Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity. Presently, there is a great demand for in vitro test systems for the elucidation of the mode of action of drug candidates and in particular for the assessment of toxic effects of compounds 1-3. The reasoning for this demand is diverse but several points are crucial in this context: Tests based on animals do not fully reflect all the aspect of human nature, animal tests are expensive and the throughput achievable is rather small 4,5. Finally, ethical concerns have a growing impact on the overall discussion of this subject. This is widely acknowledged and as a result, enormous efforts have been made in recent years to develop methods that do not only give meaningful results but are robust in order to enable a thorough validation. The bulk of recent work has been dedicated to two key components, the cell models 6-9 and the formats of the reactors 10-12 , the cells are implemented in. The former is motivated through the necessity to use cells that mimic essential functional details of the related tissue or organs. The format of the cell reactor must enable three major functions: Firstly, it has to provide an environment that is able to maintain status and the vitality of the cells. Secondly, the fluidic system should ensure the supply of medium, the availability of important soluble molecular factors and the controlled immersion of cells with the compounds of interest. Thirdly, access of powerful characterization methods to the cells is crucial for a detailed analysi...

Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond.On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.

Physiologically relevant in vitro tumor models are crucial in any research setting from drug development to individualized treatment predictions. Patient-derived 3D cell culture models (PD3D®) are validated cancer models which recapitulate the biology of the donor tissue from genotype to phenotype. A multitude of PD3D® replicates can be generated and these are suitable for high-throughput-screening. However, the influence of media conditions on specific drug sensitivity profiles widely remains elusive and thus the predictive value of screenings is obscured. Here we have investigated pathway activities in dependence of different media compositions and mapped out differences in response to targeted therapies and distinct growth media. We assessed the response of PD3D® models derived from colorectal carcinoma (CRC) patients towards two targeted drugs aiming at different levels of the EGFR/Ras/Raf/Mek/Erk axis in defined, serum-free culture media. One media composition contained growth factors in concentration prevalently used in published cell culture protocols, while the other composition is geared to better match physiological levels of relevant supplements. We observed a significant impact of media conditions on sensitivity towards targeted cancer drugs. However, these differences were as individual as the models and the sensitivity against the treatment. To uncover the underlying mechanisms in the observed discrepancy we used DigiWest - a targeted proteomics technology - to assess pathways activity in the different models. We applied a panel of over 250 (phospho-)antibodies against proteins in the MAPK/ERK/RAS, PI3K/AKT and mTOR pathways as well as cell cycle and proliferation. Using functional pathway analysis, we mapped out the difference in response to targeted therapies and distinct growth media. In conclusion, media composition has great impact on the response to targeted drugs in vitro and therefore needs to be harmonized to ensure relevant results. Combination of PD3D® models and DigiWest functional pathway analysis represents a powerful tool to identify the molecular mechanisms underlying differential drug response. Further development of this collaborative approach will lead to a better understanding of the drug resistance or sensitivity and potentially even identification of signatures related to them. Citation Format: Jürgen Loskutov, Gerrit Erdmann, Anja Arndt, Quirin Graf Adelmann, Przemyslaw Dudys, Marie Flechner, Madeleine Nadolny, Katja Osman, Ulrike Pfohl, Christoph Reinhard, Markus Templin, Katja Uhlig, Lena Wedeken, Christian Regenbrecht. Let’s get physiological! Impact of media conditions on drug response to targeted therapies in CRC organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 187.

Colorectal carcinomas (CRC) are one of the most common malignant tumors. Due to its heterogeneity and high probability to metastasize, the disease is often incurable and novel, improved therapies are needed. Enhanced, physiologically relevant in-vitro tumor models significantly increase the success rate in new drug development, reduce the number of animal experiments and result finally in better therapies for cancer patients. Furthermore, they have the potential to offer new approaches to personalized oncology. In recent years, 3D cell cultures were established as relevant preclinical cancer models, suitable for high-throughput-screening. However, their capacity to model and/or measure physiological processes can still be enhanced. In this project, we developed a tumor organoid-on-chip platform (TumOC) by combining most recent technological advances: We integrated patient-derived 3D cell cultures (organoids) from colorectal carcinomas and microsensor particles measuring oxygen concentration and therefore allowing assessment of cell vitality in real time in a microfluidic system. The resulting TumOC platform allows for defined exposition of cytostatic drugs including dynamic treatments. Measurements of cell vitality in real time enables analysis of not only the final effect of a drug treatment, but also the kinetics of drug response. At the same time, utilizing organoids allows for recapitulating tumor architecture and heterogeneity. Here we report the first proof of principle results generated with this system. We measured the cell vitality in real time over 72 hours during treatment with classic chemotherapeutics or targeted cancer drugs and compared the results to end-point measurements on the same organoids in a static system. In conclusion, our TumOC platform with its ability to recapitulate tumor heterogeneity in combination with dynamic treatments and real-time cell vitality assessment is an in-vitro tumor model closely recapitulating the physiological situation in a patient. TumOC provides an impressive opportunity to test and, consequently, predict the effectiveness of anti-cancer therapies. Therefore, this system is of interest not only for pre-clinical drug development but also for personal oncology. Citation Format: Marie Flechner, Juergen Loskutov, Ulrike Pfohl, Katja Osman, Christian R. Regenbrecht, Katja Uhlig, Lena Wedeken. TumOC: A tumor organoid-on-chip platform for real-time cell vitality measurements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6209.

Enhanced, physiologically relevant in-vitro tumor models significantly increase the success rate in new drug development, reduce the number of animal experiments and result finally in better therapies for cancer patients. Furthermore, they have the potential to offer new approaches to personalized oncology. In recent years, 3D cell cultures were established as relevant preclinical cancer models, suitable for high-throughput-screening. However, their capacity to model and/or measure physiological processes can still be enhanced. Colorectal carcinomas (CRC) are one of the most common malignant tumors. Due to its heterogeneity and high probability to metastasize, the disease is often incurable and novel, improved therapies are urgently needed. In this project, we developed a tumor organoid-on-chip platform (TumOC) by combining most recent technological advances: We integrated CRC patient-derived 3D cell cultures (organoids) and microsensor particles that enable assessment of cell vitality in real time by measuring oxygen concentration in a microfluidic system. The resulting TumOC platform allows for defined exposition of cytostatic drugs including dynamic and combination treatments. Measurements of cell vitality in real time enables analysis of not only the final effect of a drug treatment, but also the kinetics of drug response. At the same time, utilizing organoids allows for recapitulating tumor architecture and heterogeneity. Using the TumOC platform, we measured the cell vitality in real time over several days during treatment with classic chemotherapeutics or targeted cancer drugs and compared the results to end-point measurements on the same organoids in a static system. We assessed the effect of intra- and intertumoral heterogeneity and developed a protocol for treatment with clinically used combination regimes. In conclusion, our TumOC platform with its ability to recapitulate tumor heterogeneity in combination with dynamic treatments and real-time cell vitality assessment is an in-vitro tumor model closely recapitulating the physiological situation in a patient. TumOC provides an impressive opportunity to test and, consequently, predict the effectiveness of anti-cancer therapies. Therefore, this system is of interest not only for pre-clinical drug development but also for personalized oncology. Citation Format: Marie Flechner, Juergen Loskutov, Madeleine Nadolny, Ulrike Pfohl, Christian R. Regenbrecht, Katja Uhlig, Lena Wedeken. Personalized identification of cancer treatments in real-time: TumOC - a tumor organoid-on-chip platform for online cell vitality measurements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 190.