혈액 채취에 대해 Giessen-Marburg 대학병원의 임상팀에 감사드립니다. 이 작업은 Justus Liebig 대학에서 자금을 지원했습니다.

미토콘드리아 생물 에너지 분석을 위한 PBMC 수집 및 냉동 보관

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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. 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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. 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저자는 이해 상충이 없음을 선언합니다.

이 프로토콜은 생체 에너지 분석에 적합한 방식으로 인간 혈액에서 말초 혈액 단핵 세포(PBMC)를 분리하고 동결 보존하는 수단을 제공합니다. 설명된 방법은 PBMC를 부드럽게 대량으로 분리할 수 있는 가능성을 제공하며, 높은 생존율과 생체 에너지 측정을 위한 충분한 세포를 제공합니다. 최소한의 중단에도 긴 분리가 발생하지만 후속 동결 보존을 통해 생체 에너지학의 시간 독립적인 측정이 가능...

인간 말초 혈액 단핵 세포(PBMC)는 노화 과정이나 퇴행성 질환과 관련된 면역 및 생체 에너지 문제 연구를 포함하여 많은 과학 분야의 다양한 응용 분야에 사용됩니다 1,2. PBMC는 구성이 이질적이며 림프구(B 세포, T 세포 및 NK 세포), 단핵구 및 수지상 세포로 구성됩니다. 세포는 때때로 피험자 내에서 큰 개인차와 편차를 보이기 때문에 이러한 세포를 다루기 위한 표준화된 절차가 필요합니다. 분리물의 생존력 및 순도와 같은 중요한 매개변수는 취급을 위한 기본 요구 사항이며 수집 시간, 멜라토닌 수치, 피험자의 공복 여부 등과 같은 환경 요인의 영향을 추가로 받습니다 3,4.
PBMC의 생체 에너지학에 대한 연구를 바탕으로 여기서는 다른 방법에도 적합한 PBMC의 분리, 동결 보존 및 배양 방법을 설명합니다. 근육 생검은 미토콘드리아 에너지 대사의 황금 표준으로 간주되지만5 혈액 세포 검사는 빠르고 최소 침습적인 절차입니다. 이 외에도 점점 더 많은 연구에서 노화와 알츠하이머병(AD)의 미토콘드리아 기능 변화가 뇌뿐만 아니라 말초에서도 발생한다는 것을 시사합니다 6,7,8,9,10. 이 방법은 또한 당뇨병 및 비만을 포함한 다른 상태 및 질병에 대한 조사를 가능하게 한다 11,12,13. 다발성 경화증 환자의 유전자 발현 패턴은 일반적으로 면역 기능 및 영향을 분석할 수 있습니다 14,15,16.
PBMC는 일반적으로 산화적 인산화(OXPHOS)에 의존하여 아데노신 삼인산(ATP)17,18을 생성합니다. 따라서 PBMC는 대리자로서 광범위한 응용 분야를 포괄합니다. 이전 보고에서, PBMC의 에너지 대사는 미토콘드리아 기능의 초기 심부전19, 패혈성 쇼크20 또는 성별 관련 차이4와 같은 장기 기능 장애를 해결하는 데 사용되었다. PBMC의 동결 보존 분리 및 배양을 위한 일반화된 방법은 다른 기관에서 얻은 결과의 비교 가능성에 이점이 있습니다. 각 단계(21,22)에 대한 프로토콜에는 많은 변이가 있으며, 이 방법의 목표는 PBMC에서 생체 에너지 측정에 대한 지침을 제공하는 것이다.
이 기사에서는 PBMC에서 생체 에너지 파라미터를 측정하는 방법에 대해 설명합니다. 인간 혈액에서 PBMC의 생체 에너지를 분리, 동결 보존 및 측정하는 방법을 설명합니다. 이 방법은 환자의 생체 에너지 매개변수를 결정하고 임상 맥락에서 평가하는 데 사용할 수 있습니다. 이러한 측정을 적용하기 위해 연구자들은 신선한 혈액 샘플을 얻을 수 있는 환자 집단에 접근할 수 있어야 합니다.

<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

진핵 세포의 생리적 기능은 주로 미토콘드리아가 제공하는 에너지에 의존합니다. 미토콘드리아 기능 장애는 대사 질환 및 노화와 관련이 있습니다. 산화적 인산화는 에너지 항상성 유지에 중요하기 때문에 결정적인 역할을 합니다. PBMC는 미토콘드리아 기능을 측정하기 위한 최소 침습 샘플로 확인되었으며 질병 상태를 반영하는 것으로 나타났습니다. 그러나 미토콘드리아 생체 에너지 기능의 측정은 인간 샘플의 여러 요인에 의해 제한될 수 있습니다. 제한 사항은 채취한 샘플의 양, 종종 며칠에 걸쳐 분산되는 샘플링 시간 및 위치입니다. 수집된 샘플의 냉동 보존은 샘플의 일관된 수집 및 측정을 보장할 수 있습니다. 측정된 매개변수가 냉동 보존된 세포와 새로 준비된 세포 간에 비교할 수 있도록 주의해야 합니다. 여기에서는 이러한 세포에서 미토콘드리아의 생체 에너지 기능을 분석하기 위해 인간 혈액 샘플에서 PBMC를 분리하고 냉동 보존하는 방법을 설명합니다. 여기에 설명된 프로토콜에 따라 동결 보존된 PBMC는 갓 수확한 세포와 비교하여 세포 수 및 생존력, 아데노신 삼인산 수치 및 측정된 호흡 사슬 활성에서 약간의 차이만 보여줍니다. 설명된 제제에는 8-24mL의 인간 혈액만 필요하므로 임상 연구 중에 다중앙에서 샘플을 수집하고 현장에서 생체 에너지를 측정할 수 있습니다.

