ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32 1151 2016 © The Japan Society for Analytical Chemistry

Reviews -independent Cell Adhesion Substrates: Possibility of Applications for Mechanobiology Research

Takashi HOSHIBA*,**† and Masaru TANAKA*,***

*Frontier Center for Organic Materials, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992–8510, Japan **International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305–0044, Japan ***Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi, Fukuoka, Fukuoka 819–0395, Japan

Cells can mainly sense mechanical cues from the via . Because mechanical cues can strongly influence cellular functions, understanding the roles of integrins in the sensing of mechanical cues is a key for the achievement of tissue engineering. The analyses to determine the roles of integrins in the sensing of mechanical cues have been performed by many methods based on molecular- and cell-biological techniques, atomic force microscopy, and optical tweezers. Integrin-dependent cell adhesion substrates have been also used for this purpose. Additionally, the cells can adhere on several substrates via integrin-independent mechanisms. There are two types of integrin-independent cell adhesion substrates; 1) the substrates immobilized with ligands against the receptors on cell surface and 2) the substrates suppressing protein adsorption. Cells can exhibit specific functions on these substrates. Here, the examples of integrin- independent cell adhesion substrates were reviewed, and their possible applications in mechanobiology research are discussed.

Keywords Integrin, cell adhesion, extracellular matrix, receptor, protein adsorption, signal transduction

(Received June 30, 2016; Accepted September 1, 2016; Published November 10, 2016)

1 Introduction 1152 substrate surface 2 The Substrates that Promote 3·4 A comparison of the two types of Integrin-dependent Adhesion 1153 integrin-independent cell-adhesive substrates 3 The Substrates that Allow 3·5 Perspectives on the application of Integrin-independent Cell Adhesion 1153 integrin-independent cell adhesion substrates 3·1 The importance of the substrates used for for mechanobiology research integrin-independent cell adhesion 4 Conclusions 1157 3·2 The substrate-immobilization of ligands against 5 Acknowledgements 1157 non-integrin receptors on the cell membrane 6 References 1157 3·3 Suppression of protein adsorption onto the

Takashi HOSHIBA received his Ph.D. Masaru TANAKA is a professor at Kyushu degree in 2007 from Tokyo Institute of University. He worked for TERUMO Co. Technology. He worked at National and designed novel biocompatible Institute for Materials Sciences (NIMS) polymers and commercialized (World as a PostDoc fellow (2007 – 2009) and as shear No. 1). In 2000 he moved to a Research Fellow of the Japan Society Hokkaido University and became a JST for Promotion of Science (2009 – 2011). PRESTO researcher, and then moved to He has been in Yamagata University as a Tohoku University. Medical devices Research Assistant Profesor (2011 – 2014) covered with the 3D films are and also been as a Research Associate commercially available in the world Professor (2014 –). Also, he has been as a clinical market. In 2009, he has full Guest Researcher in NIMS (2013 –). His professorship at Yamagata University. He current research interests are extracellular became a leader of Funding Program for matrix and . Next Generation World-Leading Researchers (2011 – 2014). He has received the Awards including SPSJ Asahi Kasei Award for the intermediate † To whom correspondence should be addressed. water concept based on the role of interfacial water at the materials E-mail: [email protected] interphases. 1152 ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32

