title: 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.
abstract: Recombinant viral vectors are widespread tools for transfer 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 current flow 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 approach shows 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.
authors.affiliation: Physiology Unit, Technische Universität München, Research Center for Nutrition and Food Science, Weihenstephaner Berg 3, 85350 Freising-Weihenstephan, Germany. jakob.mueller@wzw.tum.de
authors.forename: Jakob
authors.initials: J
authors.lastname: Müller
authors.forename: Christian
authors.initials: C
authors.lastname: Thirion
authors.forename: Michael W
authors.initials: MW
authors.lastname: Pfaffl
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D000818:Animals; D017209:Apoptosis; D001681:Biological Assay / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation*; D002460:Cell Line; D003199:Computer Systems; D003217:Conductometry / Q000295:instrumentation*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000502:physiology* / Q000821:virology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005822:Genetic Vectors / Q000008:administration & dosage*; D013552:Swine