Keppner 2018 MiP2018

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Gloria Keppner
Effects of cryopreservation of peripheral blood mononuclear cells.

Link: MiP2018

Keppner G, Bayer F, Skurk T, Klingenspor M (2018)

Event: MiP2018

COST Action MitoEAGLE

Peripheral Blood Mononuclear Cells (PBMCs) are a collection of different blood cells with the similarity to have a round nucleus. These include myeloid cells such as Monocytes (10-29%) and Dendritic cells (1-2%) as well as lymphoid cells particularly B cells (10-15%), T cells (50-80%), and NK cells (10-15%). Since PBMCs are easily accessible by isolating them from a blood sample, they serve as an ideal source to study mitochondrial function of human cells. Right now PBMCs have to be measured immediately after isolating them from the whole blood, according to the standard protocol for mitochondrial respiration of PBMCs. Collecting many blood samples on the same day is not possible since there is only a short time-window during which mitochondrial activity can be assessed. In the present project a protocol was established for the cryopreservation and thawing of PBMCs for high-resolution respirometry. Consequently, the effect of cryopreservation was analyzed regarding the mitochondrial function of PBMCs. Whole blood was obtained by venipuncture with Lithium-Heparin Monovettes to prevent blood clotting and PBMCs were isolated by density gradient centrifugation. Freshly isolated PBMCs were either cryopreserved at – 80°C for at least 7 days or directly measured by high-resolution respirometry.

Our results show comparable respiration rates of freshly isolated and cryopreserved PBMCs. It is possible to cryopreserve PBMCs under optimal conditions, which means to thaw quickly, dilute slowly and pipette very gently. Otherwise cryopreserved PBMCs show a reduced uncoupled respiration under FCCP, which can also be prevented by adding Cytochrome C. We present here a possibility to cryopreserve PBMCs for high resolution respirometry.


Bioblast editor: Plangger M, Kandolf G O2k-Network Lab: DE Freising Klingenspor M


Labels: MiParea: Respiration, Instruments;methods 

Stress:Cryopreservation 

Tissue;cell: Blood cells  Preparation: Intact cells 


Coupling state: ET 

HRR: Oxygraph-2k 

PBMCs 

Affiliations

Keppner G(1,2), Bayer F(1), Skurk T(2,3), Klingenspor M(1,2)

  1. Chair Molecular Nutritional Medicine, Technische Univ München, Else Kröner-Fresenius Zentrum Ernährungsmedizin. - gloria.keppner@tum.de
  2. ZIEL– Inst Food and Health, Technische Univ München
  3. Chair Clinical Nutritional Medicine, Technische Univ München, Else Kröner-Fresenius Zentrum Ernährungsmedizin