To understand the mechanisms of cell function regulation by 1 Introduction biochemical cues, the mechanisms by which integrins and intracellular signaling proteins at the integrin-ECM adhesion The extracellular microenvironment, particularly the sites transduce the biochemical cues provided by the ECM into extracellular matrix (ECM), is one of the most important points the cells have been actively examined.13,23–25 There are several of study in the fields of tissue engineering and regenerative methods for modulating integrin-dependent cell adhesion that medicine because cells can sense information contained in ECM have been used to determine these mechanisms. Molecular- and and modulate their functions in cell proliferation, migration, cell biology-based methods have been frequently used for this morphogenesis and differentiation.1,2 There have been many purpose (Table 1). For example, integrin-dependent cell efforts to regulate cell function by biochemical cues in the past adhesion can be promoted by the induction of integrin over- few decades. ECM proteins and cell-adhesive peptides (e.g., the expression. On the other hand, integrin-dependent cell adhesion Arg-Gly-Asp peptide) have been frequently immobilized to cell can be inhibited by the knock-out or knock-down of integrins culture substrates to provide biochemical cues to the cells.3–6 In and by the use of function-neutralizing antibodies against addition to biochemical cues, the ECM can provide mechanical integrins.6,26–28 In addition to these molecular- and cell biology- cues to the cells to regulate cell function.7–9 Durotaxis is one of based methods, integrin-specific ligands immobilized to the the first phenomena to be reported as a cell function regulated substrates have also been frequently used to promote integrin- by mechanical cues from the ECM.7 Durotaxis is a mode of cell dependent cell adhesion. These substrates have been applied for migration regulated by the elasticity of the ECM; that is, most the analyses of the roles of integrins in the transduction of normal cells prefer to migrate onto stiffer ECM. More biochemical cues provided by the ECM (Table 1).13,23,29 There importantly, the differentiation of stem cells is strongly are also several substrates that allow integrin-independent cell influenced by the stiffness of the ECM.8 Therefore, mechanical adhesion. These approaches that use substrates have also been cues have been a focus of studies in tissue engineering and used for the analyses of the roles of integrins in the transduction regenerative medicine. of biochemical cues provided by the ECM (Table 1).30,31 It has been reported that cell adhesion sites to ECM are highly In contrast to the analyses of the roles of integrins in the important for sensing the biochemical cues provided by the transduction of biochemical cues, the methods are limited for ECM. Cells mainly adhere to the ECM via receptor proteins on the analyses of the roles of integrins in the transduction of the cell membrane called integrins. Integrins are heterodimers mechanical cues provided by the ECM. For this purpose, the composed of α and β transmembrane subunits that have analyses have been performed using molecular- and the cell extracellular domains that can bind to many ECM proteins.10 biology-based methods (e.g. genetic mutation technique and The binding of integrins to ECM proteins leads to the clustering Förster resonance energy transfer (FRET)-based molecular of integrins on the cell membrane. This clustering leads to the tension sensors)32,33 and mechanical cell manipulation methods accumulation of many intracellular proteins (e.g., focal adhesion (e.g. cell manipulations with atomic force microscope and kinase (FAK), talin, paxillin, vinculin and p130Cas) and actin optical tweezer techniques).34,35 In addition to these approaches, fibers close to the intracellular domains of integrins for the the substrates were developed to measure traction forces exerted transduction of biochemical cues provided by the ECM.10–12 by the cells. Microbeads embedded elastic substrates have been There are many types of α and β subunit associations which can used to measure the force exerted by the cells to the substrates, activate different intracellular signaling pathways and induce which is called traction force microscopy.36 Additionally, the different cell functions.10,13 substrates possessing micropillars on the surface have been also In addition to biochemical cues, mechanical cues from ECM used to measure the force exerted by the cells.37 These substrates are also sensed by integrins and intracellular signaling proteins allow the cells to interact and adhere through integrin-dependent at integrin-ECM adhesion sites although mechanical cues from mechanism. The substrates that allow integrin-independent cell extracellular milieu can be sensed by many receptors such as adhesion have been hardly used. Using substrates that allow cadherin and ion channels.14–17 The cells adhere to the substrates integrin-independent cell adhesion might pave the way toward and exert traction forces. When the cells adhere to substrates developing a new method for determining how integrins with different stiffnesses, they exert traction forces with different transduce mechanical cues from the ECM. In this review, we strengths.18 The exertion of traction forces with different summarize examples of the substrates that allow integrin- strengths leads to changes in the conformation, accumulation independent cell adhesion after a brief discussion of the and phosphorylation levels of intracellular signaling proteins at substrates that promote integrin-dependent cell adhesion. The integrin-ECM adhesion sites in response to the mechanical cues possible applications of using substrates that allow integrin- provided by the ECM.19–22 Integrins seem to play central roles independent cell adhesion in mechanobiology research are also in the transduction of the mechanical cues. Therefore, integrins discussed. The conceptual figure of integrin-independent cell should be studied for understanding how the mechanical cues adhesion substrates is shown in Fig. 1. provided by ECM are transduced into the cells.

