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Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

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In vitro Culture of Human PBMCs

Immunology > Immune cell isolation > Lymphocyte
Authors: Santosh K Panda
Santosh K PandaAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page: a221
 and Balachandran Ravindran
Balachandran RavindranAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence: ravindran8@gmail.com
Bio-protocol author page: a222
Vol 3, Iss 3, 2/5/2013, 45539 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.322

[Abstract] Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

Materials and Reagents

  1. Freshly collected heparinised blood
  2. Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771 )
  3. Sterile PBS or Dulbecco's modified eagle medium (DMEM) (catalog number: P-04-03590 )
  4. Pencillin-streptomycin solution (Sigma- Aldrich, catalog number: P4333 )
  5. W. B. C. diluting fluid (Qualigen, catalog number: 42425 )
  6. Fetal bovine serum (Pan Biotech, catalog number: 1302-P100402 )
  7. Trypan blue (Mediatech, catalog number: 193 )
  8. DMEM supplemented with 1% of Pencillin-streptomycin solution and 10% FBS (see Recipes)

Equipment

  1. Centrifuge machine with swing-out bucket rotors (Eppendorf, catalog number: 5810 R )
  2. Heparin vials (BD Biosciences, catalog number: 367886 )
  3. Sterile 15 ml centrifuge tube
  4. Auto pipettes
  5. 200 µl and 1 ml tips
  6. 24 well cell culture plate (TPP Techno Plastic Products, catalog number: 20090123 )
  7. Haemocytometer
  8. Tissue culture hood
  9. CO2 incubator
  10. Microscope

Procedure

  1. Collect about 4 ml of human venous blood sample in heparinised vials and mix well by gently inverting the tubes several times.
  2. Isolate human PBMCs by gradient centrifugation using Ficoll-Histophaque.
  3. Wash cells (centrifuge at 100 x g for 10 min) with 10 ml of sterile DMEM (without FBS) twice.
    Note: Cold DMEM is not used routinely for washing lymphocytes from culture cavities while setting up cultures. Rather when monocytes bound tightly to plastic cavities are needed to be harvested pre-chilled DMEM can be used.
  4. Discard medium and re-suspend the cell pellet in 1 ml of sterile Dulbecco's modified eagle medium.
  5. Count cells by haemocytometer using W.B.C. diluting fluid: Add 10 µl of cell suspension to 190 µl of W.B.C. diluting fluid and mix well. Load the cell suspension in a haemocytometer and count the cells. Adjust cell concentration at 1 x 106 cells/ml with Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution and 10% FBS. The approximate yield of cells from 4 ml of blood varies between 107-108.
  6. Seed 500 µl of cell suspension in a 24 well culture plate.
    Note: Monocytes in PBMCs get attached to the plastic in about 2-3 h when incubated at 37 °C. Longer incubation will result in firm attachement. Lymphocytes are not glass adherent and they will be mostly in suspension and can be removed by mildly flushing the wells with medium and/or buffer. Such treatment will keep the monocytes firmly attached to the surface of culture plates.
  7. Cells can be treated with different antigens for different period of times and the supernatants can be analysed for cytokine levels. The cells can be analysed for phenotypic change, apoptosis or proliferation.
    Note: PBMCs are primary cells and cannot be cultured for more than one passage under normal conditions. Lymphocytes of PBMCs can be made to proliferate in vitro by mitogens e.g., Phytohaemagglutin or Concanavin-A etc over a period to 72-96 h. Monocytes generally are end cells and do not proliferate. In absence of mitogens the proliferation of PBMCs will be negligible.

Recipes

  1. Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution and 10% FBS

Technical notes

  1. Viability of the isolated PBMCs needs to be monitored add 200 µl of cell suspension to 200 µl of 0.4% of trypan blue solution and incubate for 15 min. Cheque for viability of cells by trypan blue staining and score under a microscope using haemocytometer (cells taking up a blue stain are dead cells).
  2. Calculate percentage viability as follows:
    Cell Viability = total Viable cells/total cells x 100
  3. Cell suspension having more than 95% viability should be used for culture.

Acknowledgments

The laboratory protocol was evolved over time in the senior authors’ laboratory using a template that was published in 1986 in Handbook of Experimental Immunology / edited by D. M. Weir; co-editors, L. A. Herzenberg, Caroline Blackwell, Leonore A. Herzenberg. Blackwell Scientific Publications. Institute of Life Sciences is funded by Department of Biotechnology, Govt of India and SP was supported by a fellowship grant from Indian Council of Medical Research.

