for smaller EB volume.
   9. Cultivate EBs in suspension for three days (37 °C, 5% CO₂, 5% O₂). Exchange 50% of the medium (same composition as in step 1.3.7) every day (use V-shaped sedimentation rack). Prepare medium fresh every day prior to feeding.
4. Cardiac differentiation
   1. Prepare new surfactant-coated vessels the day before and handle as stated above (1.3.2).
   2. After three days of mesodermal induction, let EBs settle for up to 5 min on sedimentation rack. Remove most of the medium and wash with RPMI 1640 supplemented with 0.5% penicillin/streptomycin and 10 mM HEPES.
   3. Transfer 10 mL of EB suspension to graduated 15 mL tube and estimate EB volume after sedimentation.
   4. Resuspend EBs carefully in cardiac differentiation medium consisting of RPMI 1640 supplemented with 0.5% penicillin/streptomycin, 10 mM HEPES, 0.05% human serum albumin, 5 µg/mL transferrin, 5 ng/mL sodium selenite, 0.1% lipid mix, 250 µM phospho-ascorbate, 1 µM Y-27632, 100 nM Wnt-inhibitor DS-I-7¹³.
   5. Transfer EBs to new surfactant-coated cell culture flasks (~200 µL EBs in 46 mL/T175).
   6. Incubate for three days (37 °C, 5% CO₂, 21% O₂) with 50% medium change (medium composition see step 1.4.4) on the second and third day. Do not change the medium within the first 48 h.
   7. Incubate in medium consisting of RPMI 1640 supplemented with 500 µM 1-Thioglycerol, 10 mM HEPES, 0.5% penicillin/streptomycin, 1 µM Y-27632, 2% serum-free neural cell culture supplement (e.g. B27) with insulin, 100 nM Wnt-inhibitor DS-I-7 with daily exchange of 50% medium for the next four days. EBs should start beating within this period.
   8. After one week of cardiac differentiation, switch to the same medium without Wnt inhibition and with serum-free neural cell culture supplement. Change 50% of the medium every day, except for the first day.
5. Dissociation of human cardiomyocytes
   NOTE: After two weeks of differentiation protocol, EBs should show spontaneous contractions (Figure 1C). If so, start dissociation and prepare collagenase solution.
   1. Prepare collagenase solution by weighing and solving 200 U/mL in calcium-free Hanks' balanced salt solution without calcium/magnesium (HBSS) and add 1 mM HEPES, 10 µM Y-27632, 30 µM N-benzyl-p-toluene sulphonamide (BTS). Sterilize by filtering (0.2-µm filter) and store aliquots at -20 °C. Thaw aliquots at 4 °C over night.
   2. Settle EBs on sedimentation rack, aspirate medium and replace with 20-25 mL pre-warmed HBSS. Repeat this washing step once more.
   3. Aspirate HBSS and replace with pre-warmed collagenase solution (15 mL/T175). Incubate for 4 h in the cell culture incubator (37 °C, 5% CO₂, 21% O₂).
   4. Prepare blocking buffer with DMEM supplemented with 1% penicillin/streptomycin and DNase (12 µg/mL).
   5. Tap the cell culture flasks on the bench, dissociated EBs should disintegrate and fall apart (if not, increase incubation time). Gently triturate cell suspension and transfer to a conical 50 mL tube. Wash flasks with blocking buffer (15 mL/T175) and transfer to tube.
   6. Centrifuge for 10 min at 100 x g. Resuspend cells in warm DMEM, triturate and count cells.

