Protocol Online logo
Top : Forum Archives: : Cell Biology

Passaging stable transfected cells after thawing - (Aug/23/2007 )

Dear all

I've been working with stable transfected HEK-293 cells. These cells, which stable express my interested protein, would be selected by mean of G418 selection. I normally do freez the cell at early passage (not over passage 10) and continue culture this cell line until passage 50 before I discard the cell from culturing. My problems and questions are

After thawing of this cell line, the less numbers of cells are survived and grown and they seem to grow quicker when they getting older. Does G418 or Pen/Strep have an influence on this process because my freezing medium does not contain G418 and Pen/Strep but my culture medium does?

Can I count the cell passage after thawing as null not continue count the passage from what the cells were frozen, if the cells are still young and thier morphology are not at all different from before freezing and after thawing?

For this kind stable transfected cell line, when should I get rid of the cell?

Thank you

-L_Lio-

When we freeze stable cell lines, we donot add any antibiotics. And when we thaw them, we have them in the media with the antibiotics.

Cell passage number is just a guide to know how many times they have been split or for how long they have been cultured. If you missed counting few times, doesnt really matter. As long as they behave normally thats what matters.

We usually donot count thawing as splitting.

-scolix-

"After thawing of this cell line, the less numbers of cells are survived and grown and they seem to grow quicker when they getting older. Does G418 or Pen/Strep have an influence on this process because my freezing medium does not contain G418 and Pen/Strep but my culture medium does?"
-It is quite common that cells grow fast when they reach certain density.

"Can I count the cell passage after thawing as null not continue count the passage from what the cells were frozen, if the cells are still young and thier morphology are not at all different from before freezing and after thawing?"

- It is an interesting question. I do have some cases when cells go through one freeze-thaw cycle, they end up in better shape. I guess you can start the counting when you thaw a new vial and pass it until you feel they dont look as good as it should be.

"For this kind stable transfected cell line, when should I get rid of the cell?"

Its your call when you feel they dont look or behave formally.

-genehunter-1-

The freezing procedure does not need antibiotics like G418 and or peni/streto. Protocols using serum +10%DMSO are often used. The fact cells are growing quicker may just come from the confluency when cells reattach. May be a clue for your passaging steps (ie, you may split them at higher ratio).
If you want strickly count passage, then a freezing should be counted as a passage because you applied a detachement procedure. BUT, i would rather neglect the passage mention, and add as mention for this culture (1freeze/thaw) step (in additions to the passage)

I regulary check the transfected cell line bt a marker : i mean regulary add antibiotic at first, and for ex, if it's for recombinant protein, i check the presence of this recombinant protein regulary.

-fred_33-

Thanks guys, all your advices help a lot to give me the confidence in performing my experiments !!!
It could very well be that I split the cells in a very high ratio normally 1:10. This maybe the reason of cells come so quick to the high confluence.
Well, I'm going to note and add one passage after thawing the cell just for the right doccumentation, but for the passage number I will use as guide in culture of the cells
I normally check the level of expression of my proteins from stable transfected not directly by mean of western plot analysis or real-time PCR, but I perfrom the experiment so called patch-clamp to check the activities of the channels, which are formed by stable transfection plus selection of G418. Interestingly, some time the cells, which have been cultured for more than passage 50 still give exactly same activity of the channel as yong passage cell (unchange), so this might be a well conclusion for keeping the cells for the experiment?

-L_Lio-