I'm having some troubles making lentiviral vectors in mammalian cells. According to the protocol, I have to change medium before transfection but when I do this, my cells look awful, like they are going to detach from the plate surface. That makes me suspect about the medium, and the only difference between this new medium and the older ones is the FBS that was decomplemented for more time that the recommended (56ºC for 30 minutes, this one was decomplemented for 56º for 2 hours).
So, my question is: is it detrimental for my cells to be in a medium with FBS decomplemented for too long?
Dear Natalia,
Actually, I think it’s OK to use this FBS. Long time incubation at 56º may lead to the degradation of nutrients in serum. There nutrients are enough for 293T cells to be alive.
However, if your 293T cells are always in conditions with good nutrients, it may be not very good to substitute your media with bad nutrients abruptly before transfection.
Genemedi got a rich experience in lentivirus production and construction of stable cell lines, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
Do you have some serum around that was inactivated for 30 min to be able to test side by side?
When you change your media before the transfection, is the replacement media different than the media used before (except that it is fresh)?
Good point, I forgot that - but if you are working with immune cells you can probably work without inactivation. Freeze-thaw might be another problem.
I'm working with 293FT. We thought about the freeze-thaw problem and we cannot exclude it from the list. About the control, we haven't done it, and about the replace medium, it is exactly the same that the one I used to seed the cells.
So, Manju, you say that heating the FBS for too long could be less nourishing for the cells, but do you think this could be the reason for my stressed cells right before the medium change?
I worked quite a lot with the 293T and in my experience this is a cell line which is quite robust and easy to work with.
You can do a simple comparision: Transform cells as your protocol says, treat another batch the same without adding the transformation mixture (e.g. change media and so on) and let a third one just grown longer. Than compare the three and see how they look like. With this test you can see if your serum/medium change is the problem or the transfection.
Another thing: How dense are your cells when you change them?
Do your cells stay attached after changing the media (in my experience its quite easy to wash 293T of the flasks).
When you heat inactivate serum for longer time its possible that you are compromising the quality of the serum ( i mean all the growth factors necessary for cellular activities present in the serum may be affected).Like others suggested you can try with serum heat inactivated for 30 minutes parallely.
Prewarming to 37 degrees for 30 minutes is sufficient to inactivate the complement proteins 56 degrees for two hours will greatly reduce the nutritional components of your serum. Another question is are you changing media as you do transfections? If so, the crappy appearance of your cells is probably due to the plasmid and generation of viral particles. I went through the same process with my lentiviral preps. It is pretty hard on the cells, that along with nutrient and growth factor depleted FBS might be why you are having these problems.
It shouldnot be problem...but may be somehow ur fbs get contaminated....i would like to suggest filter ur fbs using 0.22 mu filter and try in culture again...and also heat that following protocol again and try again... So that u could at least guess what went wrong...
In transfected cells the quality of the medium is always a concern. Most serum does not need to be decomplemented (see comments above). Decomplementation in standard plastic flask in a simple water bath is anyway a not well standardized procedure. Keep in mind that beside denaturing the relatively large complement proteins other growth stimulating or adherence mediating proteins will be destroyed as a side effect. The longer you heat inactivate your FBS the more profound such undesired side effects will be. Thus your 2h treated medium could not be perfect any more.
If you plan to find out, test cell growth in a good medium and compare it to your 2h treated batch. This however is time and labor intensive and FBS relatively cheap. A simpler approach is thus to get good (e.g. commercial FBS, not necessarily decomplemented) somewhere in your institution and test if your experiment works. If yes you order some of this FBS (same lot!).
Best,
RAS
I ever decomplemented for more than 1 hour. For insect cell and vero cell can't growth very well. so, I agree with previous comment, inactivate serum for longer time can reduce the nutritional components of FBS.
The possible reasons are:
*Medium is not correct Quality/variability of FBS
*Passage number of cells is too high or Overgrowth of cells
*Infection of cells with pathogens (e.g., mycoplasma)
*cells are stressed.
2 hours at 56 °C could destroy the proteins of FBS that are necessary to support the cell growth ( they are cooked). Half an hour at 56 °C is enough.
If the FBS was at -20 or -80 °C before it has to be decomplemented, put it at 37 °C to thaw it before 30 mn at 56 °C.
Thank you for all the answers. Well, it resulted that we had two problems: the overheated serum, plus a mycoplasma infection. Both problems are solved for now, so my cells are no longer detached after the transfection!
For certain cells, heat inactivating for longer than 30 mins at 56˚C can have an adverse effect on your cells.
The most accurate google search result leads me here. I kept the FBS at 56 degrees for one hour. I didnt pre-warm it. Took it from 4 degrees and kept it into 56 degrees straight away (might have taken a few minutes to reach 56 degress). After heating, I poured the heated FBS directly into medium without allowing for gradual cooling (To cut short the time). Would it be a problem? Should I discard and prepare fresh? Please let me know. Anyone. If the thread is still active!
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