Applications

Wound Healing Assay

The ECIS Method

The Traditional Assay

Wound healing assays have been carried out in tissue culture for many years to estimate the migration and proliferation rates of different cells and culture conditions. These assays generally involve first growing a confluent cell monolayer. A small area is then disrupted and a group of cell destroyed or displaced by scratching a line through the layer with, for example, a needle. The open gap is then inspected microscopically over time as the cells move in and fill the damaged area. This "healing" can take from several hours to over a day depending on the cell type, conditions, and the extent of the "wounded" region.

ECIS Automated Wound Healing Assay Diagram

NRK Cells Time post Wounding

  • NRK cells time post wounding: 6 hours
    6 hours
  • NRK cells time post wounding: 12 hours
    12 hours
  • NRK cells time post wounding: 23 hours
    23 hours

The New ECIS Assay

Scrape Method: The Traditional Cell Wound Healing Method ECIS has now been modified (Patent pending) such that automated assays of this sort can be performed. In normal ECIS measurements, a current of less than a microampere is normally used. This is undetected by the cells, and, in its measuring mode, ECIS essentially eavesdrops on cell behavior electrically. When the current is boosted 1000 fold to a milliampere, the resulting voltages across the cell membranes result in electroporation. If this is applied for only a few milliseconds, the cells recover and it is possible to insert impermeable molecules including DNA constructs into the cytoplasm. When the high current is applied for several seconds, cell death ensues due to severe electroporation and possible local heating effects.

The ECIS wound is very well defined, as it includes only those cells on the 250 µm diameter electrode. Death can be verified both with the ECIS measurement and with vital staining.

Typical ECIS data involving this assay is shown in the figure. Here BSC1 cells were first grown as complete monolayers and the impedance traces from four confluent wells can be seen on the graph. At the arrow, an elevated field was applied to two of the wells, wounding the cells on the small electrode and causing the impedance to drop to that of an open electrode. Over time these two traces return to control values, as the healthy cells outside of the small electrode migrate inward to repopulate the wounded area and replace their dead cohorts (healing). These types of data are highly reproducible and respond to culture conditions.

ECIS Automated Wound Healing Assay Diagram

MDCK Cells wounded with ECIS Automated Cell Wounding The figure to the right shows an ECIS electrode covered with MDCK cells 24 hours after the elevated field wounding took place. Note the subtle radial patterns indicated by the arrows as the cells have migrated inward. MDCK Cells wounded with ECIS Automated Cell Wounding.

This is a completely automated assays requiring a minimum of labor. Both cell wounding and measurements of the subsequent healing process are carried out under computer control without opening the door of the incubator.

Wounding & Migration: Related ECIS Publications

Saxena, N.K., Sharma, D., Ding, X. Lin, S., Marra, F., Merline, D. Anania, F., "Concomitant Activation of the JAK/STAT, P13K/AKT and ERK Signaling is Involved in Leptin-Mediated Promotion of Invasion and Migration of Hepatocellular Carcinoma Cells." [PDF] Cancer Research: 2497-2507 (2007).

Earley, S., Plopper, G.E., "Disruption of focal adhesion slows transendothelial migration of AU-565 breast cancer cells." Biochemical and Biophysical Research Communications: 405-412 (2006).

Sapper, A., Wegener, J., Janshoff, A., "Cell motility probed by noise analysis of thickness shear mode resonators." Anal Chem. 15;78(14):5184-91, (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).

Waters, C.M., Long, J., Gorshkova, I., Fujiwara, Y., Connell, M., Belmonte, K.E., Tigyi, G., Natarajan, V., Pyne, S., Pyne, N.J., "Cell migration activated by platelet-derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1-phosphate receptor-1." The FASEB Journal Express Article 10.1096/fj.05-4810fje (2005).

Charrier, L., Yan, Y., Driss, A., Laboisse, C.L., Sitaraman, S.V. and Merlin, D.,"ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers." AJP Gastrointest Liver Physiol. 288:346-353, (2005).

Kucharzik, T., Lugering, A., Yan, Y., Driss, A., Charrier, L., Sitaraman, S., Merlin, D., "Activation of epithelial CD98 glycoprotein perpetuates colonic inflammantion", Laboratory Investigation 1-10 (2005)

Charrier, L., Yan, Y., Driss, A., Laboisse, C.L., Sitaraman, S.V., Merlin, D. "ADAM-15 Inhibits Wound Healing in Human Intestinal Epithelial Cell Monolayers." Am J Physiol Gastrointest Liver Physiol. [Epub ahead of print] (2004).

Keese, Charles R., Wegener, Joachim, Walker, Sarah R., and Giaever, Ivar. "Electrical Wound-healing assay for cell in vitro". [PDF] PNAS 101: 1554-1559 (2004). Download pdf file

Hug, T.S. "Biophysical methods for monitoring cell-substrate interactions in drug discovery." Assay Drug Dev Technol. 1(3):479-88. (2003).

Lundien, M.C., Mohammed, K.A., Nasreen, N., Tepper, R.S., Hardwick, J.A., Sanders, K.L., Van Horn, R.D., Antony, V.B. "Induction of MCP-1 expression in airway epithelial cells: role of CCR2 receptor in airway epithelial injury." J Clin Immunol. 22(3):144-52 (2002).

Wegener, J., Keese, C.R., Giaever, I, "Recovery of Adherent Cells After In Situ Electroporation Monitored Electronically" Bio Techniques 33(2), 348ff (2002)

Hadjout, N., Laevsky, G., Knecht, D.A., Lynes ,M.A., "Automated real-time measurement of chemotactic cell motility", Biotechniques, 31 (5): 1130-1138 (2001).

Lo, C.M., Linton, M., Keese, C.R., Giaever, I. "Correlated motion and oscillation of neighboring cells in vitro." Cell Commun Adhes. 8(3):139-45 (2001).

Burns, A.R., R.A. Bowden, S.D. MacDonell, D.C. Walker, T.O. Odebunmi, E.M. Donnachie, S.I. Simon, M.L. Entman, and C.W. Smith. "Analysis of Tight Junctions During Neutrophil Transendothelial Migration", J. of Cell Sci, 113:45-75. (2000)

Huang, C.N., Lo, C.M., Hsu, T.C., Tsay, G.J. "Sera from patients with scleroderma inhibit fibroblast micromotions monitored electrically." J Rheumatol. 26(6):1312-7 (1999).

Noiri, E., Lee, E., Testa, J., Quigley, J., Colflesh, D., Keese, C., Giaever, I. and Goligorsky, M., "Podokinesis in endothelial cell migration: role of nitric oxice", Am. J. Physiol. 43, 236C (1998).

Moy A.B., Van Engelenhoven,J., Bodmer, J., Kamath, J., Keese, C.,Giaever, I., Shasby,S., and Shasby, D.M., "Histamine and Thrombin Modulate Endothelial Focal Adhesion Through Centripetal and Centrifugal Forces",J Clin. Inv. (1996)

Burns, A., Walker, D., Brown, E., Thurmon, L., Bowden, R., Keese, C., Simon, S., Entman, M.and Smith, W., "Neutrophil Transendothelial Migration Is Independent of Tight Junctions and Occurs Preferentially at Tricellular Corners", J. of Immunol. 22, 2893-2903 (1997).st. 97, 1020-1027 (1996).

Lo, C.M., Keese, C.R., Giaever, I. "Monitoring motion of confluent cells in tissue culture." Exp Cell Res. 204(1):102-9 (1993).