Cell Attachment and Spreading

The ECIS Method
(ECIS Application Note - Cell-ECM Interactions)

One of the most direct ECIS measurements is that of the attachment and spreading behaviors of cells. These measurements allow one to study and quantify the interaction of cultured cells with extracellular matrix (ECM) proteins and other macromolecules continuously and in real time.

Traditionally, cell attachment and spreading measurements are labor intensive, requiring many manipulations of the cultures for microscopic evaluation of cell behavior. With ECIS, these same measurements can be made in an automated approach without opening the door of the incubator.

Different cell line behavior

The graph shows the attachment and spreading behaviors of two different cell types -namely BSC1 (African Green Monkey Kidney) and NRK (Normal Rat Kidney) cells. Cells were inoculated in ECIS wells at time zero in sufficient number to form a confluent cell layer (100,000 cells per cm). The more rapid spreading dynamics of the BSC1 cells is clearly evident; however upon reaching confluence, these two lines have very similar final impedance values. Each cell line studies in this manner will have its own characteristic shape and final values. In these data, the electrodes were not treated in any special manner before the inoculation but are coated with proteins adsorbed from the fetal bovine serum in the culture medium.

Spreading on defined protein coats

The gold film making up the ECIS electrodes is hydrophilic and can readily be coated with defined adsorbed protein layers for cell studies. Here we see data of WI-38/VA13 cells attaching and spreading on electrodes pre-coated with different proteins. These protein layers were established by simply placing a droplet of protein solution (100 micrograms per ml in 0.15M NaCl) on the active electrode, allowing 10 minutes for adsorption, and then rinsing the wells before the addition of the cell suspension. Since only the 250 µm diameter is observed in these measurement, exceedingly small amounts of solution (only a few microliters) are needed for the protein coats saving valuable protein solutions.

Measurements made at 40,000 Hz

The cell attachment data shown thus far was gathered using AC currents at 4000Hz. The impedance values obtained at this intermediate ECIS frequency depend not only on the fraction of the electrode area covered with the spreading cells but also on the height of the spaces between the electrode and the basal cell membrane. When the capacitive portion of the impedance is studied at 40,000Hz, the measurements essentially report only the fraction of the electrode covered with cells and so mimic the data obtained with normal microscopy. This is shown here with the attachment of MDCK cells to electrodes coated with different protein layers as indicated (J. Wegener, et al, Exp. Cell Res. 259, 2000).


In another example, the attachment and spreading behavior of MDCK cells on fibronectin pre-coated electrodes is attenuated with the addition of RGDS tetrapeptides to the culture medium.



Cell Attachment and Spreading
Related ECIS Publications

Characterisation of cell adhesion in airway epithelial cell types using electric cell–substrate impedance sensing. I. H. Heijink, S. M. Brandenburg, J. A. Noordhoek, D. S. Postma, D-J. Slebos, and A. J. M. van Oosterhout. Eur. Respir. J. 2010; 35:894-903. 

Focal-adhesion targeting links caveolin-1 to a Rac1-degradation pathway. Micha Nethe, Eloise C. Anthony, Mar Fernandez-Borja, Rob Dee, Dirk Geerts, Paul J. Hensbergen, André M. Deelder, Gudula Schmidt, and Peter L. Hordijk. J. Cell Sci. 2010; 123:1948-1958. 

Vladislava O. Melnikova, Krishnakumar Balasubramanian, Gabriel J. Villares, Andrey S. Dobroff, Maya Zigler, Hua Wang, Frederik Petersson, Janet E. Price, Alan Schroit, Victor G. Prieto, Mien-Chie Hung, and Menashe Bar-Eli. Crosstalk between Protease-activated Receptor 1 and Platelet-activating Factor Receptor Regulates Melanoma Cell Adhesion Molecule (MCAM/MUC18) Expression and Melanoma Metastasis. J. Biol. Chem. 2009; 284:28845-28855.

Charrier, L., Yan, Y., Nguyen, H.T.T., Dalmasso, G., Laboisse, C., Gewirtz, A.T., Sitaraman, S.V., Merlin, D., “Adam-15/Metargidin Mediates Homotypic Aggregation of Human T Lymphocytes and Heterotypic Interactions of T Lymphocytes With Intestinal Epithelial Cells.” [PDF] J. Biol. Chem. (2007).

Liu, H.B., Plopper, G., Earley, S., Chen, Y., Ferguson, B., Zhang, X.C., “Sensing minute changes in biological cell monolayers with THz differential time-domain spectroscopy., Biosens Bioelectron,” (2006).

Ten Klooster, J. P., Jeffer, Z.M., Chernoff, J., Hordijk, P.L. “Targeting and activation of Rac1 are mediated by the exchange factor B-Pix.” J. Cell Biology. 172 (5): 759-769 (2006).

Sapper, A., Reiss, B., Janshoff, A., Wegener, J.”Adsorption and fluctuations of giant liposomes studied by electrochemical impedance measurements.” [PDF] Langmuir. 22(2):676-80 (2006).

Ren, J., Xiao, Y., Singh, L.S., Zhao, X., Zhao, Z., Feng, L., Rose, T.M., Prestwich, G.D., Xu, Y.,”Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells.” [PDF] Cancer Res 2006; 66: (6) (2006).

Barker, T.H., Baneyx, G., Cardo-Vila, M., Workman, G.A., Weaver, M., Menon, P.M., Dedhar, S., Rempel, S.A., Arap, W., Pasqualini, R., Vogel, V., Sage, E.H. “SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity,” .J Biol Chem 28;280(43):36483-93, (2005).

