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Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)
使用Vacutainer®细胞制备管(CPTTM)分离外周血单核细胞   

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Abstract

Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that separates the Ficoll solution from the blood to be drawn. Once blood is drawn into CPTs, they can be centrifuged to separate the PBMC, then shipped (if desired) to a processing lab. The processing lab removes the PBMC from the upper compartment of the tube (above the gel plug), washes the PBMC, and can cryopreserve them using DMSO-containing media, as detailed in this protocol.

Keywords: PBMC(PBMC), Cell preparation(细胞制备), CPT(CPT), Ficoll(聚蔗糖), Blood(血液), Stabilization(稳定)

Background

Isolation and cryopreservation of peripheral blood mononuclear cells (PBMC) is common practice in clinical studies that employ cellular immune assays. Cryopreservation allows for batching of samples, which is convenient and improves the comparability of data. Cryopreservation also allows cells to be stored for unknown future purposes. Because erythrocytes and granulocytes are much more fragile to freezing and thawing, PBMC isolation is a common prerequisite to cryopreservation of blood cells; and the most common method for PBMC isolation is density gradient centrifugation using Ficoll-Hypaque, a high molecular weight carbohydrate solution.

Cellular Preparation Tubes (CPTs), which contain Ficoll-Hypaque, simplify the standard procedure for density gradient centrifugation in two ways: (1) blood is collected into the same tube that is then used to isolate the PBMC; and (2) the tube is pre-loaded with Ficoll-Hypaque, which is separated by a gel plug so that it is not disturbed by the entry of blood into the tube. After blood draw, the tubes are centrifuged, and the PBMCs and plasma become separated from the erythrocytes and granulocytes by the gel plug (see Figure 1). This allows the spun tubes to be shipped, maintaining the PBMC in an isolated environment from the erythrocytes and granulocytes, which may improve their viability and function.

CPTs are ideal for use in studies which collect whole blood across multiple sites and ship to a central processing laboratory (Ruitenberg et al., 2006); this reduces variability in PBMC isolation between technicians. Studies have found no significant difference in PBMCs isolated using the CPT system or by traditional layover methods via density gradient separation (Corkum et al., 2015; Ruitenberg et al., 2006). While material cost can be high, CPTs reduce the length of processing time in addition to decreasing inconsistency between operators; both of these are key to reducing cost and increasing sample quality and consistency. Furthermore, when used with studies or sites which ship blood samples overnight, PBMCs from CPTs have a higher purity and less infiltrate from other (contaminating) cell types, such as red blood cells, in contrast to samples shipped in standard blood collection tubes over a 24-48 h period (Schlenke et al., 1998).


Figure 1. Empty CPT (left), after blood draw (middle), and after centrifugation (right). Location of gel plug and sample layers after centrifugation are shown.

Materials and Reagents

  1. 1.8 ml Nunc (or similar) cryovials
  2. BD Vactuainer® Mononuclear Cell Preparation Tubes (CPTTM), 8 ml, with sodium heparin (BD, catalog number: 362753 )
  3. P10 and P200 pipette tips (any vendor)
  4. P10 and P200 mechanical pipettors (any vendor)
  5. 50 ml Falcon (or similar) conical polypropylene tubes
  6. Serological pipettes (assorted volumes)
  7. BioCision CoolCell® (BioCision, catalog number: BCS172 ) – alcohol free controlled-rate freezing container
  8. Foam packing material and shipping containers
  9. Phosphate buffer saline (PBS),Ca2+ and Mg2+ free (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  10. Trypan blue (e.g., GE Healthcare, HycloneTM, catalog number: SV30084.01 or Sigma-Aldrich, catalog number: 93595 )
  11. Human AB Serum (e.g., Valley Biomedical, catalog number: HP1022 )
  12. Dimethyl sulfoxide (e.g., Sigma-Aldrich, catalog number: D8418 )

Equipment

  1. A centrifuge capable of reaching speeds of 1,800 x g (e.g., Beckman Coulter, model: Allegra X-14 Series )
  2. 50 ml conical adapters and buckets for centrifuge
  3. Pipette gun (any vendor)
  4. Automated cell counter or microscope and hemacytometer (any vendor)
    Note: Cell counting methods vary in their throughput, cost, and flexibility for different cell types. For consistency, the same counting method should be used throughout a study.
  5. Biosafety cabinet level A2 (BSC) (any vendor)
  6. -80 °C freezer (for initial cryopreservation) (any vendor)
  7. Liquid nitrogen (LN2) freezer (for long-term cryopreservation) (any vendor)
    Note: LN2 systems range from small and simple to large and highly automated. Auto-filling and self-monitoring systems are recommended, especially for larger studies.

