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 se....

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% ....

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.

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Materials

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