Hello researchers, recently i am starting adapting hek293 adherent cell line to suspension culture. i tried direct adaption, which is accutase cells then seed them directly to F17 complete medium in shaker flask. cells viability keep dropping daily, 89%-->20%, the online article's data shows like the viability should above 70% . i am struggling repeat that, anyone have any suggestions? Thank you so much in advanced!!!
here's the protocol:
The “Do-or-Die” Approach or Direct Adaptation
1. Expand the cultures like CHO K1 or HEK293 in currently used medium.
2. Centrifuge at 200 x g for 5 minutes a sufficient number of cells that will give at least 4-6 x 10⁵ cells/mL for inoculation.
3. Resuspend cells in Xell medium (if necessary, include 6-8 mM L-glutamine and/or growth factor).
4. Passage cells or change medium every 2-4 days depending on cell density.
5. Continue cell cultures until viability stabilizes at > 90% and growth rates remain constant over 3-5 passages.
6. For optimal performance adapted cells should be inoculated at 2-5 ·10⁵ cells/mL in Xell cell culture medium. Cultures should be diluted every 2-4 days. Due to aggregation of cells, cultures should be stirred or shaken, using shake flasks, bioreactor systems or similar cultivation systems.
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