人外周血单核细胞的冷冻保存与生物能量评价
Summary
分离的外周血单核细胞可用于分析免疫功能和疾病、代谢疾病或线粒体功能。在这项工作中，我们描述了一种从全血制备PBMC和随后冷冻保存的标准化方法。冷冻保存使这个时间和地点独立。
Abstract
真核细胞的生理功能主要依赖于线粒体提供的能量。线粒体功能障碍与代谢疾病和衰老有关。氧化磷酸化起着决定性的作用，因为它对于维持能量稳态至关重要。PBMC 已被确定为测量线粒体功能的微创样本，并已被证明可以反映疾病状况。然而，线粒体生物能量功能的测量可能受到人类样本中几个因素的限制。限制是采集的样本量、采样时间（通常分布在几天内）和位置。对收集的样品进行冷冻保存可以确保样品的一致收集和测量。应注意确保测得的参数在冷冻保存的细胞和新制备的细胞之间具有可比性。在这里，我们描述了从人类血液样本中分离和冷冻保存PBMC的方法，以分析这些细胞中线粒体的生物能量功能。与新鲜收获的细胞相比，根据此处描述的方案冷冻保存的PBMC在细胞数量和活力，三磷酸腺苷水平和测量的呼吸链活性方面仅显示出微小的差异。所述制剂只需要 8-24 mL 人血，因此可以在临床研究期间多中心收集样本并在现场测定其生物能量学。
Introduction
人外周血单核细胞 （PBMC） 用于许多科学领域的各种应用，包括研究免疫学和生物能量问题，例如与衰老过程或退行性疾病相关的问题 1,2。PBMC 的组成是异质的，由淋巴细胞（B 细胞、T 细胞和 NK 细胞）、单核细胞和树突状细胞组成。这些细胞有时会在受试者中表现出巨大的个体差异和变异，因此需要处理这些细胞的标准化程序。分离物的活力和纯度等重要参数是其处理的基本要求，并且还受到环境因素的影响，例如收集时间、褪黑激素水平、受试者是否禁食等 3,4。
基于对PBMCs生物能量学的研究，我们在此描述了一种适用于其他方法的PBMCs的分离、冷冻保存和培养方法。虽然肌肉活检被认为是线粒体能量代谢的金标准5，但血细胞检查是一种快速、微创的手术。除此之外，越来越多的研究表明，衰老和阿尔茨海默病（AD）中线粒体功能的变化不仅发生在大脑中，还发生在外周6,7,8,9,10。该方法还允许调查其他病症和疾病，包括糖尿病和肥胖症11,12,13。可以分析多发性硬化症患者的基因表达模式，或一般分析免疫功能及其影响14,15,16。
PBMC 通常依靠氧化磷酸化 （OXPHOS） 产生三磷酸腺苷 （ATP）17,18。因此，PBMC 作为替代物涵盖了广泛的应用。在以前的报道中，PBMC 的能量代谢已被用于解决器官功能障碍，例如早期心力衰竭19、感染性休克20 或线粒体功能的性别相关差异4。用于冷冻保存、分离和培养PBMC的通用方法在不同研究所获得的结果的可比性方面具有优势。每个步骤21,22的方案存在很大差异，该方法的目的是为PBMC中的生物能量测量提供指南。
在本文中，我们描述了一种测量PBMC中生物能量参数的方法。我们解释了从人血液中分离、冷冻保存和测量PBMC生物能量学的方法。该方法可用于确定患者的生物能量参数，并在临床环境中对其进行评估。为了应用这些测量方法，研究人员需要访问可以从中获得新鲜血液样本的患者群体。
Protocol
本手稿中描述的所有采血、分离和分析方案均已由德国吉森大学机构审查委员会审查和批准。获得了患者同意将他们的样本纳入研究。分离和细胞培养的所有步骤均在生物安全柜下进行。 1. 静脉穿刺 准备采血所需的所有设备，包括消毒喷雾、无菌拭子、带 80 毫米管和多功能适配器的采血插管、止血带/血压袖带、Monovette 9 mL 肝素锂。注意：EDTA作为抗凝…
Representative Results
细胞活力和数量为了实现成功的分离和冷冻保存，细胞计数和活力应尽可能高。在冷冻保存之前和之后，对细胞进行计数，并确定其活力，以确保细胞的健康和质量。 图 3 是冷冻保存前后 PBMC 的代表性说明，细胞计数和活力几乎没有差异。这表明PBMC已成功分离和保存。 <img alt="Figure 3" class="xfigimg" src="/files…
Discussion
该协议提供了一种以适合生物能量分析的方式从人血液中分离和冷冻保存外周血单核细胞（PBMC）的方法。所描述的方法提供了温和和大量分离PBMC的可能性，具有高活力和足够的细胞用于生物能量测量。它的缺点是，即使中断最小，也会发生长时间的分离，但随后的冷冻保存允许对生物能量学进行与时间无关的测量。使用这种方法，可以在以后的时间点收集和测量样品。它还适用于在多中心试验?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢吉森-马尔堡大学医院的临床团队的采血。这项工作由尤斯图斯·李比希大学资助。
Materials
| 0.1 M Triethanolamine-HCl-Buffer (pH = 8,0) | Self-prepared | – | |
| 0.5 M Triethanolamine-HCl-Buffer | Self-prepared | – | |
| 1.0 M Tris-HCl-Buffer (pH = 8,1) | Self-prepared | – | |
| 1.01 mM DTBB | Self-prepared | – | |
| 10 % Triton X-100 | Self-prepared | – | |
| 10 mM Oxalacetat | Self-prepared | – | |
| 14–20 G sterile blood draw needles Multi Adapter Sarstedt Safety-Multifly | Sarstedt | 156353_v | |
| 37% HCl | Carl Roth GmbH & Co. KG | – | |
| 70% Ethanol (EtOH) | Self-prepared | – | |
| Acetyl-CoA | Pancreac Applichem | A3753 | |
| ADP | Sigma-Aldrich | A5285 | |
| Alcohol wipes | (70% isopropyl alcohol) | ||
| Antimycin A | Sigma-Aldrich | A8674 | |
