Invited Speaker

On-Chip Pre-Clinical Cardiac Toxicity: Testing Compounds Beyond hERG and QT using hES/hiPS Cardiomyocyte Cell Network Re-Entry Model on a Chip
Kenji YASUDA, Tomoyuki KANEKO, Fumimasa NOMURA, Atsushi SUGIYAMA
Japan

QT prolongation is a major safety concern for selecting and developing candidate compounds. The current integrated assay systems using hERG-transfected HEK-293/CHO-cells (hERG assay), isolated animal tissues (APD or MAP assay) and conscious and/or anesthetized whole animals (QT or MAP assay) may identify the QT prolongation, but still cannot fully predict the potential lethal arrhythmia including Torsades de Pointes (TdP) or ventricular fibrillation (VF) of drug candidates. Such limitations of conventional hERG assay and QT prolongation testing for accurate prediction of TdP and VF by compounds revealed us the necessity of new approach to evaluate global cardiac safety.

Understanding the importance of spatial and temporal regulation of cellular orientation, community size and shape, variety and interactions is one of the keys to resolve the mechanism of epigenetic processes in the higher complexity of cellular system like tissue and organ.(4) For example, the community size of cardiomyocyte cell group is important for the maintenance of stable beating intervals, and the difference of community size also gives us different results by same compounds.(5-6) To study the meaning of the spatial distribution of cells, we have developed the on-chip cell network cultivation system, and extra-cellular signals (field potentials: FP) of mouse embryonic cardiomyocytes in geometrically patterning chambers have been recorded with on-chip multi electrode array (MEA) system. Then, we functionally reconstructed the normal and abnormal re-entry model of cardiomyocytes network loop from the view point of propagation of contraction. If we can include the characteristics of heart into the chip like the functional spiral re-entry model by closed loop cell network circuit formation with constructive single cell-based approach, on-chip cardiomyocyte cell network assay is expected to be one of the candidates having the potential to measure the TdP and VF probability as pre-clinical testing for cardiac safety.

The on-chip cardiomyocyte cell network assay consists of three main units; cell network cultivation unit, MEA measurement unit which can measure the time course of single cell level FP change caused by ion channels’ opening/closing, and optical measurement unit which can measure the volume change of cardiomyocyte cells to estimate cardiac contraction. In the cell network cultivation unit, a series of 10-µm electrodes are lined-up on the chip surface, and each cardiomyocyte cell is put on the electrode one-by-one to be able to be measured the FPs of neighboring cells in the closed loop network simultaneously. The propagation velocity in the network can be controlled by replacing a part of cardiomyocyte cells in the network into fibroblast cells to represent hypertrophied heart model. Agarose microstructures are coated on the cultivation chip, and are used to keep cells’ positions and those interactions for the maintenance of the cell-electrode contacts and the loop network shape during long term cultivation. Time resolution of MEA measurement is 1 µsec, and is enough to distinguish Na, Ca, and K ion channels’ conditions, and to compare neighboring cells in the cell network loop, i.e. we can measure both the macroscopic propagation signal in cell network loop and the microscopic single cells’ field potential changes simultaneously.

Our strategy of on-chip assay for providing further insight into the extrapolation of preclinical data to human clinical settings or for replacing existing in vitro and in vivo cardiotoxicity models is as follows; i) abnormal triggering (temporal dispersion) causing lethal arrhythmias is estimated by analyzing the time course field potential dispersion of single cells in loop network using Poincaré plotting, ii) spatial dispersion of cells causing spiral re-entry is modeled by using wider width of cell network loop which can choose a different propagation pathways of cells among neighboring circulations, and iii) human ES / (LQT) iPS cell-based cardiomyocyte is used for cell network formation.
In this meeting, we present the system set-up and then, as described above, possible application of this system for drug toxicology.

References:

1. Antzelevitch C: Cardiology J. 15: 100-121, 2008.
2. Sugiyama A: Br. J. Pharmacol. 154: 1528-1537, 2008.
3. Bass AS. et al: Br. J. Pharmacol. 154: 1491-1501, 2008.
4. Yasuda K: Lab-on-Chips for Cellomics (ed. Andersson H and van den Berg A), 225-256, 2004. Kluwer Academic Publishers, Netherlands.
5. Kojima K, Kaneko T, Yasuda K: Biochem. Biophys. Res. Commun. 351: 209-215, 2006.
6. Kaneko T, Kojima K, Yasuda K: Biochem. Biophys. Res. Commun. 356: 494-498, 2007.
7. Suzuki I, Yasuda K: Biochem. Biophys. Res. Commun. 356: 470-498, 2007.