Sensors and Actuators for 2D and 3D Cell Culture Models based on Oxygen Sensitive Culture Substrates (2018)

Eukaryotic cells need oxygen to sustainably produce the metabolite ATP as their main source of energy. Next to glucose, oxygen is one of the key metabolites which can give us insight into cellular processes and conveys important information of parameters like cellular viability, metabolic activity and physiology. Thus, knowledge about cellular oxygen consumption is an essential parameter not only when studying pathophysiological conditions, such as metabolic disease, cancer or stroke, but also in drug and cytotoxicity screening. Methods to assess oxygenation levels and oxygen consumption of cells, spheroids and tissue range from electrochemical detection to the use of magnetic resonance and radioisotope techniques. However, in recent years the detection of oxygen by luminescence quenching of indicator dyes has emerged as a useful technique to study oxygenation of eukaryotic cells. Thus, one of the main projects in this work was to use planar oxygen sensitive culture substrates based on luminescence quenching as a novel means to estimate and image the oxygen consumption in 2D and 3D cell culture models. The substrates allowed the cultivation of adherent mammalian cells directly on the surface which provides the possibility to measure oxygen content directly beneath the cell monolayer. The combination of planar oxygen sensor foils with a suitable camera system enabled non-invasive, online monitoring of the spatial and the temporal oxygen distribution beneath the cell layer. The first project addressed the measurement of the oxygen consumption rate of adherent mammalian monolayer cells. Therefore, an imaging system based on microscopy optics (VisiSens TD mic) was developed in cooperation with PreSens GmbH, Regensburg. The system fits inside a standard cell incubator (48 l) and allows imaging the spatial and temporal distribution of oxygen with high resolution. Planar, polymer based oxygen sensitive foils (SF-RPSu4, PreSens GmbH) for a ratiometric fluorescence readout were used as growth substrates which allowed monitoring the oxygen content directly underneath the adherent cells. The sensor foils exhibited high photostability and biocompatibility which allowed their usage for long term cell experiments. The novel imaging system was used for first proof-of-concept studies to assess oxygen consumption rates in dependence on culture vessel volume and cell seeding density yielding highly reproducible results. Additionally, the novel ratiometric imaging system allowed the successful monitoring of the influence of medium composition, extracellular pH, drugs and toxins on respiratory activity of adherent cells. The high spatial resolution of the camera was used for long term imaging of oxygen gradients caused by a metabolically active spot of monolayer cells. Multiparametric monitoring of cells is a useful approach to obtain multiple information in one measurement. Thus, the second project focused on combining the optical oxygen sensing with impedimetric monitoring to obtain information about cell metabolic activity and cell morphology changes. Therefore, gold film electrodes were sputtered on planar, ratiometric oxygen sensor foils using a laser cut mask. A PDMS chamber was glued on top of the foil that served as cultivation chamber for the adherent mammalian cells. The dual ECIS-O2 sensor allowed simultaneous, non-invasive online recording of impedance and pO2 using a standard impedance analyzer and a commercial oxygen imaging system (VisiSens A1). Impedance changes were monitored for the cell population residing on the linear gold film electrodes, while oxygen levels were measured for those cells on the electrode-free sensor film. The dual sensor was characterized regarding its biocompatibility, signal stability and most sensitive impedimetric monitoring frequency. First studies conducted with the dual chip allowed simultaneous monitoring of the oxygen consumption and the impedance during cell adhesion. Moreover, the ECIS-O2 chip was used to investigate the influence of uncouplers and blockers of oxidative phosphorylation with impedance measurements providing information about changes in cell morphology and electrode coverage, while the ratiometric fluorescence readout reports on the cellular oxygen consumption rate. 3D tissue models such as multicellular tumor spheroids are considered useful in vitro tools in biomedical and clinical research. Due to their 3D structure, they close the gap between simple 2D monolayer cell culture and native tissue structures. Multicellular spheroids mimic physiological conditions found in tissue more closely compared to standard 2D cell culture, while handling and cultivation is easier compared to tissue. This makes multicellular tumor spheroids an attractive means in biomedical research and drug screening. Long-term studies on oxygenation in live spheroids are scarce because most assays are either endpoint-based or invasive such as microneedle or particle based approaches. Thus, the third project applied the novel imaging system VisiSens TD mic for quantitative monitoring of oxygenation levels within live MCF-7 tumor spheroids under standard growth conditions. Originally spherical spheroids were allowed to attach and spread on planar, biocompatible oxygen sensor foils forming half-spherical cell aggregates and oxygen content beneath the spheroid was monitored online with high spatio-temporal resolution. The imaging set-up allowed recording the detailed 2D distribution of oxygen gradients caused by metabolically active spheroids with higher resolution compared to commercially available imaging systems. Further studies were conducted investigating the effect of drugs blocking or uncoupling oxidative phosphorylation on oxygen gradients established in live and metabolically active MCF-7 spheroids. Wound healing assays are often used to study collective cell migration in vitro. The general concept of such assays is to introduce an artificial wound into a confluent layer of adherent cells and to study the migration of non-wounded cells from the periphery into the wound. The aim of the fourth project was the development of a novel wound healing assay based on oxygen sensitive culture substrates. The assay uses planar, polymer-based culture substrates with an embedded oxygen sensor dye as culture substrates for adherent cells. The functional layer was deposited on coverslips by spin-coating providing a transparent composite substrate that is addressable by light microscopy. Upon illumination with a suitable wavelength the dye molecules were excited. The 1O2 produced by luminescence quenching was used to selectively kill cells residing on areas of the functional coating that were exposed to the illumination light generating wounds with a highly reproducible geometry. The oxygen sensitive substrates designed for wound healing were analyzed regarding their biocompatibility and the influence of parameters such as light source, microscope filter settings and medium composition on wounding efficacy was tested. The novel wound healing assay was used to probe collective cell migration under various experimental conditions and it was compared to other wound healing assays such as scratch, barrier and ECIS® assay regarding parameters such as cost, throughput, reproducibility or wound morphology. Similar to the ECIS ® assay, wounds produced by the novel optical wound healing assay mimic the physiological conditions more closely, as dead cells remain as debris in the wound. Additional studies were performed investigating wound healing behavior of epithelial cells exposed to different extracellular pH values which resulted in reduced wound healing rates at low pH. Furthermore, it was shown that serum starvation and cytochalasin C reduced wound closure rate as well.