Name: Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation
Uploaded: 2023-10-20T13:30:00
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Agradecemos à equipe clínica do Hospital Universitário Giessen-Marburg pela coleta de sangue. Este trabalho foi financiado pela universidade Justus Liebig.

Todos os protocolos descritos neste manuscrito para coleta, isolamento e análise de sangue foram revisados e aprovados pelo Comitê de Ética em Pesquisa da Universidade de Giessen, Alemanha. Obteve-se o consentimento dos pacientes para inclusão de suas amostras no estudo. Todas as etapas de isolamento e cultura celular são realizadas sob um gabinete de segurança biológica.
1. Punção venosa
<ol><li>Preparar todo o equipamento necessário para a coleta de sangue, inclu...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

<ol>
	<li>Mancuso, M., 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=Mitochondria,+cognitive+impairment,+and+Alzheimer's+disease.">Mitochondria, cognitive impairment, and Alzheimer's disease.</a> Int J Alzheimers Dis. 2009, 951548 (2009).</li><li>Haas, R. H. <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+aging+and+diseases+of+aging.">Mitochondrial dysfunction in aging and diseases of aging.</a> Biology. 8 (2), 48 (2019).</li><li>Kleiveland, C. R., Verhoeckx, K., Cotter, P., Lopez-Exposito, I., 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+blood+mononuclear+cells.">Peripheral blood mononuclear cells.</a> The Impact of Food Bioactives on Health. 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. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Indoleamine+2,3-Dioxygenase+production+and+its+association+to+inflammatory+cytokines+in+peripheral+blood+mononuclear+cells+culture+of+type+2+diabetes+mellitus.">Altered Indoleamine 2,3-Dioxygenase production and its association to inflammatory cytokines in peripheral blood mononuclear cells culture of type 2 diabetes mellitus.</a> Int J Tryptophan Res. 13, 1178646920978236 (2020).</li><li>Cortez-Espinosa, N., 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=CD39+expression+on+Treg+and+Th17+cells+is+associated+with+metabolic+factors+in+patients+with+type+2+diabetes.">CD39 expression on Treg and Th17 cells is associated with metabolic factors in patients with type 2 diabetes.</a> Hum Immunol. 76 (9), 622-630 (2015).</li><li>Mahmoud, F., 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=Effect+of+Diabetea+tea+&#8482;+consumption+on+inflammatory+cytokines+and+metabolic+biomarkers+in+type+2+diabetes+patients.">Effect of Diabetea tea &#8482; consumption on inflammatory cytokines and metabolic biomarkers in type 2 diabetes patients.</a> J Ethnopharmacol. 194, 1069-1077 (2016).</li><li>Volman, J. 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. M. <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+to+assess+mitochondrial+function+in+permeabilized+and+intact+cells.">High-resolution respirometry to assess mitochondrial function in permeabilized and intact cells.</a> J Vis Exp. (120), e54985 (2017).</li><li>Wang, W., Zhao, F., Ma, X., Perry, G., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+dysfunction+in+the+pathogenesis+of+Alzheimer&#8217;s+disease:+recent+advances.">Mitochondria dysfunction in the pathogenesis of Alzheimer&#8217;s disease: recent advances.</a> Mol Neurodegener. 15 (1), 30 (2020).</li><li>Chaturvedi, R. K., Flint Beal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+of+the+brain.">Mitochondrial diseases of the brain.</a> Free Radic Biol Med. 63, 1-29 (2013).</li></ol>

Este protocolo fornece um meio de isolar e criopreservar células mononucleares do sangue periférico (CMSP) do sangue humano de forma adequada para análises bioenergéticas. O método descrito oferece a possibilidade de isolar CMSP suavemente e em grandes quantidades, com alta viabilidade e células suficientes para medidas bioenergéticas. Tem a desvantagem de que, mesmo com interrupções mínimas, ocorrem longos isolamentos, mas a criopreservação subsequente permite uma medição independente do tempo da bioenerg?...

As células mononucleares do sangue periférico humano (CMSP) são utilizadas para diversas aplicações em diversos campos científicos, incluindo o estudo de questões imunológicas e bioenergéticas, como aquelas relacionadas a processos de envelhecimento ou doenças degenerativas 1,2. As CMSP são heterogêneas em composição e consistem de linfócitos (células B, células T e células NK), monócitos e células dendríticas. As células às vezes mostram grandes diferenças individuais e variações dentro de um sujeito, então procedimentos padronizados para lidar com essas células são necessários. Parâmetros importantes como viabilidade e pureza do isolamento são os requisitos básicos para seu manuseio e são adicionalmente influenciados por fatores ambientais como o tempo de coleta, o nível de melatonina, se o indivíduo está em jejum, entre outros 3,4.
Com base em estudos sobre bioenergética de CMSP, descrevemos aqui um método para o isolamento, criopreserva e cultivo de CMSP que também é adequado para outros métodos. Enquanto a biópsia muscular é considerada o padrão-ouro para o metabolismo energético mitocondrial5, o exame das células sanguíneas é um procedimento rápido e minimamente invasivo. Além disso, cada vez mais estudos sugerem que as alterações da função mitocondrial no envelhecimento e na doença de Alzheimer (DA) ocorrem não só no cérebro, mas também na periferia6,7,8,9,10. O método também permite a investigação de outras condições e doenças, incluindo diabetes mellitus e obesidade11,12,13. Padrões de expressão gênica em pacientes com esclerose múltipla podem ser analisados, assim como a função imune e suas influências em geral 14,15,16.
As CMSP geralmente dependem da fosforilação oxidativa (OXPHOS) para gerar adenosina trifosfato (ATP)17,18. Portanto, os PBMCs cobrem uma ampla gama de aplicações como substitutos. Em relatos anteriores, o metabolismo energético das CMSP tem sido utilizado para tratar disfunções orgânicas, como na insuficiência cardíaca precoce19, choque séptico20 ou diferenças associadas ao sexo4 na função mitocondrial. Um método generalizado para isolamento criopreservacional e cultivo de CMSP teria vantagens na comparabilidade dos resultados obtidos em diferentes institutos. Há grande variação nos protocolos para cadaetapa21,22, o objetivo desse método é fornecer uma diretriz para medidas bioenergéticas em CMSP.
Neste artigo descrevemos um método para mensuração de parâmetros bioenergéticos em CMSP. Explicamos os métodos para isolar, criopinar e medir a bioenergética de CMSP do sangue humano. Este método pode ser usado para determinar parâmetros bioenergéticos em pacientes e avaliá-los em um contexto clínico. Para aplicar essas medidas, os pesquisadores precisam ter acesso a uma população de pacientes a partir da qual amostras de sangue fresco podem ser obtidas.

