Lymphoblastoid cell culture - (Jan/30/2012 )

Hello,
2 weeks ago I started a lymphoblastoid cell culture. I am facing a problem to get them grow. I started from 5,000,000 cells in 2.5 ml RPMI (FBS, L-Glu supplemented) (protocol from another lab.). I guess most of the cells died. Now, I have only 400,000 cells. I am not sure why they are not growing properly. I know the cell density plays a crucial role. Any idea, on what can be going wrong?

If you need more info please let me know!
Thanks
Hussein

Hi,

For Lymphoblastoid cells, I used to culture them in Advanced RPMI 1640 + 10% FBS and L-glut wich is indeed a very rich medium (in my case it was B cells). An important point when you start a new culture, put your T25 flask in a vertical position, so cells will be in closer contact than in horizontal position. Next do not use lots of medium, cells need to have access to oxygen, so if you have a more than 1,5 cm between bottom and top of the medium, it can be a problem. What I can suggest you is 24h after restarting the culture, remove half of the medium by carrefuly pipeting and then add fresh medium (i guess this can of advice can be used for all cell lines...)

Also check out the cell line culture condition from those who provide you cells.

I hope it will help

Thibaut

Hussein El Saghire on Mon Jan 30 11:10:45 2012 said:

Dear Hussein,

5 million cells in 2.5ml of growth medium is your problem. With cell densities, especially if the cells are in suspension, the maximum that I can grow many cell lines is 700-800,000/ml. So you are nearly 8 times the level of cells that is healthy.

I would:
Check for any contamination....bacterial/fungal
Have a starting cell density of 400-500,000 cells/ml. If the cells grow too quick it is easy just to re-dilute them days later
Where is your serum from....i.e. what quality of serum do the cells need


Hope this helps

Kindest regards

Uncle Rhombus

Hello Thibaut,

Thanks a lot for your reply. Actually, my 400,000 cells are now in 2.5 ml of medium, which I guess is too much. Tomorrow, I will try to replace the medium but with lower volume, around 1 ml only. Actually, the medium color is turning yellow so fast, but I don't see any growth in the number of cells (so either they are dying so slow or growing so slow). The medium I am using RPMI GlutaMAX with 15% FBS. I don't add any additional L-Glu, as I suppose GlutaMAX should contain the enough 2mM needed for growth.

Thanks again!
Hussein

Hussein El Saghire on Mon Jan 30 13:29:40 2012 said:

Just seen your latest post and this would confirm to me that you have to many cells in too small a volume.

The only other explaination is that your CO2 level in the incubator is far too high i.e. above 20%.......have you checked the internal CO2 concentration in the incubator?..................Do you know how??


Kindest regards

Uncle Rhombus

Hello Uncle Rhombus,
Thanks a lot for your reply. I contacted the lab which provided me with the cells, and they said that they always start using 5 million cells in 2.5 ml. Unfortunately, they are not growing this cell line anymore. So, I have to figure out myself how it works. It seems that not a lot of medium is needed to grow these lymphobalstoid cells. Now I had the cells (400,000 cells over the weekend, under the microscope, I can see that the density of the cells is almost the same).
I am using FBS as serum.

Greetings and thanks again
Hussein

rhombus on Mon Jan 30 13:33:45 2012 said:

Hello!
Actually the CO2 is 5%. It will give an alarm if it trespass this level.
Regards
Hussein

Hello!

This is the link that provides info about the culture of lymphoblastoid: http://ccr.coriell.org/sections/support/global/lymphoblastoid.aspx?pgid=213

I don't have an experience in cell culture, so if you can figure out an error I am doing I would appreciate it a lot!

Hussein

Hussein El Saghire on Mon Jan 30 13:40:44 2012 said:

Dear Hussein,

Just looked at your link:

What are the basic culture conditions for lymphoblasts?

Recommended Medium: RPMI 1640 2mM L-glutamine 15% fetal bovine serum Culture Conditions: T25 tissue culture flask with 10-20 ml medium upright position
37°C under 5% carbon dioxide



Cells in 10-20 ml....it says in the SOP

CO2 levels on the incubator display may say 5%......in fact the level could be 20-30% if the incubator is out of calibration

If you have never done cell culture before then there should be somebody supervising you!!!!!!!

Kindest regards

Uncle Rhombus

Dear Uncle Rhombus,

I have noted these requirement before, but I followed the instructions of the lab that provided me with the cells. Actually, the incubator continuously checked and maintained. But indeed, it could be the cellular density that is not correct, especially the starting 5 million cells were not in cultured in enough volume. Nobody before in the lab used these cells before, that is why it was difficult to start it. So, in principle, I have to wait and see if the remaining 400,000 cells will grow or I have to start from a new batch. But indeed, I have to figure out the correct density.

Greetings and thanks again!
Hussein