I was sub culturing one cell line and after 3/4 days. When I count the cell, I find 30% viability. I think something is wrong with my medium or incubation system. Now I wonder what is the standard percentage of viability? Can someone elaborate all possibility?
Hello,
A good cell viability is anywhere between 80-90% in most of the cell lines. It would be easier for me to advice you if you could elaborate about the cell line and the culture method you used.
Hi Nur as Sajjeev has mentioned the viability should be between 80-95% for a healthy culture. Non-adherent lines are generally a bit less than adherent lines (as dead cells can be rinsed off adherent lines with PBS before trypsinisation/counting but they tend to remain in the culture for non-adherent) but should still be >80% and typically 85%.
30% is very low and they are clearly struggling. The most likely reason for growing problems for any line is the nutrients you are using (media & FBS).
Personally I would:
a) Check I am using the correct media formulation for the cells (I.e. RPMI or DMEM etc).
b) Check if the cells require any additional GF's or cytokines (such as IL-2)
c) Check that you are growing the cells at the correct confluence/split levels (i.e. for non-adherent the cells may have problems if their density is too low or has reached too high - for adherent cells they may struggle if split to spare/hard such as a 1:10 split).
d) Try growing the cells in good quality FBS (Gibco, ATCC etc).
Unless you provide more details I'm afraid I can't be much more help!
Good luck,
Gary
Buying cheep FBS is a false economy in cell culture as cheep FBS can come from animals that have been stressed, are infected or have had a lot of antibiotics etc.
Ideally cells should be grown in a variety of FBS sources to find the most optimal (i.e. batch testing).
As
Thank you Sajjev and Gary for your kind information. Actually I am using myeloma cell line ANA6, IH1, OH2, ANBL6, KJON1, U266, RPMI8226, JJN3 cell lines. All haematopoetic cells so suspension cultured. Sometimes I am getting very low viability like 55%-70%. Is that ok for them?
Hi Nur - in brief - no.
The viability for suspension should be ~80-90%. I am not familiar with those lines and sometimes certain lines do struggle but 70% or less means they are not happy.
Just looking at on-line info for one of them (JJN3) I see that the minimum density (seeding) should be 0.2-0.4x10e6 cells/ml and max 2 x10e6 (so I would seed at ~ 0.4x10e6/ml). The cells should double 20-35 hours (so if you do not get doubling in 48 hours something is wrong).
I also notice that this line should be grown in 20% FBS (which I assume you are doing?). http://www.dsmz.de/catalogues/details/culture/ACC-541.html?tx_dsmzresources_pi5%5BreturnPid%5D=192
I would double check the details for each line (seeding, d.t., %FBS, media, cytokines etc) just to be sure no one has given you wrong information on their cultivation.
As mentioned I suspect the cells may not like the FBS. Check also if you are heat inactivating the FBS. This is a "hang-over" from the bad old days of low quality FBS - if you are using good stuff (such as Gibco or ATCC) the FBS does not need heat inactivating (in fact it is recommended that you do not do this as it can destroy GF's etc).
Personally I would buy a small quantity of good quality FBS (100 ml) and make up some media (at recommended % for one of your lines - the one that is "struggling" the most) and grow some cells alongside the FBS/media you are using and see if you notice a difference in d.t. and % viability.
I would also perform a mycoplasma test on all of you lines (just in case this is the problem). I would recommend performing a mycoplasma test on all cells in culture once every 3 months or so (or if any new cells esp. primary cells come into the lab).
Suspension cells (in general) are easy to grow and maintain but some lines (esp. lymphocytic) can be a pain to grow (I never could get my CEM cells to expand).
Good luck!
Gary
Hi,
usually viability should be around 90%. Check if the composition of your medium is ok ( in terms of % of FBS, low or high glucose,according to ATCC). Also check the % of CO2 and temperature of your incubator and your flasks or plates. Maybe your cells require specific material in which you have to grow them.
Best wishes!
Hello people, do you have any article that might confirm the percentages you said as ideals? Mariangela Garofalo Gary Morley
tnks
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