<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

As funções fisiológicas das células eucarióticas dependem da energia fornecida principalmente pelas mitocôndrias. A disfunção mitocondrial está ligada a doenças metabólicas e ao envelhecimento. A fosforilação oxidativa desempenha um papel decisivo, pois é crucial para a manutenção da homeostase energética. PBMCs foram identificados como uma amostra minimamente invasiva para medir a função mitocondrial e demonstraram refletir condições da doença. No entanto, a mensuração da função bioenergética mitocondrial pode ser limitada por vários fatores em amostras humanas. As limitações são a quantidade de amostras coletadas, o tempo de amostragem, que muitas vezes é distribuído por vários dias, e os locais. A criopreservação das amostras coletadas pode garantir a coleta e a medição consistentes das amostras. Deve-se tomar cuidado para garantir que os parâmetros medidos sejam comparáveis entre células criopreservadas e recém-preparadas. Aqui, descrevemos métodos de isolamento e criopreservação de CMSP de amostras de sangue humano para analisar a função bioenergética das mitocôndrias nessas células. As CMSP criopreservadas de acordo com o protocolo aqui descrito mostram apenas pequenas diferenças no número e viabilidade celular, nos níveis de trifosfato de adenosina e na atividade da cadeia respiratória medida em comparação com células recém-colhidas. Apenas 8-24 mL de sangue humano são necessários para as preparações descritas, tornando possível coletar amostras durante estudos clínicos multicentralmente e determinar sua bioenergética no local.

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.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Criopreservação e Avaliação Bioenergética de Células Mononucleares do Sangue Periférico Humano

With the help of this method, we want to describe a procedure which is suitable to cryopreserve PBMCs in such a way that after thawing, the bioenergetic parameters are unchanged. One of the major experimental challenges of cryopreservation is that the freezing and thawing process can damage PBMCs, which affects the following bioenergetic measurements. With our protocol, we try to show a way to preserve PBMCs reliably without damage to the cells so that the measured bioenergetic parameters are not influenced.The possibility of preserving PBMCs allows samples to be collected from different centers and measured at one location. It also makes measurement more time independent. Cryopreservation of PBMCs allows studies to be performed simultaneously at multiple sites where the cells are collected and preserved.The collected samples can then be measured centrally. This streamlines the process and planning of studies.

Viabilidade e número de células Para obter sucesso no isolamento e criopreservação, a contagem e a viabilidade celular devem ser as mais altas possíveis. Antes e depois da criopreservação, as células são contadas, e sua viabilidade é determinada para garantir a saúde e a qualidade das células. A Figura 3 é uma ilustração representativa das CMSP antes e após a criopreservação, a contagem de células e a viabilidade dificilmente diferem. Isso indica o isol...

Watch this Scientific Journal Video about Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation at JoVE.com

Células mononucleares isoladas do sangue periférico podem ser usadas para a análise de funções e distúrbios imunológicos, doenças metabólicas ou funções mitocondriais. Neste trabalho, descrevemos um método padronizado para o preparo de CMSP a partir de sangue total e posterior criopreservação. A criopreservação torna esse tempo e lugar independentes.

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

Com a ajuda deste método, queremos descrever um procedimento adequado para criopreservar CMSP de tal forma que, após o descongelamento, os parâmetros bioenergéticos permaneçam inalterados. Um dos grandes desafios experimentais da criopreservação é que o processo de congelamento e descongelamento pode danificar as CMSP, o que afeta as seguintes medidas bioenergéticas. Com nosso protocolo, tentamos mostrar uma maneira de preservar as CMSP de forma confiável sem danos às células para que os parâmetros bioenergéticos medidos não sejam influenciados.A possibilidade de preservação dos PBMCs permite que amostras sejam coletadas de diferentes centros e medidas em um único local. Também torna a medição mais independente do tempo. A criopreservação de CMSP permite que os estudos sejam realizados simultaneamente em múltiplos locais onde as células são coletadas e preservadas.As amostras coletadas podem então ser medidas centralmente. Isso agiliza o processo e o planejamento dos estudos.