References

  1. Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V. and Ravindran, B. (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.


How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Panda, S. K. and Ravindran, B. (2013). In vitro Culture of Human PBMCs. Bio-protocol 3(3): e322. DOI: 10.21769/BioProtoc.322; Full Text
  2. Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V. and Ravindran, B. (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.




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    4/22/2016 7:17:25 AM  

    Ioanna Eleftheriadou
    Aristotle University of Thessaloniki

    How can I check PBMCs viability after treatment with a substance( carvacrol) ? I have tried MTT assay but I think that is not enough! So, i want to try with Trypan blue 0,4%. I have my cells in 96 well plate. After 24h incubation with carvacrol how can I check viability with trypan blue?

    4/25/2016 7:02:51 AM  

    Santosh Panda (Author)
    Infectious Disease Biology,Institute of Life Sciences

    Hi Loanna
    You can remove some cells from the plate and can use trypans blue. otherwise you can also stain them by DAPI and check by FACS.
    thanks

    4/25/2016 2:54:02 PM  

    Ioanna Eleftheriadou
    Aristotle University of Thessaloniki

    Thank you very much. I have one more question: Do the PBMCs proliferate without mitogen?I seed 150000cells/well and incubate with a plant extract. 20 hours later, i count more cells. Which is the proliferation rate of PBMCs?

    4/26/2016 6:18:48 AM  

    Santosh Panda (Author)
    Infectious Disease Biology,Institute of Life Sciences

    Hi
    Loanna
    My boss Dr. Ravindran answer this to a previous query. here it is, Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.
    hope this will be helpful.
    thanks

    Reply

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    3/24/2015 9:23:43 AM  

    Shadia Nada
    University of Toledo

    is it possible to freeze the PBMC and what is the best media to be use for freezing

    3/26/2015 12:19:40 PM  

    Santosh Panda (Author)
    Infectious Disease Biology,Institute of Life Sciences

    Dear Shadia, Can you please clarify why you want to freeze??

    3/26/2015 12:31:16 PM  

    Shadia Nada
    University of Toledo

    I’m purifying the PBMC from human blood and I use the cells right away I’d like to ask if I can freeze the rest of the cells for next experiment, and if I froze them, are they going to be function as the fresh cells if I thaw and use them again and what is the best media for freezing. Thank you for your help and time

    3/26/2015 12:35:27 PM  

    Santosh Panda (Author)
    Infectious Disease Biology,Institute of Life Sciences

    We never tried in this way. If I have to advice you based on my experience it is not going to function physiologically equivalent to fresh cells. You can freeze them for DNA isolation or cell lysate preparation but not for any immune response study.

    Reply

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    2/11/2015 2:48:45 PM  

    Thao Tran
    Texas A&M

    Mohamed,

    Did you find way to make the cells to grow?

    Thanks

    2/11/2015 8:32:58 PM  

    Balachandran Ravindran (Author)
    Infectious Disease Biology,Institute of Life Sciences

    Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.

    2/11/2015 8:49:07 PM  

    Thao Tran
    Texas A&M

    I activated PBMC with either PHA 5 ug/ml or LPS 1 ug/ml. I used medium IMDM with 20% bovine serum. After 4 days, nothing worked, the cell population decreased! Now I increase the PHA and LPS up to 10 times concentration. I hope it will woek (after the first day, I checked but it still has not been working well). How long is the wait? Any one could provide me suggestions?

    2/19/2015 9:31:04 PM  

    Balachandran Ravindran (Author)
    Infectious Disease Biology,Institute of Life Sciences

    Can you clarify when you say 'nothing worked?!' I guess there was no proliferation of cells PHA or LPS. How did you score proliferation? How sure you are about the quality of PHA or LPS? Adding 10 times more PHA or LPS could actually be toxic to cells.

    2/20/2015 6:03:30 AM  

    Thao Tran
    Texas A&M

    I counted by hemocytometer. Over the time, the cell population decreased. I am using LPS from sigma and PHA ftom Gibco.

    Reply

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    7/24/2014 3:29:14 PM  

    mohamed ashour
    saddat

    How can I set up culture from PBMC?

    1- I separate blood using ficoll
    2- used RPMI+ 10%FBS+ Lglu+ strep/pencellin
    3- culture the cell in 96 well plate (10000 cells/well) and in flask at 1000000 cell/ml

    the cells is not growing and the counting is down every day. How can I set up a culturing condition which could give me replicating PBMC to test these cells for drug developments


    Regards,

    Reply

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