Cizmarova 2017 MiP2017
 The cryopreserved PBMC as a model in functional mitochondrial diagnosis. |
Link: MiP2017
Velika B, Garcia-Souza LF, Volani C, Menz V, Burtscher M, Gatterer H, Lundby C, Karabatsiakis A, Sumbalova Z, Gnaiger E (2017)
Event: MiP2017
The mitochondria play a crucial role in cellular bioenergetics. The 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 promising model for the study of mitochondrial respiration as a biomarker for mitochondrial function. Measurement of mitochondrial function in blood cells may provide a low-invasive test when compared to invasive sampling of muscle biopsies.
In our work, we focused on the development of the method for cryopreservation of freshly isolated PBMCs and their usage for High-Resolution FluoRespirometry (HRFR). The study involved 8 healthy female volunteers (26.3 ± 5.7 years old) on controlled diet and physical activity restricted to a light 1h walk per day. Samples of 18 ml whole blood were collected every morning for 4 days in Innsbruck, Austria. PBMCs were separated from whole blood by centrifugation on Ficoll-Paque™ PLUS density medium using 50 ml Leucosep tubes [1]. After the first washing of PBMC-platelet layers with DPBS (25 mL, 120 g, RT), the PBMCs were washed two times with ice-cold DPBS+2% FBS (50 mL, 300 g, 4°C). The isolated PBMC fraction was counted with a SYSMEX XN-350 haematology analyser. A subsample of freshly isolated cells was used for respiration and the remaining cells were cryopreserved [2]. Freshly isolated or cryopreserved blood cells (3 mill of PBMCs) were added into the 2-mL chambers of the O2k-FluoRespirometer (Oroboros Instruments, Innsbruck, Austria) containing mitochondrial respiration medium MiR06-Kit (Oroboros, AT) at 37°C. For HRFR coupling-control and cell viability protocol (CCVP) was used [3]. The respiration of PBMCs was corrected for contribution from contaminating platelets.
Our results showed, that the viability of PBMCs determined by O2k-FluoRespirometer and CCVP protocol for HRFR (in average 93.9% for fresh and 79.6% for cryopreserved PBMCs) corresponded well to the viability of PBMCs determined by Luna™ automated cell counter (96.2% and 85.4% correspondingly for fresh and cryopreserved PBMCs). The flux control ratios of freshly isolated and cryopreserved cells did not differ, and the individual reproducibility of respiration of freshly isolated PBMCs was similar to individual reproducibility of respiration of cryopreserved PBMCs.
In conclusion, respiration of cryopreserved PBMC when using an intact cell protocol reflects well the respiration of freshly isolated PBMCs. Therefore, cryopreserved PBMCs can be used as a model for functional diagnostic tests. Further studies including a standardization of methods for PBMCs isolation, counting and cryopreservation are necessary for quality control and data comparison between laboratories.
• Bioblast editor: Kandolf G
• O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Burtscher M, CH Zurich Lundby C, CH Zurich University of Zurich Physiology, DK Copenhagen Lundby C, DE Ulm Karabatsiakis A, SK Bratislava Sumbalova Z
Labels: MiParea: Respiration, mt-Medicine, mt-Awareness
Stress:Cryopreservation Organism: Human Tissue;cell: Blood cells Preparation: Intact cells
HRR: Oxygraph-2k
PBMCs
Affiliations
- Velika 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. – beata.velika@upjs.sk
