I have an insect cell line, Sf21 and I need to transition to serum free medium. I have tried to do this before but the growth rate decreased from 24 to 50 hours. I have new cells so what should I do.


Insect cells, Sf21 or Sf9 or HighFive have special growth requirements. Remember the media is not CO2 dependent and the optimum temperature is 27-29C with 95% relative humidity. Insect cells also seem to be special because they have a memory of their conditions and if something is wrong they don’t recover. Also always grow the insect cells is shaker flasks without baffles and shake at no faster than 125 rpm. Insect cells grow better in suspension and have much higher viability in suspension. The only time I grow adherent insect cells is when I do a plaque assay or when I do the initial transfection to make virus. The other thing is that your doubling time should be around 18-20 hour so the long time you mentioned indicates that your cells are not very happy for some reason. I also do not use antibiotics or anything like that and if you do I recommend using half strength since insect cells are very sensitive to chemicals.

Additionally, I am surprised that the new cells you purchased are not pre-adapted to SFM.

If the growth conditions I mentioned above are correct, here is how to transition to serum free medium. Lets assume you have frozen cells….Thaw the cells rapidly to 28C in a water bath then use ethanol to sterilize…next add the cells to 28C media A (usually about 5-10 x 106 cells in 25 mls of pre-warmed media in a shaker flask. Continue to grow the cells and split them when they reach about 2 x 106 cells/ml. When you split the cells do not go below 300,000 cells/ml and don’t go above 3 x 106 cells/ml. Grow the cells for several passages in this media A. When the cells are growing well in media A it is time to switch to media B.

Maintain a growth curve for the cells and look at the slope of the line and make one if possible for your cells growing in old media A
Now the next time you split your cells use 75% media A and 25% new media B…continue to grow the cells until they grow at a max rate for 2-3 passages (based on the slope of the growth curve). Once the growth rate is steady then go to 50%/50% and repeat the process using the growth curve as your guide until you are in 100% new media. This process can take a month or two but it is worth it. Let me know when this works and good luck.

Start with old media A and transition to new media B

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