Table 1 Methods for the modulation of integrin-dependent cell adhesion Promotive modulation Inhibitory modulation

Molecular- and cell biology-based Over expression of integrins Knock-down or -out mutations methods Function-neutralizing antibodies Modulation methods with substrates ECM protein immobilization Immobilization of ligands specific for cell membrane receptors Cell-adhesive peptide immobilization Suppression of protein adsorption onto the substrate surface Anti-integrin antibody immobilization ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32 1153

Fig. 1 The conceptual figure of integrin-independent cell adhesion substrates.

Table 2 The substrate-immobilized molecules that promote integrin-dependent cell adhesion Immobilized Example Advantage Disadvantage molecules ECM proteins Fibronectin Complete mimicking of integrin-ECM Limited specificity to integrin subfamily Laminin protein interaction Cell adhesive RGD Easy and stable chemical immobilization Limited specificity to integrin subfamily peptides GFOGER onto the substrates Antibodies against Anti-α5 integrin antibody Specific interactions with the integrin Non-physiological interactions with integrins integrins Anti-β1 integrin antibody subfamily

with specific integrin heterodimers. For example, integrin 2 The Substrates that Promote Integrin-dependent heterodimers such as α3β1, α5β1, α8β1, αvβ1, αvβ3 can bind Adhesion to the RGD peptide, which is the most widely used cell-adhesive peptide.41 Therefore, as with substrate-immobilized ECM The immobilization of integrin-specific ligands to substrates is proteins, it is difficult to analyze how specific integrin usually used to promote integrin-dependent cell adhesion. There heterodimers transduce the signals provided by the ECM using are mainly three types of the ligands used for immobilization: 1) substrate-immobilized cell-adhesive peptides. ECM proteins (e.g., fibronectin and collagen), 2) cell-adhesive Antibodies against integrins have also been used to promote peptides derived from ECM proteins and 3) antibodies against integrin-dependent cell adhesion.13 In contrast to ECM proteins integrins. Each method that uses a particular ligand has both and cell-adhesive peptides, these antibodies can bind specific advantages and disadvantages (Table 2). The appropriate integrin subunits or heterodimers and can activate downstream method should be selected depending on the purpose of each intracellular signals. Therefore, the substrate-immobilized study. antibodies against integrins have been used for the analyses of The immobilization of ECM proteins can completely mimic the roles of specific integrin subfamilies and heterodimers in the the physiological interaction of the ECM with integrins. transduction of signals from the ECM.13 However, the However, it is difficult to determine interactions with specific interaction between integrins and these antibodies is not integrin heterodimers because ECM proteins have binding physiological, although the antibodies can activate intracellular abilities to multiple types of integrin heterodimers.10 For signals by inducing integrin clustering. In the case of signal example, fibronectin can bind to the integrin heterodimers α4β1, transduction of mechanical cues from the ECM, conformational α5β1 and αvβ3.32 Several laminins can bind to the integrin changes in proteins are occasionally important.10,43 When heterodimers α3β1, α6β1 and α6β4.38 Additionally, laminin integrins bind to the ECM proteins, integrin conformation can bind to non-integrin cell adhesion receptors, such as dynamically changes and these changes activate downstream syndecans and dystroglycan.39,40 Therefore, it is difficult to intracellular signals.10 Therefore, using substrate-immobilized analyze how specific integrin heterodimers transduce the signals antibodies against integrins might possibly omit the signals provided by ECM using substrate-immobilized ECM proteins. activated by conformational changes. Cell-adhesive peptides derived from ECM proteins have also been immobilized to promote integrin-dependent cell adhesion. There are several cell adhesive peptides, which include Arg- 3 The Substrates that Allow Integrin-independent Gly-Arg (RGD) and Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER; Cell Adhesion Hyp and O indicate hydroxyproline).6,41,42 These peptides can somewhat mimic the physiological interaction between integrins 3·1 The importance of the substrates used for integrin- and ECM proteins. It is also easy to chemically immobilize independent cell adhesion these peptides to the substrates in a stable manner. Therefore, To analyze the roles of integrins in the transduction of signals these peptides have been widely used. In contrast to these from the ECM, the inhibition of integrin-dependent cell adhesion advantages, it has been difficult to determine the interactions has been widely performed. Specific integrin-knockdown or 1154 ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32

Fig. 2 Two approaches for preparing the substrates that allow integrin-independent cell adhesion.

