Category: Automation and High-Throughput Technologies
Human iPSC-derived cardiomyocyte (hiPSC-CM) is considered a novel promising tool in preclinical assessment of drug-induced cardiotoxicity as well as in cardiovascular disease research. Among various platforms and methods have been examined to detect drug-induced cardiotoxicity such as arrhythmic risks and contractility changes using hiPSC-CMs, measurement of Ca2+ transients with calcium-sensitive fluorescent dyes has a great potential to become a standard method of cardiotoxicity screening, since it can be easily performed in the high-throughput manner with a lower running cost with a kinetic plate reader. FDSS/μCELL is a kinetic plate reader that measures kinetics of fluorescence signals of all 96/384/1536 wells in a microplate simultaneously and has been widely used to measure intracellular Ca2+ signals in drug discovery. Recently, we have developed various significant functions to make the FDSS/μCELL an instrument to measure and analyze Ca2+ transients-waveforms in hiPSC-CM accurately in the physiological condition. These are, (1) Sampling rate: hiPSC-CMs spontaneously beat at rate of approximately ~0.5 Hz, and can reach 2 Hz under stimulated conditions. Under these situations, it is shown that the sufficient higher sampling rate is important for accurate measurement and analysis of Ca2+ transients, which is synchronized with the beat of cardiomyocyte. We equipped a high speed acquisition function (up to 200 Hz sampling rate) using Hamamatsu camera technology. (2) Temperature control: It has been shown that beating of cardiomyocytes depends on temperature. To make beating of cardiomyocyes stable during the measurement, the strict temperature control is needed. (3) pH control of the medium: It has been shown that intracellular pH could be one of the major determinants of the changes in myocardial contractility. It would be important to keep the pH of the medium during the measurement to make the hiPSC-CMs healthy and stable. (4) Electric Field Stimulation: Electric Field Stimulation (EFS) has been used for pacing the beatings of cardiomyocytes. We recently developed a 96 channel Electric Field Stimulation system that adds EFS to all 96 wells in a plate simultaneously during the fluorescence measurement. (5) Waveform analysis; we developed a software to estimate beating rate, peak width, rising/falling slopes, etc., from the Ca2+ transients-waveforms. Also, we have been developing an analysis method to detect irregular peaks automatically. Another obstacle to perform cardiotoxicty screenings using hiPSC-CMs is the cell cost. We obtained the results in which the measurement in the 384-well small volume plate (4,000 cells/well) in FDSS/μCELL gives the same results in drug-induced arrhythmic assessment as the measurement in 96-well plates (40,000 cells/well). With these functions and results, it can be considered that Ca2+ transient measurement with a kinetic plate reader would greatly develop the cardiotoxicity screening using hiPSC-CMs.
Natsumi Kato– Application Engineer, Hamamatsu Photonics K.K., Hamamatsu City, Shizuoka, Japan
Hamamatsu Photonics K.K.
Hamamatsu City, Shizuoka, Japan
Natsumi Kato is an application engineer at Hamamatsu in Japan since 2011. She studied Molecular Biology at the Nagoya University before joining Hamamatsu, and obtained her PhD degree in Photonics Industry Management from the Graduate School for the Creation of New Photonics Industries in 2016.