Cytolysis

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Tony Rook
Tony Rook's picture
Cytolysis

How common, or is it common at all for mouse (mammal cells) to cytolyse (by osmolarity or hypotonic death?) when incubated in the same MEM solution for 72 hours? Or would they lyse at all, being they are in a saline environment....could they shrivel up and die since their internal water would be flowing out to the solution?

Any help on understanding how these cells might die would be great.

marcus muench
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They should not die because

They should not die because of any osolarity imbalance. Media are designed to be isotonic. However, cells require more than just medium and if you deprive them of growth and survival factors that are often present in serum then they will die. I don't know what cells your referring to, but I can't think of any cell type that would survive more than a few days in just plain medium.

Edit: Obviously cells in the body are not suspended in a salt solution, but are in the presence of a lot of protein etc. The presence of all the non-water molecules clearly has an effect on the structure and function of individual proteins etc. I'm not sure how the lack of protein affects cells at large, but it could be part of the reason cell viablity crashes in medium alone. However, I think my answer above is the main reason. I think were you see the effect, in lab work, of protein concentration is when working with purified proteins. It is why we store proteins at high concentration or with glycerol or sucrose. My logic is that if changes in hydrophobicity affect purified proteins, then it could also affect cell surface proteins.

davidj
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I would be helpful to know

I would be helpful to know what kind of cells you are trying to grow and if you are growing them in T-flasks, shake flasks, the incubator (tem and CO2), etc.

In general, you will need some sort of growth factor source, whether it be from serum or a specific cocktail to have the cells survive. You can adapt some cell lines to grow in just media, but i would not recommend it.

Your cells have likely progressed through apoptosis in the 72 hours they have been incubated, growth factor withdrawl is a classic method for driving it.

If i get an idea of what your are trying to grow, and your goal for the cells, I may be able to point you in a couple of directions.

Tony Rook
Tony Rook's picture
davidj wrote:I would be

davidj wrote:

I would be helpful to know what kind of cells you are trying to grow and if you are growing them in T-flasks, shake flasks, the incubator (tem and CO2), etc.

In general, you will need some sort of growth factor source, whether it be from serum or a specific cocktail to have the cells survive. You can adapt some cell lines to grow in just media, but i would not recommend it.

Your cells have likely progressed through apoptosis in the 72 hours they have been incubated, growth factor withdrawl is a classic method for driving it.

If i get an idea of what your are trying to grow, and your goal for the cells, I may be able to point you in a couple of directions.

The cells are a ATCC L-929 mouse fibroblast cells; in Eagle' Minimum Essential Medium (E-MEM), supplemented with 5%(v/v) FBS and antibiotics. They were incubated at 37 deg C and 5% CO2. It is used for a cytotoxicity test, to test for toxicity of chemical residual extracts.
I hope this is enough information for you to respond with, please let me know if you need additional infomation.

macbride
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How confluent are your cells?

How confluent are your cells? If you seeded them heavily and then let them go 72 hours, they may be very overcrowded. That can definitely lead to cell death in some cell lines.

Tony Rook
Tony Rook's picture
macbride:

macbride:

Thank you for your input! However, I do not believe these plates were seeded extraordinarily heavy. It might be something to investigate.

Is cell death due to culture or seeding conditions a common issue in cell culture?

samm
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This detracts some from your

This detracts some from your original post, but seeding densities are often critical to the well-being of cultured cells. Too much OR too little density can both have deleterious consequences, ranging from non-expression of a protein of interest to death.
In addition, extraneous medium factors secreted by the cells also play a role - hence the use of "conditioned" medium.
Haven't actually worked with the L929s, but have the impression that they are quite hardy cells (true for most fibroblasts) - do you have some kind of contamination? Is your medium pH changing very rapidly? If so, adding HEPES to your medium may help.

montgomj
montgomj's picture
You said that you were using

You said that you were using 5% FBS. ATCC recommends (and it has been published elsewhere) 10% horse serum. You may want to try this. Also if you had just thawed the cells you may want to try replacing the media after 24h. I find this helps with the health of some of my cells.