isolation and cryopreservation of human peripheral blood mononuclear cells for bioenergetic studies

用于线粒体生物能量学分析的 PBMC 采集和低温储存

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.

作者聚焦：冷冻保存后外周血单核细胞中生物能量参数的保存 

人外周血单核细胞的冷冻保存与生物能量评价

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.

Watch this Scientific Journal Video about Isolation and Cryopreservation of Human Peripheral Blood Mononuclear Cells for Bioenergetic Studies at JoVE.com

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

用于生物能量研究的人外周血单核细胞的分离和冷冻保存

首先，将 8 毫升 DPBS 加入无菌 50 毫升锥形管中。为此，加入 8 毫升血液，并使用 3 毫升塑料巴斯德移液管将内容物充分混合。将 15 毫升淋巴细胞分离培养基加入另一个锥形管中。将管子倾斜 20 到 30 度。然后使用三毫升巴斯德移液管将第一层血液DPBS混合物轻轻涂抹在隔离培养基上。将剩余的混合物小心地铺在管的侧壁上。慢慢地将管子直立，将剩余的血液分层到血液层上。在室温下将试管在摆动桶中以1000G离心10分钟，并关闭制动器。血液PBMC混合物将分为四个不同的层。使用巴斯德移液管，取出三分之二的血浆层，然后使用一毫升移液管收集PBMC。 将PBMC转移到新的50毫升管中，直到收获整个层。然后用 DPBS 使体积达到 25 毫升，以去除残留的隔离介质和其他残留物。在室温下以100G离心管10分钟，并打开制动器。然后用真空泵除去上清液。接下来，将沉淀重悬于一毫升 DPBS 中，并加入更多 DPBS 以弥补体积至 25 毫升以清洗并重悬沉淀。将冷冻容器冷却至四摄氏度。然后将胎牛血清放在冰上。将含有分离的PBMC的细胞沉淀重悬于一毫升胎牛血清中。接下来，将DMSO与冷却的血清以1：5的比例在冰上混合。将细胞悬液转移到两毫升标记的冷冻管中，每个收集管使用一个管。使用自动细胞计数仪，确定细胞计数和活力。使用一毫升移液管将一毫升DMSOFBS混合物滴入试管中。然后将试管放入预冷的冷冻容器中。将容器放入零下 80 摄氏度的冰箱中 24 小时。第二天，将试管从冰箱中取出并将它们储存在液氮的气相中。冷冻保存前后的细胞计数和活力一致，表明分离和保存成功。

isolation-cryopreservation-human-peripheral-blood-mononuclear-cells

Research

JoVE Journal

Immunology and Infection

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

204 次观看.  Justus-Liebig-University of Giessen. 首先，将 8 毫升 DPBS 加入无菌 50 毫升锥形管中。为此，加入 8 毫升血液，并使用 3 毫升塑料巴斯德移液管将内容物充分混合。将 15 毫升淋巴细胞分离培养基加入另一个锥形管中。将管子倾斜 20 到 30 度。然后使用三毫升巴斯德移液管将第一层血液DPBS混合物轻轻涂抹在隔离培养基上。将剩余的混合物小心地铺在管的侧壁上。慢慢地将管子直立，将剩余的血液分层到?...

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

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.

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 ...

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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.