By 2nd day of culturing HEK293 and mESCs, a lot of precipitates are found in the dish and affect the cell health badly (worse in the case of mESCs). By 4th or 5th day of culture all the cells are dead. Can anyone please explain what is happening with my cultures and the way to reduce the precipitaiton ?
If you are just routinely culturing the cells and are getting precipitation and cell death, something must be lysing your cells. The precipitate is probably cell debris, but it is difficult to say for certain without seeing your cells. It is most likely caused by contamination. Have you checked that you are using the appropriate media for those cell types? How did you thaw your cells? Do you have an image of the precipitation you can post?
Your cultures are either contaminated or you over-trypsinize them if you used trypsin to split the cultures. Try to leave trypsin with cells for less time and use cells that you are sure not have been contaminated.
HEK 293 cells dont require prolonged trypsinisation. Make sure when you trypsinise cells, simply rinse the monolayer and discard the remaining trypsin in flask. Keep the flask as such at 37. Over trypsinisation may cause cell death which may be a cause of appearance of cell debris. Secondly, these cells are very fragile and grow robustly. Ensure that cell culture medium pH is not acidic in any stage. keeping cells even in moderately acidic medium would cause cell sloughing in the form of sheets..
Good luck.
- Badges
- Science method