Name: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (Video) | JoVE
Uploaded: 2023-10-20T13:30:00
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We would like to thank the clinical team of the University Hospital Giessen-Marburg for the blood collection. This work was funded by the Justus Liebig university.

All protocols described in this manuscript for blood collection, isolation and analysis have been reviewed and approved by the Institutional Review Board at the University of Giessen, Germany. The consent of the patients to include their samples in the study was obtained. All steps for isolation and cell culture are carried out under a biological safety cabinet.
1. Venipuncture
<ol>
	<li>Prepare all equipment needed for blood collection including disinfection spray, sterile ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

<ol>
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In Vitro and Ex Vivo Models. , (2015).</li><li>Silaidos, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-associated+differences+in+mitochondrial+function+in+human+peripheral+blood+mononuclear+cells+(PBMCs)+and+brain.">Sex-associated differences in mitochondrial function in human peripheral blood mononuclear cells (PBMCs) and brain.</a> Biol Sex Differ. 9 (1), 34 (2018).</li><li>Acin-Perez, R., Beninc&#225;, C., Shabane, B., Shirihai, O. S., Stiles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+human+samples+for+assessment+of+mitochondrial+bioenergetics:+Gold+standards,+limitations,+and+future+perspectives.">Utilization of human samples for assessment of mitochondrial bioenergetics: Gold standards, limitations, and future perspectives.</a> Life. 11 (9), 949 (2021).</li><li>Schindowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+aging.">Impact of aging.</a> NeuroMol Med. 4 (3), 161-177 (2003).</li><li>Migliore, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+the+role+and+the+most+suitable+biomarkers+of+oxidative+stress+in+Alzheimer's+disease+and+in+other+neurodegenerative+diseases.">Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer's disease and in other neurodegenerative diseases.</a> Neurobiol Aging. 26 (5), 587-595 (2005).</li><li>Leutz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+trophic+support+enhances+apoptosis+in+PC12+cells+expressing+Alzheimer&#8217;s+APP+mutation+and+sensitizes+cells+to+staurosporine-induced+cell+death.">Reduction of trophic support enhances apoptosis in PC12 cells expressing Alzheimer&#8217;s APP mutation and sensitizes cells to staurosporine-induced cell death.</a> J Mol Neurosci. 18 (3), 189-201 (2002).</li><li>Leuner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+mitochondrial+dysfunction+in+Alzheimer&#8217;s+disease:+Focus+on+lymphocytes.">Peripheral mitochondrial dysfunction in Alzheimer&#8217;s disease: Focus on lymphocytes.</a> Mol Neurobiol. 46 (1), 194-204 (2012).</li><li>Leuner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+apoptosis,+oxidative+stress+and+mitochondrial+dysfunction+in+lymphocytes+as+potential+biomarkers+for+Alzheimer&#8217;s+disease.">Enhanced apoptosis, oxidative stress and mitochondrial dysfunction in lymphocytes as potential biomarkers for Alzheimer&#8217;s disease.</a> J Neural Transm Suppl. 2007 (72), 207-215 (2007).</li><li>Kartika, R., Wibowo, H., Purnamasari, D., Pradipta, S., Larasati, R. 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J., Ramakers, J. D., Plat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+modulation+of+immune+function+by+&#946;-glucans.">Dietary modulation of immune function by &#946;-glucans.