Eukaryotic cell culture

FredsMom

Hello,

I am carrying out eukaryotic cell culture. I have found that when I seed to 48 well plates the cells in the well at the edge appear to round up and die, they also show high cytotoxicity. Does anyone know what could be causing this? is it because the cells are at the edge of the plate..something to do with pH maybe?

Any suggestions as to reasons are more than welcome!!

Thanks

adamski8

i would say there is so many things that could be happening but you'd need to give alot more information, what cells, what density, what media, where did u get your culture protocol, how are you checking cytoxicity, why are you using 48 well plates, are you stimulating them with something, are you using round of flat bottom plates?

Kevster

What do you even mean by them showing high cytotoxicity? Something that is cytotoxic is capable of killing cells themselves, but yoiu're implying that the cells are cytotoxic to themselves?

What do you mean by 'round up'? too? Do you mean that they - like swell up individually?; or they clump together? If they clump together, what chemicals are you using the the substrate? EDTA will obviously cause them to clump together, but you haven't indicated using this.

kevin

CramCycle

If the treatments are correct it is most likely your seeding densities. Alot of cell types do not like to attach near the edge (can't remember why but there is a reason), and immortal cells do start to kill each other off at high densities, this is common across nearly all cell types. Try a significantly lower density eg if your using 10^5 try 10^4, although again some cells also need a minimum proximity and therefore density as well (sharing of specific hormones/GF etc.), anyone else in your lab using these cells? or what specific cell type are they?

Kevster

CramCycle wrote: »

...and immortal cells do start to kill each other off at high densities, this is common across nearly all cell types.

Thanks - that explains what she meant by them exhibiting cytotoxicity. I culture different breast cancer cells but some strains just seem to grow 'all over each other', or so it appears under the 'scope. Others grow much less aggresively and indeed die off if they become crowded. This must be merely due to a lack of nutrients though, and nothing more. Mortal or immortal cells cannot be cytotoxic to each other in the general sense of the word...?

Sorry to be friggin' pedantic, but I want to be sure on this.

CramCycle

Overcrowding is what generally kills the cells but this is for various reasons and varies cell line to cell line. We have a kidney cell that will grow in mounds when overcrowded and will survive for quite a time but other pancreatic cell lines we have die off after they become 80% confluent. Reasons for this include, as you said, starvation but this is rarely the case as you would be refeeding the cell line before they used all media supplements.

Induction of programmed cell death due to signals from the cells surronding it as they become to confluent.

For alot of cell lines once a monolayer is formed they will die off rapidly as they need to adhere for whatever reason to the plate/flask and cannot.

Usually in this case some will detach or newly formed ones will detach and eventually die. When they lyse in the media they change the pH of the media which will kill of the rest of the cells.

There are alot more reasons that people know of but I honestly have not looked into it too much before, it's one of those things that you accept in cell culture.

Just to point out those cell lines that survive past a monolayer often give unreliable/unpublishable results and you should definitely investigate the protocol of previous investigators in similar cell lines, for reported issues.

Kevster

Thanks. Despite the research I've got myself into, cell biology was never really covered in my undergraduate back in Ireland. Growing cancer cells is quite demanding though, as they grow rapidly. There are some cell lines that could be almost 100% confluent after 24-hours of incubation (in a T-75 flask), for example.

Kevin

CramCycle

Kevster wrote: »

Thanks. Despite the research I've got myself into, cell biology was never really covered in my undergraduate back in Ireland. Growing cancer cells is quite demanding though, as they grow rapidly. There are some cell lines that could be almost 100% confluent after 24-hours of incubation (in a T-75 flask), for example.

While splitting try to maybe seed the cells at different densities, to get an idea of what you need to seed them at for getting them confluent on different days. You have to do this with most cell lines to seperate out your experiments.

Some cell lines also need this so as to become accustomed after splitting as they can find it traumatic and they need time to return to their resting metabolic states, others do not. Maybe you could try seeding them in T-175s.

When you say you are growing cancer cells, do you mean your cells are based on carcinomas or they are immortal cell lines for the study of cancer or are they primary cell lines that are cancers? Just for ideas on possible problems you maybe having.