Hybridoma cultures - floaters? - (May/21/2008 )
Hoping for some insight:
I've been growing up a number of hybridoma (SP2/mouse fusions) cell lines and have noticed if I scale up too quickly to larger volumes (starting from 24-well plates gradually to larger and larger flasks), the cells sometimes don't become confluent or I find lots of floaters (usually I find both at the same time). Is there a seeding concentration I should be aware of? (cells/surface area?)
I've also noticed certain clonal lines act a little differently. Some are harder than others at scaling up. I'm relatively new to growing up the hybridoma lines for mAb purification but need to resolve this before I work with many more lines.
Even if the cells aren't completely confluent, Ab should still be produced. May not be maximum yields but certainly mAb is secreted. But what about the floaters? Is this a common problem to scaling volumes up too quickly?
(I even went as far as leaving a few little flasks over the course of 2-3 weeks, untouched, just to see....and sure enough, nothing ever really changed. Cells were never fully confluent and the floaters remained. The cells were still viable, just not expanding out. I can only assume the culture are static. Of course, I saved the media as I know mAb is present).
Thoughts?
-BioDoc-
I've been growing up a number of hybridoma (SP2/mouse fusions) cell lines and have noticed if I scale up too quickly to larger volumes (starting from 24-well plates gradually to larger and larger flasks), the cells sometimes don't become confluent or I find lots of floaters (usually I find both at the same time). Is there a seeding concentration I should be aware of? (cells/surface area?)
I've also noticed certain clonal lines act a little differently. Some are harder than others at scaling up. I'm relatively new to growing up the hybridoma lines for mAb purification but need to resolve this before I work with many more lines.
Even if the cells aren't completely confluent, Ab should still be produced. May not be maximum yields but certainly mAb is secreted. But what about the floaters? Is this a common problem to scaling volumes up too quickly?
(I even went as far as leaving a few little flasks over the course of 2-3 weeks, untouched, just to see....and sure enough, nothing ever really changed. Cells were never fully confluent and the floaters remained. The cells were still viable, just not expanding out. I can only assume the culture are static. Of course, I saved the media as I know mAb is present).
Thoughts?
-BioDoc-
Being doing lots of monoclonal prurification and hybridoma culture but never really consider thos points some clones adhere more than others but even very few cells can produce lots of antibody and vice versa for teh bad producers there's no real rules in biology
Just avoid to let them overgrow to very yellow medium if you want to maintain the line, if you want the max ab's titer just let the cells die and collect the sup
One more thing remember that L glutamine is needed and get exusted in a medium after 15 days so supplement back your medium every now and then
Scaling up too quickly (or to slowly, for that matter) can leave your cell line in poor condition. I think the cells themselves secrete growth factors that help them stay in good shape and if you dilute them too much they start to grow poorly.
Unfortunately, there is no set rule on how the cells will behave, but in time you will develop a feeling for what is best for a given cell line.
Unfortunately, there is no set rule on how the cells will behave, but in time you will develop a feeling for what is best for a given cell line.
Thanks for the input. I will look to see if I can figure out a "safe" seeding amount...I too believe that growth factors have a role here. All my best!
I've been growing up a number of hybridoma (SP2/mouse fusions) cell lines and have noticed if I scale up too quickly to larger volumes (starting from 24-well plates gradually to larger and larger flasks), the cells sometimes don't become confluent or I find lots of floaters (usually I find both at the same time). Is there a seeding concentration I should be aware of? (cells/surface area?)
I've also noticed certain clonal lines act a little differently. Some are harder than others at scaling up. I'm relatively new to growing up the hybridoma lines for mAb purification but need to resolve this before I work with many more lines.
Even if the cells aren't completely confluent, Ab should still be produced. May not be maximum yields but certainly mAb is secreted. But what about the floaters? Is this a common problem to scaling volumes up too quickly?
(I even went as far as leaving a few little flasks over the course of 2-3 weeks, untouched, just to see....and sure enough, nothing ever really changed. Cells were never fully confluent and the floaters remained. The cells were still viable, just not expanding out. I can only assume the culture are static. Of course, I saved the media as I know mAb is present).
Thoughts?
-BioDoc-
Being doing lots of monoclonal prurification and hybridoma culture but never really consider thos points some clones adhere more than others but even very few cells can produce lots of antibody and vice versa for teh bad producers there's no real rules in biology
Just avoid to let them overgrow to very yellow medium if you want to maintain the line, if you want the max ab's titer just let the cells die and collect the sup
One more thing remember that L glutamine is needed and get exusted in a medium after 15 days so supplement back your medium every now and then
Good call on the L glutamine. Will definitely keep that in mind. Thanks for the help!