Need help on thawing primary cells - Cells dead!!! (Mar/04/2005 )
maybe someone can give me some suggestions to my protocol.
I am relatively new in ACC field. Recently i just got 3 vials of Pig bone marrow cells (primary cells). As far as i know, those vials were frozen for about... 5 months in - 80'C. Will that affect the cells viability since they are stored in -80'C for so long and also they were taken in & out of the -80'C occasionally?
When i thawed the cells which i was given and observed them, they were like..... DEAD! omg... i'm not sure whether is the protocol or if is just ME.
i thawed the cells according to the protocol:
take vials out from -80'C
thaw quickly in 37'C water bath
transfer content to 15ml centrifuge tube
add 10ml complete media dropwise to content
spin @1500rpm, 5mins
discard media (with DMSO as cryopreservative)
resuspend pellet with 10ml media
cell count & viability check
seed suspension to T25 and incubate.
When i did a viability check and cell count... i have got something like 45% viability for my cells, but after the incubation overnight... they did not attach to flask and were still in suspension. Its my first time dealing with primary cells and i'm not too sure why they always die after the incubation? Never had such problems with cell lines.
I think it might be that freeze thaw cycle that the cells had been through.
Do you add certain growth factors in your medium to support BM growth?
If not, you need to do so. We have a formula in my lab if you need.
I did not add any growth factors to my media. But I am using advance DMEM; high glucose from Gibco. Why is it necessary to add growth factors? It would be great if you can share the formula with me! Thanks
Well the cells will probably not grow if there aren't any growth factors, but you tjeck for viability and get none??
They could be dead before you thaw them. Freeze thaw cycles kill cells, or they should have been kept in liqiud nitrogen. One thing you could do differently is the centrifugation, that ca be rather harsh on the cells. Maybe the protocol you have got uses another centrifuge the G is different in different centrifuges, I can only use 800 RPM in mine.
its not necessery using growth factors (actually in some cases it even change parts of your cells).
pay attention on time when u put the vails in the 37c bath, make sure that your cells still have ice in side them, the dmso is ruing the cells- the more time dmso (while thawing) stays in the cells the more dead cells u'll get.
Are you sure the cells are not suspension cultured, I know nothing about bone marrow cells, but it could be that they are supposed to be in suspension.
Also as in other posts, check the conditions of growth, you may need some growth factors or to use a special medium for growth... if the place that supplied the cells to you is using a different medium to you, then the cells need time to wean onto the medium you are using. i.e. grow them in the medium that your supplier is using, then slowly start to wean them onto your medium.
Cells stored in -80 freezers for long peroids of time (months) will eventually die, as apparently cellular processes are still working slowly. Preferentially store them in LN2 gas phase for extended periods.
Not knowing anything about pig bone marrow as I work with mice only but ....
If this is whole bone marrow it is likely that the majority of the cells are supposed to be suspension as bob1 pointed out. Also bone marrow cells often do not like to be grown on plastic and need a feeder layer of stromal cells. Depending on what cell type you want to expand I expect you need to add factors particular for that application. What is your application or objective ultimately with these cells? This will indicate what media you might want to culture them with.
Let me know if you need more information.