How to handle EBV transformed lymphoblasts? - (Jun/18/2007 )
Hello!
In some days EBV transformed lymphoblasts will arrive which I will need to cultivate. I know the composition of the needed media but thats it. Does anybody know how and how often I should subcultivate them??? And are there any special saftey things I need to consider because of the EBV???
Any suggestions would be helpful!
Himbiu
Hi Himbiu,
LCLs grow in suspension and in clumps/groups of cells from 3-5 cells up to 20-30 cells. They can look like a fungal contaminate because you can see large lumps of cell clusters in the medium. They're normally cultured in RPMI with L-glutamine, pen/step (both standard concs) and 10% FCS and you probably need to change the medium twice a week (Monday and Friday) or more often if the medium becomes yellow. You can freeze them down by resuspending 5-10ml dense culture medium in 3ml 10% DMSO, 50% FCS, and 40% RPMI complete medium then putting the cryovials (1.5ml per vial) into an isopropanol freezing container at -70C overnight and transfering them to liquid nitrogen for long term storage. To thaw put them in a 37C waterbath and as soon as the tube is thawed transfer to a 20ml universal and add 10ml pre-warmed complete RPMI dropwise, then centrifuge at 1200rpm for 5mins and resuspend them in 10ml complete RPMI and transfer to a T25 flask. At low cell density or after getting them out of nitrogen they might take a while to grow at a fast rate and so it's better to grow them with the flask upright so the cells settle at the bottom of the flask one they start growing you can lay the flask down.
Although EBV is associated with human malignancies, there are other risk factors involved in their causation and a high percentage of the world's population are infected with EBV (infection is life-long). Also the amount of virus through the lytic cycle produced by LCLs varies between different cell lines and some LCLs are tightly latent with viral gene expression (latent genes) but no virus actually being produced. Having said all that you probably should try the cell lines as infectious use in a flow cabinet and bleach your culture waste before disposal. These are the precautions we took in the lab I did my PhD in which used alot of EBV positive cell lines.
Good luck,
Ceri
Dear Ceri!
Thanks a lot for your help. It is just what I was hoping for!
Best regards
himbiu
LCLs grow in suspension and in clumps/groups of cells from 3-5 cells up to 20-30 cells. They can look like a fungal contaminate because you can see large lumps of cell clusters in the medium. They're normally cultured in RPMI with L-glutamine, pen/step (both standard concs) and 10% FCS and you probably need to change the medium twice a week (Monday and Friday) or more often if the medium becomes yellow. You can freeze them down by resuspending 5-10ml dense culture medium in 3ml 10% DMSO, 50% FCS, and 40% RPMI complete medium then putting the cryovials (1.5ml per vial) into an isopropanol freezing container at -70C overnight and transfering them to liquid nitrogen for long term storage. To thaw put them in a 37C waterbath and as soon as the tube is thawed transfer to a 20ml universal and add 10ml pre-warmed complete RPMI dropwise, then centrifuge at 1200rpm for 5mins and resuspend them in 10ml complete RPMI and transfer to a T25 flask. At low cell density or after getting them out of nitrogen they might take a while to grow at a fast rate and so it's better to grow them with the flask upright so the cells settle at the bottom of the flask one they start growing you can lay the flask down.
Although EBV is associated with human malignancies, there are other risk factors involved in their causation and a high percentage of the world's population are infected with EBV (infection is life-long). Also the amount of virus through the lytic cycle produced by LCLs varies between different cell lines and some LCLs are tightly latent with viral gene expression (latent genes) but no virus actually being produced. Having said all that you probably should try the cell lines as infectious use in a flow cabinet and bleach your culture waste before disposal. These are the precautions we took in the lab I did my PhD in which used alot of EBV positive cell lines.
Good luck,
Ceri