Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

低温保存 生物能学 外周血单个核细胞 线粒体 氧化磷酸化 细胞生物学 生物 三磷酸腺苷 男科 免疫学 生物化学 医学 体外 胚胎
作者
Fabian Dieter,Jordon G. Grube,Tim Birkenhauer,Alice Quentin,Gunter P. Eckert
出处
期刊:Journal of Visualized Experiments [MyJoVE Corporation]
卷期号: (200)
标识
DOI:10.3791/65730
摘要

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.
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