A whole-cell biosensor as in vitro alternative to skin irritation tests Ute Hofmann a , Stefanie Michaelis b , Thomas Winckler c , Joachim Wegener b , Karl-Heinz Feller a,n a Department of Medical Engineering and Biotechnology, University of Applied Sciences Jena, Carl-Zeiss-Promenade 2, 07745 Jena, Germany b Institute for Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitaetsstraße 31, 93053 Regensburg, Germany c Department of Pharmaceutical Biology, Institute of Pharmacy, University of Jena, Semmelweisstraße 10, 07743 Jena, Germany article info Article history: Received 10 April 2012 Received in revised form 5 July 2012 Accepted 13 July 2012 Available online 8 August 2012 Keywords: Lab on a Chip Cell-based assay Whole-cell biosensor Heat shock protein Reporter gene Time-lapse microscopy Electric cell-substrate impedance sensing abstract This study presents the time-resolved detection of chemically induced stress upon intracellular signaling cascades by using genetically modified sensor cells based on the human keratinocyte cell line HaCaT. The cells were stably transfected with a HSP72-GFP reporter gene construct to create an optical sensor cell line expressing a stress-inducible reporter protein. The time- and dose-dependent performance of the sensor cells is demonstrated and discussed in comparison to a label-free impedimetric monitoring approach (electric cell-substrate impedance sensing, ECIS). Moreover, a microfluidic platform was established based on mSlidesI 0,4 Luer to allow for a convenient, sterile and incubator-independent time-lapse microscopic observation of the sensor cells. Cell growth was successfully achieved in this microfluidic setup and the cellular response to a cytotoxic substance could be followed in real-time and in a non-invasive, sensitive manner. This study paves the way for the development of micro-total analysis systems that combine optical and impedimetric readouts to enable an overall quantitative characterization of changes in cell metabolism and morphology as a response to toxin exposure. By recording multiple parameters, a detailed discrimination between competing stress- or growth-related mechanisms is possible, thereby presenting an entirely new in vitro alternative to skin irritation tests. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Many chemicals can induce irritant contact dermatitis. Several irritation models ranging from single-cell assays, epidermal equivalents, skin equivalents to excised skin have been described (Netzlaff et al., 2005; Schafer-Korting et al., 2008). Keratinocytes, the predominating cell type of the human skin, are the first to encounter chemicals that come into contact with the skin; they are involved in the immune reaction, for example, by producing cytokines. Therefore, keratinocytes represent a valuable model cell type to screen for dermatological effects in response to skin- damaging agents. However, this does not mean that all dermato- logical effects of the human skin can be studied within this particular cell culture model, because the three-dimensional tissue architecture of the in vivo environment and some of its unique transport pathways, e.g. penetration of the stratum corneum, are missing. Nevertheless, we need to establish standard operating procedures for future in vitro assays whose relevance and reliability should be based on internationally recognized protocols. Equally important to the type of model used to investigate irritancy is the type of biomarker used as readout parameter. Currently those approaches are in focus that aims to investigate the early irritation response, preferably via real-time monitoring before visible cell damage occurs, to provide an alternative to conventional endpoint measurements (Gibbs, 2009). Among these techniques in vitro cell culture in microfluidic devices has attracted considerable interest and has led to a substantial impact on cell biology research (Sung and Shuler, 2010; Yeo et al., 2011). Different microfluidic platforms have been developed for on- chip cell culture (Zhang et al., 2009), allowing quantitative read- outs of cell-based assays to study cellular responses to chemical gradients (Hung et al., 2005) or cell/reagent manipulation (Wang et al., 2008) even for incubator-independent conditions (Petronis et al., 2006). Using genetically modified cells that carry reporter genes under the control of stress-sensitive promoters provide functional information about the impact of cytotoxic reagents on cell physiology (El-Ali et al., 2006). The use of reporter genes, includ- ing their individual merits and limitations, has been extensively reviewed (Ghim et al., 2010; Palmer et al., 2011). It has been reported repeatedly that inducible heat shock protein (HSP) genes are up-regulated in mammalian cells by very different forms of cellular stress via binding of heat shock factor to the heat shock element (Morimoto, 1998). This strategy of cell Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.075 n Corresponding author. Tel.: þ49 3641 205621; fax: þ49 3641 205622. E-mail address: feller@fh-jena.de (K.-H. Feller). Biosensors and Bioelectronics 39 (2013) 156–162