I used RPMI w 10% autologous serum, non-essential aa, glutamine and antibiotics, I used to measure prolif with tritiated thymidine or MTT and they proliferated as expected. Try a change of medium!

The medium I use is RPMI-1640 with 1mM Sodium pyruvate, 2mM L-Glutamine, 4.5 gms Glucose per litre, 10mM HEPES and 2 gms per litre Sodium bicarbonate.

Are you are using freeze stored cells?

For cells stored @ -80 Degree C, please thaw gradually and carefully by keeping in -20 degree for one day then 4 degree for one day and then @ room temperature. In this case, you can increase the quantity of FBS little more. Change the medium in 3days.

Good luck

Yes, I thawed the cells by using the company's instruction. Then I counted by Trypan blue. They were good. The only thing is they did not grow to reach the quantity of 10^6/ml.

Did you seed the cells at 10^5/ml and harvest at 10^6/ml after 3 days?

I may change to use your recommended medium. By the way, I do not have sterile glucose. Is it possible that I make glucose solution and then autoclave at 121oC, for 20 mins?

Thank you!

If you want them to proliferate you need to activate them with phytohemagluttinin or other activation protocol. As far as I remember there are Abs against CD3 that may do the work, but you need to activate them, since they are in G0. (for Peripheral Blood Mononuclear Cells=PBMC that are not a cell line). Your media is OK for me.

I agree with Pavel Montes de Oca B, i think they do not  proliferate because they are not activated. phytohemagluttinin (PHA ), or any other mitogene, can help you activate these and induce their proliferation after 3  to 5 days from the stimulation, they should be about 3 times the original number. 

P.S if you don't have a sterile solution you can filtrate it with a 0,2µm filter, to sterilize it for culture use.

Good luck.

Ok. Thanks you all for your helps. I plan to try with PHA 5ug/ml and LPS 1ug/ml too compare how they work. Hopefully!

You can activate them also by lipopolysaccharide

I tried with PHA 5ug/ml and LPS 1ug/ml but nothing worked! After 4 days, the number of cells decreased! I used IMDM with 20% Bovine serum. I do not why??? 

Now i am trying with the same medium but I increased PHA and LPS treatments up to 50ug/ml and 10 ug/ml respectively.

I think the mistake is not in the concentration of  PHA. OR  LPS, it is in the steralization of glucouse  because  autoclaving damages all sugars.  Sugars are steralized  by  tyndalization or. Filtration. Avoid higher temperature more than 100c 

I changed to use IMDM which includes glucose in it.

I activated them with LPS 100ug/ml. Finally, I saw they proliferated. Thank you for you all helps. Here are other things that concern me:

1/ I started from 1.2*10^5 cells/ml. After 3 days, I counted and knew that they proliferated but slowly (about maximum twice as the beginning. I tried to leave medium unitl 5 days and 1 week. The cell concentration showed very little increase before starting to decline. I hope they could increase to 10^6 cells/ml. But they never reached to a million. Can anyone suggest me ways to boost growth?

2/ After 2 passages, it seems that the cells stopped growing even being activated with mitogen. Any ideas?

I appreciate your sharing thoughts and experiences. Thanks a lot!

I used RPMI w 10% autologous serum, non-essential aa, glutamine and antibiotics, I used to measure prolif with tritiated thymidine or MTT and they proliferated as expected. Try a change of medium!

I used to use autologous serum, and it worked ok.

PBMCs are not supposed to grow over culturing as far as I know.

Supplementing the complete media with IL2 (at concentration range of 10 to 100 U/mL) acts as anti-apoptotic and increases the survival of T-cells. So, I always supplement the culture with IL2 whenever culturing/maintaining PBMCs. I agree with Ahmed, that PBMCs show very low metabolic activity, evident by no change in color of media or non-increasing cell density and doesn't behave like other established cell cultures.

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. 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.

From https://bio-protocol.org/e322

Hello everyone,

I realize I'm a bit late to the thread. I have a very quick question regarding general viability cells. I normally isolate my PBMCs from fresh blood on Day 0 and then seed and plate them as needed and leave them overnight in a standard incubator. I then conduct my functional tests (phagocytosis, migration etc) the next day. Is this an ok timeline to follow or is there likely to be a huge change in viability and pronounced cell loss overnight?

Thanks

Nayana

Mr. P. Tran ,

PBMC's are mostly mature in form and couldn't be cultured further. If you are intended to evaluate the affect of any agent or regulatory factor in lineage specific differentiation and maturation of PBMC, you need to start with Haematopoietic stem cells ( HSC ). HSC can be stimulated and guided towards the lineage specific differentiation by multiple cytokines . Downregulation or upregulation of any factor can have immense impact on the way of their differentiation. For example, Pax5 for B cells maturation.

Please feel free to knock me for further details ( if needed ) as i work on HSC to PBMC differentiation.

Reference- Cobaleda, C., Schebesta, A., Delogu, A. and Busslinger, M., 2007. Pax5: the guardian of B cell identity and function. Nature immunology, 8(5), p.463.

Best regards,

Babu M.

Hi,

You need to add IL-2 and/ or PHA for stimulate them.

Good Luck!