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.

저자 스포트라이트: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (냉동 보존 후 말초 혈액 단핵 세포의 생체 에너지 매개변수 보존) 

인간 말초 혈액 단핵 세포의 냉동 보존 및 생체 에너지 평가

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.

세포 생존율 및 수 성공적인 분리 및 동결 보존을 달성하려면 세포 수와 생존율이 최대한 높아야 합니다. 냉동 보존 전후에 세포를 계수하고 세포의 건강과 품질을 보장하기 위해 생존율을 결정합니다. 그림 3 은 냉동 보존 전과 후의 PBMC를 대표적으로 나타낸 것으로, 세포 수와 생존율은 거의 차이가 없습니다. 이는 PBMC의 성공적인 격리 및 보존을 나타냅니다....

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

분리된 말초 혈액 단핵 세포는 면역 기능 및 장애, 대사 질환 또는 미토콘드리아 기능의 분석에 사용할 수 있습니다. 이 연구에서는 전혈에서 PBMC를 준비하고 후속 냉동 보존을 위한 표준화된 방법을 설명합니다. 냉동 보존은 이 시간과 장소를 독립적으로 만듭니다.

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

이 방법의 도움으로 해동 후 생체 에너지 매개변수가 변경되지 않는 방식으로 PBMC를 동결 보존하는 데 적합한 절차를 설명하고자 합니다. 냉동 보존의 주요 실험 과제 중 하나는 냉동 및 해동 과정이 PBMC를 손상시킬 수 있으며, 이는 다음과 같은 생체 에너지 측정에 영향을 미친다는 것입니다. 당사의 프로토콜을 통해 측정된 생체 에너지 매개변수가 영향을 받지 않도록 세포에 손상을 주지 않고 PBMC를 안정적으로 보존하는 방법을 보여주려고 합니다.PBMC를 보존할 수 있기 때문에 여러 센터에서 샘플을 수집하고 한 위치에서 측정할 수 있습니다. 또한 측정 시간을 더 독립적으로 만듭니다. PBMC의 냉동 보존을 통해 세포를 수집하고 보존하는 여러 부위에서 동시에 연구를 수행할 수 있습니다.수집된 샘플은 중앙에서 측정할 수 있습니다. 이를 통해 연구 프로세스와 계획을 간소화할 수 있습니다.

cryopreservation-bioenergetic-evaluation-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

842 조회수.  Justus-Liebig-University of Giessen. 이 방법의 도움으로 해동 후 생체 에너지 매개변수가 변경되지 않는 방식으로 PBMC를 동결 보존하는 데 적합한 절차를 설명하고자 합니다. 냉동 보존의 주요 실험 과제 중 하나는 냉동 및 해동 과정이 PBMC를 손상시킬 수 있으며, 이는 다음과 같은 생체 에너지 측정에 영향을 미친다는 것입니다. 당사의 프로토콜을 통해 측정된 생체 에너지 매개변수가 영향을 받지 않도록 세포?...

비디오: 저자 스포트라이트: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (냉동 보존 후 말초 혈액 단핵 세포의 생체 에너지 매개변수 보존) 

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.

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.

연구

면역학 및 감염병학

Isolation and Cryopreservation of Human Peripheral Blood Mononuclear Cells for Bioenergetic Studies

To begin, add eight milliliters of DPBS into a sterile 50 milliliter conical tube. To this, add eight milliliters of blood and use a three milliliter plastic Pasteur pipette to mix the contents well. Add 15 milliliters of lymphocyte isolation medium into another conical tube.Tilt the tube by 20 to 30 degrees. Then use a three milliliter Pasteur pipette to apply the first layer of the blood DPBS mix gently over the isolation medium. Layer the remaining mixture carefully over the sidewall of the tube.Slowly bring the tube into an upright position to layer the remaining blood onto the blood layer. Centrifuge the tube at 1000G for 10 minutes at room temperature in a swinging bucket with the brakes off. The blood PBMC mix will separate into four distinct layers.With a Pasteur pipette, remove two thirds of the plasma layer, then use a one milliliter pipette to collect the PBMCs. Transfer the PBMCs into a new 50 milliliter tube until the entire layer has been harvested. Then make the volume up to 25 milliliters with DPBS to remove residual isolation medium and other residues.Centrifuge the tube at 100G for 10 minutes at room temperature with the brakes on. Then remove the supernatant with a vacuum pump. Next, resuspend the pellet in one milliliter DPBS and add more DPBS to make up the volume to 25 milliliters to wash and resuspend the pellet.Cool a freezing container to four degrees celsius. Then place fetal bovine serum on ice. Resuspend the cell pellet containing the isolated PBMCs in one milliliter of fetal bovine serum.Next mixed DMSO with cooled serum on ice at a ratio of one to five. Transfer the cell suspension to two milliliter labeled cryotubes, using one tube per collection tube. With an automated cell counter, determine the cell count and viability.Use a one milliliter pipette to drop one milliliter of the DMSOFBS mixture into the tube. Then place the tubes in the pre-cooled freezing container. Place the container in a freezer at minus 80 degrees Celsius for 24 hours.The next day, remove the tubes from the freezer and store them in the gas phase of liquid nitrogen. Cell counts and viability were consistent before and after cryo-preservation indicating successful isolation and preservation.