High-Throughput CMOS MEA System with Integrated Microfluidics for Cardiotoxicity Studies

Abstract

Event Abstract Back to Event High-Throughput CMOS MEA System with Integrated Microfluidics for Cardiotoxicity Studies Thomas Pauwelyn1, 2, Beatrice Miccoli1, 2, Aravinthan Velnayagam1, Robert Jan Boom3, Maciej Skolimowski3, Elwin Vrouwe3, Dries Braeken1 and Veerle Reumers1* 1 Interuniversity Microelectronics Centre (IMEC), Belgium 2 KU Leuven, Belgium 3 Micronit Microfluidics (Netherlands), Netherlands High-throughput screening is essential for current pharmaceutical drugs development where the number of target compounds to be screened is in the range of 105 – 106 trials and constantly increasing (Macarron et al., 2011). Specifically, cardiotoxicity is one of the main causes of drug rejection and withdrawal from the market. Additionally, current in vitro research based on animal models does not truly predict the response of the human heart (Mathur et al., 2015). Multi-electrodes arrays (MEAs) represent a powerful tool for in vitro electrophysiological studies of both animal or human cardiac models. They allow non-invasive and long-term monitoring of cardiac action potentials, including both extra- and intra-cellular recordings. Therefore, they represent an effective strategy for high-throughput and high-resolution cardiotoxicity studies. To further increase the throughput of MEAs, microfluidic devices can separate the surface of chips into multiple, independent cell culture chambers. Microfluidic devices enable parallelization and automation as well as the reduction of reagents and cell consumption. This leads to a decrease in the final cost of the assay. Nevertheless, the integration of microfluidics on complementary metal-oxide semiconductors (CMOS) integrated circuits presents numerous technological challenges that need to be thoroughly assessed and faced (Huang and Mason, 2013). To overcome these challenges, we developed a novel strategy to integrate a polystyrene (PS)-based microfluidic system on top of a CMOS MEA (Lopez et al., 2018). A thin perforated glass interposer was bonded onto the CMOS MEA, while the PS-based microfluidic system was glued on top. The PS-device determined the height of the microfluidic chamber and their locations. The microfluidic chip separated the chip surface into 16 independent cell culture chambers. Therefore, up to 16 different experiment conditions are possible per device. The microfluidic cell culture chambers (area 0.62 mm^2, height 1.25 mm) are addressed by an individual inlet and an individual outlet. Each chamber contains 1,024 recording electrodes that are grounded by on-chip integrated reference electrodes. Therefore, no external reference electrodes were required. The electrodes in each chamber are independently addressable for individual measurements. The chip architecture allows for 6 different types of measurements on the same chips: extracellular recordings, intracellular recording, constant voltage stimulation, constant current stimulation, impedance monitoring at a fixed frequency and impedance spectroscopy between 10 Hz and 1 MHz (Lopez et al., 2018). At first, we optimized the protocol for the microfluidic culture of rat ventricular cardiomyocytes in prototype devices. In the first-generation prototypes, the CMOS MEA chip was replaced with a transparent glass slide for easier optical access. External syringe pumps controlled the perfusion of fluids through the microfluidic chambers. Flow rates were optimized for each of the steps of the cell culture, including sterilization, coating, seeding, and nutrients delivery. Cardiomyocytes were successfully cultured for up to 9 days in vitro (DIV). Cardiac functionality was confirmed both by visualizing cardiac contractions using phase-contrast microscopy and by lens-free imaging for in situ monitoring. Cell viability was assessed through calcein and Hoechst stainings. Specifically, from the fluorescent staining a cell density of 1640 ± 280 nuclei/mm^2 was measured inside the microfluidic chambers at 2 DIV. In the second-generation prototypes, the glass slide was replaced with a passive high-density MEA. The optimized protocol for culturing microfluidic cardiac cell cultures was successfully transferred and clear extracellular recordings were detected in the electrodes of size 11x11 µm^2 from DIV 3 – 5. An average peak-to-peak voltage amplitude of 601 ± 16 µV was measured at 4 DIV with a beating rate of 51.5 ± 1.1 bpm. Therefore, the presented approach is compatible with Si-based MEAs. Currently, the optimized protocols developed in the prototype device are being applied to a microfluidic system integrated onto the CMOS MEA substrate. As syringe pumps are bulky and not scalable, they will be replaced by embedded peristaltic pumps to internally control the fluids flows. Human induced pluripotent stem-cells will be then used to assess drug toxicity assays exploiting the high-throughput 16 wells configuration. Our approach is compatible with the 96 well plate standard. Moreover, both the number of CMOS MEAs and of microfluidic chambers per system can be extended so to further increase the throughput and reach a 96 wells configuration. 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). The fabrication of the microfluidic devices and their integration on the CMOS chip was performed by Micronit Micro Technologies B.V. (Enschede, Netherlands). The passive electrical read-out instrumentation was loaned by Multichannel Systems (Reutlingen, Germany). References Huang, Y., and Mason, A. J. (2013). Lab-on-CMOS integration of microfluidics and electrochemical sensors. Lab Chip 13, 3929. doi:10.1039/c3lc50437a. Lopez, C. M., Chun, H. S., Berti, L., Wang, S., Bulcke, C. Van Den, Weijers, J., et al. (2018). A 16384-Electrode 1024-Channel Multimodal CMOS MEA for High-Throughput Intracellular Action Potential Measurements and Impedance Spectroscopy in Drug- Screening Applications. in 2018 International Solid-State Circuits Conference, 4–6. Macarron, R., Banks, M. N., Bojanic, D., Burns, D. J., Cirovic, D. A., Garyantes, T., et al. (2011). Impact of high-throughput screening. Nature 10, 188–195. doi:10.1038/nrd3368. Mathur, A., Loskill, P., Shao, K., Huebsch, N., Hong, S. G., Marcus, S. G., et al. (2015). Human iPSC-based cardiac microphysiological system for drug screening applications. Sci. Rep. 5, 1–7. doi:10.1038/srep08883. Keywords: CMOS MEA, Microfluidics, High-Throughput Screening, drug screening, cardiotoxicity 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, Miccoli B, Velnayagam A, Jan Boom R, Skolimowski M, Vrouwe E, Braeken D and Reumers V (2019). High-Throughput CMOS MEA System with Integrated Microfluidics for Cardiotoxicity Studies. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00009 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Veerle Reumers, Interuniversity Microelectronics Centre (IMEC), Leuven, Belgium, Veerle.Reumers@imec.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Thomas Pauwelyn Beatrice Miccoli Aravinthan Velnayagam Robert Jan Boom Maciej Skolimowski Elwin Vrouwe Dries Braeken Veerle Reumers Google Thomas Pauwelyn Beatrice Miccoli Aravinthan Velnayagam Robert Jan Boom Maciej Skolimowski Elwin Vrouwe Dries Braeken Veerle Reumers Google Scholar Thomas Pauwelyn Beatrice Miccoli Aravinthan Velnayagam Robert Jan Boom Maciej Skolimowski Elwin Vrouwe Dries Braeken Veerle Reumers PubMed Thomas Pauwelyn Beatrice Miccoli Aravinthan Velnayagam Robert Jan Boom Maciej Skolimowski Elwin Vrouwe Dries Braeken Veerle Reumers Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.