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Sludge when growing insect cells


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#1 imm

imm

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Posted 23 March 2005 - 07:20 PM

Hi
I have been growing and expressing proteins in insect cells for aprox 4 years. When I grow my cells in shaker cultures, I get a 'sludge' ring of cells that stick to the glass flask, just slightly higher than the media level.
After a few hours of placing cells in a new flask, I can see the start of this cell loss. As these cells are not in contact with the media they die.
From this I have 2 problems
1. Cell viabilty drops
2. A lot of the cells I seed into the flask end up on the glassware, so my cell number is much lower than it should be. (as a result I am getting very low expression of my protein since not enough cells in culture).
This problem seems to have gotten worse and I have afeeling it is related to the way I am growing the cells (no mycoplasma and have this problem with Sf21 and High 5 cells).

Anyone know what might be happening ??
Thanks in advance




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