Procedure

  1. Aliquot tube preparation
    Print out labels containing the study name, study number, de-identified patient number and visit number, and paste on cryovials.
  2. Sample preparation
    1. Mix the blood by gentle inversion several times to ensure a homogeneous suspension.
    2. Centrifuge the tubes at 1,800 x g for 20 min (brake can be on) at room temperature. Be sure that the tubes can clear the rotor in swinging-bucket configurations; some tube locations can cause collision with the rotor and result in broken tubes.
    3. If shipping to another location, wrap in protective foam and ship via same-day or overnight courier to the processing lab at room temperature.
      Note: Shipping sample at 4 °C (or on wet ice), can result in platelet activation and other unwanted physiological and phenotypic changes.
  3. PBMC isolation
    1. In BSC, gently invert the CPT tubes several times, then pipette the plasma and PBMCs into a sterile 50 ml conical tube.
    2. Fill tube containing the plasma and PBMCs with PBS for a final volume of 50 ml in conical. Centrifuge at 250 x g for 10 min at room temperature with the brake on.
    3. Aspirate the supernatant carefully to not disturb cell pellet.
      1. A Pasteur pipette and vacuum can be used for aspirating as well as manually aspirating with serological pipettes and a pipette gun.
    4. Gently re-suspend the cells in 10 ml of PBS and remove an aliquot for cell counting. Use a 10 μl sample for a microscope using trypan blue exclusion, or amount needed for automated cell counter.
    5. After removing the aliquot for counting, fill the tube to final volume of 50 ml with PBS and centrifuge for 10 min at 250 x g with the brake on.
  4. Determining the number of aliquots
    1. Depending on your cell count methods (manual or automated counter), determine the total amount of cells. This will be used to calculate how many aliquots of PBMCs are frozen down.
    2. Cells should be frozen in approximately 107 cells per ml of freezing media; when the total cell count is divided by 107, the result is the total number of aliquots.
    3. Place the appropriate number of cryovials on ice or in a cooling rack in the 4 °C to begin chilling the vials in preparation for freezing.
  5. Determining the volume of freezing media
    1. Freezing media is broken into two parts: ‘A’ and ‘B’. Part ‘A’ is made up of 100% Human AB Serum and Part ‘B’ is made up of 80% Human AB Serum and 20% DMSO.
      Note: Human AB Serum must be heat inactivated prior to use.
    2. Your samples will be frozen in a 50:50 mixture of Part ‘A’ and Part ‘B’, totaling a final concentration of 10% DMSO.
    3. Each aliquot will have a total of 1 ml of freezing media. Divide your total number of aliquots in half to determine what volume of each media you will need.
    4. Keep both parts of the freezing media on ice (or at 4 °C) until use.
  6. Freezing PBMC ALiquots (for viable cryopreservation)
    1. Aspirate the supernatant from the cells and re-suspend them with the volume of ‘A’ calculated in step 5c.
    2. At an approximate rate of 1 drop per second, and while swirling the sample, add the necessary volume of ‘B’.
    3. Do not re-freeze unused ‘B’ media.
    4. After all the freezing media has been added, gently pipette the sample up and down a couple times to ensure re-suspension. Over pipetting can result in cell rupture.
      Note: It is important to be efficient through this process, as warming DMSO is toxic to the PBMCs.
    5. Add 1 ml of the cell suspension to each pre-chilled cryovial and place the vials in a CoolCell or Mr. Frosty.
    6. Place the cells in a -80 °C freezer for 24 h.
    7. After 24 h, remove cells to a long-term LN2 freezer.
      Note: PBMC can be kept for days or even weeks at -80 °C before transfer to LN2; however, there is progressive loss of viability and function with longer storage at -80 °C. Also, repeated cycling between -80 °C (or dry ice) and LN2 will negatively affect viability and function. Thus, if cells are to be shipped on dry ice, it’s preferable to store them short-term at -80 °C, ship, then move to LN2.