Table 3 A partial list of substrate-immobilized ligands against non-integrin receptors on the cell membrane Immobilized molecule Cell adhesion mechanism Cellular function Reference E-Cadherin E-Cadherin Maintenance of ES cell stemness 45, 46 Maintenance of primary hepatocyte functions 47 LIF LIF receptor Maintenance of ES cell stemness 48 Activation of the ERK signal EGF EGF receptors Spheroid formation of primary hepatocytes 49 Activation of the ERK signal 50, 51 HGF c-Met Activation of the ERK and Akt signals 52 N-Cadherin N-Cadherin Maintenance of neural stem cell stemness 53 Insulin-like growth factor Wnt receptor (the complex of Frizzled 8 Cardiac differentiation of ES cells 54 binding protein 4 and LRP6) GlcNAc Vimentin localized to on the cell membrane Maintenance of hepatic stellate cells in a quiescent state 55 Galactose Asialoglycoprotein receptor Maintenance of primary hepatocyte functions 56, 57

integrin-knockout cells have been used. The phenotypes and against non-integrin receptors on the cell membrane, and the intracellular signal activities of these cells have been observed. cells exhibit distinct behaviors depending on these substrates These studies showed that integrins play pivotal roles in the (Table 3 and Fig. 2A). There are two types of molecules transduction of biochemical cues from the ECM.26–28 In addition immobilized on substrates: 1) bioactive molecules that can to integrin knockdown or integrin-knockout cells, cells treated activate intracellular signals directly and 2) non-bioactive with function-neutralizing antibodies and cell-adhesive peptides molecules that cannot activate intracellular signals directly. (e.g., RGD peptide) that competitively inhibit the interaction Mouse embryonic stem (ES) cells adhered on an E-cadherin- between integrins and their ligands have also been used. These immobilized substrate in an E-cadherin-dependent but integrin- treatments suppress integrin-dependent cell adhesion and the independent mechanism. On this substrate, the ES cells were transduction of biochemical cues.6,44 However, the above- scattered, and they maintained their stemness.45,46 In contrast, mentioned methods inhibit cell adhesion to the substrates and mouse ES cells formed colonies on a gelatin-immobilized most of the cells are kept in suspension. Because mechanical substrate, which allowed the cells to adhere via integrin.45,46 cues from the ECM are sensed during interactions with the Mouse ES cells also maintained their stemness on substrates ECM, the cells should be adhered to the substrate during the immobilized with leukemia inhibitory factor (LIF), which analysis. Therefore, substrates that allow integrin-independent allowed the cells to adhere via the LIF receptor.48 Primary cell adhesion will be helpful for the understanding of the roles hepatocytes maintained their specific functions on substrates of integrins in the transduction of the mechanical cues from the immobilized with epidermal growth factor (EGF).49 In addition ECM. to cell functions changing, the activities of intracellular signals To prepare the substrates that allow integrin-independent cell were also changed depending on the type of substrate- adhesion, there are mainly two approaches: 1) the substrate- immobilized bioactive molecules. Substrate-immobilized with immobilization of ligands against the non-integrin receptors on EGF or LIF can activate extracellular signal-regulated kinase the cell membrane and 2) the suppression of protein adsorption (ERK) for longer periods than soluble forms of EGF or LIF.48,50,51 onto the substrate surfaces (Fig. 2). We summarize examples of ERK and Akt were also continuously activated in HepG2 cells substrates designed according to the above-mentioned concepts that adhered on substrates immobilized with hepatocyte growth in the sections below. factor (HGF) via c-Met, a receptor against HGF.52 In addition to the substrate-immobilization of bioactive 3·2 The substrate-immobilization of ligands against non- molecules, it is also possible to immobilize non-bioactive integrin receptors on the cell membrane molecules on substrates to promote integrin-independent cell The use of substrate-immobilized ligands against non-integrin adhesion. For example, primary hepatocytes adhered on receptors on the cell membrane to allow integrin-independent substrates immobilized with galactose via the endocytosis cell adhesion has been well-studied in research. receptor protein, asialoglycoprotein receptor (ASGP-R), which There are many examples of substrate-immobilized ligands clears old proteins from the blood.56–58 On substrates ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32 1155

Fig. 3 A comparison of survival mechanisms between primary hepatocytes adhered on either substrates immobilized with ECM proteins or PVLA-coated substrates.

