Sludge when growing insect cells - (Mar/23/2005 )
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
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
I haven't done insect cell culture - but I think if you have stuff sticking to your glass vessels, you want to find a way to make the glass vessel less sticky. In animal cell culture we used to siliconize the interior of our growth vessels - we used Sigmacote (available from Sigma). It worked very well and the silicon coating lasted through several cleaning/sterilization cycles.
If you do decide to try this - start with a very clean vessel, make sure that you have a well-ventilated area to work in (Sigmacote is a heptane solution), and give yourself enough time to let the vessel dry thoroughly after you treat it. Then give it a good cleaning before you use it the first time.
Katherine