help! 293F cell line

Nancy Hurtado-Ziola nhurtado at UCSD.Edu
Fri Jan 25 19:25:50 EST 2008


First of all, I want to thank all my fellow flow-ers for your  
wonderful help regarding this issue.  Most individuals who used 283F  
cells were quite successful in not only keeping the cells alive, but  
getting good protein production.  One of you handed over some very  
useful tips on handling the cells (see below) right after thawing and  
for the next few days in order to insure cells survival.   Many of  
you thought that the cells may have been contaminated with  
MYCOPLASMA! If that's the case, they came from the vendor that way.   
The last batch died in less than two weeks!

I did the following with these cells:  FS293 Media, 37 deg C, 8% CO2,  
orbital shaker about 110 rpm as suggested by the mfg., have NOT  
checked for mycoplasma (but will next time I get some of these from  
Invitrogen - they are replacing them at no cost.)

One individual (again see below) was having exactly the same problem  
that I was and did not get resolution from the vendor.	In fact they  
finally chose to go back to another cell line for their experiments.   
I've appended this email with all the answers about this topic.

Once again, you guys are the best.  THANKS!

Nancy


ORIGINAL QUESTION:
Hello fellow flow-ers,

I have a soluble protein that's really difficult to express. I  
mentioned it to someone and they said I should use 293F cells to  
solve this problem. Well, I need some information on these cells. I'm  
having trouble growing 293F cells. I purchased them directly from  
Invitrogen. The first batch started out OK. I did everything the  
supplier said to do (use their SF media, use warmed media, keep cells  
at 37deg c, with 8% CO2 level, check viability with tripan blue,  
etc., etc.) but the cell population diminished and there was so much  
debris. I got to pass 3, but never had enough cells after that to do  
another pass let alone freeze down or for a transfection. I've never  
seen a photo of what these cells are supposed to look like after the  
culture takes off. Do cells in suspension typically have a lot of  
debris?

I called Invitrogen and asked for help 3 different times during my  
attempt to grow these cells. They finally sent me a new vial of  
cells. Again, the cells started out ok. The first pass, went ok, but  
now the cells seem to be dying and there is an increase in cellular  
debris. (I also thought it might be the media so I gave them the lot  
number, but there didn't seem to be any reported problems.) Has  
ANYONE seen this before? If so, did you figure out what the problem  
was? Were you able to resolve it and get a good culture going?

As always, I appreciate any help you might have to offer.

ANSWERS TO THE ORIGINAL QUESTION:


On Jan 24, 2008, at 10:58 AM, David Ko wrote:

> That could also be it.  There’s a mycoplasma detection kit from  
> Sigma called VenorGem that I have been using to test various cell  
> lines for mycoplasma before we bulk them up.	The below is the link  
> to this product:
>
> http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/MP0025
>
> Hope that helps.
>
> Thanks,
> David
> Flow Cytometry Core Facility
> BC Cancer Research Centre
> BC Cancer Agency, Vancouver, Canada
>
> -----Original Message-----
> From: Gilman-Sachs, Alice [mailto:Alice.Gilman- 
> Sachs at rosalindfranklin.edu]
> Sent: Wednesday, January 23, 2008 12:47 PM
> To: Cytometry Mailing List
> Subject: RE: help! 293F cell line
>
> Mycoplasma
>
>
> From: Joel Tabb [mailto:jtabb at agavebio.com]
> Sent: Monday, January 21, 2008 11:44 AM
> To: Cytometry Mailing List
> Subject: RE: help! 293F cell line
>
> Nancy,
>   We are having almost identical problems with 293F cells from  
> Invitrogen.  Like you, we have contacted Tech support several  
> times, and gotten different answers each time.  Once they said that  
> multiple groups have complained and they traced it down to a  
> problem with a specific lot of media.  Alas, they have replaced our  
> cells 3x and our media 2x, and still no improvement.	We have given  
> up and gone back to adherent 293 cells.  We have started to obtain  
> cells that express more protein the old-fashioned way—carefully  
> trying different expression vectors and patiently screening/ 
> selecting high expressing clones.  It’s been slow and tedious, but  
> we are finally starting to get results.
>
> Regards,
>
> Joel Tabb, Ph.D.
> Principal Scientist
> Agave BioSystems
> 401 E. State Street
> Ithaca, NY  14850
> 607-272-0002
> www.agavebio.com
>