2. Generation of Engineered Heart Tissue (EHT)

1. Autoclave commercially available PDMS racks and polytetrafluorethylene (PTFE) spacers (see Materials and Equipment spreadsheet; Figure 2) and prepare the following solutions under sterile conditions, one day prior to EHT generation.
   1. Prepare 2% agarose by weighing and dissolving in appropriate volume of PBS, autoclave, immediately store at 60 °C.
   2. Prepare aprotinin (33 mg/mL) by weighing and dissolving in appropriate volume of aqua ad iniectabilia, filter sterile (0.22-µm filter), aliquot and store at -20 °C.
   3. Prepare fibrinogen (200 mg/mL) by dissolving sterile fibrinogen in appropriate volume of sterile 0.9% NaCl (lukewarm), aliquot and store at -20 °C.
   4. Prepare thrombin (100 U/mL) by dissolving sterile thrombin in three parts sterile PBS and 2 parts aqua ad iniectabilia, aliquot portions of 3 µL in small tubes and store at -20 °C.
   5. Prepare 10x DMEM by dissolving 670 mg 10x DMEM powder in 5 mL aqua ad iniectabilia, filter sterile and store at 4 °C.
   6. Prepare 2x DMEM by mixing 2 mL 10x DMEM with 2 mL heat-inactivated horse serum, 0.2 mL penicillin/streptomycin and 5.8 mL aqua ad iniectabilia, filter sterile and store at 4 °C.
   7. Prepare EHT-casting medium (ECM) by mixing DMEM with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin and 1% L-glutamine (200 mM), store at 4 °C.
2. Prepare casting molds in 24-well plates by pipetting 1.6 mL warm agarose (2%, PBS) and placing the PTFE spacer (Figure 2A) into the wells.
   NOTE: Place spacer immediately after pipetting into a maximum of 8 wells - agarose solidifies very fast. Do not leave the agarose at room temperature for longer than 5 min.
3. After agarose solidification (~10 min, agarose will turn opaque), remove the PTFE spacers (Figure 2C) and place the PDMS racks (Figure 2B) into the casting molds so that pairs of PDMS posts reach into each mold (Figure 2D).
4. Resuspend the cardiomyocytes in ECM with a minimum concentration of 15 x 10⁶ cells/mL.
5. Prepare 110% of the estimated volume of reconstitution mix in a round bottom 15-mL tube on ice.For 24 EHTs, pipette the reconstitution mix listed in Table 1 for human, rat or mouse cardiomyocytes, respectively (10% extra is included).
   NOTE: Cell concentration of the reconstitution mix is different for human (10 x 10⁶ cells/mL), rat (4.1 x 10⁶ cells/mL) and mouse (6.8 x 10⁶ cells/mL) EHTs. Depending on the concentration of the cell suspension, add additional ECM to a final volume of 2,640 µL. Fibrinogen has to be lukewarm for pipetting. Slowly fill the tip of the pipet and do not touch the surface of the cold reconstitution mix with the fibrinogen-containing pipet when adding fibrinogen to the reconstitution mix - the viscosity of the fibrinogen in the pipette tip would increase immediately.
6. Triturate the reconstitution mix 10 - 15 times with a serological pipette until the fibrinogen clot is dissolved. Check the reconstitution mix in the pipette for homogeneity.
7. For each EHT, pipet 100 µL reconstitution mix in one thrombin aliquot (3 µL in 200 µL tube), mix and pipet the mixture into the agarose-slots as quickly as possible (Figure 2E). Use a new filter tip for each EHT. Resuspend the reconstitution mix (trituration with serological pipette 5-10 times) after every 8 EHTs.
8. Once all slots are filled with reconstitution mix, close the lid of the cell culture dish and incubate at 37 °C, 7% CO₂ for 2 h.
9. Remove the plate from the incubator and place under a sterile hood. Cover each well with a small amount of medium (e.g. DMEM, approximately 200 - 500 µL), shake the plate gently and incubate again at 37 °C for at least 10 min. The addition of DMEM will ease the removal from fibrin gels from agarose casting molds.
10. Prepare a new 24-well plate with 1.5 mL EHT-medium per well consisting of DMEM supplemented with 10% heat-inactivated horse serum, 1% penicillin/streptomycin, 10 µg/mL insulin, 33 µg/mL aprotinin.
11. Carefully remove the PDMS racks from the agarose casting molds and transfer them to the medium-filled 24-well plate (Figure 2F). Place the dish back in the incubator (37 °C, 7% CO₂, 40% O₂ for human and rat EHTs, 21% O₂ for mouse EHTs) and feed Mondays, Wednesdays and Fridays with freshly prepared EHT-medium. EHTs should show spontaneous contractions deflecting the posts of the PDMS racks 7-10 days after casting (Figure 1D, 2G).
    1. For mouse EHTs add 25 µg/mL of 5-cytosine βDarabinofuranoside to culture medium for 48 h to limit fibroblast proliferation.

3. Contraction Analysis

NOTE: The contraction analysis is based on video-optical recording in a commercially available EHT analysis instrument (see Table of materials). The central unit of this instrument is an incubation chamber (Figure 3A). The software provided with the instrument calculates contraction force based on deflection of the PDMS posts with known elastic modulus and geometry¹¹ (Supplementary Figure 1).

