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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-23 17:46 |
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Conference: Bucharest University Faculty of Physics 2017 Meeting
Section: Biophysics; Medical Physics
Title:
Comparison between automated and manual patch-clamp platforms in assessment of proarrhythmogenic effects of drugs via hERG channels blockade
Authors:
Răzvan AIRINI (1,2), Alexandru-Florian DEFTU (1,2), Florin Bogdan EPUREANU (1,2), Bogdan AMUZESCU (1,2)
*
Affiliation:
1) Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest
2) Research Group in Molecular Physiology & Pharmacology, Life, Environmental and Earth Sciences Division, Research Institute of the University of Bucharest
E-mail
airinirazvan@gmail.com
Keywords:
Automated patch-clamp, cytocentering, hERG channel, cardiac safety drug testing, voltage-clamp, activation, inactivation, dose-response curve
Abstract:
The aim of our study was to compare results obtained via classical and automated patch-clamp in assessment of pharmacological effects of compound E-4031 on human ether-à-go-go related gene (hERG) channels stably expressed in HEK293T cells. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, up to a surface density of approximately 70%, in Petri dishes (for manual patch-clamp) or 25 cm^2 flasks (for automated patch-clamp). Cells for automated patch-clamp were detached with trypsin, centrifuged and resuspended in extracellular solution; they were further placed in a polyethylene tube, from where a tiny amount (~ 3 microliters) was collected by the dispensing needle of the CytoPatch™ 2 platform and applied into a microfluidic chip with two quartz pipette tips of 2 micrometers inner diameter. The experiments showed that hERG potassium channels block occurred in a concentration and time-dependent manner, and wash-out of the compound was extremely slow and incomplete. Using manual patch-clamp we proved similar effects, building a dose-response curve fitted with a Hill function.
References:
Amuzescu B., Airini R., Ghica L., Epureanu F.B., Deftu A.F., Cucu D.M., Ristoiu V.P., Mihailescu D.F., Radu B.M. (2017): Novel approaches to proarrhythmogenic risk testing using automated patch-clamp platforms, Rom. J. Biophys. 27(1) (in press) http://www.rjb.ro/number_articles.php?id_num=79
Acknowledgement:
This study was funded from Competitiveness Operational Programme 2014–2020 project P_37_675 (contract no. 146/2016), Priority Axis 1, Action 1.1.4, co-financed by the European Funds for Regional Development and Romanian Government funds.
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