Expanding the Scope of Impedance Spectroscopy for the Analysis of Adherent Cells: Electrode Material, Electrode Design, and Data Analysis

This thesis focused on the advancement of impedance-based cellular assays using the electric cell-substrate impedance sensing (ECIS) approach. Three linchpins of ECIS were addressed: electrode material, electrode design, and data analysis. Typically, ECIS measurements are performed by application of ac currents with low amplitude between coplanar gold-film electrodes with adherent cell monolayers growing on top. Different chapters of this work discussed the replacement of the electrode material gold by a conducting polymer, the use of a single bipolar electrode instead of two separate electrodes, and the data analysis by means of the derivatives of impedance spectra. The first part of this work dealt with the fabrication and characterization of screen printed polymer electrode arrays. The arrays, called PDT, were based on the conducting polymer PEDOT:PSS as electrode material, silver leads to reduce the lead resistance, and a silicone passivation layer to delineate the electrode dimensions. The electrodes showed high adhesion stability in aqueous environment at 37 °C, electrical stability in the analytically relevant voltage range between 10 mV and 300 mV, and long-term stability when stored in ambient air in the dark. In comparison with gold electrodes, PEDOT:PSS exhibited a greatly enhanced interface capacitance due to its hydrogel properties. This improved the sensitivity of the impedance magnitude |Z| and the real component of the impedance R at lower frequencies for cell analysis, which is relevant for the analysis of the barrier function and the cell-substrate interaction of adherent cell monolayers. Cell proliferation and adhesion assays are commonly conducted by monitoring the capacitance at 32 kHz or 40 kHz. In this frequency range, the sensitivity of the polymer electrodes surpassed the gold electrodes. A disadvantage of PEDOT:PSS was its high resistivity and thus the high lead resistance, reducing the sensitivities for the parameters |Z| and R at higher frequencies. Alterations in the cell-substrate contact as well as the adhesion and spreading kinetics compared to commercial gold electrodes were discussed with respect to the lower substrate stiffness of PEDOT:PSS compared to gold. Several common analytical assays investigating the micromotion, proliferation, cytotoxicity, and electroporation performance of the PEDOT:PSS electrodes were successfully conducted on both electrode materials, yielding overall similar results. Nevertheless, several differences were established. Due to the high interface capacitance, the PEDOT:PSS electrodes showed an extremely low noise level during micromotion experiments compared to the gold electrodes. This resulted in accordingly high signal-to-noise ratio and sensitivity. The proliferation assay provided more data scattering on PEDOT:PSS electrodes, which could be either attributed to the smaller electrode size or an inhomogeneous distribution of the substrate stiffness across the electrode surface. Due to a reduced generation of cytotoxic species at the electrode surface, the electroporation at lower frequencies (1.5 kHz) was found to be less invasive on PDT electrodes. Different cell lines with individual cell parameters were shown to retain their specific characteristics concerning barrier function, cell-substrate contact, and membrane capacitance on PEDOT:PSS. A remaining challenge is the electrode drift that occurred over the course of several days during the measurement. The drift was less pronounced in the impedance magnitude and appeared more prominently in the capacitance. In the second part of this work, a novel bipolar electrode for impedimetric analysis and manipulation of living cells was developed. The bipolar electrode consisted of a high-resistance conduction path showing a potential gradient along its length. The potential at the respective position on the electrode surface depended on its distance from the contact pads. Thus, upon application of elevated electric fields of 5 V for 60 s, the bipolar electrodes caused a gradient of increasing electric field strength along the conduction path. This property of the bipolar electrodes was explained by an increasing lead resistance with growing distance from the contact pads. The system is suggested to be suitable for the optimization of wounding and electroporation experiments as the position on the electrode indicates the optimal voltage amplitude required for the respective application. In a second approach, bipolar electrodes were fabricated comprising a passivation layer with a small working and a large counter electrode like in the ECIS layout. For this layout, a mathematical model based on the ECIS model was developed. Using this model, it was shown that the low frequency part of the impedance magnitude spectrum was determined by the inherent resistance of the bipolar electrode. Compared to ECIS electrodes, the bipolar electrodes showed a sensitivity shift from the real part to the imaginary part of the complex impedance. The third part of this work aimed to improve the accuracy of data analysis for impedance spectroscopy by calculating the derivatives of the spectra. Therefore, a set of methods was developed to analyze the applicability of derivative impedance spectroscopy (DIS) for the analysis of impedance-based cellular assays. Artificial raw data with known electrode and cell parameters were created by overlaying simulated impedance spectra with computer-generated noise. The derivative orders zero to three of these spectra were fitted using different settings of the fitting conditions and weighting methods. For a quantitative evaluation, the parameter Qrel was introduced, which is a direct measure for the quality of a fit. For certain fitting conditions the first derivative displayed some improvement compared to the zero-order spectrum. However, the overall tendency was a strong decline of the fit quality with increasing derivative order. This was found to be due to insufficient reduction of the noise, which is amplified after each differentiation. In a second approach, DIS was used to discriminate between spectral changes caused by alterations of either one of the cell parameters, α or Rb. Fixed frequencies were found, where simulations showed more distinct differences in the first or second derivative than in the zero-order spectrum. In particular, the first and second derivative exhibited opposing tendencies for the two cell parameters at 400 Hz and 1 kHz, respectively. Moreover, the migration of the zero-points of the second derivative when varying the cell parameters might become useful in a similar fashion. The high-frequency zero-point (~ 10 kHz) was shown to migrate only upon alteration of Rb and was unaffected by α. The results presented in this chapter are only valid for the investigated system and are not applicable to other cell lines

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion