I have cultured A4/Fuk cell lines and it looked healthy in T75 flasks. I seeded for drug screening and growth curve. The cell count in growth curve in next day showed that there is no growth of cell lines with the count which is almost none. I repeated the seeding twice already and the data is coming same for growth curve which is none.
I would really appreciate any responses.
Thank you in advance!
Hi Sadhana,
Sorry but it is not clear from the context if the growth is hampered after treating with the drug or without treatment (seeding before the treatment). If it is after treatment, does the drug inhibit or accelerate the growth of the cells?
And if you find that the growth is not increasing after seeding to the plate, checking the following aspects might help:
- Contamination: Mostly, contamination is the biggest reason for drastically hampered cell proliferation. Fortunately, most of the bacterial contamination (major source of contamination) are detectable under microscope (may need staining). Also, check if there is abnormal depletion of medium (change in pH: Colour). This will confirm bacterial contamination. But in case of micoplasma contamination, you can not detect under simple microscope. You must perform PCR based detection technique. In micoplasma contamination, cells may look healthy (like you said), but they cease the growth. Also, you may throw all the old medium/reagents and everything you used, throw the things inside the hood, clean everything with antiviral/antibacterial agents and try with fresh cell line in culture. Note that sometime antibacterial/antimicotic fails to stop contamination, even though they are present in medium.
- Cell line too old: Though your line is malignant, highly rare, but they might stop growing after numerous/threshold population doublings. As I use stem cells, this happens most of the time. If this is the case, you need to use new cell line.
- Culture condition are not optimal: Every cell line has different need of culture conditions. Check if you have maintained everything needed for your cell line.This include culture medium, FBS concentration, if any supplement necessary, if growth factor needed and CO2 concentration.
- Seeding number: Number of cells used for seeding is another important factor. If you seed too less cells, they do not grow because cells need growth signal from neighboring cells (Paracrine signalling). If you seed too much cells, they won't grow either, because of cell clustering and hence, contact inhibition. It is upto you to decide the optimal number of cells for your line.
- Doubling time: I always have a hard time to determine the doubling time for the cells, specially if the cell is extracted from a subject. Some cell type doubles faster, whereas other stubborn cells need a lot of time to double. But most malignant lines double alot faster compared to non-immortal lines. I am sorry that I can not help you in this as I have never cultured A4/Fuk line though I know that it is derived from acute B cell leukemia.
Hope these points help. Best wishes and happy culturing!
Hi Prabin,
Thank you for your reply.
I have one plate with drug treated and another without treated which is used to see their normal growth without drug treatment. So I can compare the growth if the inhibition was deu to drug or the cell lines itself. But the plate without drug treatment is showing no response when done CTG so I was wondering if the cell line is metabolically inactive.
Contamination: the cells in flasks look healthy and since its suspension line. I can look for micoplasma contamination and will also change media just to be on safer side. The media has FBS, Pens Strep and other growth factors as needed in media.
Cell line is in early passage.
I was just wondering if the cell viability assay was giving false result. But CTG is a reliable assay. Also to double check, you can use manually count cells using hemocytometer or digital cell counter everyday, just to ensure if there is increase in cell number in unit volume of homogeneous cell+medium mixture. Also medium becomes more cloudy if there is increase in cell number.
The closest inference I can predict is that there might be micoplasma contamination. Check for it. To double check, clean/sterilise everything, change everything and start a fresh batch of culture.
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