Historically, scientists have relied heavily on animal studies to determine if a medical product (i.e., drug or medical device) is safe before testing it on humans. While animal testing is still justified in many situations, finding alternatives is highly desirable. With recent advances in science and engineering, the development of alternatives to animal testing is more feasible than ever. A renewed focus on the development of human in vitro alternative methods for cardiac safety assessment is at least partially attributable to advances in induced pluripotent stem cell (iPSC) technology. Human heart cells can be generated in a lab using a simple blood or skin sample from a patient, yielding human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) suitable for robust high-throughput testing. Progress in 3D tissue bioengineering, microelectrode array technologies, live cell imaging, and other technologies have also been instrumental to the development of the protocols included in this collection.
The protocol by Gerges et al.1 applies a non-invasive optical method (MyoBLAZER) to assess changes in contractility in adult human primary ventricular cardiomyocytes. The cells are electrically paced, and image analysis measures sarcomere shortening across multiple cells in parallel. This method can collect concentration-response curves every 30 min per compound per device, and provides structure-activity relationship data. This non-invasive optical method helps preserve the physiology and pharmacology of adult human cardiomyocytes during high-throughput screening. Additionally, the use of human adult cardiomyocytes can provide a critical translational piece to predicting contractility.
The methodology by Lickiss et al.2 presents a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform by using a soft, flexible, silicon-based cell culture substrate. The approach proved successful by re-establishing the physiological positive inotropic response to isoproterenol in commercially available healthy hiPSC-CMs, which is knowingly absent in the standard (stiff substrate) culture without the need for a 3D system. The hybrid system allows for the direct measurement of contraction force (mN/mm2), beat rate, as well as cell monolayer density and integrity. It also addresses the challenges of a traditional 3D system (i.e., low throughput, considerable training needs), reducing time and costs necessary to complete the assay.
The collection also includes work by Feaster et al.3, who demonstrate an in vitro, high-throughput, non-invasive method to evaluate cardiac contractility modulation (CCM) therapy in 2D human stem cell cardiomyocytes plated on a flexible Matrigel mattress, using probe-free video-based microscopy. The authors highlight acute effects of CCM on the contractile properties of healthy and diseased hiPSC-CMs. This tool offers a low-cost method to understand the safety or efficacy of CCMs, and could reduce the dependence on animal studies and assist in the regulatory decision making of cardiac electrophysiology medical devices.
The refined protocol by Schaefer et al.4 describes a novel extension to the standard microelectrode array (MEA) system that normally records extracellular field potential in hiPSC-CMs, allowing for intracellular-like action potential recordings by opening cell membranes with nanosecond laser beam pulses. This device not only includes standard MEA advantages (i.e., signal propagation monitoring, acute and chronic experiments), but also permits an insight into intracellular-like action potential shape without the use of strong electric field pulses for cell electroporation.
The protocol by Berry et al.5 describes a new platform that enables reproducible fabrication of 3D engineered muscle tissues (EMTs) for direct contractility force measurements. The instrument can detect micronewton changes in contractility force, thus making it a powerful tool for dose-dependent compound screening. Contractility in hiPSC-based cardiac tissue, as well as skeletal muscle tissues, can be recorded in up to 24 tissues simultaneously, and the data can be analyzed longitudinally over weeks or months. Therefore, minimal additional skills or training is required for the researchers.
Finally, the publication by Zhao et al.6 describes an array of functional assays (extracellular field potential, action potential, contractility, and calcium) optimized for use with cardiomyocytes that can be generated in house by user labs. This can be done using previously published differentiation protocols and iPSCs available from the Stanford University Cardiovascular Institute Biobank (https://med.stanford.edu/scvibiobank/request-cells.html), providing a wide range of “diseased” and control cells. This is a complete set of methods for the main cardiac contractility and electrophysiology recordings, including standard approaches (patch clamp, microelectrode arrays, calcium-sensitive fluorescence probes, and video-based contractility measurements).
In conclusion, while the current methods collection does not claim to be complete (and continues to grow), it is already a relatively comprehensive set of methods illustrating many current challenges in contractility and electrophysiological recordings in human cardiomyocytes. It includes protocols for primary human cardiomyocytes1, commercially available hiPSC-CMs derived from healthy donors2,3,4, as well as protocols optimized for cells carrying a signature of congenital heart disease3,6. These methods span across various cell culture conditions, from single cells for patch clamp experiments6, to conventional hiPSC-CM 2D monolayers on stiff substrates1,4, 2D cardiomyocyte monolayers on soft, flexible substrates2,3, and finally, 3D engineered cardiac tissues5. The included methods use different approaches for the recording of the most relevant cardiac physiology parameters, such as contractility (measured either indirectly with video-based assays1,3,6 or directly with contractile force2,5), action potential (in a single cell using patch clamp6, a surrogate extracellular field potentials with an MEA4,6, or by using a novel approach to porate the cells to record action potential-like recordings with the standard MEA system4), and calcium transients (using calcium-sensitive probes6). Taken together, these methods provide not just detailed protocols that can be reproduced in other laboratories, but also illustrate some of the challenges with human cardiac in vitro methods, such as: hiPSC-CM immaturity, especially when using a standard 2D culture on stiff substrate; unwanted effects of fluorescent voltage- or calcium-sensitive probes; low throughput of the conventional action potential recordings; difficulties in the interpretation of standard field potential duration recordings; and lack of assays for the assessment of medical devices (e.g., vs. drugs). It is inspiring to see how many laboratories are working on improving these methods, which will inevitably lead to the wide adaptation of these methods in the future.
The authors declared no competing interest for this work.
This article reflects the views of the authors and should not be construed to represent the US Food and Drug Administration’s views or policies. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.
The authors have no acknowledgements.
- Gerges, N. A., et al. Measurement of heart contractility in isolated adult human primary cardiomyocytes. Journal of Visualized Experiments. (186), e64394 (2022).
- Lickiss, B., et al. Hybrid cell analysis system to assess structural and contractile changes of human iPSC-derived cardiomyocytes for preclinical cardiac risk evaluation. Journal of Visualized Experiments. (188), e64283 (2022).
- Feaster, T. K., Casciola, M., Narkar, A., Blinova, K. Evaluation of cardiac contractility modulation therapy in 2D human stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (190), e64848 (2022).
- Schaefer, J., Danker, T., Gebhardt, K., Kraushaar, U. Laser-induced action potential-like measurements of cardiomyocytes on microelectrode arrays for increased predictivity of safety pharmacology. Journal of Visualized Experiments. (187), e64355 (2022).
- Berry, B. J., et al. Preclinical drug testing in scalable 3D engineered muscle tissues. Journal of Visualized Experiments. , e64399 (2022).
- Zhao, S. R., Mondéjar-Parreño, G., Li, D., Shen, M., Wu, J. C. Technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (186), e64265 (2022).