The use of 3D tissue models as in vitro models provides a powerful tool for numerous cell-based assays in biomedical research and drug screening. Based on their characteristic microenvironment, spheroids mimic tumor behavior more effectively than conventional two-dimensional monolayer cultures and are therefore applied in preclinical screening experiments for assessing drug safety while identifying potential cancer therapeutics. A challenging aspect in this context is the development of applicable techniques and measurement setups capable of extracting significant biological information from such models and detecting morphological or biochemical changes upon stimulation. This thesis presents the design of a newly developed impedance-based device for the non-invasive and label-free analysis of spheroids. The measurement setup is based on an existing flow channel device developed by C. Hupf (Hupf, 2018), in which the orientation of the system was shifted from a horizontal to an upright position. As a result, an upright funnel-shaped micro trap with a central opening between two electrodes was produced. For the purpose of setup characterization, various aspects such as specific dimensions of the funnel-based micro trap, the choice of the manufacturing material, or the overall measurement principle were investigated in detail and adjusted to achieve optimal settings. As a result, spheroid measurements could be conducted in a highly sensitive and reproducible manner, detecting the impedimetric responses of up to eight spheroids respective experiments in parallel. When a spheroid is inserted into the device, it is retained at the aperture and seals the opening by adapting to the smaller diameter of the funnel hole through gravitational forces. In this way, EIS analysis of the trapped spheroid is enabled by forcing the electric current to flow through the 3D tissue model, thereby yielding a significant impedance signal increase due to spheroid positioning in a time resolved manner. In addition, the overall impedance contribution of the system and signal changes obtained by intrinsic spheroid changes are addressed. To quantify the dielectric properties of spheroids, a transfer function is used that describes the equivalent circuit model of a spheroid in the EIS device, representing both the extracellular and intracellular resistance combined with the capacitance contribution of the tissue model. The impedance signaling of the newly developed funnel-based measuring device was determined in relation to basic spheroid features. Therefore, the spheroids derived from breast cancer and brain tumor cells were characterized in a comprehensive microscopic study, investigating the spheroid formation procedure, their growth behavior, and the kinetics of their adhesion to a surface. In addition, fundamental model studies on three different aspects of the tissue model conditions, namely spheroid size, spheroid age, and pH, were performed by analyzing their influence on the impedimetric parameters. As a result, the optimal setting for the tissue model conditions in the context of this thesis was obtained while investigating the signal output and result interpretation of the new measurement setup. In addition to the design and characterization of the new setup, the application of the device has to be evaluated. Since the setup has the crucial advantage of allowing stimulation studies during an impedance measurement, the respective compound solution was applied via the upper compartment to induce cellular responses without removing the spheroid from the trap. The cellular response to the varied stimuli mimicking different cell death scenarios was analyzed based on spheroid impedance measurements and complementary time-lapse microscopy, impedance measurements on cell monolayer cultures, and biochemical endpoint assays determining cell viability. This provided a comprehensive overview of the effects of each stimulation from different perspectives, such as cytotoxic specification, impedimetric characterization, and morphological properties, by summarizing the specific measurement setup signals into a single key parameter. In this context, the sensitivity and applicability of the newly developed funnel-based device were evaluated by considering its advantages and limitations of use. In conclusion, the funnel-based impedance measurement device provides a non-invasive, label-free, and time-resolved novel sensing method for monitoring 3D tissue models and their changes during biomedical experiments.
Simone Bäumler (Thu,) studied this question.