Cizmarova 2017 Abstract MITOEAGLE Barcelona
Optimization of cryopreservation of human peripheral blood mononuclear cells and platelets for high-resolution respirometry.
Event: MitoEAGLE Barcelona 2017
Peripheral blood mononuclear cells (PBMC) represent a heterogeneous cell population including monocytes, lymphocytes (T- and B- cells, natural killer cells), and dendritic cells, in contrast to the relatively homogenous population of blood platelets (PLT). These cells provide a model of sensitive biomarkers, as far as they reflect systemic changes representative of a range of other organs in health and disease. Further establishing the use of blood cells may stimulate the entire field of mitochondrial (mt) medicine, beyond the specific pathologies for which a link has been established to mt-function in PBMC or PLT. Cryopreservation of cells is commonly used in various biomedical areas (fertility, stem cells research, cord blood) and provides a promising although not unambiguously successful approach to separate the sampling of blood from the diagnostic respirometric assays [1,2]. The aim of our study was to optimize cryopreservation protocols not only for PBMC but including PLT, applying respirometric protocols specifically developed for diagnostic monitoring.
Human blood cells were isolated in 50 mL Leucosep® tubes with 15 mL of Ficoll-Paque™ . 18 mL full blood were diluted 1:1 with sterile DPBS and centrifuged at 1,000 g (first centrifugation: room temperature, 10 min, acceleration 6, no brakes). The PBMC layer was collected, washed twice with 25 mL DPBS (120 g; 10 min), resuspended in 0.5 mL DPBS and counted (Sysmex XN-350, Japan). For isolation of PLT, 5 mL diluted plasma obtained in the first centrifugation were combined with 25 mL supernatant obtained in the first PBMC washing step, and EGTA was added at 10 mM final concentration. After centrifugation at 1,000 g (10 min) the cell pellet was washed with 5 mL DPBS containing 10 mM EGTA (1,000 g; 5 min), resuspended with 0.5 mL DPBS containing 10 mM EGTA and the PLT were counted. For cryopreservation the cells were resuspended in ice-cold freezing medium (PBMC: FBS+10% DMSO; PLT: autologous plasma+5% DMSO) and counted. Cryovials were placed in pre-cooled (4 °C) CoolCell® containers and stored at -80 °C. After one week storage, cells were thawed in a 37 °C water bath, diluted in 10 mL of pre-warmed 37 °C medium (PBMC: DPBS or DPBS+2%FBS ; PLT: DPBS+10 mM EGTA), centrifuged (PBMC: 300 g; 10 min (twice), resuspended in 0.25 mL DPBS; PLT: 1,000 g; 10 min, resuspended in 0.25 mL DPBS+10 mM EGTA). Stock suspensions of cryopreserved or freshly isolated cells were titrated into the 2-mL chambers of the Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria) containing MiR05.
The optimum PBMC density for cryopreservation was 5∙106 cells/mL freezing medium. Respiratory capacity of intact cells was fully preserved after thawing the cells in DPBS+2%FBS. Pyruvate added to MiR05 did not affect ROUTINE respiration (R), but increased ETS capacity (E). The apparent excess E-R capacity factor, (E-R)/E, of intact fresh and cryopreserved PBMCs was higher with 10 mM pyruvate in MiR05 than in RPMI. ET capacity of fresh and cryopreserved intact PLT was higher in MiR05 than in M199. After cryopreservation, respiratory capacity and (E-R)/E were depressed, and the respirometric cell viability test (stimulation of oxygen flux by addition of succinate to rotenone inhibited cells) indicated cell damage of PLT by cryopreservation. Further optimization of cryopreservation protocols for blood cells provide new tools for storage and effective use of these cells in mitochondrial physiology, extending the scope for diagnosis of mitochondrial diseases.
Affiliations and support
- Cizmarova B(1,2), Garcia-Souza LF(3), Sumbalova Z(1,4), Karabatsiakis A(5), Gnaiger E(1,6)
- Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria
- Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Slovakia
- Institute of Sport Science, University Innsbruck, Austria
- Pharmacobiochemical Laboratory, 3rd Depatrment of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia
- University of Ulm, Clinical and Biological Psychology, Germany
- Oroboros Instruments, Innsbruck, Austria
Figure 1. High-resolution respirometry with intact human PBMC (fresh controls) applying a coupling control protocol (CCP_1R,2U,3P,4Glc,5Rot,6S,7Ama): 1R: ROUTINE respiration; 2U: Uncoupler titrations with CCCP, 3P: Pyruvate 5 mM, 4 Glc: Glucose 11 mM, 5Rot: Rotenone 0.5 µM, 6S: Succinate 10 mM, 7Ama: Antimycin A 2.5 mM.
- Karabatsiakis A, Böck C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa IT (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Transl Psychiatry 4:e397.
- Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723–33.
- Sumbalova Z, Hiller E, Chang S, Garcia-Souza LF, 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. - MiPNet21.17_BloodCellsIsolation
Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Blood cells, Platelet Preparation: Intact cells
Coupling state: ROUTINE, ET
HRR: Oxygraph-2k Event: B3 MitoEAGLE