Cizmarova 2017 MiP2017
Dysfunction of mitochondria plays a central role in the pathophysiology of a variety of diseases. Freshly isolated and cryopreserved peripheral blood mononuclear cells (PBMC) are a promising model for the study of mitochondrial respiration as a biomarker for mitochondrial dysfunction. PBMC can be isolated easily from blood samples which provide a low-invasive approach when compared to invasive sampling of muscle biopsies.
We focused on the evaluation of the method for cryopreservation of freshly isolated PBMC and their usage for high-resolution respirometry (HRR). The study involved 8 healthy female volunteers (26.3 ± 5.7 years) on a controlled diet and physical activity restricted to a light 1-h walk per day. 18 mL of whole blood was collected in the morning for 4 days at low altitude (575 m, Innsbruck, Austria). PBMC were separated from whole blood by centrifugation on Ficoll-Paque™ PLUS density medium using 50 mL Leucosep tubes . After the fist washing of the PBMC-platelet layer with DPBS (25 mL, 120 g, RT), the PBMC were washed two times with ice-cold DPBS+2% FBS (50 mL, 300 g, 4 °C). The isolated PBMC fraction was counted on SYSMEX XN-350 haematology analyser. A subsample of freshly isolated cells was used for respiration and the remaining cells were cryopreserved . Freshly isolated or cryopreserved blood cells (3∙106 PBMC) were added into the 2-mL chambers of the O2k-FluoRespirometer (Oroboros Instruments, Innsbruck, Austria) containing mitochondrial respiration medium MiR05-Kit with catalase (Oroboros, AT) at 37 °C. The coupling-control and cell viability protocol (CCVP) was used . Respiration of PBMC was corrected for the contribution by contaminating platelets.
The viability of PBMC determined by the CCVP averaged 93.9% for fresh and 79.6% for cryopreserved cells, corresponding well with the viability determined by the Luna™ automated cell counter (96.2% and 85.4% for fresh and cryopreserved cells). The flux control ratios of freshly isolated and cryopreserved cells did not differ. The individual reproducibility of respiration of freshly isolated PBMC was similar to that determined in cryopreserved PBMC.
In conclusion, respiration of cryopreserved PBMC reflects well the respiration of freshly isolated PBMC. Therefore, cryopreserved PBMC can be used as a model for functional diagnostic tests. Further studies including standardization of methods for PBMC isolation, counting and cryopreservation are necessary for quality control and data comparison between laboratories.
Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Blood cells Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET
Affiliations and support
- Cizmarova B(1,2), Garcia-Souza LF(1,3), Volani C(4), Menz V(3), Burtscher M(3), Gatterer H(3), Lundby C(5), Karabatsiakis A(6), Sumbalova Z(1,7), Gnaiger E(1,8)
- Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
- Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik Univ Košice, Slovakia
- Inst Sport Science, Univ Innsbruck, Austria
- Dept Internal Medicine II, Medical Univ Innsbruck, Austria
- Center Physical Activity Research, Univ Hospital Copenhagen, Denmark
- Clinical Biol Psychol, Univ Ulm, Germany
- Pharmacobiochemical Lab, 3rd Dept Internal Medicine, Fac Medicine, Comenius Univ, Bratislava, Slovakia
- Oroboros Instruments, Innsbruck, Austria. – [email protected]
- Supported by Action Austria-Slovakia (BV) and K-Regio project K-Regio MitoFit (CL, EG, HG, LFGS, MB, VM, ZS). Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.
- Sumbalova Z, Hiller E, Chang S, Garcia L, Droescher S, Calabria E, Volani C, Krumschnabel G, Gnaiger E (2016) Isolation of blood cells for HRR. Mitochondr Physiol Network 21.17(02):1-15. »Bioblast link»
- Cizmarova B, Garcia-Souza LF, Sumbalova Z, Karabatsiakis A, Gnaiger E (2017) Optimization of cryopreservation of human peripheral blood mononuclear cells and platelets for high-resolution respirometry. »Bioblast link»
- Garcia-Souza LF, Velika B, Sumbalova Z, Menz V, Burtscher M, Gnaiger E (2017) Assessment of mitochondrial respiratory function in cryopreserved platelets. MiPschool Obergurgl 2017 »Bioblast link»