Serum-Free Conditions

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.

This is a good question and those of you what are using VERO cell lines are lucky. GIbco's VP-SFM is a media designed for growing VERO cells in suspension. VP--SFM is a serum-free media that requires little or no adaptation for VERO cells. The media will also grow other types of cells such as BHK-21 cells and COS-7, but some like COS-7 will require adaptation. We can talk about adaptation later on, but to answer your question directly, you can start with low passage cells that are highly viable. Gently pellet your cells grown in the old media. I would not centrifuge faster than about 1000 rpm. Leave a small amount of media above the cel pellet and use it to gently loosen the pellet. After pellet resuspension you can add the fresh SFM containing L-Glutamine or GLutaMAX. and grow your cells as you normally would in suspension. It is often helpful in the beginning to add a higher starting cell density than normal.

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

I guess the first question is...were the cells growing in the lab of the collaborator and did you switch the growth method. Use a shaker flask without baffles. Also the best thing to do in the future is to split the cells into your media instead of pelleting them. I try not to pellet cells if possible. I would not try and continue growing those cells that you have. Get more cells from the collaborator. Let say you get 50 mls of culture. I would let the 50 mls in a sterile falcon tube or equivalent sit for a while to allow the cells to settle. Then remove the medial above the cells but leave some for safety and conditioning. Then re-suspend those cells and the little bit of media they are in in some volume of fresh media. Do not let your cells get below 500,000/ml for the first several passages. I hope this helps. I suspect the centrifugation was the problem?

Serum-Free media are expensive but do outperform serum containing media. To be honest with you I have used Gibco's Sf900. Sf900II and Sf900III and several other manufactures serum-free media and the new Sf900 III media is the best I have used. The problem you will face if you switch is adapting your cells to a new media. I went over adaptation in a previous question today that is archived on this site. I have done studies comparing media and when I say outperform I mean with both virus titer when amplifying your virus and also for recombinant protein expression. I think you stated it correctly that good and inexpensive don't go together when talking about a SFM culture media.

This is a difficult question to answer without more information. The low viability may de due to the isolation process or/and the culture process. Below is a link to an article in the journal Blood that you may find helpful.
http://bloodjournal.hematologylibrary.org/content/103/8/3175.long