| Aqua (bidest.) | With MilliQ Academic (self-made) | – | |
| Ascorbate | Sigma-Aldrich | A4034 | |
| ATP-Standard | Sigma-Aldrich | 6016949 | |
| Biocoll Seperating Solution | Biochrom | 6115 | |
| Biological safty cabinet MSC Advantage | Thermo Fisher Scientific Inc. | ||
| Carbonylcyanid-p-trifluoromethoxy-phenylhydrazon (FCCP) | Sigma-Aldrich | C2920 | |
| Cell counter TC20 Automated Cell Counter | Bio-Rad | ||
| Centrifuge Heraeus Megafuge 16 R | Thermo Fisher Scientific Inc. | ||
| Counting slides, dual chamber for cell counter | Bio-Rad | 1450016 | |
| Cryotube Cryo.S | Grainer Bio-One | 126263-2DG | |
| Digitonin | Sigma-Aldrich | 37008 | |
| Dimethylsulfoxid (DMSO) | Merck | 102952 | |
| Disinfection spray | |||
| Disposable gloves latex, rubber, or vinyl. | |||
| Distrips (12.5 ml) DistriTips | Gilson | F164150 | |
| Dulbecco’s Phosphate Buffered Saline (DPBS; 10x) | Gibco (Thermo Scientific) | 15217168 | |
| Ethanol (EtOH 100%) | Carl ROTH GmbH & Co. KG | 9065.3 | |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F9665 | |
| Frezer (-80°C) | Thermo Fisher Scientific Inc. | ||
| Glutamate | Sigma-Aldrich | G1626 | |
| Holder/adapter | |||
| Incubator Midi 40 CO2 | Thermo Fisher Scientific Inc. | ||
| Injection syringe | Hamilton | ||
| Malate | Sigma-Aldrich | M-1000 | |
| MIR05 | Self-prepared | – | |
| Mr. Frosty Freezing Container | Thermo Fisher Scientific Inc. | 10110051 | |
| Multireader CLARIOstar | BMG Labtech | ||
| Nitrogen tank Locator 6 plus | Thermo Fisher Scientific Inc. | ||
| Oligomycin | Sigma-Aldrich | O4876 | |
| Oxalacetate | Sigma-Aldrich | – | |
| Oxygraph-2k | Orobororus Instruments | ||
| Penicillin-Streptomycin | PAA | 15140122 | |
| Pipettes Performance Pipettor 10 μL, 100 μL, 1000 μL | VWR | ||
| Roswell-Park. Memorial-Institute-Medium (RPMI-1640) | Gibco (Thermo Scientific) | 11530586 | |
| Rotenone | Sigma-Aldrich | R8875 | |
| Saccharose | Carl ROTH GmbH & Co. KG | 9286.2 | |
| Sodium azide | Sigma-Aldrich | S2002 | |
| Succinate | Sigma-Aldrich | S2378 | |
| Tetramethylphenylendiamin (TMPD) | Sigma-Aldrich | T3134 | |
| Tourniquet/ Blood pressure cuff | |||
| Tris(hydroxymethyl)amino-methane | Sigma-Aldrich | 108382 | |
| Triton X-100 | Sigma-Aldrich | 108643 | |
| Trypanblau | Biochrom | T6146 | |
| Vacuum pump | Vaccubrand GmbH & Co. | ||
| ViewPlate-96 | Perkin Elmer | 6005181 | |
| Water bath WNB22 | Memmert GmbH & Co. KG |
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Cite This Article
Dieter, F., Grube, J., Birkenhauer, T., Quentin, A., Eckert, G. P. Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells. J. Vis. Exp. (200), e65730, doi:10.3791/65730 (2023).