Difference between revisions of "Cizmarova 2017 MiP2017"

Latest revision as of 15:25, 21 February 2024

PBMC cryopreservation and evaluation for functional mitochondrial diagnosis.

Link: MiP2017

Cizmarova B, Garcia-Souza LF, Volani C, Menz V, Burtscher M, Gatterer H, Lundby C, Karabatsiakis A, Sumbalova Z, Gnaiger E (2017)

Event: MiP2017

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 a promising model for the study of mitochondrial respiration as a biomarker for mitochondrial dysfunction. PBMC can be isolated easily from blood samples which provide a low-invasive approach when compared to invasive sampling of muscle biopsies.

We focused on the evaluation of the method for cryopreservation of freshly isolated PBMC and their usage for high-resolution respirometry (HRR). The study involved 8 healthy female volunteers (26.3 ± 5.7 years) on a controlled diet and physical activity restricted to a light 1-h walk per day. 18 mL of whole blood was collected in the morning for 4 days at low altitude (575 m, Innsbruck, Austria). PBMC were separated from whole blood by centrifugation on Ficoll-Paque™ PLUS density medium using 50 mL Leucosep tubes [1]. After the fist washing of the PBMC-platelet layer with DPBS (25 mL, 120 g, RT), the PBMC were washed two times with ice-cold DPBS+2% FBS (50 mL, 300 g, 4 °C). The isolated PBMC fraction was counted on 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∙10⁶ PBMC) were added into the 2-mL chambers of the O2k-FluoRespirometer (Oroboros Instruments, Innsbruck, Austria) containing mitochondrial respiration medium MiR05-Kit with catalase (Oroboros, AT) at 37 °C. The coupling-control and cell viability protocol (CCVP) was used [3]. Respiration of PBMC was corrected for the contribution by contaminating platelets.

The viability of PBMC determined by the CCVP averaged 93.9 % for fresh and 79.6 % for cryopreserved cells, corresponding well with the viability determined by the Luna™ automated cell counter (96.2 % and 85.4 % for fresh and cryopreserved cells). The flux control ratios of freshly isolated and cryopreserved cells did not differ. The individual reproducibility of respiration of freshly isolated PBMC was similar to that determined in cryopreserved PBMC.

In conclusion, respiration of cryopreserved PBMC reflects well the respiration of freshly isolated PBMC. Therefore, cryopreserved PBMC can be used as a model for functional diagnostic tests. Further studies including standardization of methods for PBMC isolation, counting and cryopreservation are necessary for quality control and data comparison between laboratories.

• Bioblast editor: Gnaiger E • O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Burtscher M, DK Copenhagen Lundby C, DE Ulm Karabatsiakis A, SK Bratislava Sumbalova Z

Labels: MiParea: Respiration

Stress:Cryopreservation Organism: Human Tissue;cell: Blood cells Preparation: Intact cells

Coupling state: LEAK, ROUTINE, ET

HRR: Oxygraph-2k

MitoEAGLE, PBMCs

Affiliations and support

Cizmarova 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

Supported by Action Austria-Slovakia (BV) and K-Regio project K-Regio MitoFit (CL, EG, HG, LFGS, MB, VM, ZS). Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.

References

Sumbalova Z, Hiller E, Chang S, Garcia L, 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. »Bioblast link»
Cizmarova B, Garcia-Souza LF, Sumbalova Z, Karabatsiakis A, Gnaiger E (2017) Optimization of cryopreservation of human peripheral blood mononuclear cells and platelets for high-resolution respirometry. »Bioblast link»
Garcia-Souza LF, Velika B, Sumbalova Z, Menz V, Burtscher M, Gnaiger E (2017) Assessment of mitochondrial respiratory function in cryopreserved platelets. MiPschool Obergurgl 2017 »Bioblast link»

