Garcia-Souza 2017 MiPschool Obergurgl
Event: MiPschool Obergurgl 2017
Peripheral-blood mononuclear cells (PBMC) and platelets (PLT) are potentially powerful models for the diagnosis of mitochondrial respiratory function and dysfunction, offering a minimally invasive approach in comparison to tissue biopsies. Rapid isolation of platelets and respiratory measurement without delay are required to avoid post-blood harvesting stress and cellular activation followed by metabolic alterations. Although it is well known that PLT are sensitive to temperature fluctuations, tending to activate the cells below 18 ºC , we considered cryopreservation as a promising strategy to preserve mitochondrial function in PLT comparable to PBMC . Cryopreservation of PLT is successful for the study of inflammatory properties [3,4]. Therefore, the objective of our study was to optimize this cryopreservation protocol for the measurement of respiratory mitochondrial properties of human PLT.
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: 10 min, room temperature, RT, acceleration 6, no brakes). The PBMC-PLT layer was collected and washed with 25 ml DPBS (120 g; 10 min, RT). The supernatant was combined with 5 ml of diluted plasma obtained from the first centrifugation, and EGTA was added at 10 mM final concentration. After centrifugation at 1,000 g (10 min, RT) the cell pellet was washed with 5 ml DPBS containing 10 mM EGTA (1,000 g; 5 min, RT), resuspended with 0.5 ml DPBS containing 10 mM EGTA and the PLT were counted (SYSMEX XN-350). Cells were resuspended in autologous plasma+5% DMSO for cryopreservation. Cryovials were placed in pre-cooled (4 °C) CoolCell® containers and stored at -80 °C. After one, two and four weeks storage, cells were thawed in a 37 °C water bath, diluted in 10 ml of pre-warmed 37 °C medium (DPBS+10 mM EGTA), centrifuged (1,000 g; 10 min, RT) and 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 Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) containing mitochondrial respiration medium MiR05 (Oroboros MiR05-Kit) or culture medium M199. An extended substrate-uncoupler-inhibitor titration (SUIT) protocol was developed using MiR05 and adding cells (ce) for measuring endogenous ROUTINE respiration (1ce), with sequential addition of pyruvate (2P; exogenous substrate), uncoupler (3U; ET capacity), glucose (4Glc; Crabtree effect), malate (5M; no stimulation expected in intact cells), rotenone, (6Rot; residual oxygen consumption, ROX, except for stimulation by PM in permeabilized cells), succinate (7S; stimulation of permeabilized cells), digitonin (8Dig; permeabilization of the plasma membrane in the entire cell population, providing a reference level of 100% nonviable cells in the respirometric cell viability test), cytochrome c (8c; test of outer mt-membrane integrity), antimycin A (9Ama; ROX), ascorbate&TMPD (10AsTm; CIV activity plus autooxidation), and azide (11Azd; chemical background to correct for autooxidation).
There was no significant difference in ROUTINE respiration, R, of freshly isolated PLT in M199 and MiR05. Pyruvate addition resulted in a significant increase in R in MiR05. Cold storage stress was detected as an overall decrease in mitochondrial respiration in both media, without a difference in the 1- to 4-weeks cryopreservation groups (MiR05). Flux control ratios (FCR) of cryopreserved and freshly isolated PLT were preserved, suggesting a global decrease in mitochondrial respiration per cell, without qualitative changes detected in our SUIT protocol. Cell viability of cryopreserved platelets decreased by 20% based on succinate respiration normalized by digitonin titration. This is consistent with an over-all loss of mitochondrial function in the non-viable cells, if permeabilization of these cells occurs in high-Ca2+ medium during rewarming at 37 ºC.
These results illustrate that cryopreservation imposes a substantial damage on PLT. Modifications, such as DMSO concentration and freezing medium cell density, will be investigated in order to improve cell viability and improve cryopreservation of mitochondrial respiratory function.
Affiliations and support
- Garcia-Souza LF(1,2), Cizmarova B(2,3), Sumbalova Z(2,4), Menz V(1), Burtscher M(1), Gnaiger E(2,5)
- Inst Sport Science, Univ Innsbruck, Austria
- 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
- Pharmacobiochemical Lab, 3rd Dept Internal Medicine, Fac Medicine, Comenius Univ, Bratislava, Slovakia
- Oroboros Instruments, Innsbruck, Austria. - [email protected]
Figure 1. Representative experiment of extended substrate-uncoupler-inhibitor titration (SUIT) protocol. Blue line corresponds to oxygen concentration (µM) in the chamber and red line corresponds to oxygen flow of intact platelets (pmol •s-1•10-8 cells). The titration events and marks are represented as follows: ROUTINE respiration (R) is initially measured in intact cells (1ce) in mitochondrial respiration medium (MiR05-Kit); single titration of pyruvate (2P, 10 mM) to supplement MiR05 medium with oxidative substrate; uncoupler (3U*, titration of CCCP) is used for determination of ET capacity; glucose (4Glc, 11 mM) to compare Crabtree effect with culture media; malate (5M, 2 mM) stimulation is observed only in permeabilized cells; rotenone (6Rot, 0.5 mM) inhibiting Complex I (CI) in intact cells; succinate (7S, 10 mM) stimulation of mt-respiration is observed in non-viable cells only; digitonin (8Dig) is used for permeabilization of the eintire cell population, providing a reference state of succinate-stimulated ET capacity; cytochrome c (8c) titration test provides information about outer mt-membrane integrity; antimycin A (Ama, 2.5 mM) inhibiting Complex III; ascorbate 2 mM & TMPD 0.5 mM (10AsTm) stimulation of Complex IV plus chemical background; azide (11Azd, 200 mM) as an inhibitor of cytochrome c oxidase provides information about the oxygen consumption chemical background subtracted from flux in 10AsTm.
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Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Blood cells, Platelet Preparation: Intact cells, Permeabilized cells
Coupling state: ROUTINE, ET Pathway: S, CIV, ROX HRR: Oxygraph-2k Event: C1, Oral PBMC, MitoEAGLE