immobilized with galactose, primary hepatocytes can survive interaction and other non-specific interactions (i.e., integrin- and maintain liver specific functions such as albumin independent cell adhesion).63–65 Although cells can adhere on secretion.56,57 Additionally, primary hepatocytes formed substrates via non-specific interactions, serum-derived and spheroids on substrates immobilized with galactose in the cell-secreted proteins can be adsorbed onto substrate surfaces. presence of EGF and HGF.57,59 Adsorbed proteins can mask substrate surfaces to inhibit Similar to substrates immobilized with galactose, Ise et al. non-specific interactions with substrates and induce the cells prepared substrates immobilized with N-acetyl glucose to interact via integrins (Fig. 4A).66,67 Therefore, it seems that by coating poly[N-p-vinylbenzyl-O-2-acetoamid-2-deoxy-β-D- the suppression of protein adsorption can allow the cells to glucopyranosyl-(1-4)-2-acetoamide-2-deoxy-β-D-gluconamide] adhere on the substrates via integrin-independent mechanisms (PVGlcNAc) on the substrate surface.55,60 PVGlcNAc-coated (Fig. 4B). substrates allowed mesenchymal cells to adhere via intermediate There are several polymers that can suppress protein filament proteins such as desmin and vimentin that appeared on adsorption onto the surfaces. Polyethylene glycol (PEG), the cell membrane surface.60 In particular, Hepatic stellate cells poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2- can adhere on PVGlcNAc-coated substrates via these methacryloyloxyethyl phosphorylcholine) (MPC) have been intermediate filament proteins. The expression of α-smooth frequently used to suppress protein adsorption.68–70 In particular, actin, a marker of activated hepatic stellate cells, was suppressed PHEMA-coated substrates are sometimes used in cell culture to on PVGlcNAc-coated substrates suggesting that hepatic stellate suppress interactions with integrins.69,71 Primary hepatocytes cells can maintain a quiescent state.55 and a hepatocarcinoma cell line, HepG2, exhibited round shapes When substrates immobilized with non-bioactive molecules when cultured on PHEMA-coated substrates.69,71 Additionally, were used, intracellular signals were not activated directly by primary hepatocytes maintained their specific functions when the immobilized molecules, though many cell functions were adhering on PHEMA-coated substrates.69 However, the low altered. Cell survival was especially believed to be maintained numbers of adherent cells cultured on these polymer-coated by the activation of survival signals followed by the binding of substrates made the analyses difficult.68,70,71 integrin to the ECM proteins.61,62 However, cells can survive on Recently, we reported that substrates coated with poly(2- the substrates adhered on via integrin-independent mechanisms.63 methoxyethyl acrylate) (PMEA) and its analogous polymers, Hoshiba et al. investigated the survival mechanisms of primary which suppress protein adsorption, allowed the cells to hepatocytes adhering to the substrates coated with poly-[N-p- adhere.71–74 On PMEA-analogous polymer-coated substrates, vinylbenzyl-O-4-β-D-galactopyranosyl-D-gluconamide] (PVLA) the cells can adhere even in the presence of for the immobilization of galactose on the surface.30 Two ethylenediaminetetraacetic acid (EDTA), an inhibitor of integrin- important survival signal molecules, ERK and Akt, were not dependent cell adhesion. The few focal adhesions formed in activated in primary hepatocytes adhering on these PVLA- cells cultured on PMEA-analogous polymer-coated substrates coated substrates, but both ERK and Akt were activated in the also indicate that the cells adhered via integrin-independent cells adhering on collagen- and fibronectin-coated substrates. mechanisms.72,73 The cells exhibited different functions on However, the expression level of fas, which induces apoptosis PMEA-analogous polymers-coated substrates compared with upon cell detachment, were similarly decreased on PVLA-, conventional culture substrates (e.g., tissue culture polystyrene). collagen- and fibronectin-coated substrates (Fig. 3).30 In particular, HepG2 cells were able to adhere on a PMEA- coated substrate via integrin-independent mechanisms and form 3·3 Suppression of protein adsorption onto the substrate a round shape that increased hepatocyte-specific functions such surface as albumin expression. Actin monomers were not assembled. Cells are capable of adhering on substrates via electrostatic Moreover, Yes-associated protein (YAP) was located in a cytosol 1156 ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32

Fig. 4 Integrin-independent cell adhesion using the substrates suppressing protein adsorption.

