I am working on PUREC cells which need to grow 20%FBS. When I grow those cells in FBS free medium, cells seems to be stop proliferation and growing some inert matter like particles. please see photos. please help me if some one know what is this inert matter like particles in culture? it seems to be not contaminations.
I am working on PUREC cells which need to grow 20%FBS. When I growing those cells in FBS/ serum free medium, cells seems to be stop proliferation and growing some inert matter like particles. (please see photos). Please help me if some one know what is this inert matter like particles in culture? it seems to be not contaminations.
Thanks
Hi,
my first guess when looking at the picture would indeed be a bacterial contamination, too. However if you can rule that out, consider that those particles may be vesicles shed by the cells under stress, which you most certainly induced by serum starvation. If you also obeserve reduced viability in the culture in general, the small particles can also be apoptotic bodies, formed when cells disintegrate into smaller vesicles. Serum-starved cultures in general are well known to eventually go into apoptosis in many cases. The larger particles remind me of protein aggregates frequently observed when you heat-inactivate your serum, but as it is completely omitted in your culture, you can rule that out.
these are not contamination. . As Patrick Heinrich said these may be vesicles produce by the cells.
Cultures as born of the uterus terroot of the sanubari, creator, the extortion, and the Watchers
Thank you very much Dr. Patrick Heinrich and all others for your valuable reply for me. Could you please advise me how to avoid this problem?. It's actually not Bacterial contamination as I already check it.
My culture condition is; Thawing cells at 37C/2min, PBS wash X1, centrifuge 800g/5min, culture with our laboratory cell culture medium ASC002, 20%FBS and 2ng/ml b-FGF. When medium change (every 3 day), I will not wait until medium become RT. Please advise me how to culture cells with stress free? Are their any problem with my culture procedure? How to avoid it?
I think you change the medium every day. It will sure reduce within few days. After disappearance of all these particle you can change the medium at every 3 days interval.
From your initial question, did I get it right that you omitted serum supplementation and then the particles appeared? Because in your description of the culture conditions, you now say you add 20% FBS.
If this is the case, the simplest solution is to just add serum in recommended concentrations. If you cannot do this for other reasons, you may try to find a protocol for serum-free cultivation for your particular line (check https://fcs-free.org/) or try to develop one yourself, but be aware that both in general will be expensive and elaborate approaches. You would have to figure out which component(s) in the FBS your cells require and specifically supplement those to the medium.
Also, if you didn't warm up your medium before adding it to the cells, the cold shock may also be the reason for the stress observed; always bring your medium to the appropriate cultivation temperature before adding it to the cells.