1. Turn on the heating device of the machine 2 h prior to the measurement. Turn on gas supply and electronic supply of the axis system immediately prior to the contraction analysis.
2. For baseline recording (reference point in the analysis), place the 24-well-plate with the EHTs in EHT-medium into the EHT analysis instrument and start the contraction analysis software according to supplier manual.
   1. Click the "Protocol" tab on the right panel and enter relevant information regarding study name, user, species, cell type, age, recording length and additional remarks.
   2. Click the "Camera view" tab on the left panel and start camera live mode with automatic figure detection mode. Click the "Setup" tab on the right panel andadjust the camera position with the xyz coordinates for each well in the 24-well-dish. Make sure the EHT is in the center of the window and in focus of the camera.
      1. Make sure the lid of the cell culture dish is not foggy as this will impair figure recognition and contraction analysis.
         NOTE: The figure recognition algorithm is based on detecting high contrast areas and monitoring their change in position. In the software these areas of figure recognition will be marked with a blue cross that moves with the contractions of the EHT.
      2. Make sure that the positions of the blue crosses are at both the top and bottom ends of the EHTs and moving only vertically matching the contractions of the EHTs (Figure 3B). Save setup positions of each well by pressing the button "position ok".
   3. Next, click the "Parameters" tab on the left panel. The parameters displayed here define the criteria by which the software identifies a contraction peak and marks it with a green square. These values are very important for correct identification of contraction peaks (Figure 3C) and no false data points (Figure 3E-F).
      NOTE: A list of species dependent parameters and examples is depicted in Figure 3H. For further details, please consult the software manual.
   4. Click the "Automatic" tab on the right panel and initiate the analysis by activating the "Start" button. The software will move sequentially from one well to the next, record EHT contractions and calculate force during recording (displayed in the "Real time" tab on the left panel). At the end of the analysis, a pdf report summarizing the results will be generated automatically.
3. Save and analyze the pdf report the system generated to summarize the results.
4. Monitor the contractile parameters by repeated measurement over several days. The EHT will increase in contraction force until it has reached a plateau (usually around day 15 of culture).
5. Drug screening
   1. Prepare protein free Tyrode solution one day prior to measurement and equilibrate in 24-well plates (2 mL/well) in the cell culture incubator (37 °C, 7% CO₂, 40% O₂) over night: 120 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 0.1-5 mM CaCl₂, 0.4 mM NaH₂PO₄, 22.6 mM NaHCO₃, 5 mM glucose, 0.05 mM Na₂EDTA, 25 mM HEPES. Add calcium up to 1.8 mM or the previously determined [EC₅₀] to investigate positive inotropic effects.
   2. Weigh and dissolve the drug in DMSO and filter sterile, if necessary. Prepare 6 different 1000x-sub-stock solutions (in DMSO) of the working concentration (e.g. 0.3, 1, 3, 10, 30, 100 µM for the respective nanomolar range).
   3. Dilute the respective stock in the Tyrode plates (2 µL in each 2 mL well) and mix thoroughly. Place the plates back in the incubator until needed.
   4. Start the baseline measurement by taking the first Tyrode-filled 24-well-plate under the sterile hood and position carbon electrodes in the wells. Transfer the PDMS racks with the EHTs placed on top of carbon electrodes, close the lid, put the plate back in the incubator and wait for an equilibration period of ~ 30 min.
   5. Transfer the EHTs from the incubator to the interlock in the EHT analysis instrument. Close the instrument and record spontaneous baseline contractility (20 s/EHT).
   6. Connect the carbon electrodes to the pacing cable and start electrical stimulation of the EHTs (2.5 V, 4 ms impulse duration, human: ~1 Hz, rat: ~2 Hz, mouse: ~4 Hz). Record the "paced" baseline (10 s/EHT). Ensure that all EHTs are properly paced. Stimulation frequency and voltage might vary from cell line to cell line.
   7. After baseline recording, remove the EHTs from the instrument and transfer the electrodes with the PDMS racks on top, to the 24-well-plate with the first drug concentration. Transfer the EHTs back onto the interlock of the EHT analysis instrument immediately and incubate there (standard drug incubation time e.g. 20 min).
   8. Reconnect pacing cables, adjust figure recognition and record EHT contractions responding to the first drug concentration.
   9. For the following drug concentrations, repeat step 3.5.7 and 3.5.8 with the 24-well-plates containing higher drug concentrations.
   10. Save all pdf recordings. Check each recording for figure recognition, correct positioning of green squares identifying contraction peaks and artifacts (see 3.2.1 and Figure 3).
   11. Perform additional offline analysis if needed.
       1. Choose the respective run from the "Runs" database on the left panel by clicking "Select run". In the "Parameter" tab on the left, change parameter for contraction analysis. Now choose "Offline analysis" in the "Offline" tab on the right panel to reanalyze the videos.
       2. To adjust figure recognition and therefore record a new video file, choose "Sample review" after adjusting the figure recognition in the "Real time" tab. NOTE: Sample review can only reanalyze one well at a time, whereas offline analysis can apply new parameter for all wells by clicking the button "Use parameter on all wells" in the "Parameter" panel on the left. Both types of offline analyzes will automatically create new data files and pdf displaying the results.
   12. Analyze the experiment by combining the results of the biological replicates and summarize the data in concentration response curves for frequency, contraction force, contraction time and relaxation time (Figure 5D) and/or graphical display of average contraction peaks (Figure 5C).
       NOTE: PDMS racks and PTFE spacer can be used repeatedly (PDMS racks max. 5 times, and PTFE spacer endlessly). After use, clean them manually with water, boil twice in demineralized water and autoclave. Be aware of potential carry-over effects, when re-using the racks, as drugs might be absorbed by the PDMS.

