I am culturing the raw cells 264.7 from the last 1 year in the passage of 18-20. But now I am facing a problem, they are growing but not reached in 80% confluencey. After 3-4 days cells are floated, confluency not reached to 50%. They don't have contamination, cells are healthy but maximum cells are floating and low confluency. Before they are not like this. I changed the media every time when i see the cells are floated and the media is slightly yellow but they are not growing very well
The thing about RAW 264.7 cells is that unlike most other infinite cell lines, they do not form 80-90% confluence just about ever. 60-75% is the highest I have observed when I have worked with them.
Moreover, sometimes the RAW cells may be semi-adherent in nature (depends on culture technique and requirement but generally they can be semi- adherent).
You need to be careful since the moment the cells mature, they will begin to float in the flask.
Please note, it is definitely not because they are dead.
Sometimes if during preservation, if most cells preserved were mature, they may end up as a mass of floating cells many a times even after being revived.
It has been suggested that the media decanted (containing the mature floating cells) can be subject to low speed centrifugation and then introduced to the tissue culture flask to continue with culture. However, I do not follow that.
Try this, at the end of day 2 or 3 at the most when the first few cells begin to float, subculture it. Either do a 1 in 2 culture, discard half the volume retaining volume sufficient for 1 flask
OR
Rock the flask hard (or scrape using a cell scraper) to detach the cells; add sufficient media to mix well. Do a 1 in 1 type of culture and transfer the suspension to a fresh flask.
It is not advisable to retain mother flasks generally as the cells in the mother flask grow and mature faster than those of the daughter flasks.
Do run a test nonetheless to check for any kind of contaminants such as mycoplasma etc,.
I also encountered recently with similar problem. After recovery from liquid nitrogen (passage 15) they stopped to proliferate and detached at confluence of 30%. However, after 5-day incubation in DMEM with 20-% FBS they started to proliferate. Addition of piryvate and GM-CSF should also be stimulating for their growth. My expertise with these cell line determined that changing of growth medium every 3 days yields more cells than daily medium change.
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