My work involves culturing PBMCs (majorly lymphocytes) to later treat them with Lithium and perform few assays.
I went through the various protocols available only about culturing PBMCs but they all involve mitogen addition.
1. I want to know for how long I can culture PBMCs without adding any additional mitogen.
2. I have read that we can culture these upto 72 to 96 hours but while culturing, should i perform media change? Since these are primary cells, they will not divide after passage one, so is media change recommended?
I am planning to seed the PBMCs in 24 well plate for 24 hours and then remove the supernatant and re-seed it in new well so as to remove the adherent monocytes (which i dont need). Then, I can take cell count with 1:10 dilution every 24 hours and check the cell death. Our AIM for this experiment is to check for how long lymphocytes remain alive and healthy without mitogens or antigens.
If anyone has PBMC culturing experience, please feel free to guide me.
It would depend on your experimental objective. PBMCs are fragile. There are a million protocols out there and they have all been optimized for the author’s particular situation.
You could culture PBMCs in the absence of stimulation probably for a couple of days. However, I have seen significant cell death starting 24 hours. Immunological assays using PBMCs are done on fresh cells that may have been rested for a max 1-2 hours. The longer you leave the cells, the more they would die. Some of the more sensitive cell subsets may start dying faster or at increased frequencies. Therefore, if you are measuring cell death globally (i.e. not by looking at individual cell subsets), you may not see the subtle changes or missing populations. By removing certain cell subsets, you may also be accelerating cell death. All this would have an impact on your downstream assays.
If your experimental objective allows it, I would recommend using fresh cells. You could rest them for a couple of hours and then leave cells either untreated/ treat them with Lithium.
I have done PBMC culture without stimulation up to 8 days without/with minor cell death. U need to change the media every 2/3 days. Also, u may check the cell loss by CTG assay. where you seed the cells on the first day and get the reading and on the final days (in my case day 8), compare the CTG count should match (plus minor 10%), day8 divide by day1 should be close to 1. You may keep the media change as recommended and perform CTG if you would like to go beyond 8 days culture.
Harsimar Bhatt Consider not adding antibiotic to your culture media while culturing PBMC since they have anti-mitogenic effects on some cells. As such, try as much as possible to practice aseptic technique and maintain a sterile culture environment so that you will not need to add any antibiotics. I hope this suggestion helps you. Good luck with your research.
Hi Harsimar Bhatt Did you manage to culture it for a week without Mitogens? I am trying to do something similar.
Hi Mohamad Fairus Abdul Kadir , could you please say what supplements you have added to culture them without mitogens for 8 days?
Hi Ashwin Nandakumar , for 8 days assay without mitogen in 96 wells plate, I just used RPMI1640 media + 10%FBS. There will be a one-time media change in between (on day 4). Hope this helps.
Hi Ashwin Nandakumar yes i am able to culture PBMCs without adding mitogen. I observed more cell death with FBS, so I switched to autologous serum. Media change every alternate day.