Materials and Reagents

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

Equipment

A centrifuge capable of reaching speeds of 1,800 x g (e.g., Beckman Coulter, model: Allegra X-14 Series )
50 ml conical adapters and buckets for centrifuge
Pipette gun (any vendor)
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.
Biosafety cabinet level A2 (BSC) (any vendor)
-80 °C freezer (for initial cryopreservation) (any vendor)
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

Aliquot tube preparation
Print out labels containing the study name, study number, de-identified patient number and visit number, and paste on cryovials.
Sample preparation

Mix the blood by gentle inversion several times to ensure a homogeneous suspension.
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.
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.

PBMC isolation

In BSC, gently invert the CPT tubes several times, then pipette the plasma and PBMCs into a sterile 50 ml conical tube.
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.
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.
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.
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.

Determining the number of aliquots

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.
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.
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.

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.
Freezing PBMC ALiquots (for viable cryopreservation)

Aspirate the supernatant from the cells and re-suspend them with the volume of ‘A’ calculated in step 5c.
At an approximate rate of 1 drop per second, and while swirling the sample, add the necessary volume of ‘B’.
Do not re-freeze unused ‘B’ media.
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.
Add 1 ml of the cell suspension to each pre-chilled cryovial and place the vials in a CoolCell or Mr. Frosty.
Place the cells in a -80 °C freezer for 24 h.
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

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.
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

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 (CPT^TM) and standard density gradient. BMC Immunology 16:48.
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.
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.