I got a growing 293 line grown in suspension in Freestyle-293 from a collaborator. I spun them down, resuspended in fresh Freestyle at 3E5/ml, and incubated at 100rpm 4 days. Cells were at 5E5/ml on day 3 and 4E5/ml day 4. I’m not seeing a lot of death but they don’t seem to be growing. Any suggestions?


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?

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