Data analysis

Typical assessment of a PBMC isolation protocol includes monitoring yield and purity. To some degree, the latter can be determined visually, as erythrocyte contamination will redden the pellet of PBMC acquired after centrifugation. This is sometimes seen with CPT, to a greater extent than traditional Ficoll protocols. However, these contaminating erythrocytes are lost after downstream procedures such as cryopreservation, and have not been observed to influence function (Ruitenberg et al., 2006).
Yield is easily determined by either manual or automated cell counting, and has also been reported to be roughly equivalent between CPT and manual Ficoll methods (Ruitenberg et al., 2006). An example is shown below in Figure 2 from one of our own lab’s studies.

Representative data



Figure 2. Comparison of cell recovery from CPTs versus conventional Ficoll procedure (using SepMate tubes, Stem Cell Technologies). Data were derived from two different studies, one drawing 4 x 8-cc CPT, the other 4 x 10-cc sodium heparin tubes. Because of the different maximum draw volumes, there is a wider range of blood volumes for the SepMate study; but recovered cells/ml were very similar with CPT and SepMate tubes. Only samples in good condition (no visible clumping or erythrocyte contamination) with > 20-cc blood volume were included for comparison.

Notes

  1. Shipping Temperature. When using CPT in the context of a multisite study, we have seen that gel plugs can loosen and the cell separation can fail when tubes become too cold during shipping. For this reason, an insulated shipping container is highly recommended, especially in cold winter climates.
  2. Consistency of centrifugation step. Similarly, when multiple sites are involved, it is critical to ensure success that the correct centrifugation G force is calculated and implemented across sites with potentially different instrumentation. Both spin time and G force are important to success with the procedure.

Acknowledgments

We thank BD Biosciences for development of the CPT method, on which this protocol is based.

References

  1. Corkum, C., Ings, D., Burgess, C., Karwowska, S., Kroll, W. and Michalak, T. (2015). Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPTTM) and standard density gradient. BMC Immunology 16:48.
  2. Ruitenberg, J. J., Mulder, C. B., Maino, V. C., Landay, A. L. and Ghanekar, S. A. (2006). VACUTAINER CPT and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples. BMC Immunol 7: 11.
  3. Schlenke, P., Klüter, H., Müller-Steinhardt, M., Hammers, H. J., Borchert, K. and Bein, G. (1998). Evaluation of a novel mononuclear cell isolation procedure for serological HLA typing. Clin Diagn Lab Immun 5(6): 808-813.

简介

外周血单核细胞(PBMC)分离通常通过Ficoll-Hypaque密度梯度离心来进行,Ficoll-Hypaque是一种劳动密集型程序,需要熟练的技术人员并且可以有助于样品变异性。细胞制备管(CPT)是含有Ficoll-Hypaque的Vacutainer采血管和将Ficoll溶液与待吸取血液分离的凝胶塞。一旦血液吸入CPT,就可将其离心分离PBMC,然后运送(如果需要)到加工实验室。处理实验室从管的上部隔室(凝胶塞上)除去PBMC,洗涤PBMC,并可以使用含DMSO介质冷冻保存,如本方案所述。

背景 外周血单核细胞(PBMC)的分离和低温保存在采用细胞免疫测定的临床研究中是常见的做法。冷冻保存允许样品的批量化,这是方便的并且提高了数据的可比性。冷冻保存还允许将细胞存储在将来未知的未知目的。因为红细胞和粒细胞比冻融融化要脆弱得多,所以PBMC分离是血液细胞冷冻保存的常见先决条件。 PBMC分离最常见的方法是使用高分子量碳水化合物溶液Ficoll-Hypaque进行密度梯度离心。
 含有Ficoll-Hypaque的细胞制备管(CPT)以两种方式简化了密度梯度离心的标准方法:(1)将血液收集到同一管中,然后用于分离PBMC;和(2)管预装有Ficoll-Hypaque,其由凝胶塞分离,使得其不会被血液进入管中的干扰。抽血后,离心管,PBMC和血浆通过凝胶塞与红细胞和粒细胞分离(见图1)。这样可以运送纺丝管,将PBMC保持在与红细胞和粒细胞分离的环境中,这可以提高其生存力和功能。
  CPT适用于在多个地点收集全血并运送到中央加工实验室的研究(Ruitenberg et al。,2006);这减少了技术人员之间PBMC隔离的变异性。研究发现,使用CPT系统分离的PBMCs或通过密度梯度分离法(Corkum等人,2015年; Ruitenberg等人,2006)分离的PBMCs没有显着差异)。虽然材料成本可能高,但CPT减少了处理时间的长短,同时减少了操作员之间的不一致;这两者都是降低成本和提高样品质量和一致性的关键。此外,当与用于血液样品过夜的研究或部位一起使用时,来自CPT的PBMC具有比其他(污染)细胞类型(例如红细胞)更高的纯度和更少的渗透,与在标准采血管中运送的样品相反, 24-48小时期间(Schlenke等人,1998)。