We have had these cells in the laboratory for a few years and found  
them to be excellent for protein production and for expressing  
recombinant receptors. A few tricks we have learned are thaw 10^7  
cells in 20 ml of Freestyle media into a 125 ml Erlenmeyer flask (we  
use flasks with a filtered cap from Corning cat # 431143), change the  
media the day after thawing, spin the cells down @ 100xg (this is  
important as they don't do well when spun down hard), resuspend the  
cells no lower than 0.2 x 10^6 c/ml, do not allow the culture to get  
above 2.5 x 10^6 c/ml  and use your regular cell freezing media,  
don't make up the recipe that Invitrogen recommends using Freestyle  
media.
Rich Hastings



I culture 293T and HEK-293 on a regular basis, without any problem. I  
use DMEM+10% FBS, with 37C and 5% CO2.
First thing to know is that those are adherent cells; so if you try  
to work with the cells in suspension,  you are basically working with  
dying cells...
To detach the cells, I used trysin/EDTA for a very short incubation:  
30secondes for 293T cells (10 sec for 293-HEK).
Then I remove the excess of Trypsin/EDTA and detach the cells by  
resuspending them in new media.
It works great for me.
Isabelle G. Solman




Nancy,
I have used this cell line years ago for transient transfections, but  
I don’t remember using 8% CO2, I believe we used 5%,and they seemed  
to do just fine in DMEM w/10% Hi FCS + 1%Pen/strep. Hope that helps,
K. Melissa Keays



Hi Nancy,
We have used these successfully in the past.  You didn’t mention  
this, are you keeping them on a shaker?  We kept them in the  
Invitrogen custom medium, 37 degrees, 8% CO2, on a shaker…  and they  
were great.  I don’t recall seeing a lot of debris.
Kerry



Hi,
I have a lot of expertise with 293f cells. We grow them in suspension  
uising spinner flasks. This flask keep the medium moving to avoid the  
clamping that really lead to the death of the cells. I usually have  
enough cells for transfection after pass 3, them they work great  
until pass 25-30. There is another trick, that is the cleanning of  
the spinner flasks with sodium hydroxide and the number of rpm for  
the sppiner flasks. It really works great if your vector or your  
protein has a signal peptide.




Not sure if these cells are very fancy, but HEK 293 cells for  
tranfecting are very robust cells.  I used to culture these guys like  
mad in simple 10% DMEM media in a regular incubator at 7% C02.	If  
they are having troubles with debris, you might have a bacteria  
infection, or it simply does not like the SF media.
The cells hopefully are adapted to them since they are sending it to  
you, but I certainly wouldn't put it past that they were/are.
Try to bump it in some regular serum rich media and see if the growth  
is any better.	If it is, then you know they simply haven't been  
adapted.
Good luck, this shouldn't be too difficult at all.
Steve




Have you checked for contamination?
Mycoplasm will sometimes kill cell cultures without obvious signs.



Hi Nancy,
For me the problem seems serious - check all cell cultures in your  
lab. The probable explanation is mycoplasma contamination transferred  
via aerosols under the cell culture cabinet. The cells in suspension  
do not usually produce much debris (unless their density - i.e. cell  
concentration -  is too high - check also for optimal denisty for  
your cell line).
Best greetings,
Blazej




I handled 293F and 293T (suspension) cell lines for over 2.5 years  
and I hope I can help.	I haven't had any problem with using 293T or  
293F to express soluble proteins, but I like to use 293T better  
because the 293F does not adhere well.	The media I use for growing  
293T/F is the Freestyle media from Gibco, the % FBS is 2%, and the %  
CO2 I use in the incubator is 5%.  As for the cell debris, I do see  
some from time to time (but not a lot and not too frequently).	Try  
changing media when that happens again, because that definitely  
helped me.  By that I mean changing your culture media to fresh  
media, and NOT to throw away your stock bottle and replace with a new  
bottle/batch.
How often do you passage or split your cells?  I usually keep them at  
around 70% confluent for 293F and around 500,000cells/ml for 293T  
(with rocker/shaker).  Also, I split my 293T three times a week.   
Yes, it is a lot of work but I have never ever had dramatic or  
unexpected cell deaths.  In fact, they just wouldn't stop growing!
Anyway, I hope that helps and I'll definitely email you if I can  
think of anything else.




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