@@ Line 1: / Line 1: @@
 {{Abstract
-|title=[[Image:VelikaBeata.JPG|left|90px|Beate Velika]]
+|title=[[Image:VelikaBeata.JPG|left|90px|Beate Cizmarova ]] PBMC cryopreservation and evaluation for functional mitochondrial diagnosis.
-The cryopreserved PBMC as a model in functional mitochondrial diagnosis.
 |info=[[MiP2017]]
-|authors=Velika B, Garcia-Souza LF, Volani C, Menz V, Burtscher M, Gatterer H, Lundby C, Karabatsiakis A, Sumbalova Z, Gnaiger E
+|authors=Cizmarova B, Garcia-Souza LF, Volani C, Menz V, Burtscher M, Gatterer H, Lundby C, Karabatsiakis A, Sumbalova Z, Gnaiger E
 |year=2017
 |event=MiP2017
 |abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]
-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.
+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 a promising model for the study of mitochondrial respiration as a  biomarker for mitochondrial dysfunction. PBMC can be isolated easily from blood samples which provide a low-invasive approach 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.
+We focused on the evaluation of the method for cryopreservation of freshly isolated PBMC and their usage for high-resolution respirometry (HRR). The study involved 8 healthy female volunteers (26.3 ± 5.7 years) on a controlled diet and physical activity restricted to a light 1-h walk per day. 18 mL of whole blood was collected in the morning for 4 days at low altitude (575 m, Innsbruck, Austria). PBMC were separated from whole blood by centrifugation on Ficoll-Paque™ PLUS density medium using 50 mL Leucosep tubes [1]. After the fist washing of the PBMC-platelet layer with DPBS (25 mL, 120 ''g'', RT), the PBMC were washed two times with ice-cold DPBS+2% FBS (50 mL, 300 ''g'', 4 °C). The isolated PBMC fraction was counted on 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∙10<sup>6</sup> PBMC) were added into the 2-mL chambers of the O2k-FluoRespirometer (Oroboros Instruments, Innsbruck, Austria) containing mitochondrial respiration medium MiR05-Kit with catalase (Oroboros, AT) at 37 °C. The coupling-control and cell viability protocol (CCVP) was used [3]. Respiration of PBMC was corrected for the contribution by 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.
+The viability of PBMC determined by the CCVP averaged 93.9 % for fresh and 79.6 % for cryopreserved cells, corresponding well with the viability determined by the Luna™ automated cell counter (96.2 % and 85.4 % for fresh and cryopreserved cells). The flux control ratios of freshly isolated and cryopreserved cells did not differ. The individual reproducibility of respiration of freshly isolated PBMC was similar to that determined in cryopreserved PBMC.
-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.
+In conclusion, respiration of cryopreserved PBMC reflects well the respiration of freshly isolated PBMC. Therefore, cryopreserved PBMC can be used as a model for functional diagnostic tests. Further studies including standardization of methods for PBMC isolation, counting and cryopreservation are necessary for quality control and data comparison between laboratories.
-|editor=[[Kandolf G]]
+|editor=[[Gnaiger E]]
-|mipnetlab=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
+|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck Burtscher M, DK Copenhagen Lundby C, DE Ulm Karabatsiakis A, SK Bratislava Sumbalova Z
 }}
 {{Labeling
-|area=Respiration, mt-Medicine, mt-Awareness
+|area=Respiration
 |injuries=Cryopreservation
 |organism=Human
 |tissues=Blood cells
 |preparations=Intact cells
+|couplingstates=LEAK, ROUTINE, ET
 |instruments=Oxygraph-2k
-|additional=PBMCs
+|additional=MitoEAGLE, PBMCs
 }}
-== Affiliations ==
+== Affiliations and support ==
-::::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)
+:::: Cizmarova 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
@@ Line 37: / Line 37: @@
 ::::#Pharmacobiochemical Lab, 3rd Dept Internal Medicine, Fac Medicine, Comenius Univ, Bratislava, Slovakia
 ::::#Oroboros Instruments, Innsbruck, Austria. – beata.velika@upjs.sk
+:::: Supported by Action Austria-Slovakia (BV) and K-Regio project [[K-Regio MitoFit]] (CL, EG, HG, LFGS, MB, VM, ZS). Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 [[MitoEAGLE]].
 == References ==
-::::#[[MiPNet21.17_BloodCellsIsolation]]
+::::#Sumbalova Z, Hiller E, Chang S, Garcia L, 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 |»Bioblast link»]]
-::::#[[Velika_2017_Abstract_MITOEAGLE_Barcelona]]
+::::#Cizmarova B, Garcia-Souza LF, Sumbalova Z, Karabatsiakis A, Gnaiger E (2017) Optimization of cryopreservation of human peripheral blood mononuclear cells and platelets for high-resolution respirometry. [[Velika_2017_Abstract_MITOEAGLE_Barcelona |»Bioblast link»]]
-::::#[[Garcia-Souza_2017_MiPschool_Obergurgl]]
+::::#Garcia-Souza LF, Velika B, Sumbalova Z, Menz V, Burtscher M, Gnaiger E (2017) Assessment of mitochondrial respiratory function in cryopreserved platelets. MiPschool Obergurgl 2017 [[Garcia-Souza_2017_MiPschool_Obergurgl |»Bioblast link»]]