I am currently establishing Expi293F cells in my lab using the spinner flasks and not the orbital shaker, but I'm having a few issues:
I do not know the best rpm for growth,
Cell viability is decreasing post thaw,
I can not always get a pellet after centrifugation,
My cells have not reached optimum growth since thawing the original vial.
I'm sure that I am making a minor mistake that is disturbing my growth rate and cell viability but I have no idea where I'm going wrong. I just need a basic protocol for the spinner flasks because the manual is focused on the orbital shaker.
Hi Dominique,
I can suggest some points for your problem.
- After thawing your cells, put them into 15 ml sterile falcon tube with 8 ml fresh prewarmed medium to clean DMSO by centrifugation at 1500 rpm for 5 minutes from your cells before the inoculation in ExpiMedium (which is special for this cells). DMSO could be hazardous to your cells. If your cells are very few, you can inoculate your cells into fresh medium directly then next day change your medium.
- If you do not use erlenmayer flasks with ventilated caps, loose your caps during incubation. Keep CO2 level 8%.
- Keep your rpm low like 60 rpm per minute, first keep your cells alive, after that you can increase the rpm to 125.
- Watch your cells if their shape is shrunken, this means your cells are unhealthy. They must be in a shape of coins. Try to separate healthy cells from the unhealthy population. Unhealthy or dead cells send dead signals to healthy ones, after a couple of days healthy cells also become dead. So check the morphology of the cells, maybe your vial contains too much unhealthy cells after thawing. If it does, transfer your cells into a 150 mm Petri culture dish, let them grow 2-3 days, collect healthy colonies by pippeting and transfer them into an erlen mayer flask ( if you have, small ones are better, 5-10 ml).
I hope this works
Cigdem
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