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METAbolism analysis made easy for CELL Translational Medicine

Periodic Reporting for period 2 - METACELL-TM (METAbolism analysis made easy for CELL Translational Medicine)

Reporting period: 2017-11-01 to 2019-04-30

Drug failure and high development cost (est. €2.3bn/new drug) have driven enormous growth in market need for scientific tests and disease models that narrow the gap between bench and clinic. This will be achieved by increasing the physiological relevance of the models used in in vitro testing. However, the critical tests necessary for such analysis are either currently unavailable or are beyond the reach of many researchers due to a requirement of expensive dedicated instrumentation. In addition, there is a public demand for a reduction in animal testing and a drive towards better models of in vitro (laboratory or controlled environment) rather than in vivo tests.

The MetaCell-TM project H2020 FTI funding enables the consortium of Agilent Technologies Ireland Ltd (formally Luxcel Biosciences Ltd), BMG LABTECH GmbH and Ncardia (formally Axiogenesis AG and Pluriomics) to develop a physiologically relevant cell-based assay platform for in vitro metabolism analysis in preclinical drug discovery and development, creating the Gold Standard in microplate-based metabolic analysis and providing new insights into our understanding of the interplay between cell metabolism, cell environment and related disease progression. These new insights will be driven by academic collaboration from, Oxford University, UK and Imperial College London, UK, taking tools and technologies developed by SME partners and combining them to answer key research questions in the fields of Cancer Metabolism and Cardiovascular biology
WP1: METACELL-TM ASSAY KIT PRODUCTISATION; Multiple Metabolic Flux Kits, associated data and resources developed. Advanced ischemia reperfusion model assay developed combining novel cellular oxygenation measurement with CLARIOstar ACU (rapid de- & re-oxygenation), as well as metabolically conditioned cardiomyocytes and cardiomyocyte contractility kit.

WP2: METACELL-TM PRODUCT SCALE-UP; Up-scaled manufacture of core fluorescent reagents, assay controls & buffers, metabolic modulators, as well as upscaled bioreactor produced cardiomyocytes.

WP3: HARDWARE, DASHBOARD & ANALYTICS DEVELOPMENT; CLARIOstar ACU (atmospheric control unit) instrument with gas ramping & advanced software control developed for rapid reoxygenation post rapid deoxygenation underpinning the Ischemia / reperfusion model. As well as MetaCell dashboard and data analytics packages.

WP4: DEMONSTRATION; Tech transfer and MetaCell Kit Demonstration at academic partners including CLARIOstar ACU, MitoXpress-FAO, Stress Kits and Hypoxia Kits, Ischemia, reperfusion model and combined metabolism & contractility measurements. Progress and advancement partly driven through partner feedback.

WP5: BUSINESS SOLUTION EXPLOITATION & DISSEMINATION: Commercial launch of MitoXpress® FAO Kit, pH-Xtra® Stress Test Kit & MitoXpress® Stress Test Kit, exploited by Distributors as well as OEM partners (LUX), 2018 commercial release of Contractility kit (Ncardia), 2019 commercial Launch of CLARIOstar ACU (atmospheric control unit) instrument with gas ramping & advanced software control (BMG). As well as associated applications notes, fliers, conference posters, social media posts, workflows, and peer reviewed publications. Successful training workshops for both end-users and Distributors completed.
Some of the technical achievement outlined above are a clear advance beyond the state of the art. These include the capacity developed by the project to model ischemia reperfusion injury on the bench through the development and combination of (a) ACU with Gas ramping including rapid reoxygenation (WP3) and (b) Real-time cellular oxygenation analysis combined with monitoring of mitochondria membrane potential, glycolytic flux and reactive oxygen species production (WP1), all applied to beating hiPS derived cardiomyocytes (WP1). The combination of the oxygen ramping capability of the CLARIOstar ACU with MitoXpress-Intra enabled cellular oxygenation measurements which facilitates precise control of an ischemic insult whereby instrument [O2] can be modulated (21% to 0.1% O2) to provide the desired depth and duration of hypoxia.

The measurements facilitated here are essential to a proper IR characterisation due to the significant impact cell respiration can have on cellular oxygenation, while multiplexing MitoXpress-Intra and JC-1 facilitates parallel monitoring of MMP and cellular oxygenation in the same cells with parallel monitoring of ROS (DHE). This facilitates detailed metabolic characterisation of the short-term metabolic implications of reperfusion, offering a means by which the efficacy of model therapeutic interventions can be investigated, and is a clear advance beyond the state of the art.
A further technical achievement was the development of multiplexed ‘mix-and-measure’ cytotoxicity and mitochondrial function assay using Calcein AM & MitoXpress workflow. This multiplexed Assay while providing flexibility for the researcher, helps contextualize observed metabolic perturbations of long term treatments, thereby facilitating a more complete characterization of compound toxicity without the need to run additional assays.
The combination of these novel assay and instrument technologies allows the in vitro modelling of tumor hypoxia, whereby ambient O2 can be modulated to physiologically relevant conditions, actual cellular oxygenation can be monitored and the related alterations to cellular metabolism assessed. Work conducted within the project has revealed that metabolic poise can cause significant differences in the levels of cellular oxygenation and have illustrated the importance of knowing the actual level of cellular oxygenation when assessing related signalling pathways, including the HIF pathway. Such levels however cannot be inferred from applied O2 and if ignored, lead to a flawed understanding of the relationship between O2 concentration, Hif stabilisation and associated metabolic adaptations.
Furthermore, the development of a substrate preference assay, allows convenient high throughput characterization of cellular metabolic substrate utilization (oxidation) on a plate reader with MitoXpress detection. This can deliver an informative assessment of the contribution of the three key metabolic pathways: glucose, long chain fatty acid, and glutamine metabolism, allowing for the delineation of both those pathways individual and combined contributions to mitochondrial respiration. This characterization is impactful in several fields of research helping to understanding of metabolic phenotypes, including cancer research where metabolic vulnerabilities can be exploited to develop novel therapeutic drugs, as substrate availability and preference has been shown drive proliferation and cell differentiation. It is also a tool to help understand and establish a more physiological relevant conditioned culture media which can be applied to different disease models including cardiovascular cell model.
These advances exemplify the progress of the MetaCell project, offering integrated technology solutions that allow researchers to better reflect the in vivo condition, thereby contributing to a narrowing of the gap between bench and clinic. Advances in this area are expected to have a significant positive impact in the ability of biological research to develop more effective therapeutic intervention. Importantly, the advances are also underpinning new commercial activity across all three SME partners.