help! 293F cell line

Hastings, Richard C richard.hastings at
Wed Jan 23 16:50:11 EST 2008

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 

 -----Original Message-----
From: Nancy Hurtado-Ziola [mailto:nhurtado at UCSD.Edu]
Sent: Thursday, January 17, 2008 8:42 PM
To: cyto-inbox
Subject: help! 293F cell line

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.


Nancy Hurtado-Ziola, Ph.D.

Varki Laboratory

Department of Cellular and Molecular Medicine

University of California, San Diego

9500 Gilman Dr.

CMM-E 1087, Mail Code 0687

La Jolla, CA 92093

Lab Phone: (858) 534-1346

Fax: (858) 534-5611

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