Impedance Spectroscopy for Toxicity Testing Martin Dominguez Cronus Technologies Ltd.

Barrier Forming Tissue Epithelial and endothelial cell layers form selectively permeable barriers

The Concept Growing cell layers on permeable membranes

The Concept Measuring the electrical impedance of epithelial and endothelial cell layers under physiological conditions

Understanding Impedance Spectra Contributions stemming from CPE and R MED

Understanding Impedance Spectra The formation of a plateau at mid-range frequencies is attributable to the cell layer’s resistance TER and capacitance C CL

Understanding Impedance Spectra An increase in the resistance TER shifts the plateau upwards

Understanding Impedance Spectra An increase in the capacitance C CL narrows the plateau

Time-Lapse Impedance Spectroscopy

The Setup

cellZscope Automated cell monitoring device

Monitoring of MDCK-II Cells Formation of a differentiated cell monolayer with established cell-cell junctions

cellZscope vs. „Chopstick“ Electrodes Comparing TER values measured on MDCK-II cell layers

Long Term Monitoring of Caco-2 Cells Comparing growth dynamics of different cell lines

EDTA Treatment of MDCK-II Cells Effects on the tight junction network

MBCD Treatment of MDCK-II Cells Dose dependent response in TER and C cl to cholesterol depletion

Testing the Toxicity of Nanoparticles Exposure of MDCK cells to different types of particles + Triton X100 Control Carbon Black * ZrO 2 * ZnO - NP * + Particles * 10 µg/cm² Time (h) TER (%) Data courtesy of A. Kroll, J. Schnekenburger, University of Münster, Germany

Testing the Toxicity of Nanoparticles Exposure of NRK52E cells to ZnO Control ZnO * ZnO NP * * 10 µg/cm² TER (%) + ZnO+ Triton X100 Time (h) Data courtesy of A. Kroll, J. Schnekenburger, University of Münster, Germany

cellZscope Automated cell monitoring device

Compatibility with Standard Cell Culture Inserts Manufacturer Part No.MembraneInsert 24-Well Size12-Well Size6-Well Size MaterialPore SizePore DensityOpticsType Corning PC0.4 µm1.0×10 8 /cm 2 translucenthanging PET0.4 µm4.0×10 6 /cm 2 transparenthanging PC3.0 µm2.0×10 6 /cm 2 translucenthanging PET3.0 µm2.0×10 6 /cm 2 transparenthanging Greiner Bio-One PET0.4 µm1.0×10 8 /cm 2 translucenthanging PET0.4 µm2.0×10 6 /cm 2 transparenthanging PET1.0 µm2.0×10 6 /cm 2 transparenthanging PET3.0 µm2.0×10 6 /cm 2 translucenthanging PET3.0 µm6.0×10 5 /cm 2 transparenthanging BD Biosciences PET0.4 µm1.6×10 8 /cm 2 translucenthanging PET0.4 µm1.6×10 6 /cm 2 transparenthanging PET1.0 µm1.6×10 6 /cm 2 transparenthanging PET3.0 µm2.0×10 6 /cm 2 translucenthanging PET3.0 µm8.0×10 5 /cm 2 transparenthanging Millipore PIHT 12R 48PIHT 15R 48PIHT 30R 48PET0.4 µm1.0×10 8 /cm 2 translucenthanging PIRP 12R 48PIRP 15R 48PIRP 30R 48PET1.0 µm2.0×10 6 /cm 2 transparenthanging PISP 12R 48PISP 15R 48PISP 30R 48PET3.0 µm2.0×10 6 /cm 2 translucenthanging Nunc PC0.4 µm8.5×10 7 /cm 2 translucentstanding PC3.0 µm1.7×10 6 /cm 2 translucentstanding

Software for Data Acquisition and Analysis

Effects of Hydrocortisone on Endothelial Cells adapted from Wegener et al., BioTechniques 37, 590 (2004) Transepithelial electrical resistance (TER) of primary cultured endothelial cells derived from porcine brain microvessels incubated in serum-free medium supplemented with and without hydrocortisone

Effects of Serum-free Media on Epithelial Cells adapted from Wegener et al., BioTechniques 37, 590 (2004) Capacitance (C cl ) of primary cultured epithelial cells derived from porcine choroid plexus incubated in serum-free and serum-containing medium

Inflammatory Response to TNF-   Transepithelial electrical resistance (TER) of porcine brain capillary endothelial cells

Response to TNF-   Suppressed by Hydrocortisone Transepithelial electrical resistance (TER) of porcine brain capillary endothelial cells