Simultaneous electrical and contractile monitoring of cardiomyocytes on silicon microelectrode arrays
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1
Interuniversity Microelectronics Centre (IMEC), LST, Belgium
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2
KU Leuven, Department of Physics and Astronomy, Belgium
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3
Interuniversity Microelectronics Centre (IMEC), Imeclink, Belgium
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4
Johns Hopkins University, Institute for NanoBioTechnology, United States
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5
KU Leuven, Faculty of Medicine, Belgium
Drug-induced cardiotoxicity is one of the main causes of drug development termination and drug market withdrawal. To better predict proarrhythmic risks, the focus of in vitro cardiotoxicity assays is shifting from the activity of a single ion channel in hERG blockage assays to the full recording of electrical activity of primary and hiPSC-derived cardiac cells. In such assays, microelectrode arrays (MEA) non-invasively record the electrical activity of cardiac cells to evaluate drug-induced effects on action potential duration and ion channel activity. However, evaluating drug-induced effects on cardiac cells solely by electrical measurements only offers partial insight into adverse reactions on the heart. The combined monitoring of electrical and contractile activity of in vitro cardiac cells would thus offer unique insight into the mechanisms of drug-induced side effects. Nevertheless, this is a challenging experiment to perform. So far, recording electrical and contractile activity was mainly achieved by combining MEAs with traditional microscopy2 or with impedimetric techniques3. Nevertheless, these systems suffer from disadvantages such as bulky components, small field-of-view (FOV), poor signal quality and limited read-out sites (in case of impedance).
To overcome these issues, we developed a multi-parametric platform that combines MEAs with reflection-based lens-free imaging (RLFI). This platform allows simultaneous monitoring electrical activity and contractility of cardiac cells without the use of a label. Single-cell intracellular activity and extracellular activity was recorded by a CMOS-based MEA with 16k TiN sub-cellular sized electrodes. For contractility measurements, we developed a novel RLFI setup that is compatible with opaque substrates such as Si-based chips4. The setup only consisted of a coherent laser source (wavelength of 638 nm at 15 mW), a sample, an image sensor (CMOS CMV2000), and an I/O pulse for synchronization of electrical and optical signals. Image processing scripts were used to analyse the unprocessed interference patterns through motion vectors over a large FOV of maximally 57 mm2. The obtained values were further processed to obtain the relative cellular deformation (RCD) and relative cellular deformation rate (RCDr) in an area of 0.124 mm2 and with a temporal resolution of 6 ms. The RCD signal was of high quality and its signal-to-noise ratio was 49.
The RLFI module was compact as there are limited set of optical components and could thus easily be integrated onto the high-density MEA system. Measurements were carried out inside an incubator at physiologically relevant conditions. As RCD contractility measurements could be extracted from unprocessed RLFI interference patterns, hardware and software requirements of the setup were significantly simplified. Moreover, contractility was monitored at high speed over a FOV 19x larger than a conventional microscope with 2x magnification.
Initial system optimization was carried out using rat ventricular cardiomyocytes. First, the relationship between RCD measurements and cardiac contractility was validated with simultaneous fluorescent calcium imaging. Both RCD and intracellular calcium transients were detected concurrently. Second, the RLFI system was implemented on the high-density MEA to detect electrical and contractile activity simultaneously. This system could be used to directly monitor the electromechanical window (i.e. the interval between the termination of contractile activity and electrical activity). The system also was able to detect unlinking of the electro-mechanical cellular coupling after exposing the cells to 5 µM of blebbistatin, a selective inhibitor of the myosin protein responsible for cardiac contraction. No contractile activity was detected despite continuing detection of action potentials. Additionally, the RLFI system detected a dose-dependent reduction of contraction, contraction rate, and relaxation rate. Interestingly, the IC50 was lower for the contraction rate than relaxation rate IC50 (381 nM vs 612 nM, respectively). Third, the system monitored the propagation of the excitation wave throughout the cardiac monolayer. Even though both electrical and optical techniques could detect the flow of an excitation wave, the RLFI monitored it over a much larger FOV. The RLFI system was then used to reveal conduction disturbances induced by 1-octanol, as well as the concentration dependent reduction of excitation propagation velocity (IC50 was 45 µM).
The developed platform offers unique insight into drug-induced cardiotoxicity and was implemented in a compact device. It not only offers insight into contractility and electrical activity of in vitro cardiac cells, but also their underlying relationship. The system monitored the electro-mechanical window as well as the unlinking of the electro-mechanical coupling. Moreover, the system identified drug-induced reductions in contractility and conduction blocks. Therefore, the combined RLFI-MEA strategy offers multi-parametric and non-invasive read-out of microscopic and macroscopic properties of cardiac cells for cardiotoxicity screening and drug development. Moreover, we envision these sensors in compact heart-on-a-chip devices, simultaneously monitor cardiac cells through electrical activity and contractility.
Acknowledgements
This work was supported by the “Agency for Innovation by Science and Technology in Flanders” (IWT) and Electronic Components and Systems for European Leadership (ECSEL) “InForMed” (No. 2014-2-662155).
References
1. Vargas, H. M. A new preclinical biomarker for risk of Torsades de Pointes: Drug-induced reduction of the cardiac electromechanical window. British Journal of Pharmacology 161, 1441–1443 (2010).
2. Hayakawa, T. et al. Image-based evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: Correlation and complementarity with extracellular electrophysiology. J. Mol. Cell. Cardiol. 77, 178–191 (2014).
3. Qian, F. et al. Simultaneous Electrical Recording of Cardiac Electrophysiology and Contraction on Chip. Lab Chip 17, 1732–1739 (2017).
4. Pauwelyn, T. et al. (in press). Reflective lens-free imaging on high-density silicon microelectrode arrays for monitoring and evaluation of in vitro cardiac contractility. Biomed. Opt. Express 9, (2018).
Keywords:
cardiotoxicity,
CMOS-MEAs,
Lens-free imaging,
Electro-mechanical window,
Heart-on-a-chip
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Oral Presentation
Topic:
Microphysiological systems
Citation:
Pauwelyn
T,
Stahl
R,
Mayo
L,
Zheng
X,
Lambrechts
A,
Janssens
S,
Lagae
L,
Reumers
V and
Braeken
D
(2019). Simultaneous electrical and contractile monitoring of cardiomyocytes on silicon microelectrode arrays.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00017
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Received:
17 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Thomas Pauwelyn, Interuniversity Microelectronics Centre (IMEC), LST, Leuven, 3001, Belgium, thomas.pauwelyn@imec.be