I am trying to grow some Sf9 cells but after incubation at 27 degrees and shaking at 125rpm for 2 days, they all die or at least majority of them are dead.
I grew a 3mL culture (SF900II media) in at 125mL Baffled Erlenmeyer Flasks with initial density of 1.5mil cells/mL (from frozen stock) so after diluting it to 3mL, the cell density becomes 500000 cells/mL. The SF900II media that I used had gentamycin added to it (I added 200uL of 10mg/mL Gentamycin to 1L of media).
Before growing them in the flask as suspension culture, I counted the cells and most of them are alive with some dead. Then, I centrifuged them at 500g for 5mins, removed the supernatant to get rid of the DMSO and resuspended them in 3mL of SF900II medium with gentamycin in it. I then transfer them into a 125mL Baffled Erlenmeyer flask and incubate them in a incubator at 27 degree, shaking at 125rpm for 2 days.
After 2 days, I took 10uL of the cells and added 10uL of Trypan Blue and counted the cells immediately after that. However, most of them were dead (most of them were stained blue) and I am left with around 40000 cells/mL.
Can anyone tell me why is this happening and how I can solve it?
Thank you very much.
To everyone who saw my question and was wondering the same thing, I was told a couple of reasons why my cells are dying.
Firstly, 3 mL was way too little. So Ideally, the volume should be no more than 40% of the flask's volume (but still 3mL in a 125mL flask was way too low). So, what was suggested to me was that I should start with around 2X10^7 cells/mL and dilute it to 1-2 million cells/mL. So if you don't have enough cells to begin with, I suggest you buy some cells or find some source of obtaining them.
Secondly, as insect cells are very fragile, using a baffled flasks may shear them while shaking. Hence, non-baffled flasks are better at growing them. Also, sometimes you may see fewer cells in total (alive or dead) compared to when you seed them. This may be due to the fact that the cells are dead and lysed, so you don't see them at all.
After changing these 2 conditions, my cells are now happily growing (although for some reason they can't seem to grow to 8 million cells/mL even after 5days, but one step at a time. I can now grow up to 4 million cells/mL (50mL total, that's 200 million cells!) from 1 million cells/mL.
Hope this helps.
Cells must demonstrate that they can be recovered as healthy logarithmically growing cells within 2 to 3 days after thawing
Do you grow 3ml in a 125ml flask?
the volume is too low. Should be ~20ml in the flask.
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