suspension culture of lymphoblastic cells - (Nov/21/2007 )

dear all,
I am new in cell culture and I am working on the lymphoblastic cells that is derived from ALL patients.
in the first days they were growing fast and happily,but it is about one week that they grow very slow.have this cells a limited lifetime?or did I do sth wrong?
then I defreezed one of my kryotubes but there is too much of wrong shapes.I mean there was something like the residue of the cells.
I thank you all so much in Advance

Hi acute,
you could try to reduce the volume of the medium. how is the colour of the medium in cell flasks after the first week? is it yellow
or red again? these cells havent limited halftime. they become old but are almost "immortal".
if you need help dont esitate

vittorio

hi,
after 4 days the colour of my medium is still red...but it seems that they are growing!!!
just another question,...how can I subculture my cells?
at the first day that I derived them from peripheral blood,they were growing fast and ok,but now after 2 subculture my cells are growing slow and they become a little bit smaller than before...
tnx for guidance
acute

dear acute, are you working with lymphoblastoid (infected with EBV) cell lines or lymphocites? how do you odtained the subculture?

dear vit72
my cells are lymphoblasts (I mean the precursors of lymphocytes)that i obtain from preipheral blood of the ALL (acute lymphoblastic leukemia) patients.I have sublutured them only once and with the same protocol for adherent cells.after that time the medium is still pink and my cells are alittle bit smaller.

dear acute,
i'm working with lymphoblastoid cell lines obtained after EBV infection. so i think that its the same. i dont know the division rate of your cells but i think that it is the same then mine. is the cells are growing very slowly you could add serum to cell culture and reduce the volume of the medium. the optimal density of suspension cell culture is 0.5- 1 X 10^6 cells/ml.

vittorio

Dear Vittorio,
thanks for your help. I have now another problem with my cells.
after a while their morphology changed.at first they were round in suspension and now they are adherent and they changed to a rectangular long cells.
is it normal?I mean are these cells change their morphology like this and become adherent?
thanks in advance
regards
arash

dear arash,
i think that now you have lymphocites in culture and not lymphoblasts. peraphs cells are too much old and changed in mature lymphocites. you must work with "fresh" cell culture. i think that you must perform your experiments within 2 or 3 weeks after cell withdrawal.

vittorio