Summary of Re:293 cells with transformed phenotype

Lloyd Graham graham at dbe.csiro.au
Mon Mar 24 20:09:48 EST 1997


In a bionet posting, Lloyd Graham wrote:

>I am in the difficult position of having made numerous stable 293
>transfectants (using pRc/CMV derivatives) which adopted a 'transformed'
>non-adhesive phenotype during the selection procedure, and have stayed that
>way ever since. My (untransfected) stock flasks also switched to a
>non-adhesive phenotype soon afterwards, so the phenotype change is unlikely
>to be due to the transfection itself. I gather that 293s do spontaneously
>transform to a poorly adhesive form at high passage number; my cells were
>at passage ATCC+12.
>
>Since I cannot just go back to earlier passages of the transfectants, I'm
>hoping that I can work around their lack of adhesion and passage them like
>suspension cultures. However, I don't know if the growth requirements of
>non-adhesive 293s are any different to those for the normal 293s, or even
>whether these non-adhesive cells are stable to subculture.  Do you have any
>ideas?

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I have receieved many helpful responses by email, and reproduce them here 
for the benefit of other bionet subscribers. In the meantime I have found 
that my cells will spread on gelatin-coated plates, and do even better on
fibronectin-coated plates.

REPLIES FOLLOW:
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From: vando006 at mc.duke.edu

     I have been culturing Hek 293 cells now for about four years and I can 
     tell you that this is fairly normal for this celline. Somewhere 
     between passage 10 and 20 they seem to transform and grow like you see 
     them. I always advise people to choose another celline if they do not 
     have to use these cells.
     What you could do is freeze a very early passage in ten tubes (or 
     hundred if you feel especially dedicated) take one tube of these and 
     divide into ten tubes again (one passage up) freeze these and go back 
     to this frozen stock every time. In this way you have a lot of times 
     to go back to the first passage.
     A few tips that keep them in passage longer:
     1. Never split them without trypsin, once you do they loose their 
     adherence receptors and you will see they will not stick to your plate 
     anymore.
     2. Keep the same lot of serum if you can, they are extremely sensitive 
     for certain badges of serum. Try a new lot out first.
     3. Lower the CO2 tension in your incubator, they seem to do better in 
     4.5 CO2, they are extremely PH sensitive.

   From: vando006 at mc.duke.edu

     You might want to try to use collagenase or fibronectin on the dishes 
     that you culture with. I have had very good results with both of those 
     when I cultured them on glass coverslips. 
     I saw on the newsgroup somebody suggested mycoplasma contamination but 
     I really think that is not the case. I just recently went to the 
     neuroscience meeting and spoke with several labs that cultured Hek 
     cells ant they basically said the same thing happened to them. There 
     seems to be a different Hek celline around now that works better but I 
     have not tried them out yet.
     My suggestion would be to split them out really thin on fibronectin 
     and see if you can get them to grow normal that way.
     Try the CO2 tension too, it worked great for me.

     Good luck, Margon 

     
     Margon Vandongen
     Duke University
     919-681-4863
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From: anthonyp at scripps.edu (Anthony J. Pelletier  Ph.D.)

Someone else on the net suggested that you check for mycoplasma.  This is
always a good idea whenever there is a change in phenotype, of course.
But, if they are growing normally otherwise (rapid doubling time, not alot
of junk in the medium) I doubt that is the problem.  Also, remember that
293s are not the most adherent cell line in the first place.

I have noticed over the years (I have had 293's and derivatives in culture
almost continuously for 6 years) that if they are allowed to get over
confluent, they tend to pile up on one another, forming foci, then little
balls of cells, that come off the plate easily.  Once they take on that
phonotype, they tend to keep it.

If you are comfortable re-cloning them, plate them for single colonies and
look for those that behave "properly,"  that is, how you want them to, and
grow those up.  I work on cell-adhesion molecules (integrins) so I find it
important to keep them uniform.  But, depending on what you want them for,
you may do fine with your transfectants in the less adhered form.

At Genentech, many people used to suspension-adapt the cells and grow them
in spinner flasks to get alot of protein.  I never did since i was, as I
said, studying adhesion molecules.  however, as far as I know, they are
fine in the less adhered state.

If you tell me what you are doing, perhaps I can come up with better
suggestions.

-tony

Anthony J. Pelletier, Ph.D.
Assistant Professor
The Scripps Research Institute
La Jolla, Ca
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From: Ong Siew Hwa <mcbongsh at leonis.nus.sg>

That's how my 293 cells grow, wildtype and stable tranfectants.  I have
derivatives of them and they are all the same.  I think that's how they
are.

Siew Hwa
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From: Caroline Szymeczek-Seay <seay at niehs.nih.gov>

Many people in our lab have had the same experience.  I have escaped
this problem so far.  The only thing I can determine that I am doing
differently than other folks in our lab is 1) i always split my cells
very densely (1:3 or 1:5 2 or 3 times a week and 2) I use heat
inactivated fetal calf serum at 10%.  I do not use the media recommended
by ATCC,  but i use DMEM instead.  

this is small help, i know, but good luck.

Caroline SEay

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