Shear stress-dependent cell detachment from temperature-responsive cell culture surfaces in a microfluidic device Zhonglan Tang, Yoshikatsu Akiyama, Kazuyoshi Itoga, Jun Kobayashi, Masayuki Yamato, Teruo Okano * Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan article info Article history: Received 16 May 2012 Accepted 26 June 2012 Available online 20 July 2012 Keywords: Poly(N-isopropylacrylamide) Temperature-responsive cell culture surface Microfluidic system Peeling model abstract A new approach to quantitatively estimate the interaction between cells and material has been proposed by using a microfluidic system, which was made of poly(dimethylsiloxane) (PDMS) chip bonding on a temperature-responsive cell culture surface consisted of poly(N-isopropylacrylamide) (PIPAAm) grafted tissue culture polystyrene (TCPS) (PIPAAm-TCPS) having five parallel test channels for cell culture. This construction allows concurrently generating five different shear forces to apply to cells in individual microchannels having various resistance of each channel and simultaneously gives an identical cell incubation condition to all test channels. NIH/3T3 mouse fibroblast cells (MFCs) and bovine aortic endothelial cells (BAECs) were well adhered and spread on all channels of PIPAAm-TCPS at 37  C. In our previous study, reducing culture temperature below the lower critical solution temperature (LCST) of PIPAAm (32  C), cells detach themselves from hydrated PIPAAm grafted surfaces spontaneously. In this study, cell detachment process from hydrated PIPAAm-TCPS was promoted by shear forces applied to cells in microchannels. Shear stress-dependent cell detachment process from PIPAAm-TCPS was evalu- ated at various shear stresses. Either MFCs or BAECs in the microchannel with the strongest shear stress were found to be detached from the substrate more quickly than those in other microchannels. A cell transformation rate constant C t and an intrinsic cell detachment rate constant k 0 were obtained through studying the effect of shear stress on cell detachment with a peeling model. The proposed device and quantitative analysis could be used to assess the possible interaction between cells and PIPAAm layer with a potential application to design a cell sheet culture surface for tissue engineering. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction An intelligent surface, which can control cell adhesion and deadhesion by changing temperature, has been proposed by our laboratory [1e3]. Poly(N-isopropylacrylamide) (PIPAAm), a well- known temperature-responsive polymer [4], is introduced on the surfaces of tissue culture polystyrene (TCPS) dishes (PIPAAm-TCPS) by electron-beam irradiating polymerization. At 37  C where PIPAAm surface is hydrophobic, cells can adhere and spread on the surface, and grow to confluence. By decreasing temperature to be 20  C, because the surface turn to hydrophilic, cells detach them- selves from the surface spontaneously and form an intact cell sheet [5,6]. It is a new approach for tissue engineering, and many kinds of cell sheets are harvested successfully and used in several clinical applications such as corneal reconstruction [7,8], cardiac tissue reconstruction [9,10], and periodontal regeneration [11,12]. Furthermore, the surface performance is improved by introducing hydrophilic or hydrophobic component to achieve a rapid cell detachment or a pattern surface for cell co-culture [13e15]. In our previous studies, the surface properties of PIPAAm-TCPS are eval- uated by contact angle measurement, total reflection Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), cell attach- ment and detachment assay, and fluorescent labeling [6,13e17]. These techniques focused on either the surface properties or a cell attachment and detachment behavior. In this study, cellematerial interactions were quantitatively evaluated at first time. Various kinds of techniques have been developed to evaluate cellematerial interactions by measuring the force or energy of detachment of either many or a single cell in previous studies. One strategy was to generate a shear force, usually utilizing a fluid flowing through cell-adhered substrates. The generated shear force was caused to suppress the adhesion of cells, and the percentage of cell detached from the substrate was monitored. A rotational flow, which includes the rotational flow between a cone and a plate, or between parallel disks, was developed for studying a bulk cell * Corresponding author. Tel.: þ81 3 5367 9945x6201; fax: þ81 3 3359 6046. E-mail address: tokano@abmes.twmu.ac.jp (T. Okano). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.06.077 Biomaterials 33 (2012) 7405e7411