Bioprinting-based automated deposition of single cancer cell spheroids into oxygen sensor microelectrode wells.

Three-dimensional (3D) cell agglomerates, such as microtissues, organoids, and spheroids, become increasingly relevant in biomedicine. They can provide in vitro models that recapitulate functions of the original tissue in the body and have applications in cancer research. For example, they are widely used in organ-on-chip systems. Microsensors can provide essential real-time information on cell metabolism as well as the reliability and quality of culture conditions. The combination of sensors and 3D cell cultures, especially single spheroids, is challenging in terms of reproducible formation, manipulation, and access to spheroids, precise positioning near sensors, and high cell-to-volume ratios to obtain meaningful biosignals in the most parallel approach possible. To overcome this challenge, we combined state-of-the-art bioprinting techniques to automatically print tumour spheroids directly into microwells of a chip-based electrochemical oxygen sensor array. We demonstrated highly accurate and reproducible spheroid formation (diameter of approx. 200 µm) and a spheroid deposition precision of 25 µm within a volume of 22 nl per droplet. Microstructures and hydrogel-coated microwells allowed the placement of single MCF-7 breast cancer spheroids close to the sensor electrodes. The microelectrode wells were sealed for oxygen measurements within a 55 nl volume for fast concentration changes. Accurate and stable amperometric oxygen sensor performance was demonstrated from atmospheric to anoxic regions. Cellular respiration rates from single tumour spheroids in the range of 450-850 fmol min-1 were determined, and alterations of cell metabolism upon drug exposure were shown. Our results uniquely combine bioprinting with 3D cell culture monitoring and demonstrate the much-needed effort for facilitation, parallelization, sensor integration, and drug delivery in 3D cell culture and organ-on-chip experiments. The workflow has a high degree of automation and potential for scalability. In order to achieve greater flexibility in the automation of spheroid formation and trapping, we employed a method based on drop-on-demand liquid handling systems, instead of the typical on-chip approach commonly used in microfluidics. Its relevance ranges from fundamental metabolic research over standardization of cell culture experiments and toxicological studies, to personalized medicine, e.g. patient-specific chemotherapy.

Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in 3D cell cultures.

Three-dimensional cell cultures using patient-derived stem cells are essential in vitro models for a more efficient and individualized cancer therapy. Currently, culture conditions and metabolite concentrations, especially hypoxia, are often not accessible continuously and in situ within microphysiological systems. However, understanding and standardizing the cellular microenvironment are the key to successful in vitro models. We developed a microfluidic organ-on-chip platform for matrix-based, heterogeneous 3D cultures with fully integrated electrochemical chemo- and biosensor arrays for the energy metabolites oxygen, lactate, and glucose. Advanced microstructures allow straightforward cell matrix integration with standard laboratory equipment, compartmentalization, and microfluidic access. Single, patient-derived, triple-negative breast cancer stem cells develop into tumour organoids in a heterogeneous spheroid culture on-chip. Our system allows unprecedented control of culture conditions, including hypoxia, and simultaneous verification by integrated sensors. Beyond previous works, our results demonstrate precise and reproducible on-chip multi-analyte metabolite monitoring under dynamic conditions from a matrix-based culture over more than one week. Responses to alterations in culture conditions and cancer drug exposure, such as metabolite consumption and production rates, could be accessed quantitatively and in real-time, in contrast to endpoint analyses. Our approach highlights the importance of continuous, in situ metabolite monitoring in 3D cell cultures regarding the standardization and control of culture conditions, and drug screening in cancer research. Overall, the results underline the potential of microsensors in organ-on-chip systems for successful application, e.g. in personalized medicine.

On-chip photodynamic therapy - monitoring cell metabolism using electrochemical microsensors.

We introduce a new system which combines metabolic monitoring using electrochemical microsensors with photodynamic therapy on-chip for the first time. Oxygen consumption of T-47D breast cancer cells was measured during therapy with protoporphyrin IX. We determined the efficacy of the therapy and revealed its recovery effects, which underlines the high relevance of continuous monitoring.

Accessing 3D microtissue metabolism: Lactate and oxygen monitoring in hepatocyte spheroids.

3D hepatic microtissues, unlike 2D cell cultures, retain many of the in-vivo-like functionalities even after long-term cultivation. Such 3D cultures are increasingly applied to investigate liver damage due to drug exposure in toxicology. However, there is a need for thorough metabolic characterization of these microtissues for mechanistic understanding of effects on culture behaviour. We measured metabolic parameters from single human HepaRG hepatocyte spheroids online and continuously with electrochemical microsensors. A microsensor platform for lactate and oxygen was integrated in a standard 96-well plate. Electrochemical microsensors for lactate and oxygen allow fast, precise and continuous long-term measurement of metabolic parameters directly in the microwell. The demonstrated capability to precisely detect small concentration changes by single spheroids is the key to access their metabolism. Lactate levels in the culture medium starting from 50µM with production rates of 5µMh-1 were monitored and precisely quantified over three days. Parallel long-term oxygen measurements showed no oxygen depletion or hypoxic conditions in the microwell. Increased lactate production by spheroids upon suppression of the aerobic metabolism was observed. The dose-dependent decrease in lactate production caused by the addition of the hepatotoxic drug Bosentan was determined. We showed that in a toxicological application, metabolic monitoring yields quantitative, online information on cell viability, which complements and supports other methods such as microscopy. The demonstrated continuous access to 3D cell culture metabolism within a standard setup improves in vitro toxicology models in replacement strategies of animal experiments. Controlling the microenvironment of such organotypic cultures has impact in tissue engineering, cancer therapy and personalized medicine.

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem.

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 µl min(-1)) and low volumes (15 µl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.