A physiologically relevant model for predictive toxicology screening in vitro is offered with human-induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) (Axol Bioscience). Cell contractility (impedance) and the extracellular electrical field potential (EFP) can be simultaneously recorded in a 96-well plate with the hybrid screening instrument, CardioExcyte 96 (Nanion Technologies). More accurate predictions of the risk of human clinical pro-arrhythmias could be possible when these tools are used in combination.
Data is presented here on the optimization of hiPSC-vCMs on the CardioExcyte 96. Seeding parameters are determined and the optimal time point for analysis identified. In accordance with the Comprehensive in vitro Pro-arrythmia Assay (CiPA) guidelines, an assessment was made of excitation-contraction coupling in response to three standard reference compounds:
- Verapamil, a mixed ion channel blocker, which acts on both L-type calcium channels (ICaV) and potassium channels (IKr)
- Nifedipine, a selective calcium channel (ICaV) blocker
- Dofetilide, a selective ion channel blocker for IKr.
Low pro-arrhythmic risk is exhibited in both verapamil and nifedipine but the Cardiac Safety Consortium classify dofetilide as a high-risk pro-arrhythmic compound. Vontractility and electrical excitation in the hiPSC-vCMs were altered with the addition of each of these compounds.
Demonstrated here is a non-invasive, label-free, high temporal resolution tool, the CardioExcyte 96, which, when used in conjunction with Axol hiPSC-vCMs, may be able to predict pro-arrythmic risk in vitro.
Materials and methods
Cardiomyocyte culture: In accordance to the manufacturer’s protocol, human iPSC-Derived Ventricular Cardiomyocytes (ax2505, Axol Bioscience) were thawed on Fibronectin (ax0049)-coated plates, and cultured for the initial 24 hours in Cardiomyocyte Maintenance Medium (ax2530) + 10% FBS. Cells were switched to serum-free Cardiomyocyte Maintenance Medium the following day. The medium was then switched every 2 days after that.
Plating and recording: Cells were dissociated with Axol Unlock (ax0044) after 4 days and re-plated at 30,000 cells per well on the CardioExcyte 96 Sensor Plate. The following drug compounds were applied after one week: dofetilide (10nM), nifedipine (100nM) and verapamil (200 nM).
Immunocytochemistry: Cells were fixed in 3% PFA, permeabilized with 0.2% Triton X-100 and blocked with BSA. Primary antibody was incubated overnight at 4 ̊C before a secondary antibody coupled to Alexa Fluor® dyes (Invitrogen) was applied for 2 hours.
Western blot: 30 μg protein run on 10% SDS-PAGE gel for 70 minutes at 130 V and transferred to PVDF membrane. Membranes were incubated with primary antibody overnight at 4 ̊C before being washed and incubated with secondary antibody for 1 hour.
Impedance and EFP recordings from the same monolayer of cells are combined with the CardioExcyte 96 (CE 96), which is a hybrid system. Recordings are performed in an incubator at physiological temperature or within the environmental chamber. The CardioExcyte 96 can be used in cardiac safety screening on a variety of hiPSC-CMs to examine the effects of CiPA-relevant compounds. Complementary data to other assays such as automated patch clamping are provided by the CardioExcyte 96.
CardioExcyte 96 Technology
Figure 1: CardioExcyte 96 sensor technology
The CardioExcyte 96 combines impedance and Extracellular Field Potential (EFP) recordings to measure contractility and ion channel activity. A: The 96-well sensor plate contains gold electrodes embedded in each well. B: Manual overlay of impedance (uppe rtrace) and EFP (lower trace) trace data recorded from an autonomously beating cardiac preparation. Image Credit: Axol Bioscience
Table 1. Source: Axol Bioscience
|Number of channels
|Impedance and EFP measurements
|Cell adhesion and spreading assays
|Operation in standard incubator
|Environmental control (for use outside incubator)
|Temporal resolution (Imp/EFP)
||1 ms / 0.1 ms
|Dedicated analysis and recording software
|Integration with CYBERNANO analysis
Characterisation of Axolventricular iPSC-CMs
Figure 2: Molecular and physiological characterization of Axol hiPSC-vCMs
A: Top Immunocytochemistry data showed the expression of ventricular cardiomyocyte markers (cardiac troponin-I; 93.5% expression). Data from Dr Christian Zuppinger, University of Bern and Prof Matt Daniels, University of Oxford. Bottom Western blot data confirmed that Axol hiPSC-vCMs express more cardiac troponin-T (cTnT) and α-Actinin than human skin fibroblasts (hSFs). Data from Abigail Robertson, University of Manchester. B: 87% of Axol hiPSC-vCMs have a ventricular phenotype determined by MLC2v expression, compared to 13% expressing atrial MLC2a (n=1). Data from Dr Christian Zuppinger, University of Bern. Image Credit: Axol Bioscience
Figure 3. The cardiotoxic effects of cardiac ion channel modulators on Axol hiPSC-vCMs
Axol hiPSC-vCMs showed compound-relevant responses (light blue) to known cytotoxic compounds. A: DMSO control, showed no change in EFP but an increase in amplitude. B: verapamil and C: nifedipine, both reduced the impedance amplitude, increased the beat rate and reduced the EFP. D: dofetilide, a selective ion channel blocker for the rapidly activating delayed rectifier potassium channel (IKr), resulted in arrhythmic events typical of IKr blockers. Image Credit: Axol Bioscience
- Specific compound-relevant responses to known cardiotoxic compounds are shown with Axol hiPSC-vCMs
- The presence of ICaV and IKr channels in Axol hiPSC-vCMs are confirmed with the impedance and EFP pharmacology shown here.
- The CardioExcyte 96 system enables the use of a dual reading technology, which facilitates the detection of compound effects on both the contractility and electrophysiological properties of a beating network of hiPSC-vCMs.
- The CardioExcyte 96 (Nanion Technologies) system, in combination with Human iPSC-Derived Ventricular Cardiomyocytes (Axol Bioscience) has been shown to be an effective system for assessing cardiac pro-arrhythmia using compounds from the CiPA validation toolbox.
Produced from materials originally authored by Priyanka Dutta-Passecker1, George Gibbons1, Krisztina Juhasz2, Zoe Allen1, Ying Shao1, Sonja Stölzle-Feix2, Corina Bot4, Ulrich Thomas2 Leo Doerr2, Matthias Beckler2, Niels Fertig2, and Yichen Shi1 from Axol Bioscience.
- Axol Bioscience Ltd., Cambridge, UK
- Nanion Technologies, Munich, Germany
- Technical University of Munich, Munich, Germany
- Nanion Technologies Inc., Livingston, NJ, USA
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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