Virus e impedancia electrica

Biosensors and Bioelectronics 26 (2011) 2000–2005

Contents lists available at ScienceDirect

Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios

Electric cell-substrate impedance sensing (ECIS) based real-time measurement of titer dependent cytotoxicity induced by adenoviral vectors in an IPI-2I cell culture model
Jakob Müller a,∗ , Christian Thirion b , MichaelW. Pfaffl a
a b

Physiology Unit, Technische Universität München, Research Center for Nutrition and Food Science, Weihenstephaner Berg 3, 85350 Freising – Weihenstephan, Germany Friedrich-Baur-Institute, Klinikum der LMU, Department of Neurology, Marchioninistrasse 17, 81377 Munich, Germany

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a b s t r a c t
Recombinant viral vectors are widespread tools fortransfer of genetic material in various modern biotechnological applications like for example RNA interference (RNAi). However, an accurate and reproducible titer assignment represents the basic step for most downstream applications regarding a precise multiplicity of infection (MOI) adjustment. As necessary scaffold for the studies described in this work we introduce a quantitative real-time PCR (qPCR)based approach for viral particle measurement. Still an implicated problem concerning physiological effects is that the appliance of viral vectors is often attended by toxic effects on the individual target. To determine the critical viral dose leading to cell death we developed an electric cell-substrate impedance sensing (ECIS) based assay. With ECIS technology the impedance change of a currentflow through the cell culture medium in an array plate is measured in a non-invasive manner, visualizing effects like cell attachment, cell–cell contacts or proliferation. Here we describe the potential of this online measurement technique in an in vitro model using the porcine ileal epithelial cell line IPI-2I in combination with an adenoviral transfection vector (Ad5-derivate). This approachshows a clear dose-depending toxic effect, as the amount of applied virus highly correlates (p < 0.001) with the level of cell death. Thus this assay offers the possibility to discriminate the minimal non-toxic dose of the individual transfection method. In addition this work suggests that the ECIS-device bears the feasibility to transfer this assay to multiple other cytotoxicological questions. ©2010 Elsevier B.V. All rights reserved.

Article history: Received 8 June 2010 Received in revised form 3 August 2010 Accepted 28 August 2010 Available online 9 September 2010 Keywords: ECIS Cytotoxicity IPI-2I Adenovirus 5 RNA interference qPCR virus titration

1. Introduction Electrochemical impedance measurements in cell-based assays represent a method which is not yet widespread but bears agreat potential for cell culture studies by providing the technical feature to monitor cell interactions, proliferation or cell death in real-time kinetics. Electrochemical impedance spectroscopy was first utilized for cell culture studies by Giaever and Keese (1984) who developed the ECIS-device. Thereby array like cell culture dishes covered with microelectrodes serve as adhesion substrate. Bydetecting the impedance change over time and/or altering the frequency of the current in the measurement, effects on cellular level can be monitored. The application can provide information about cell growth (Mitra et al., 1991), cell motility (Lo et al., 1993), cell barrier function (Tiruppathi et al., 1992) and in vitro toxicology (Ceriotti et al., 2007). It is also utilized in cancer research(Lovelady et al., 2007). Our approach was to use this technique to determine the toxicity occurring as transfection side effect while using viral vectors for

∗ Corresponding author. Tel.: +49 8161 71 3867; fax: +49 8161 71 4204. E-mail address: jakob.mueller@wzw.tum.de (J. Müller). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.08.075

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