Gnaiger 2015 Abstract MiP2015
|Mitochondrial function of cyropreserved HEK 293T cells: development of a reference sample for high-resolution respirometry.|
An increasing number of metabolic and other diseases is recognized as being linked to mitochondrial physiology and dysfunction of oxidative phosphorylation (OXPHOS), which can be analyzed effectively and quantitatively by high-resolution respirometry (HRR). Instrumental quality control is a fundamental component of HRR applied in many laboratories . Beyond the instrumental level, a standardized mt-laboratory quality management system (QMSmtf ) is required for establishing a global data base on mitochondrial function (mtf) in human cells and tissues, taking into account the variables of evolution, age, gender, life style and environment (MitoEAGLE). A QMSmtf requires the availability of a mt-reference sample which is functionally stable over time and across geographical space, as a basis of standard proficiency tests within and between reference laboratories. Mammalian mt-preparations (isolated mitochondria, tissue preparations) appear to be neither suitable for prolonged storage nor large scale production. Therefore, we focused on cryopreserved human cells . Using the widely applied cell line HEK 293T we optimized cryopreservation to maintain cell viability and stabilize respiratory characteristics of intact and permeabilized cells over variable periods of time.
HEK 293T cells were cultured under standard conditions with DMEM, were cryopreserved by cooling cells suspended in a freezing medium with fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), and stored at -80 °C for variable periods of time. Cells were thawed by addion of and careful mixing in pre-warmed PBS or MiR05. Cell counts and viability were assessed by determination of Trypan blue exclusion using an automated Countess cell counter. Respiration was measured in the Oroboros Oxygraph-2k applying standard substrate-uncoupler-inhibitor-titration (SUIT) protocols for permeabilized cells in MiR05. For HRR with intact cells, cyropreserved cells were suspended in pre-warmed DMEM. Results were compared with tests on cells cryopreserved with addition of the powerful antioxidant melatonin at 10 nM or 25 µM.
• O2k-Network Lab: AT Innsbruck Oroboros
1-Oroboros Instruments; 2-Daniel Swarovski Research Laboratory, Mitochondrial Physiology, Dept Visceral, Transplant and Thoracic Surgery, Med Univ; Innsbruck, Austria. – [email protected]
Cryopreservation did not impair cell viability for storage times up to 311 days. In intact cells both ROUTINE and Electron transfer pathway (E) but not LEAK respiration (L) were depressed, with a decline of c. 20% after 3 weeks of cryopreservation. Subsequently, respiration was stable after prolonged storage for up to 311 days. Short-term preservation for 1 to 3 weeks did not affect respiration examined in permeabilized cells with or without melatonin, but OXPHOS (P) and ET capacities were reduced compared to controls in the presence of fluorescence probes applied for simultaneous detection of mt-membrane potential (TMRM; CI- and CI&II-linked respiration) or H2O2 production (Amplex Red; tested for CII ). Storage for 1 to 12 months was without effect on CIL and CIIE compared to cells maintained in culture, but CIP, CI&IIP, and CI&IIE were reduced. CI&II-linked OXPHOS capacity of these permeabilized control cells was not stimulated by cytochrome c (10 µM c), compared to a c-flux control factor in the cryopreserved cells as low as 0.02±0.01 (SD; N=5 cultures measured in duplicate). Therefore, damage of the outer mt-membrane was not the mechanism responsible for the changes observed in these OXPHOS analyses.
In summary, cryopreserved HEK 293T cells maintained full cell viability but showed some impairment of respiration upon prolonged storage. Such impairments may even be observed after short-term storage in the presence of fluorescence probes typically applied to examine mitochondrial function. However, the overall decline of respiration in permeabilized and intact cells appear to be rather limited even after a period of up to 311 days, suggesting that with careful characterization of these changes, maintenance of strictly controlled conditions and further optimization, cryopreserved HEK 293T cells may provide a validated reference sample for HRR, applicable for standardized proficiency testing and a QMSmtf without geographical borders.
References and acknowledgements
- Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - »Bioblast link«
- Wadhwa V, Rai S, Thukral T, Chopra M (2012) Laboratory quality management system: road to accreditation and beyond. Indian J Med Microbiol 30:131-40.
- Karabatsiakis A, Bock C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa I-T (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Translational Psychiatry 4:e397. - »Bioblast link«
- Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. - »Bioblast link«
Supported by projects K-Regio MitoFit and NextGen-O2k, Tiroler Innovationsförderung.
Labels: MiParea: Respiration, Instruments;methods, mt-Membrane
Stress:Cryopreservation, Oxidative stress;RONS Organism: Human Tissue;cell: HEK Preparation: Intact cells, Permeabilized cells
Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: N, S, NS HRR: Oxygraph-2k, O2k-Fluorometer Event: C1, Oral MiP2015