</a> Physiol Behav. 94 (2), 276-284 (2008).</li><li>Reddy, M., Eirikis, E., Davis, C., Davis, H. M., Prabhakar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+lymphocyte+activation+marker+expression+and+cytokine+secretion+profile+in+stimulated+human+peripheral+blood+mononuclear+cell+cultures:+an+in+vitro+model+to+monitor+cellular+immune+function.">Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.</a> J Immunol Methods. 293 (1), 127-142 (2004).</li><li>Otaegui, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+micro+RNA+expression+in+PBMC+from+multiple+sclerosis+patients.">Differential micro RNA expression in PBMC from multiple sclerosis patients.</a> PLoS One. 4 (7), e6309 (2009).</li><li>Geltink, R. I. K., Kyle, R. L., Pearce, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+complex+interplay+between+T+cell+metabolism+and+function.">Unraveling the complex interplay between T cell metabolism and function.</a> Annu Rev Immunol. 36, 461-488 (2018).</li><li>Fox, C. J., Hammerman, P. S., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fuel+feeds+function:+energy+metabolism+and+the+T-cell+response.">Fuel feeds function: energy metabolism and the T-cell response.</a> Nat Rev Immunol. 5 (11), 844-852 (2005).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+dysfunctions+of+blood+mononuclear+cells+link+with+cardiac+disturbance+in+patients+with+early-stage+heart+failure.">Mitochondrial respiratory dysfunctions of blood mononuclear cells link with cardiac disturbance in patients with early-stage heart failure.</a> Sci Rep. 5, 10229 (2015).</li><li>Weiss, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+peripheral+blood+mononuclear+cells+in+pediatric+septic+shock.">Mitochondrial dysfunction in peripheral blood mononuclear cells in pediatric septic shock.</a> Pediatr Crit Care Med. 16 (1), e4-e12 (2015).</li><li>Higdon, L. E., Lee, K., Tang, Q., Maltzman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+global+transplant+laboratory+standard+operating+procedures+for+blood+collection,+PBMC+isolation,+and+storage.">Virtual global transplant laboratory standard operating procedures for blood collection, PBMC isolation, and storage.</a> Transplant Direct. 2 (9), e101 (2016).</li><li>Betsou, F., Gaignaux, A., Ammerlaan, W., Norris, P. J., Stone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biospecimen+science+of+blood+for+peripheral+blood+mononuclear+cell+(PBMC)+functional+applications.">Biospecimen science of blood for peripheral blood mononuclear cell (PBMC) functional applications.</a> Curr Pathobiol Rep. 7, 17-27 (2019).</li><li>Pesta, D., Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry:+OXPHOS+protocols+for+human+cells+and+permeabilized+fibers+from+small+biopsies+of+human+muscle.">High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle.</a> Methods Mol Biol. 810, 25-58 (2012).</li><li>Djafarzadeh, S., Jakob, S. 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This protocol provides a means of isolating and cryopreserving peripheral blood mononuclear cells (PBMCs) from human blood in a manner suitable for bioenergetic analyses. The described method offers the possibility to isolate PBMCs gently and in large quantities, with high viability and sufficient cells for bioenergetic measurements. It has the disadvantage that even with minimal interruptions, long isolations occur, but subsequent cryopreservation allows a time-independent measurement of bioenergetics. With this method,...

