Impedimetric Monitoring of Cell‐Based Assays

Abstract

Electrochemical impedance measurements (electric cell‐substrate impedance sensing, ECIS) have been established as a label‐free and noninvasive experimental tool to monitor and quantify the behaviour of adherent cells grown on planar gold‐film electrodes while they are exposed to chemical, biological or physical stimuli/stressors. Owing to the noninvasiveness of the measurement, impedance‐based cell observation provides continuous information about the cell response in real time. Different data acquisition modes in combination with tailored electrode layouts are used to assess the time courses of several fundamental processes in cell physiology such as cell adhesion, cell proliferation, cell migration, intracellular signal transduction or cell death. Experiments are performed entirely automated and computer‐controlled under regular cell culture conditions. State‐of‐the‐art instrumentation allows for the parallel analysis of up to 384 samples. Its multimodality and throughput suggest that ECIS can play a similar role for the analysis of cell‐based assays as flow cytometry.

Key Concepts

  • Electric cell‐substrate impedance sensing (ECIS) is a noninvasive experimental technique to monitor adherent cells in vitro along different types of biomedical or bioanalytical assays with a time resolution that is adjustable from milliseconds to hours.
  • To perform ECIS‐based assays, the cells are grown on planar gold‐film electrodes deposited on the surface of a regular cell culture dish. ECIS is compatible with regular cell culture conditions, fully automated and software controlled.
  • The approach is sensitive to (1) the fractional coverage of the electrode surface and (2) the three‐dimensional shape of the cells attached to the electrode.
  • ECIS is significantly more sensitive for cell shape changes than optical microscopy.
  • On the basis of this generic sensitivity, different assays have been developed to monitor cell adhesion to biomaterial surfaces, cell proliferation, cell migration, cell death or the stimulation of cell‐surface receptors.
  • The scope of available assay formats has been further extended using elevated electric fields for well‐defined cell manipulation integrated into continuous, impedance‐based cell monitoring (e.g. in situ electroporation).

Keywords: electric cell‐substrate impedance sensing; cell‐based assays; holistic cell monitoring; impedance‐based cell monitoring; label‐free; cytotoxicity; wound‐healing assay; in situ electroporation; micromotion

Figure 1. The basic principle of impedance‐based cell monitoring. Adherent cells are grown on planar gold‐film electrodes deposited on the bottom of a cell culture dish. The presence of the dielectric cell bodies increases the electrochemical impedance as the current is forced to flow around or through the cells. Impedance readings are sensitive to any change in cell coverage of the electrode as well as changes in the morphology of the adherent cells.
Figure 2. Time course of the normalised impedance during adhesion and subsequent manipulation of cells on the electrode surface. The initial increase in the impedance after cell inoculation mirrors the attachment and spreading of initially suspended cells upon the electrode surface. Exposing the attached cells to chemical, biological or physical challenges leads to changes in cell morphology accompanied by corresponding changes in the measured impedance.
Figure 3. Collection of established assays to determine fundamental parameters of cell physiology from ECIS readings. Starting at the top in clockwise direction: cell migration/cell proliferation/time‐resolved response profiles for all kinds of stressors or stimuli/cell motility/cytotoxicity/cell adhesion and spreading. This multimodality of the ECIS core technology is based on its generic sensitivity to electrode coverage and cell shape changes.
Figure 4. Sample data for several of the assays sketched in Figure. (a) Time course of cell spreading when initially suspended MDCK cells were seeded on ECIS electrodes with different protein coatings (FN, fibronectin; VN, vitronectin; LAM, laminin; BSA, bovine serum albumin). (b) Time course of cell proliferation when different numbers of NRK cells were inoculated into the electrode containing wells. ‘1x’ corresponds to the number of cells necessary to cover 50% of the available growth surface without any further cell division. The other cell densities used in this experiment are calculated accordingly. (c) Time course of migration and wound healing after those cells within an NRK monolayer, which resided on the electrode surface, were killed by a lethal voltage pulse. Recovery of the impedance mirrors the repopulation of the electrode by cells from the periphery. In presence of different concentrations of the drug daunorubicin, the cells' capability to migrate is seriously affected. (d) Time course of cell death after a confluent monolayer of NRK cells was exposed to increasing concentrations of cadmium chloride. The decrease in impedance mirrors the time course of cell necrosis. (e) Time‐resolved response profile when confluent layers of bovine aortic endothelial cells are treated with 10 μM of the adrenaline derivative isoprenaline (ISO) which stimulates the β‐adrenergic receptor expressed by these cells. (f) Micromotility of two clones of pancreas tumor cells with different metastatic potential. While PaTu‐S is essentially nonmetastatic, PaTu‐T cells are considered as highly metastatic. The impedance fluctuations mirror the cells' inherent motility. (g) Epithelial MDCK cells were treated with the fungal toxin cytochalasin D which is well known to disrupt epithelial and endothelial barrier function. The time course of impedance reflects this barrier disruption. (h) Time‐resolved response profile of confluent NRK cells after introduction of 100 μM Bleomycin (BM) by in situ electroporation (ISE). ISE or 100 μM BM alone do not induce a persistent cell response. Only the combination of BM and ISE causes cell death. This phenomenon is explained by the toxicity and membrane impermeability of BM.
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Further Reading

Banerjee P and Bhunia AK (2009) Mammalian cell‐based biosensors for pathogens and toxins. Trends in Biotechnology 27 (3): 179–188.

Jiang WG (2012) Electric Cell‐Substrate Impedance Sensing and Cancer Metastasis. Series: Cancer Metastasis – Biology and Treatment. Heidelberg: Springer. ISBN. ISBN: 978-94-007-4926-9.

Szulcek R, Bogaard HJ and van Nieuw Amerongen GP (2014) Electric cell‐substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility. Journal of Visualized Experiments 85. DOI: 10.3791/51300.

Wegener J (2009) Impedance analysis of cell junctions. In: Fuchs H (ed) Nanotechnology, vol. 6. Nanoprobes, pp. 325–357. Weinheim: Wiley VCH.

Wegener J and Seebach J (2014) Experimental tools to monitor the dynamics of endothelial barrier function: a survey of in vitro approaches. Cell and Tissue Research 355 (3): 485–514.

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Lukic, Sonja, and Wegener, Joachim(Sep 2015) Impedimetric Monitoring of Cell‐Based Assays. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0025710]