Results

Cardiac Differentiation and Preparation of EHT

HiPSC were expanded on reduced growth factor basement membrane matrix, dissociated with EDTA and embryoid bodies (EBs) formed in spinner flasks overnight. After mesodermal induction for three days, cardiac differentiation was initiated with the Wnt inhibitor. After ~17 days of differentiation protocol, beating EBs were dissociated into single cells with collagenase type II (Fig...

Discussion

Engineered heart tissue offers a valuable option to the tool box of cardiovascular research. EHTs in the 24-well format have proven valuable for disease modeling^8,14, drug safety screening^7,8,10,11,15, or basic cardiovascular research^16,17.

...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EHT analysis intrument	EHT Technologies GmbH	A0001	Software is included
EHT PDMS rack	EHT Technologies GmbH	C0001
EHT PTFE spacer	EHT Technologies GmbH	C0002
EHT electrode	EHT Technologies GmbH	P0001
EHT pacing adapter/cable	EHT Technologies GmbH	P0002
24-well-plate	Nunc	144530
6 well-cell culture plate	Nunc	140675
15 mL falcon tube, graduated	Sarstedt	62,554,502
Cell scraper	Sarstedt	831,830
Spinner flask	Integra	182 101
Stirrer Variomag/ Cimarec Biosystem Direct	Thermo scientific	70101	Adjust rotor speed to 40 rpm
T175 cell culture flask	Sarstedt	831,812,002
V-shaped sedimentation rack	Custom made at UKE Hamburg	na
10× DMEM	Gibco	52100
1-Thioglycerol	Sigma Aldrich	M6145
2-Phospho-L-ascorbic acid trisodium salt	Sigma Aldrich	49752
Activin-A	R&D systems	338-AC
Agarose	Invitrogen	15510-019
Aprotinin	Sigma Aldrich	A1153
Aqua ad injectabilia	Baxter GmbH	1428
B27 PLUS insulin	Gibco	17504-044
BMP-4	R&D systems	314-BP
Collagenase II	Worthington	LS004176
DMEM	Biochrom	F0415
DMSO	Sigma Aldrich	D4540
DNase II, type V (from bovine spleen)	Sigma	D8764
Dorsomorphin	abcam	ab120843
EDTA	Roth	8043.2
Fetal calf serum	Gibco	10437028
FGF2	Miltenyi Biotec	130-104-921
Fibrinogen (bovine)	Sigma Aldrich	F8630
Geltrex	Gibco	A1413302	For coating: 1:200 dilution
HBSS w/o Ca2+/Mg2+	Gibco	14175-053
HEPES	Roth	9105.4
Horse serum	Life technologies	26050088
Human serum albumin	Biological Industries	05-720-1B
Insulin, human	Sigma Aldrich	I9278
L-Glutamin	Gibco	25030-024
Lipidmix	Sigma Aldrich	L5146
Matrigel	BD Biosciences	354234	For EHT reconsitutionmix.
N-Benzyl-p-Toluenesulfonamide	TCI	B3082-25G
PBS w/o MgCl2/CaCl2	Biochrom	14190
Penicillin/Streptomycin	Gibco	15140
Pluronic F-127	Sigma Aldrich	P2443
Polyvinyl alcohol	Sigma Aldrich	P8136
RPMI 1640	Gibco	21875
Sodium selenite	Sigma Aldrich	S5261
TGFß1	Peprotech	100-21
Thrombin	Sigma Aldrich	T7513
Transferrin	Sigma Aldrich	T8158
Y-27632	Biorbyt	orb6014
hiPSC	Custom made at UKE hamburg	na
iCell cardiomyocytes kit	Cellular Dynamics International	CMC-100-010-001
Pluricyte cardiomyocyte kit	Pluriomics	PCK-1.5
Cor.4U - HiPSC cardiomyocytes kit	Axiogenesis AG	Ax-C-HC02-FR3
Cellartis cardiomyocytes	Takara Bio USA, Inc.	Y10075