The effects of confluency on cell mechanical properties Yu.M. Efremov n , A.A. Dokrunova, D.V. Bagrov, K.S. Kudryashova, O.S. Sokolova, K.V. Shaitan M.V. Lomonosov Moscow State University, Faculty of Biology, Department of Bioengineering, Leninskie Gory, 1/73, 111991 Moscow, Russia article info Article history: Accepted 21 January 2013 Keywords: AFM Vero cells Viscoelasticity Confluency Actin cytoskeleton abstract Mechanical properties of cells depend on various external and internal factors, like substrate stiffness and surface modifications, cell ageing and disease state. Some other currently unknown factors may exist. In this study we used force spectroscopy by AFM, confocal microscopy and flow cytometry to investigate the difference between single non-confluent and confluent (in monolayer) Vero cells. In all cases the stiffness values were fitted by log-normal rather than normal distribution. Log-normal distribution was also found for an amount of cortical actin in cells by flow cytometry. Cells in the monolayer were characterized by a significantly lower (1.4–1.7 times) Young’s modulus and amount of cortical actin than in either of the single non-confluent cells or cells migrating in the experimental wound. Young’s modulus as a function of indentation speed followed a weak power law for all the studied cell states, while the value of the exponent was higher for cells growing in monolayer. These results show that intercellular contacts and cell motile state significantly influence the cell mechanical properties. & 2013 Elsevier Ltd. All rights reserved. 1. Introduction Atomic force microscopy (AFM) is widely used in biological studies for visualization of biomolecules (Engel and Muller, 2000; Graham et al., 2010) and cells (Costa, 2006; Efremov et al., 2011; Rotsch and Radmacher, 2000) and for the assessment of their mechanical characteristics (Kuznetsova et al., 2007; Rico et al., 2008; Takai et al., 2005). Force spectroscopy of cells by AFM, along with other techniques like the optical stretcher (Guck et al., 2005), micropipette aspiration (Ward et al., 1991), magnetic twisting cytometry (Alenghat et al., 2000) and laser tracking microrheology (Hoffman et al., 2006), is an important source of information about cell mechanical properties. The data about cell mechanical proper- ties combined with confocal microscopy data confirm that the cytoskeleton, mainly the actin network, contributes significantly to cell stiffness (Guck et al., 2005; Mackay et al., 2012; Rotsch and Radmacher, 2000). It has been shown, that changes in cell stiffness are closely related to cell type and various external and internal conditions, like substrate stiffness (Franze et al., 2009; Mackay et al., 2012; Solon et al., 2007) and surface modifications (Takai et al., 2005), cell ageing (Starodubtseva, 2011) and disease state (Lekka et al., 2012). However, the elasticity modulus values measured by AFM additionally depend on several factors, including force loading rate, indentation depth, probe geometry, models for force curve approximation, properties of the substrates used for cell growth and others (Lekka et al., 2012; Mahaffy et al., 2000; Rico et al., 2008). Their influence on the elastic properties of cells must be considered for a proper evaluation and for further comparison of data obtained in different experiments. In vivo cells stay in close connection with each other in the tissues and continuously exchange signals with neighboring cells (Lodish et al., 2000). Cells, cultivated on a flat substrate in vitro, are deprived of some of those signals. Single non-confluent cells are deprived more than cells in a monolayer, where signaling information can be shared by neighboring cells with intercellular contacts. These molecular or mechanical signals may lead to different properties of non-motile cells in monolayer (Yeung et al., 2005) and single non-confluent cells. On the other hand, transition from a non-motile to a motile state is accompanied by polarization of cells. Various signaling and structural proteins operating together lead to the reorganization of the actin cytos- keleton (Kole et al., 2005; Ridley et al., 2003). We used the African green monkey Cercopitecus aethiops kidney cell line (Vero) as a model system to determine the effects of confluency on the mechanical properties of cells. Vero cells are widely used in various areas of biology and biotechnology. They are used as substrates for viral vaccine manufacture (Barrett et al., 2009; Ehrlich et al., 2012), for virus detection (Macfarlane and Sommerville, 1969), for detection of verotoxin (Maniar et al., 1990), as substrates Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com Journal of Biomechanics 0021-9290/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbiomech.2013.01.022 n Correspondence to: M.V. Lomonosov Moscow State University, Faculty of Biology, Department of Bioengineering, Leninskie Gory, 1/73, 111991 Moscow, Russia. Tel./fax: þ79459395738. E-mail addresses: yu.efremov@gmail.com, efremov@mail.bio.msu.ru (Yu.M. Efremov), aitsanochka-79@yandex.ru (A.A. Dokrunova), dbagrov@gmail.com (D.V. Bagrov), rekamoskva@mail.ru (K.S. Kudryashova), sokolova184@gmail.com (O.S. Sokolova), shaitan@moldyn.org (K.V. Shaitan). Journal of Biomechanics 46 (2013) 1081–1087