Hi! I've just started to work with Sf21 and already came across some problems. I thawed them according to manufacturer (gibco) protocol in SF-900 II SFM medium and kept for 6 days now (at day 3 I've changed medium as the viability was low). They are still not proliferating at all (density 4x10^5)... I will change the medium again today.
The conditions are: 27deg, 25 ml in 125ml flask with filtered cap, shaking 130 rpm.
Hi Natalia, I worked with these cells some time ago, and the protocol called for growing them in regular atmosphere (no added CO2) and no shaking since they are non-adherent.
That has changed I see, but that does not protect them from contamination. Take a flask and spin down the cells. If the media is cloudy after the spin then you have a contamination problem.
Hi Steingrimur,
there is no contamination issue here. I've checked that. Any other ideas?
N,
Hi Natalia,
I've grown these cells many times before and experienced similar issues. Keeping the cells at a high viability is critical and I've found it difficult to bring my cultures back to greater than 90% viability if the viability suddenly drops. Did you check the viability of the cells after thawing? Perhaps the thawed vial was not that great to begin with? Consider thawing a fresh vial.
Are the cells clumping? Spinning/shaking speed is critical to avoid the cells clumping, which they easily do and when they clump, they die. Check under a LM or on a hemocytometer for clumping. If you see clumping, increase the rpm.
Passage number of these cells is critical (especially if you are going to use them for baculovirus protein expression). Try keep the passage number low.
If you have baculovirus in your lab, make sure your glassware is stringently cleaned to remove any virus. I clean by 10% acetic acid wash, followed by 5x rinsing in dH20, followed by 3x autoclaving. Do not use detergents on your glassware as the sf21 cells don't like detergent residue. If you have residual virus on your glassware, then the cells will become infected and their viability will drop. Also, I add 1% antibiotic/antimycotic (Gibco), to prevent bacterial and fungal contamination.
Best of luck!
Peter.
Hi Peter,
the cells I thawed were purchased from Gibco. I didn't check the viability till day 3, when it was 73%. Then I spined them and resuspend in new media. I did everything according to protocol (thawing, supying vial with 25 ml of media and shiaking 135 rpm in 27deg.
What I see is cells proliferating either really slow or not at all.
Did you ever came across problem that after being frozen those cells went to cycle arrest?
And I use plastic bottles - is this making a difference?
Nata
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