MitoFit-Bloodcells

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MitoFit-Bloodcells

The project MitoFit highlights the benefits of mitochondrial fitness.


MitoFit Workpackages


WP5

Human blood cells as study model of mitochondrial respiration and H2O2 production


Abstract

The experimental conditions will be investigated as required for the application of human blood cells as models for evaluation of mitochondrial function and H2O2-production. Optimum conditions will be developed to collect and store blood samples for later measurements and analysis. A standard procedure will be developed for the separation of different blood cell types from a whole blood sample, allowing the individual functional study of thrombocytes, granulocytes, monocytes and lymphocytes. The separation procedure will be optimized towards requiring only a small blood volume so as to minimize the physical strain of study participants, requiring a minimum of time, and towards optimum quality of the cells harvested with as little cross-contamination between different cell types as possible. Technically, this will comprise the successive combination of various separation media for multiple density gradient centrifugation steps, at optimized volumes and centrifugation settings. Next, the most appropriate conditions for the respirometric study of the different cell types will be determined, anticipating that cells with different functions may require different respiration media and/or substrate combinations for the adequate respirometric characterization. Then, the ideal conditions for cryopreserving blood samples will be evaluated in close collaboration with our strategic partner A. Karabatsiakis. Specifically, separated cells will be suspended in freezing media of different composition and additives for stabilization, frozen at -80°C, and kept in a frozen state for a pre-defined period of time. Samples will then be thawed after specified periods and immediately examined with regard to cell vitality and mitochondrial functional characteristics using both intact and permeabilized cells. Finally, building on the then developed set of standard operating procedures (SOPs), a showcase lab-study as well as an exemplary field study will be conducted so as to validate and document the applicability of the methodological approaches. The results of these studies shall ultimately be published along with the exactly documented SOPs.


Towards a mitochondrial biomarker

Assessing fitness and performance in humans
Standardized protocols are available to assess physical fitness by performance testing of athletes and patients, e.g. with spiroergometry for monitoring the success of training or therapeutic interventions (WP 7). In addition, biomarkers in blood samples are commonly monitored for risk evaluation (glucose tolerance test, HbA1c, ß-cell function and insulin resistance, triglycerids, cholesterol – TC, HDL, LDL), inflammatory processes (C-reactive protein, IL-6 and TNF-alpha) and disturbances of oxidative balance (derivatives of reactive oxygen metabolites; biological antioxidant potential test). Available blood tests providing an index of mitochondrial function are limited by over-simplified protocols, low resolution and controversial interpretation (Chacko et al 2014). Respirometric SUIT protocols for OXPHOS analysis (Sjoevall et al 2010, 2013, Pecina et al 2014) need to be extended by combined measurement of hydrogen peroxide production to provide additional information on mitochondrial-specific oxidative stress (De Lucas et al 2014).
Diagnostic evaluation of cryopreserved samples
Karabatsiakis et al (2014) collected blood samples from patients diagnosed for major depression, separated peripheral blood mononuclear cells and froze these cells in a standard culture freezing medium for cryopreservation at -80 °C. Cells were subsequently thawed and examined by HRR. ROUTINE respiration, electron transfer-pathway capacity and coupling efficiency were diminished, in direct correlation with the severety of major depression. With this group as an International Strategic Partner, this approach will be extended for application to life style diagnostics in preventive medicine.


Progress and next steps

» MitoFit_Workshop_Blood_Cells_2016-01-08
» Sumbalova 2016b Abstract MitoFit Science Camp 2016: Human blood cells: isolation and HRR.
» Volani 2016 Abstract MitoFit Science Camp 2016: Effects of iron imbalances on mitochondrial activity in mouse liver homogenate and permabilized rat PBMCs.
» MitoEAGLE Verona 2016


Links and references

» O2k-Publications: Blood cells
» O2k-Publications: Lymphocyte
» O2k-Publications: Platelet
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