Published: Vol 7, Iss 2, Jan 20, 2017 DOI: 10.21769/BioProtoc.2103 Views: 37016
Reviewed by: Emmanuel ZavalzaMartin V KolevAnonymous reviewer(s)
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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: PBMCBackground
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
Equipment
Procedure
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
Acknowledgments
We thank BD Biosciences for development of the CPT method, on which this protocol is based.
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Puleo, A., Carroll, C., Maecker, H. T. and Gupta, R. (2017). Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM). Bio-protocol 7(2): e2103. DOI: 10.21769/BioProtoc.2103.
Category
Immunology > Immune cell isolation > Leukocyte
Cell Biology > Cell isolation and culture > Cell isolation
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