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In Clinical Capabilities

What is PBMC? The peripheral blood mononuclear cell compartment

By Zara Puckrin, BSc / 10 August 2022


PBMC meaning: what does PBMC mean?

PBMC is short for "Peripheral Blood Mononuclear Cell". Many of our immune cells are PBMCs including lymphocytes, monocytes, and dendritic cells.1 They are characterized as having a round nucleus and being less dense than the other cells found in our peripheral circulation, such as red blood cells (RBCs) and granulocytes.1


Isolating PBMCs from whole blood

Protocols for the isolation of PBMCs from whole blood are well established. If you are isolating PBMC from a buffy coat, rather than whole blood samples, you'll want to look at this protocol instead.

Protocol for isolating PBMCs from whole blood

This protocol outlines the isolation of PBMCs from 10 ml of whole blood using a standard density gradient overlay process.2

1. Dilute whole blood with sterile PBS at a 1:1 v/v ratio.

2. Prepare a tube with density gradient medium (DGM) as per the manufacturer's instructions e.g., a 50 mL tube with 15 ml DGM.

3. Gently, overlay the diluted blood onto the DGM taking care not to mix the layers.

4. Centrifuge at 1000 x g for 20 mins at room temperature and with the brake off to generate distinct plasma, PBMC, DGM, and red blood cell (RBC) layers.

diluted-blood-dgm-centrifuged
5. Insert a pipette directly through the plasma layer and carefully harvest the PBMC layer via gentle aspiration and transfer it to a fresh 50 mL tube.

6. Add wash buffer (PBS+0.5% BSA) to the harvested PBMC to give a final volume of 45 mL and mix well.

7. Centrifuge at 400 xg for 10 mins at room temperature with the brake on.

8. Carefully, remove the supernatant to leave a PBMC pellet in the base of the tube.

9. Repeat wash and centrifugation (steps 7-8).

10. Add a final volume of 10 mL wash buffer and proceed to cell count/downstream use.


Identifying a high-quality PBMC composition

Table 1 shows the cell types contained within the PBMC compartment and their typical distribution in healthy adults. A high-quality PBMC isolate has a viability >90% and a low concentration of erythrocytes and granulocytes when processed fresh.3 Cell numbers and viability can be determined by manual or automated cell count or by using a flow cytometer.4 The latter can also be used to determine the percentage of PBMCs compared with other cell types (which should be >85%) using anti-mAb antibodies.4-5 If you do not have the capacity to isolate PBMCs, PBMC isolation can be outsourced to a central laboratory service provider.

Table 1: Average composition of PBMCs in a healthy adult. Adapted from Bittersohl et al (2016)1 

Cell Type

% Number of cells isolated from 1mL of blood (x103
T Cells 60 300-1200
    T Helper Cells     70 of T cells    210-840
    Cytotoxic T Cells     30 of T cells    90-360
Monocytes/Macrophages 15 75-300
B Cells 10 50-200
Natural Killer Cells 15 75-300

 


Downstream PBMC Processing

It may be necessary to subject PBMCs to further processing for quality control, freezing, or enrichment. Below we have outlined protocols for:

  • Counting PBMCs
  • Freezing PBMCs
  • Enriching cells from PBMCs
  • Enriching lymphocytes from PBVMCs

Protocol for counting PBMCs 

In this protocol6, we will describe how you can manually estimate the number of PBMCs in your sample using trypan blue and a hemocytometer or similar chamber. 

1. Prepare a 1:1 dilution of your cells with 0.4% trypan blue in a 2 mL tube.
- Pipette the solution up and down to mix

2. Incubate cells in this mixture for 2-3 minutes.
- Prepare the hemocytometer while the cells are incubating with the mixture

3. Transfer 10 µL into the counting chamber and count the number of alive (non-blue) cells and dead cells (blue) using a light microscope.
- Use this number to estimate the total number of viable cells in the original sample

Protocol for freezing PBMCs

You may wish to cryopreserve your PBMCs for future research or biobanking. The below protocol4 uses DMSO to store your cells >-150°C. Make sure to keep all of the components on ice throughout the freezing process.

1. Prepare the freezing medium at a ratio of 90% FBS and 10% DMSO.
- Pipette up and down to mix

2. Centrifuge the PBMCs to create a pellet. 
- Resuspend the pellet in the freezing medium at a concentration of 5x106 cells/mL

3. Distribute the mixture of cells and freezing medium into 1.5 mL cryovials.
- Transfer into a -80°C freezer

4. Take the cryovials out of the -80°C freezer the following day.
- Transfer them into liquid nitrogen for long-term storage

Protocol for enriching cells from PBMCs

Immunomagnatic labeling of target cells

Immunomagnetic labelling of target cells

External magnetic field is applied
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Arrow pointing right
Separation of distinct layers

Separation of distinct layers

Collection of target cells
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Arrow pointing right
Separation of target cells

Separation of target cells

This magnetic technique uses positive selection to deplete PBMCs you do not want in your sample. In this method, magnetic beads are conjugated to antibodies that will bind to the target cells. These are exposed to a magnetic field and the non-target cells are disposed of.4 

Protocol enriching lymphocytes from PBMCs

Lymphocytes, specifically monocytes, can be isolated from a PBMC sample based on their affinity for plastic. Our scientists have outlined the methodology for this non-magnetic isolation technique below.4,5

1. Pre-prepare a tissue culture flask with a suitable monocyte adherent solution e.g., gelatin

2. Add autologous plasma and incubate for 30-60 minutes at 37°C.

3. Pipette the PBMC cell suspension into the pre-coated flask at a concentration suitable for flask size.
- Incubate the flask for 40 minutes in a CO2 incubator at 37°C

4. Dispose of the non-adherent cells, then rinse the tissue culture flask gently with warm PBS.
- Repeat this washing step up to three times

5. Detach the adherent monocytes with PBS/5mM EDTA (1:1) and shake gently.
- Aspirate the cell suspension and transfer to a 50 mL conical tube

6. Centrifuge the suspension at 200 x g for 10 minutes at room temperature.
- Dispose of the supernatant and resuspend the pellet in complete culture medium at a concentration suitable for flask size
- Incubate the flask in a CO2 incubator at 37°C




Outsourcing work to a PBMC company

If you have a large number of PBMC samples to process or lack internal resources to process, you may want to consider outsourcing your cell isolation and enrichment requirements to a central laboratory service provider. To find a reputable partner, you should consider the following:

  • Relevant sample handling and processing experience 
  • 24/7 sample receipt and processing capacity
  • Location of facilities
  • Quality and project management systems

If you need further downstream processing, it is also useful to consider whether the company offers additional services to meet your project requirements. You can find out more information about our whole blood processing services here: Whole Blood and PBMC Processing Services →




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References

  1. Bittersohl et al. Intracellular concentrations of immunosuppressants. Personalized Immunosuppression in Transplantation (2016).
  2. Bøyum et al. Isolation of mononuclear cells and granulocytes from human blood (Paper IV). Scand J Clin Lab Invest, 97, 77–89 (1968).  
  3. Sahaf et al. Redox Cell Biology and Genetics Part B. Methods in Enzymology (2002).
  4. Virgilio et al. Tumour Immunology and Immunotherapy - Molecular Methods. Methods in Enzymology (2012).
  5. Mahajan et al. Nanomedicine. Methods in Enzymology (2012).
  6. Bidmon et al. Tumor Immunology and Immunotherapy - Cellular Methods Part A. Methods in Ezymology (2020).

Editors note: This protocol is for guidance only, based on freely available information and typical protocols used in labs worldwide. REPROCELL is not responsible for the results of any work using this protocol(s). 

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