Cell-Based Assays in High-Throughput Screening for Drug Discovery

Drug screening is a long and costly process confronted with low productivity and challenges in using animals, which limit the discovery of new drugs.  To improve drug screening efficacy and minimize animal testing, recent efforts have been dedicated to developing cell-based high throughput screening (HTS) platforms that can provide more relevant in vivo biological information than biochemical assays and thus reduce the number of animal tests and accelerate the drug discovery process. Today, cell-based assays are used in more than half of all high-throughput drug screenings for target validation and ADMET (absorption, distribution, metabolism, elimination and toxicity) in the early stage of drug discovery. In this review, we discuss the uses of different types of cells and cell culture systems, including 2D, 3D and perfusion cell cultures, in cell-based HTS for drug discovery. Optical and electrochemical methods for online, non-invasive detection and quantification of cells or cellular activities are discussed. Recent progresses and applications of 3D cultures and microfluidic systems for cell-based HTS are also discussed, followed with several successful examples of using cell-based HTS in commercial development of new drugs. Finally, a brief discussion on potential applications of cell-based HTS for screening phytochemicals and herbal medicines is provided in this review

Development of a novel gene screening platform for cells exposed to flow

Flow is a key regulator of endothelial function and therefore cardiovascular disease development. Nonetheless, the application of flow to cultured cells is a non-trivial challenge. Results obtained from in vitro flow systems must be critically reviewed as several technical limitations exist within each system. Recent developments of molecular high throughput techniques are constantly changing the scientific demands of life science laboratories. In order to meet this demand, there should also be the possibility to increase the throughput for flow studies. However, at present there is no high throughput device commercially available to apply flow to cells in vitro. Therefore there is a strong need for a novel high throughput technology that can effectively monitor the impact of shear stress on cells. Endothelial cells (EC) form the inner lining of the arterial wall, thus acting as a barrier between the vessel wall and flowing blood. EC are subjected to wall shear stress (WSS), a force created by the flowing blood. Although Atherosclerosis is associated with systematic risk factors like Diabetes or smoking, disease characteristic plaques occur predominantly near branches and bends, where EC are exposed to low or bidirectional WSS; thus illustrating the physiological importance of WSS. This project aims to develop a high throughput in vitro system for functional screening of gene regulation in EC exposed to flow. The development of an in vitro device for application of flow to cells in 96 well plates will allow screening to identify genes involved in cellular responses to flow. It is envisaged that the system will be used in future studies to identify novel putative therapeutic targets for the prevention or treatment of atherosclerosis. Therefore, the proposed high throughput device is designed to be compatible with standard cell culture methods. Briefly, flow will be generated in a standard 96 well cell culture plate using 96 individual stirring cones mounted in a re-usable lid. Each cone will be rotated by a magnetic stirrer, placed underneath the 96 well plate. Due to the device’s compatibility with plate readers it will be an ideal addition to existing flow studies to perform fluorescent in situ staining

Label-free and Multi-parametric Monitoring of Cell-based Assays with Substrate-embedded Sensors

