Serum-Free Conditions – A discussion on creating and optimizing serum-free culture

By on May 30, 2013
Expert Session Summaries

Last week, we finished our Ask the Expert discussion on Serum-Free Conditions. While there is a strong desire to transition to or maintain serum-free conditions, the truth is that with some cell lines this can be quite a challenge. With so many cell culture scientists working towards a truly serum-free culture or looking to improve their existing serum-free process, we felt that this was a very relevant topic.

This session, sponsored by Life Technologies and hosted by Timothy Fawcett, Ph.D. was a lively discussion with most questions focused, as expected, on the more challenging cell lines including stem cells, insect cells, 293 cells and vero cells. Dr. Fawcett who has been in biotechnology for over 30 years, provided real hands-on suggestions that readers could implement right away.

Question topics included

  • Adaptation to serum-free media
  • Improving cell viability in serum-free conditions
  • Serum-free and xeno-free conditions
  • Improving slow growth rates
  • Improving cell density issues
  • Switching between serum-free and serum-containing media

I have selected a few of the submitted questions and answers to include below. For a full list of questions and answers, please see Ask the Expert – Serum-free Conditions.


Can I switch back and forth between Serum Free and serum containing media for short periods of time for experimental reasons?

The Answer:

Hi, This is a great question since sometimes for experimental reasons you might want to do this. Cells that are grown in a serum containing media can be transferred to a serum-free medium, D-PBS (or a serum-containing medium like D-MEM without the serum added) for about 4 hours without hurting them. Remember, if you want to keep your cells attached to the plastic you need to have calcium and magnesium in the medium at all times. Some reasons you might want remove serum is to control experimental conditions or to get the best response using some type of inducer, say for gene expression studies. This works by minimizing binding of an inducer compound to albumins contained in serum., meaning more inducer gets to the correct site of action. Note, that if you have cells conditioned to grow in a serum-free medium you should not add serum since this results in the addition of animal origin components that may be harmful downstream in another process.


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.

The Answer:

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


Please visit this week’s Ask the Expert session –

How to get the most out of your conference attendance,” hosted by Caroline Hornby, Marketing Director, Terrapinn Inc. USA

Also join us for next week’s Ask the Expert Session –

“All about CHO, everything from the basics to troubleshooting,” hosted by Tim Fawcett, Ph.D. Director of the BioTechnical Institute of Maryland (BTI). Founder and Director, BioSciConcepts.


  1. Monika

    22 February, 2014 at 1:39 AM

    I am treating HepG2 cells with Lipoprotein. I seed cells in serum media (10%). Just before treatment, I change media to serum free media. Should I maintain cells in serum free media for 12 hours prior to the treatment?

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