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 protocols4,5 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 cardiomyocytes8,9,10 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
Representative Results
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 modeling8,14, drug safety screening7,8,10,11,15, or basic cardiovascular research16,17.
…Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors are grateful to Alessandra Moretti and Dennis Schade for their kind contribution of material. We acknowledge the great support of the iPS and EHT working group at the Department of Experimental Pharmacology and Toxicology of the UKE. The work of the authors is supported by grants from the DZHK (German Centre for Cardiovascular Research) and the German Ministry of Education and Research (BMBF), the German Research Foundation (DFG Es 88/12-1, HA 3423/5-1), British National Centre for the Replacement Refinement & Reduction of Animals in Research (NC3Rs CRACK-IT grant 35911-259146), the British Heart Foundation RM/13/30157, the European Research Council (Advanced Grant IndivuHeart), the German Heart Foundation and the Freie und Hansestadt Hamburg.
Materials
| 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 |
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