PBMC Isolation: Methods for the Isolation of White Blood Cells From Whole Blood

Peripheral Blood Mononuclear Cells (PBMC)

Peripheral blood is composed of plasma, red blood cells and granulocytes (neutrophils, basophils and eosinophils) … and Peripheral Blood Mononuclear Cells (PBMC) which represent about 1% of the blood.

PBMC is a group of cells  that carry out the functional immune response. In other words, PBMC are immune cells. PBMC are mainly composed of lymphocytes (~80% including T-cells, B-Cells and NK cells), monocytes (~10%) and dendritic cells. In presence of a pathogen, PBMC are actively participating to the immune response to eradicate the threat. 1 mL of blood from adults contains 0.5 to 3 x 10⁶ PBMCs (1.5 x 10⁶ in average).

PBMC Applications

Peripheral Blood Mononuclear Cells (PBMC) are widely used in immunology and toxicology research. They are easy to isolate and well representative of the global immune system.

They can be used to evaluate the toxicity of a drug candidate, to conduct bioassays such as specific T-cell activations followed by FACS measurements of their proliferation, to perform Mixed lymphocyte reaction (MLR), or to isolate antigen-presenting cells (APC) for antigen loading… PBMC are the starting material of many bioassays.

PBMC Isolation protocol

To be used in research laboratories, PBMCs must be isolated from peripheral blood samples. It is recommended to perform isolation on fresh blood collected less than 8 hours before.

PBMC are usually isolated from blood using a density gradient centrifugation using a medium of Ficoll or similar. The medium will separate blood components according their size. Erythrocytes will go down the tube, plasma will go up and PBMCs will constitute a thin layer between erythrocytes and plasma. This thin layer will then be collected manually with a pipette.

All reagents must be at room temperature.

Dilute blood in 2 volumes or more of saline buffer such as PBS or DPBS (Dulbecco’s Phosphate Buffered Saline with 2% Fetal Bovine Serum) and mix sample by repeated pipetting.

Add 15mL of Ficoll in another 50mL centrifuge tube then add slowly 15mL of bloo/PBS solution.

Centrifuge without the brake applied at 400 g x 30 min at 20°C using.

Use a pipette to aspirate the thin layer at the interface of plasma and gradient medium. Manipulate carefully to avoid layers mixing. Alternatively, aspirate the whole plasma layer slowly to remove it and then aspirate PBMCs at the surface.

Wash by transfering PBMCs in a new tube of 50 mL and fill it with PBS buffer (3 times volume) and centrifuge 300xg for 10 min at 20°C. Then remove the supernatant and repeat 1 time if necessary.

Add PBS or media to PBMC to reach the desired concentration. You can estimate the number of cells by considering that 1mL of initial blood contains 1.5 x 10⁶ mononuclear cells and aliquote according this.

PBMC storage by cryopreservation

To store Peripheral Blood Mononuclear Cells for later use, PBMC must be cryopreserved. Add PBMCs in an appropriate cryopreservation medium that protects cells after thawing (i.e. 10%DMSO+90% FBS or commercial cryopreservants)

Prepare 20% DMSO in FBS and keep it on ice.

Centrifuge PBMCs at 300xg for 10 minutes to obtain a cell pellet and remove the supernatant with a pipette.

Resuspend PBMCs in cold FBS to a concentration of 1 – 20 x 10⁶ cells/mL. Keep on ice.

Add an equal volume of pre-made solution of 20% DMSO in FBS to obtain a final solution of 10% DMSO + 90% FBS at a concentration of 0.5 – 10 x 10⁶ cells/mL.

Transfer the desired volume into cryovials and place in vapor phase liquid nitrogen (below -135°C). Keep on ice anytime during the preparation.