Various approaches have been pursued on the basis of electrochemical or piezoelectric transducers, particularly of the quartz crystal microbalance (QCM), to monitor non-invasively and in real-time cellular states and reactions with substrate-embedded sensors. On the one hand, these comprised the technical development of piezoelectric sensors with multiple read-out spots and the integration of additional non-invasive (electro- and optochemical) sensor technologies on the QCM surface. On the other hand, a variety of studies and cell-based assays (CBAs) have been performed in order to test the sensor performances and to gain a deeper understanding of the sensors’ readout parameters with respect to their information content about the biophysical properties and the metabolic behavior of cells. Fig. 7–1 presents an overview of the different projects on the basis of QCM sensor disks presented in this thesis. In the first project (Fig. 7–1 A) a novel electrode layout was designed on the basis of commercial 5 MHz AT-cut quartz disks to implement two independent readout spots on the QCM surface. This also comprised the construction of new measurement chambers for the electrical actuation and sensing of quartz oscillations. These two-electrode QCM sensors (2ElQ) are also referred to as multichannel QCM (MQCM). The developed MQCM sensor spots on one quartz disk exhibited a strong interference, even though they were operated sequentially, which is in contrast to the results of previous theoretical calculations. The resonances could be successfully decoupled by coating half of the quartz surface and one sensor spot, respectively, with a thin and rigid film of photoresist. This quartz loading with mass caused a shift in the resonance spectra of the coated resonator to lower frequencies and efficient decoupling. The operation of the decoupled MQCM sensors demonstrated both, a sensitive and equal change in the oscillation characteristics of the two resonators upon loading of the quartz with medium. The Q-factor was not significantly different for the two resonators, qualifying the MQCM for its application in CBAs. Building on the preceding development of the double-electrode quartz disks, a novel electrode layout has been realized at the sensor surface, which enables the complementary electrochemical (impedance spectroscopical) characterization of the substrate-liquid interface in addition to its mechanical characterization by the piezoelectric transducers (Fig. 7–1 B). This layout was achieved by removing a small area of the insulating photoresist on the coated electrode in the photolithographic process. By this, a coplanar electrode arrangement of a small working electrode and a bigger counter electrode was created. This sensor combination on the basis of the MQCM is an improvement of the so-called 2nd generation QCM to what we call the 3rd generation QCM, and which is also referred to as QCM-ECIS. Various electrode layouts, varying in size and number of the working electrode(s), were fundamentally characterized microscopically and by profilometry regarding the geometrical properties and by means of impedance spectroscopy with respect to the sensing performances in QCM- and ECIS-mode. An optimal electrode layout was identified and defined as standard for subsequent applications in CBAs. In both QCM- and ECIS studies of cell-covered sensor surfaces significant changes in the characteristic sensing parameters with respect to the cell-free electrodes are measurable. In addition to the measurement of absolute signal changes, the transducer technologies of QCM and ECIS also enable to monitor the kinetic changes of the readout parameters with high temporal resolution. This allows to use the dual sensors for monitoring and analyzing the states of adherent cell cultures in any kind of assay, label-free, non-invasively, and in real-time. Mechanical (QCM-mode) and the dielectric (ECIS-mode) characteristics of cell adhesion were simultaneously measured for two different cell lines (MDCK II and NRK), with high reproducibility for each. The total and kinetic parameter changes in both sensing modes distinguished clearly and were specific for the cell lines under test. The signals from both QCM-mode and ECIS-mode recordings also reported on significant impacts of the presence/ absence of bivalent cations (Ca2+, Mg2+) on the attachment and spreading kinetics and behavior of MDCK II cells. Aside from cell adhesion studies, the cytomechanical and cell morphological reactions towards various stimuli were monitored and analyzed by QCM-ECIS in a multitude of cellular assays: systematic softening and stiffening of cells (using agents for disassembling the actin cytoskeleton and cross-linking protein structures, respectively), intracellular stimulation (using a second messenger analogue), as well as electrical manipulation (electroporation (ELPO) and wounding) of cell layers (applying invasive voltage pulses). The applicability of electrical actuation and the subsequent non-invasive, time-resolved, and dual sensing with the electrodes of the QCM-ECIS substrates has been successfully demonstrated. The monitoring of CBAs with the dual QCM-ECIS sensor chips developed in this thesis provides not only a multiplication of the information gain due to the complementarity of QCM and ECIS readout parameters. The simultaneous, time-resolved measurements also enable the kinetic correlation of the sensor signals in novel 2 D and 3 D diagrams, which offers the hitherto unprecedented opportunity for a more detailed view and analysis of the coherence or consecutiveness of mechanical and morphological/ dielectric changes of a cell layer under study. A third research project focused on the combination of optical-chemical sensors (OCS) with the piezoelectric (QCM) sensor technology. For this purpose, the quartz crystal surface was coated with a polymer film with embedded phosphorescent indicator dye for the target analyte. The luminescence properties were measured by means of fluorescence (phosphorescence) lifetime imaging (FLIM). By using a temperature-sensitive paint (TSP), an increase in temperature on the sensor surface upon high-amplitude oscillations was monitored and imaged this way in one project (Fig. 7–1 C). Based on this experimentally determined local heating on the QCM surface and the thereby generated temperature gradient in the liquid above the resonator, a thermophoretic convection in the fluid has been simulated. Theoretical considerations showed that the convection profile in the measurement vessel counteracts and even largely prevents the sedimentation of cells onto the sensor surface. It is suggested that the effect of thermophoresis is crucial especially in studies of biomolecular interactions on QCM surfaces at elevated shear amplitudes and driving voltages, respectively, which however has not been considered in literature to date. The phosphorescence quenching capability of oxygen was utilized in a second imaging project to monitor and image the local concentration and distribution of oxygen on the growth substrate of cells by means of a so-called pressure(/oxygen)-sensitive paint (PSP) (Fig. 7–1 D). A home-made experimental setup was constructed for sensor calibration and the imaging of subcellular oxygen, consisting of a FLIM setup coupled to an upright microscope and a temperature- and oxygen-controlled calibration and measurement chamber suitable for cellular applications. The cytocompatible sensor films have been characterized under various test conditions (in air, under medium, at different temperatures) regarding their sensitivity and response characteristics to different oxygen partial pressures. The oxygen consumption of cells adherently grown on the sensor film was successfully monitored and imaged by this setup. The time-resolved measurements demonstrated a significantly faster consumption of oxygen of a cell layer stimulated with a respiration chain decoupler compared to an unstimulated control cell layer. Taken together, various technical improvements of piezoelectric sensors (QCM) have been realized (MQCM, QCM-ECIS, ELPO-QCM-ECIS, QCM-OCS), which provide a significant information gain in cell-based applications. The sensors developed enable the high-content screening (HCS) of adherent cell lines in a wide range of assay formats and provide complementary physico-chemical information for obtaining a more complete picture of the state of cells and their reactions in contact to diverse stimuli. All sensor techniques share the characteristics of time-resolved, label-free, and non-invasive monitoring. This allows to disclose and analyze even the kinetics, delayed effects, recoveries, and fluctuations of physicochemical alterations of a studied cell layer, in addition to the absolute parameter changes, which is a valuable improvement compared to classical endpoint assays. The approach of combined, independent sensor systems also provides the novel possibility to bring parameters obtained by the different readout technologies from one cell layer in a temporal correlation, by which new insights into physiological relationships are possible