I am growing N-Tera 2 cells and performing CRISPR on them. They are taking up the plasmid construct well, but the problem we keep having is that both with and without CRISPR intervention, we can't seem to keep them alive at low density. This is a MUST for isolation of our construct-positive cells for a monoclonal population. Our current approach is to try using conditioned media at a 1:1 ratio with fresh media in order to try to get these cells to grow at low density, but I don't know the results from that yet and am looking for other suggestion,s either on the clonal isolation or the growth at low density. Thanks!
P.S.- We don't not have access to cell sorting (FACS), and our cells do not have an antibiotic selection marker-our positive marker is GFP.
Hi Kimberly,
This reminds me of a similar issue with stem cells, which often won't grow without support from other neighboring cells. What stem cell scientists do is use "feeder cells" - MEFs or another cell line that they irradiate or chemical treat so that they can't grow normally (so they won't take over a co-culture). These feeder cells are added to many stem cell cultures to provide the cell contact and hormone secretion that the stem cells need to properly grow and differentiate.
You could try adding feeder cells to your monoclones (or more likely, add your single cells to layers of plated feeder cells). Since the feeder cells are treated to prevent mitosis, they would eventually die out of the culture as your monoclonal population expands to support its own growth and passaging.
I'm not a stem cell scientist and haven't used feeder cells much myself, but this may be worth testing. Good luck!
One of my suggestion would also have been using e.g. Fibroblasts as feeder Cells.
If it is less about cell-cell contacts and more about growth signals etc. you the method of exchanging only half of the cell culture medium instead of the complete medium often works nicely. This way, you don't remove all of the soluble molecules, excreted by the cells and they often times stay more viable.
If this is the problem, it could possibly also help if you simply use more serum, up to 20 % are possible. This can sometimes ensure cell survival in low density cultures.
A last possible approach, if it is more about cell-cell or cell-matrix contacts, you have to find a suitable molecule for precoating your cell culture dishes. There are many available and they can strongly impact the cell survival.
just a wild thought: what if you would add some fresh N-tera-2 cells to the transfected cells like 6h-12h post-transfection? You would change the media, without detaching the transfected cells and add the fresh cells.
Then, your GFP positive transfected cells would stay appart from one another (monoclonal) but would be surrounded by non transfected cells added afterwards and used as feeders. Well...this may work.
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