Dear All,
I am trying to establish stable cell lines expressing my transgene. The transfections seemed to have worked fine and I had picked the clones few weeks back which were first growing in 96-well plates. I have been expanding them and now that I am transferring them to 12 or 6- well plates, even 24-well plates, they are forming clumps and are dying. They are not adhering to the plate surface anymore. Everything has detached, forming clumps which are swimming around. What could be the reason.
Hi Ikram,
It's really difficult to say what could be the problem, from the information you have supplied, so I've some questions that might help narrow down the problem.
What are the cells you are transfecting? How many clones have you expanded? And how many weeks have they been on selection? What is the selection? Do the cells only die when you expand the plate size, can you passage the cells into new 96 well plates? How are you removing the cells from the wells? Are you using the same batch of media/selection? Does this happen if you remove selection? Do your untransfected cells grow on the plates?
- Richard
Hi Richard
Thank you for your effort. I will answer in the sequence as the questions. These are Drosophila S2 cells. They were in selection for 4 weeks and I used 10ug/ml Puromycin. When I pick clones from the selection plate, I seed them in 96-well plates without selection media. So important to mention is that they are dying in normal S2 media. Yes the cells die when I passage them to bigger plates. However, there were few clumps even in 96-well plates, but as I expand them from smaller to bigger wells there are huge number of clumps which are swimming around. I am using a 1ml pipette, pipetting up and down few times to suspend the cells and then transfer them in new plates. I have other untransfected cells growing nicely in the same batch of media. Yes this is happening without selection.
I really appreciate your time and effort.
Thank you
Ikram
Hi Ikram,
Drosophila S2 cells are not a cell line I've used, so I'm a little short of advice as to how to proceed. It could be possible that your gene is causing the problem if it is constitutively expressed, given the untransfected cells behave normally, but why it is happening only after scale up I don't know. It might be interesting to see if you get the same effect if you maintain selection.
Sorry I can't be of more help,
- Richard
Your transfection methodology initially worked, but that does not address the main phenomenon that you are observing now. What comes to mind is a simple chance to question the toxicity of the material that was transfected, and secondly the potential toxicity of the transfection medium and methodology itself. We should always be conscious of these issues as the possible limitations of such methodologies, compounds and materials and really consider the pros and cons of such approaches. Otherwise to me science should add value to our endeavors, not one step up and two or more steps back and not achieve anything in the end that can benefit science and society.
Vilmar
Vilmar Villa
Researcg Biologist
Hi Ikram,
Just an idea, have you tried adding some Anti-clumping agent to your cultures?
https://www.lifetechnologies.com/order/catalog/product/0010057AE
Also thinking a bit more about your methodology. This is (probably) not related to your problem, but I would recommend on general principal you maintain some selection pressure on your lines. Currently each line will be polyclonal, and removal of the selection can cause none expressing cells to outgrow the expressing ones. Once you have clonally selected lines, then you can remove selection if you want. Personally, I would maintain the clonal line with a lower level of selection, but not use it in scale-up expressions.
- Richard
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