图1.空CPT(左),抽血(中),离心后(右)。显示离心后凝胶塞和样品层的位置。

关键字:PBMC, 细胞制备, CPT, 聚蔗糖, 血液, 稳定

材料和试剂

  1. 1.8毫升Nunc(或类似的)冰箱
  2. BD Vactuainer 单核细胞制备管(CPT TM ),8ml用肝素钠(BD,目录号:362753)
  3. P10和P200移液器吸头(任何供应商)
  4. P10和P200机械移液器(任何供应商)
  5. 50ml Falcon(或类似)锥形聚丙烯管
  6. 血清移液管(各种体积)
  7. BioCision CoolCell ®(BioCision,目录号:BCS172) - 无酒精控制速率的冷冻容器
  8. 泡沫包装材料和运输集装箱
  9. 磷酸盐缓冲盐水(PBS),Ca 2+ +和/或不含游离的(例如,Thermo Fisher Scientific,Gibco TM) sup>,目录号:10010-023)
  10. 台盼蓝(例如,GE Healthcare,Hyclone TM,目录号:SV30084.01或Sigma-Aldrich,目录号:93595)
  11. 人AB血清(例如,,Valley Biomedical,目录号:HP1022)
  12. 二甲基亚砜(例如,Sigma-Aldrich,目录号:D8418)

设备

  1. 能够达到1,800 x g(例如Beckman Coulter,型号:Allegra X-14系列)的速度的离心机
  2. 50ml锥形适配器和离心机桶
  3. 移液枪(任何供应商)
  4. 自动细胞计数器或显微镜和血细胞计数器(任何供应商)
    注意:细胞计数方法的不同细胞类型的吞吐量,成本和灵活性有所不同。为了一致性,在整个研究中应该使用相同的计数方法。
  5. 生物安全柜A2级(BSC)(任何供应商)
  6. -80°C冷冻机(用于初始冷冻保存)(任何供应商)
  7. 液氮(LN 2)冷冻器(用于长期冷冻保存)(任何供应商)
    注意:LN 2 系统范围从小而简单到高度自动化。推荐使用自动填充和自我监测系统,特别适用于较大的研究。

程序

  1. 等分试管准备
    打印包含研究名称,研究编号,未确定的患者编号和访问号码的标签,并粘贴在冰箱上。
  2. 样品制备
    1. 通过温和倒置几次混合血液以确保均匀的悬浮液。
    2. 在室温下以1,800×g离心管20分钟(制动器可以打开)。确保管子可以摆动铲斗配置清除转子;一些管道位置可能导致与转子碰撞并导致破裂的管道。
    3. 如果运送到另一个地点,请保护泡沫,并通过同一天或隔夜快件在室温下运送到加工实验室。
      注意:在4°C(或湿冰)下运送样品可导致血小板活化和其他不必要的生理和表型变化。
  3. PBMC隔离
    1. 在BSC中,轻轻地将CPT管反转数次,然后将血浆和PBMC移液到无菌的50ml锥形管中。
    2. 用PBS填充含有血浆和PBMC的管,最终体积为50ml。在室温下以250g离心10分钟,制动器打开。
    3. 小心吸出上清液,不会干扰细胞沉淀。
      1. 巴斯德吸管和真空可用于抽吸以及用血清移液管和移液枪进行手动吸气。
    4. 轻轻地将细胞重新悬浮在10ml PBS中,并取出等分试样以进行细胞计数。使用台盼蓝排除的显微镜使用10μl样品,或自动细胞计数器所需的量。
    5. 取出等分试样进行计数后,用PBS将管填充至最终体积为50ml,并用制动器在250 x离心10分钟。
  4. 确定等分试样的数量
    1. 根据您的细胞计数方法(手动或自动计数器),确定细胞的总量。这将用于计算多少等分的PBMCs被冻结。
    2. 细胞应该在每毫升冷冻介质中约10个细胞/个细胞中冷冻;当总细胞计数除以10 7 时,结果是等分试样的总数。
    3. 将适当数量的冰箱放在冰上或在4°C的冷却架上开始冷冻小瓶以准备冻结。
  5. 确定冷冻介质的体积
    1. 冷冻媒体分为两部分:"A"和"B"。部分'A'由100%人AB血清组成,部分'B'由80%人AB血清和20%DMSO组成。
      注意:使用前必须先将人AB血清热灭活。
    2. 您的样品将在部分'A'和部分'B'的50:50混合物中冷冻,总计最终浓度为10%DMSO。
    3. 每个等分试样将具有1ml的冷冻介质。将您的总数等于一半,以确定您需要的每个媒体的数量。
    4. 将冰箱的两部分放在冰上(或4℃),直到使用。
  6. 冷冻PBMC分批(用于可行的冷冻保存)
    1. 从细胞中吸出上清液,并在步骤5c中计算出的体积"A"重新悬浮它们。
    2. 以大约每秒1滴的速度,在样品旋转的同时,添加必要的"B"体积。
    3. 不要重新冻结未使用的"B"媒体。
    4. 加入所有的冷冻介质后,将样品上下移动几次,以确保重新悬浮。超移液会导致细胞破裂。
      注意:通过这一过程,通过加热DMSO对PBMC有毒性,这一点很重要。
    5. 将1ml细胞悬浮液加入每个预先冷冻的冷冻瓶中,并将小瓶放入CoolCell或Frosty先生。
    6. 将细胞置于-80°C冷冻箱中24 h。
    7. 24小时后,将细胞移至长期的LN 2冷冻箱。
      注意:PBMC可以在-80°C保存数天甚至数周,然后转移到LN 2 ;然而,在-80℃下,存储和存储功能逐渐丧失。而且,-80℃(或干冰)和LN 2之间的重复循环将对生存力和功能产生不利影响。因此,如果电池要在干冰上运输,最好将它们短时间存放在-80°C,然后运送到LN <2>。