Human peripheral blood mononuclear cells (PBMCs) are used for various applications in many scientific fields, including the study of immunological and bioenergetic issues, such as those related to aging processes or degenerative diseases1,2. PBMCs are heterogeneous in composition and consist of lymphocytes (B cells, T cells, and NK cells), monocytes, and dendritic cells. The cells sometimes show great individual differences and variations within a subject, so standardized procedures for handling these cells are required. Important parameters such as viability and purity of the isolation are the basic requirements for its handling and are additionally influenced by environmental factors such as the time of collection, the melatonin level, whether the subject is fasting, and others3,4.
Based on studies on bioenergetics of PBMCs, we describe here a method for the isolation, cryopreservation, and cultivation of PBMCs that is suitable for other methods as well. While muscle biopsy is considered the gold standard for mitochondrial energy metabolism5, the examination of blood cells is a rapid, minimally invasive procedure. In addition to this, more and more studies suggest that the changes in mitochondrial function in aging and Alzheimer&#39;s disease (AD) occur not only in the brain but also in the periphery6,7,8,9,10. The method also allows investigations of other conditions and diseases, including diabetes mellitus and obesity11,12,13. Gene expression patterns in multiple sclerosis patients can be analyzed, or immune function and influences on it in general14,15,16.
PBMCs generally rely on oxidative phosphorylation (OXPHOS) to generate adenosine triphosphate (ATP)17,18. Therefore, PBMCs cover a wide range of applications as surrogates. In previous reports, the energy metabolism of PBMCs has been used to address organ dysfunctions, such as in early heart failure19, septic shock20 or sex-associated differences4 in mitochondrial function. A generalized method for cryopreservation isolation and cultivation of PBMCs would have advantages in the comparability of results obtained at different institutes. There is a great deal of variation in the protocols for each step21,22, the goal of this method is to provide a guideline for bioenergetic measurements in PBMCs.
In this article we describe a method for measuring bioenergetic parameters in PBMCs. We explain the methods for isolating, cryopreserving and measuring bioenergetics of PBMCs from human blood. This method can be used to determine bioenergetic parameters in patients and evaluate them in a clinical context. To apply these measurements, researchers need access to a patient population from which fresh blood samples can be obtained.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Triethanolamine-HCl-Buffer (pH = 8,0)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Triethanolamine-HCl-Buffer</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Tris-HCl-Buffer (pH = 8,1)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.01 mM DTBB</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10 % Triton X-100</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Oxalacetat</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>14&#8211;20 G sterile blood draw needles Multi Adapter Sarstedt Safety-Multifly</td>
			<td>Sarstedt</td>
			<td>156353_v</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Carl Roth GmbH &#38; Co. KG</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol (EtOH)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Pancreac Applichem</td>
			<td>A3753</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol wipes</td>
			<td></td>
			<td></td>
			<td>&#160;(70% isopropyl alcohol)</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua (bidest.)</td>
			<td>With MilliQ Academic (self-made)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP-Standard</td>
			<td>Sigma-Aldrich</td>
			<td>6016949</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocoll Seperating Solution</td>
			<td>Biochrom</td>
			<td>6115</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safty cabinet MSC Advantage</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonylcyanid-p-trifluoromethoxy-phenylhydrazon (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Heraeus Megafuge 16 R</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>1450016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube Cryo.S</td>
			<td>Grainer Bio-One</td>
			<td>126263-2DG</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>37008</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxid (DMSO)</td>
			<td>Merck</td>
			<td>102952</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection spray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable gloves latex, rubber, or vinyl.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distrips (12.5 ml) DistriTips</td>
			<td>Gilson</td>
			<td>F164150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS; 10x)</td>
			<td>Gibco (Thermo Scientific)</td>
			<td>15217168</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH 100%)</td>
			<td>Carl ROTH GmbH &#38; Co. KG</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Frezer (-80&#176;C)</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Holder/adapter&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Midi 40 CO2</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe</td>
			<td>Hamilton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-Aldrich</td>
			<td>M-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MIR05</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>10110051</td>
			<td></td>
		</tr>
		<tr>
			<td>Multireader CLARIOstar</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen tank Locator 6 plus</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>O4876</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxalacetate</td>
			<td>Sigma-Aldrich</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygraph-2k</td>
			<td>Orobororus Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>PAA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes Performance Pipettor 10 &#956;L, 100 &#956;L, 1000 &#956;L</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell-Park. Memorial-Institute-Medium (RPMI-1640)</td>
			<td>Gibco (Thermo Scientific)</td>
			<td>11530586</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Carl ROTH GmbH &#38; Co. KG</td>
			<td>9286.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylphenylendiamin (TMPD)</td>
			<td>Sigma-Aldrich</td>
			<td>T3134</td>
			<td></td>
		</tr>
		<tr>
			<td>Tourniquet/ Blood pressure cuff</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)amino-methane</td>
			<td>Sigma-Aldrich</td>
			<td>108382</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>108643</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypanblau</td>
			<td>Biochrom</td>
			<td>T6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vaccubrand GmbH &#38; Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViewPlate-96</td>
			<td>Perkin Elmer</td>
			<td>6005181</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath WNB22</td>
			<td>Memmert GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cryopreservation and bioenergetic evaluation of human peripheral blood mononuclear cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

Cell viability and number 
To achieve successful isolation and cryopreservation, cell count and viability should be as high as possible. Before and after cryopreservation, the cells are counted, and their viability is determined to ensure the health and quality of the cells. Figure 3 is a representative illustration of PBMCs before and after cryopreservation, cell count and viability hardly differ. This indicates successful isolation and preservation of PBMCs.
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Watch this Scientific Journal Video about Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation at JoVE.com

Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (Video) | JoVE

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...