Table 4 A comparison of substrate preparation approaches that allow integrin-independent cell adhesion Advantage Disadvantage

Immobilization of ligands against non-integrin Determined interaction Activation of intracellular signals by immobilized ligands receptors on the cell membrane mechanisms Possible loss of availability of immobilized ligands Suppression of protein adsorption Low activation of Low cell adhesion (PEG, PHEMA and MPC) intracellular signals Unclear adhesion mechanisms

in HepG2 cells adhering on a PMEA-coated substrate to increase analogous polymers), the amount of adsorbed protein could hepatocyte-specific functions.71 determine the level of cell adhesion contributed by integrins (Fig. 4B). 3·4 A comparison of the two types of integrin-independent For both types of substrates that allow integrin-independent cell-adhesive substrates cell adhesion, the alteration of cell adhesion mechanisms should As mentioned above, there are two approaches to prepare the be considered. Even if the substrates that can suppress protein substrates that allow integrin-independent cell adhesion. Each adsorption are used, the cells can spontaneously recruit ECM approach has several advantages over the other (Table 4). The proteins that can be deposited at the interface between the cell interactions between the cells and the substrates with membranes and the substrate surfaces after a long time in immobilized specific ligands have been clearly determined. In culture. Deposited ECM proteins can bind to integrins and contrast, the interactions between the cells and the substrates convert the cell adhesion mechanism from an integrin- that suppress protein adsorption are not clear because the cells independent to an integrin-dependent mechanism.66,78,79 If the might be adhering on these substrates via non-specific cells are adhering on the substrates immobilized with specific interactions.72 Many reports have tried to determine the adhesion ligands, then a similar alteration in the adhesion mechanism can mechanisms that occur via non-specific interactions.75,76 The also occur.66,80 Therefore, the effects of deposited ECM proteins proposed mechanisms have not been accepted broadly, so far. should be considered to interpret the obtained results after the Therefore, analyses of the signals transduced by the substrates long-term culture although the integrin dependency of cell might be easier when using the substrates immobilized with adhesion is strictly regulated in the initial period of the culture. specific ligands than when using the substrates that suppress protein adsorption. However, there are many possible ways to 3·5 Perspectives on the application of integrin-independent activate intracellular signals using the substrates immobilized cell adhesion substrates for mechanobiology research with specific ligands and with bioactive molecules, in particular. In mechanobiology research, gels composed of ECM proteins Therefore, the particular intracellular signals activated by the (e.g., collagen) and the Engelbreth–Holm–Swarm (EHS) gel immobilized ligands should be always considered to elucidate have often been used.81,82 Additionally, polyacrylamide gels the mechanisms of signal transduction from the substrates. immobilized with integrin ligands have been frequently used.81 Moreover, it is sometimes difficult to immobilize specific These gels allow the cells to adhere via integrin-dependent ligands to the substrates. In particular, the availability of mechanisms only. To investigate the roles of integrins in the bioactive molecules can be easily lost due to conformational transduction of mechanical cues from these elastic substrates, changes that might be induced during the immobilization several methods have been used (e.g. genetic mutation and process. optical tweezer-based cell manipulations).6,2–28,32–37 Although To investigate the roles of integrins in the transduction of these approaches are very effective for analyzing the roles of signals from the substrates, the level of cell adhesion contributed integrins, unexpected side effects from these treatments can by integrins should be controlled. To control the level of cell arise. For example, genetic mutations might modulate adhesion contributed by integrins, different approaches can be intracellular signals, which are independent from mechanical taken depending on which of the two types of substrates will be stimulation. Optical tweezer-based cell manipulation can used. For the substrates immobilized with specific ligands, basically load the mechanical cues at a limited area. To avoid some integrin-specific ligands could be co-immobilized with unexpected side effects, using substrates that allow integrin- non-integrin-specific ligands on the substrate surface to control independent cell adhesion can provide another analytical method the level of cell adhesion contributed by integrins.77 For the to further examine the roles of integrin in signal transduction substrates that suppress protein adsorption (e.g. PMEA activated by mechanical cues from the other viewpoints. With ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32 1157 integrin-independent cell adhesion substrates, it might also be Biotechnol. Prog., 2013, 29, 1331. possible to control the level of cell adhesion contributed by 5. U. Hersel, C. Dahmen, and H. 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