数据分析

PBMC分离方案的典型评估包括监测产量和纯度。在某种程度上,后者可以视觉上确定,因为红细胞污染将使离心后获得的PBMC颗粒变红。有时在CPT中可以看到这一点,比传统的Ficoll协议更大。然而,这些污染性红细胞在下游操作如冷冻保存后会丢失,并没有被观察到影响功能(Ruitenberg等人,2006)。
产量可以通过手动或自动细胞计数轻松确定,并且据报道在CPT和手动Ficoll方法之间大致相当(Ruitenberg等人,2006)。从我们自己实验室的一个研究中,图2中显示了一个例子。

代表数据



图2.来自CPT的细胞恢复与常规Ficoll程序的比较(使用SepMate管,Stem Cell Technologies)。 数据来自两个不同的研究,一个绘制4 x 8 cc CPT,另外4 x 10 cc肝素钠管。由于不同的最大抽取体积,SepMate研究的血容量范围更广;但回收细胞/ml与CPT和SepMate管非常相似。只有样品处于良好状态(没有可见的聚集或红细胞污染),具有>包括20cc血容量进行比较。

笔记

  1. 运输温度当在多站点研究的背景下使用CPT时,我们已经看到,当管子在运输过程中变得太冷时,凝胶塞会松动,并且细胞分离可能会失败。因此,强烈推荐使用绝缘运输容器,特别是在寒冷的冬季气候中
  2. 离心步骤一致。类似地,当涉及多个位点时,确保成功的关键是在具有潜在不同仪器的站点上计算和实施正确的离心力G力。旋转时间和G力都是成功的重要手段。

致谢

我们感谢BD Biosciences开发本协议所基于的CPT方法。

参考文献

  1. Corkum,C.,Ings,D.,Burgess,C.,Karwowska,S.,Kroll,W.and Michalak,T。(2015)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/26307036"target ="_ blank">由Vacutainer细胞制备管(CPT TM)分离的人PBMC的免疫细胞亚群及其基因表达谱,以及标准密度梯度。
    BMC Immunology 16:48。
  2. Ruitenberg,JJ,Mulder,CB,Maino,VC,Landay,AL和Ghanekar,SA(2006)。  VACUTAINER CPT和Ficoll密度梯度分离在维持来自HIV血清阳性血液样品的PBMC的质量和功能方面相当地进行。 BMC Immunol 7:11 。
  3. Schlenke,P.,Klüter,H.,Müller-Steinhardt,M.,Hammers,HJ,Borchert,K.and Bein,G。(1998)。评估血清学HLA分型的新型单核细胞分离程序。临床诊断实验室免疫 5(6):808-813。
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引用:Puleo, A., Carroll, C., Maecker, H. and Gupta, R. (2017). Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM). Bio-protocol 7(2): e2103. DOI: 10.21769/BioProtoc.2103.
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