Castaneda, F.E., Walia ,B., Vijay-Kumar, M., Patel, N.R., Roser, S., Kolachala, V.L., Rojas, M., Wang, L., Oprea, G., Garg, P., Gewirtz, A.T., Roman, J., Merlin, D., Sitaraman, S.V. “Targeted deletion of metalloproteinase 9 attenuates experimental colitis in mice: central role of epithelial-derived MMP.” Gastroenterology.129(6):1991-2008 (2005).

Adel Driss, Laetitia Charrier, Yutao Yan, Vivienne Nduati, Shanthi Sitaraman, and Didier Merlin “Dystroglycan Receptor is Involved in Integrin Activation in Intestinal Eptithelia.” Am J Physiol Gastrointest Liver Physiol, 0: 3782005 (2005).

Newbold, C., Richardson, R., Huang, C.Q., Milojevic, D., Cowan, R., Shepherd, R., “Electrical and optical characterization of thrombin-induced permeability of cultured endothelial cell monolayers on semiconductor electrode arrays.” J Neural Eng. 1(4):218-27, (2004).

Meyer, M.C., McHowat, J., “The role of platelet-activating factor in the adherence of circulating cells to the endothelium.” [PDF] Recent Research Developments in Physiology (2); 129-147 (2004).

Slaughter, G., Bieberich, E., Wnek, G., Wynne, K., Guiseppi-Elie, A.* “Improving Neuron-to-Electrode Surface Attachment via Alkanethiol Self-Assembly: An Alternating Current Impedance Study,” Langmuir, 20 (17), 7189 -7200, (2004).

De Blasio, B.F., Laane, M., Walmann, T., Giaever, I. “Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell cultures.” Biotechniques. 36(4):650-4, 656, 658 passim (2004).

Yanase, M., Ikeda, H., Matsui, A., Noiri, E., Tomiya, T., Arai, M., Inoue, Y., Tejima, K., Nagashima, K., Nishikawa, T., Kimura, S., Fujiwara, K., Rojkind, M., Ogata, I., “HMG-COA reductase inhibitor modulates collagen GEL-contraction by hepatic myofibroblast-like stellate cell line: involvement of geranylgeranylated proteins.” Comparative Hepatology 3 Suppl 1 (2004).

John H. T. Luong, Caide Xiao, Bernard Lachance, Mircea Leabu, Xiaolan Li, Shashi Uniyal and Bosco M. C. Chan. “Extended applications of electric cell-substrate impedance sensing for assessment of the structure-function of alpha 2 beta 1 integrin.” Analytica Chimica Acta, 501:61-69 (2004).

Xiao, C., Luong, J.H. “On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS).” Biotechnol Prog. 19(3):1000-5 (2003).

Schmidt, M., Chen, B., Randazzo, L., Bogler, O., “SETA/CIN85/Ruk and its binding partner ALP1 associated with diverse cytoskeletal elements, including FAKs, and modulated cell adhesion,” Journal of Cell Science 166, 2845-2855 (2003).

Rotundo, R.F., Curtis, T.M., Shah, M.D., Gao, B., Mastrangelo, A., LaFlamme, S.E., Saba, T.M., “TNF-disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin.” Am J Physiol Lung Cell Mol Physiol 282: L316-L329, (2002).

Negash S., Wang H.S., Gao C., Ledee D., Zelenka P., “Cdk5 regulates cell-matrix and cell-cell adhesion in lens epithelial cells.” J Cell Sci. 115(Pt 10):2109-17 (2002).

Sawhney RS, Zhou GH, Humphrey LE, Ghosh P, Kreisberg JI, Brattain MG. “Differences in sensitivity of biological functions mediated by epidermal growth factor receptor activation with respect to endogenous and exogenous ligands.” J Biol Chem. Jan 4;277(1):75-86 (2002).

Xiao, C., Lachance, B., Sunahara, G., Luong, J.H.T., “An in-depth analysis of electric cell-substrate impedance sensing to study the attachment and spreading mammalian cells,” Analytical Chemistry, 74 (6): 1333-1339 (2002).

Negash, S., Wang, H.S., Gao, C., Ledee, D., Zelenka, P., “Cdk5 regulates cell-matrix and cell-cell adhesion in lens epithelial cells,” Journal of Cell Science, 115 (10): 2109-2117 (2002).

Charboneau, A.L., Singh, V., Yu, T.X., Newsham, I.F., “Suppression of growth and increased cellular attachment after expression of DAL-1 in MCF-7 breast cancer cells,” International Journal of Cancer, 100 (2): 181-188 (2002).

Sawhney, R.S., Zhou, G.K., Humphrey, L.E., Ghosh, P., Kreisberg, J.I., Brattain, M.G. “Differences in Sensitivity of Biological Functions Mediated by Epidermal Growth Factor Receptor Activation with Respect to Endogenous and Exogenous Ligands.” , J. Biol. Chem. 277, 75-85 (2002).

Luong, J.H., Habibi-Rezaei, M., Meghrous, J., Xiao, C., Male, K.B., Kamen, A. “Monitoring motility, spreading, and mortality of adherent insect cells using an impedance sensor.” Anal Chem. 73(8):1844-8 (2001).

Sharma, K.V., Koenigsberger, C., Brimijoin, S., Bigbee, J.W., “Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth,” Journal of Neuroscience Research, 63 (2): 165-175 (2001).

Luong, J.H.T., Habibi-Rezaei, M., Meghrous, J., Xiao, C., Male, K.B., Kamen, A., “Monitoring motility, spreading, and mortality of adherent insect cells using an impedance sensor,” Analytical Chemistry, 73 (8): 1844-1848 (2001).

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Wegener, Joachim. Keese, R., Charles, Giaever, Ivar, “Electric Cell-Substrate Impedence Sensing (ECIS) as a Noninvasive Means to Monitor the Kenetics of Cell Spreading to Artificial Surfaces,” Experimental Cell Research 259, 158-166 (2000).

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