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METACELL-TM Report Summary

Project ID: 737978

Periodic Reporting for period 1 - METACELL-TM (METAbolism analysis made easy for CELL Translational Medicine)

Reporting period: 2016-11-01 to 2017-10-31

Summary of the context and overall objectives of the project

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, critical test is 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 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 be academic collaborates from, Oxford University, UK and Imperial College London, UK taking to tools and technologies developed by SME partners and combining them to answer key research questions in the fields of Cancer Metabolism and Cardiovascular biology.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Description of the project work performed and the main results achieved so far
Significant progress has already been made in this regard and includes the following advances:

• MitoXpress FAO Kit, pHXtra Stress Kit & MitoXpress Xtra Stress Kit and associated data and resources developed
• Reagent vial developed to facilitate dual flux measurements (O2 and pH sensitive reagent) and biological proof of concept data generated
• Data generated demonstrating feasibility of substrate preference analysis
• POC data for Hypoxia kit developed demonstrating the impact of oxygenation on glycolytic flux and its use for advanced ischemia reperfusion model
• Metabolically conditioned Cor4U cells developed. Media optimisation continuing

• Up-scaled manufacture of MitoXpress® Intra achieved through increase in production volume and vial drying capacity, yielding batch scale increase from ~80-100 vials to ~800-1000 vials.
• Up-scaled manufacture of pH-Xtra dye achieved in conjunction with subcontract-partner SYNCOM-BV based in Groningen, The Netherlands yielding ~ 2,500 vials per purification
• Processed developed for production and testing of kit buffer tablets, glucose oxidase reagent
• Processes developed for specific metabolic modulators with external source validated as contingency.

• ACU unit with gas ramping developed and launched - this innovation underpins the Ischemia reperfusion model
• Enhancement of existing software packageas exemplified with the progression of the ACU GUI which has seen 3 iterations during its development.
• The development of the imaging platform is ongoing and whilst having changed in specification for our proposed first product iteration is progressing well with high quality optics and images achieved and the basics of software control established.
• MetaCell dashboard and data analytics packages are advancing as planned

• Tech transfer and MetaCell Kit Demonstration including ACU, MitoXpress-FAO, Stress Kits and Hypoxia Kits, Ischemia, reperfusion model and combined metabolism & contractility measurements.
• Metabolic characterisation by 1H-NMR spectroscopy consistent with MitoXpress-FAO kit data output
• End-user feedback facilitating product improvement and refinement
• Early testing of metabolically conditioned hiPS cells
• Data-set illustrating the value of MitoXpress-Intra in the study of tumour metabolism, signalling (incl HIF) and metabolic plasticity.

• Launch of MitoXpress® FAO Kit, pH-Xtra® Stress Test Kit & MitoXpress® Stress Test Kit (LUX)
• Development of MetaCell-TM logo and branding – incorporated by LUX, AXIO, BMG.
• New MetaCell-TM website launched and v2.0 updated
• CLARIOstar® with ACU & Gas Ramping function launched (BMG)

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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 (i) ACU with Gas ramping (WP3) and (ii) Real-time cellular oxygenating 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 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 measurement 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 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. These advances will be further developed into period 2 through the inclusion of metabolically pre-conditioned cardiomyocytes, thereby further increasing the physiological relevance of the model.

The combination of these novel assay and instrument technologies also allows the in vitro modelling of tumor hypoxia whereby ambient oxygen 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 acan 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 oxygen concentration, Hif stabilisation and associated metabolic adaptations. These advances will be further developed into period 2.

These two advances exemplify the progress that the MetaCell project will continue to deliver into Period 2, 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.

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