Hi all,
We have adapted HEK293T to grow in suspension in serum free media. We have been working with them for 6 months without any problem. They usually grown in 125ml Erlenmeyer with vent cap, in a CO2 incubator (37ºC, 5%CO2) with an orbital shaker (19 mm orbit) inside the incubator at 150 rpm.
However, they suddenly die after defrosting without any known reason. They have around 90% viability after defrosting (as usual), but they loose viability in 3-5 days (1-2 passes). DMSO is removed by dilution and complete media exchange by centrifugation after defrosting. We have discarded contamination (Mycoplasma and any visible contamination), media and additives (we have tested different lots of everything), incubator (we have used other incubator but with the same shaker), lot of frozen vials (defrosting different freezing lots, and even different clones), and different Erlenmeyer/plate (125ml and non adherent MW6, where we have worked previously with exit) but nothing has worked so far.
Any suggestion would be more than welcome. Thank you in advance
Carlos
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