Will dead cells in culture media have any significant impact on Live cells during incubation?

Question

I am generating stable cell lines and have something like 25+ culture flasks to take care of. Ideally I can let dead cells sit in the media with the live adherent without rushing to change media constantly - Will this affect growth of live cells - I don't want to hinder them as they are already sitting in high[] Hygromycin

Answer 1

I'm not sure if this is linked to the actual dead cells, but I've found that not changing the media on plates while trying to generate lines from primary cultures can lead to contamination. Again, I haven't determined if this is because there are dead cells available for contaminants to derive nutrients from, or if it's just because changing media removes contaminants before they can grow too much. You might want to keep that in mind, though. I've found that changing media every 3 days is good enough.

Also, it depends on what you want to use your lines for. If you're hoping to study cell signaling or polarization (like if these are immune cells), it may be a problem, as the dead material will activate immune cells, and alter their signaling patterns. This has less to do with your worry about overall growth and more about you developing a good model for whatever you're doing.

To be fair, these signaling shifts can modulate proliferation in culture, but that's really going to vary from cell type to cell type. Sorry for being a bit vague! It's just that all cells are different- I happen to work with immune cells that are fairly finicky. There are other types that are a lot more robust, however, like fibroblasts, that you could probably spit on and they'd be fine.

If changing the media every 3 days really is too cumbersome, you could try splitting off small subcultures and doing a mini-experiment to see if changing the media vs not impacts confluence after a week or so? That would tell you how flexible your cells are when it comes to this.

Edit: Here are some publications that discuss the effects of dead cells in culture and differences between cell types. I also recommend that if you're using a cell line, looking up information via the ATCC website and reading their datasheet and some of the top references they site that likely describe how the line was isolated and best practices for culture.

1. "Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro" - a good example of how a lack of dead cell removal in vitro can impact cell function/phenotype and highlights the differences between different cell types.

2. "Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies" - a good review that touches on the actual molecular mechanisms to which I was alluding. I particularly recommend reading the section "Induction of cell death in mammalian cell cultures", as it summarizes and links to several primary resources that show how increases in cell death can affect things like productively, growth, and cell number. It also does a good job of touching on the different kinds of cell death, which is important to understand but may be a bit too in-the-weeds for the original question.

3. "Links between metabolism and apoptosis in mammalian cells: Applications for anti-apoptosis engineering" - similarly good as the previous source, but looks at it through a more metabolic lense, which I think is helpful when considering the realities of cell culture.