Hi,
I'm used to culturing primary human monocytes to generate macrophages in vitro, but three donors-worth of cells in a single week have had to be thrown out. I isolate PBMC form whole blood using the dextran and histopaque method which includes a final wash step that depletes the majority of the platelets. I then resuspend in serum-free RPMI and plate in 24-well suspension plates (monocytes still adhere strongly to these plates, I use them so I can detach my macrophages for flow cytometry at the end of my culture) at 9x10^5 cells per well, and incubate for 1 hour at 37C. After an hour I wash 3 times with PBS to remove the majority of lymphoctes, and replace with RPMI + 10% FBS + 50ng/mL M-CSF.
I've done this numerous times with great success, I get good looking cultures of macrophages that behave as should be expected. But this past week, using brand new media, all of my plates are sparse with adherent cells but instead have great long, stringy clumps of cells in suspension. When I swirl the media, I can see these as cloudy masses of cells with the naked eye.
Has anyone else experienced something similar? As I'm usually successful I can only think I either made a mistake, or my cells have died as a result of infection. Any thoughts would be greatly appreciated.
Thanks
Hefin
Just covering all the bases... did you heat inactivate your FBS?
Also, make sure you wash with 1x PBS (and not water by mistake). Most of the times, this occurs when you wash with a solution that is not iso-osmotic.
I agree that it could be apoptotic granulocytes and DNase treatment could help but as it never occured to you before, it seems that you have an underlying problem to resolve. DNase treatment would treat the symptom but not the cause.
Good luck,
Hi Hefin,
Just to clarify, are you using the exactly the same medium (same company/brand, just different batch) or did you change to a medium from a different company? and your FBS, are you sure it is the same batch as from your previous experiments?
Usually we don't experience much variations with RPMI medium from batch to batch, but with the FBS we do have considerable differences from brand to brand, and even sometimes from batch to batch.
Hi Iris, thanks for your answer. The RPMI is just a different bottle from the same company, the FBS is a different lot, but could something in the FBS really ruin my culture? I'll ask if anyone else has experienced something similar in the lab with this lot of serum. Thanks!
You need to add DNAse. Cells, likely myeloid/granulocytic that have apoptosised and DNA has been released forming a "stringy" aggregate. We run into this with apheresis products and develop what we call a hairy golf-ball. Of course we are working with >10e9 cells.
Just covering all the bases... did you heat inactivate your FBS?
Also, make sure you wash with 1x PBS (and not water by mistake). Most of the times, this occurs when you wash with a solution that is not iso-osmotic.
I agree that it could be apoptotic granulocytes and DNase treatment could help but as it never occured to you before, it seems that you have an underlying problem to resolve. DNase treatment would treat the symptom but not the cause.
Good luck,
Again, covering the basics: we have the problem sometimes as well and it turns out to be dust in the plastic ware. Rinse them with medium once before you add cells, should help.
In addition to Michels comment. make sure that the heat inactivation is not above an hour and at 55c max. we had similar problem when one of students did the heat inactivation at 65c.
That might be over the top, but we heat-inactivate and sterile filter our serum, for exatly that reason of aggregate formation
HI,
i think everyone's suggestions are correct.
I would try and keep an eye on the lot number of your medium. We had a similar problem once with the medium pH which was somehow affected in a batch of the media.
Also, for sensitive work we tend to batch test our serum and also either buy already heat inactivated or do it yourself 55C for 45min. Then filter sterilise before you add to your medium.
Have you considered to check any other additives you add to your medium?
How about all the solutions you used? Was the PBS molarity and pH correct?
were the cell aggregates "happy" looking or mostly miserable/dead?
if the first then some DNAse treatment and a few washes should break up the aggregates
if the later then you have a buffer issue in my opinion.
Hope that helps.
good luck
Different lots of FBS can most definitely lead to problems in cell culture and it's one of the most common issues! I agree with a lot of the suggestions here about heat-inactivating it just to be safe. You may also want to wash your cells in PBS that does not contain Ca2+ or Mg2+ as those can increase clumping (calcium-dependent cell adhesion) which may also help.
Thank you all! It was indeed a problem with the serum inactivation (or so it would seem). New medium with new serum seems to have cured the problem. Thanks!
hi hefin, has your problem solved. i have faced similar problem with monocyte isolation. my cells seem like they have adhered for few hrs (many in clumps) and later more and more cells keep being nonadherent. It would be of great help if u can reply me the exact step which solved your cell clumping problem.
Hi Deepali,
We isolate monocytes from buffy coat with ficol and percol method. We avoid any FCS while isolating (for preparing 46 % percol). But isolated cells are seeded in the flask in complete medium. We incubate them for almost 3 Hrs and wash them once with medium. It works fine. It looks like when you have FCS while isolating then you get more platelets which also play spoiler. Also do not go more than 10*6 cells /ml, this is ideal concentration for incubating them in flask or plate.
Hi Deepali and thank you everyone for your answers. My problem I think arose from the batch of FBS we were using but I couldn't say exactly what the problem was (possibly improper inactivation). A new batch of serum seemed to solve it and my cells have "behaved" ever since!
Good luck
Hefin
- Badges
- Science method