DMSO usage in cell culture - (May/26/2009 )

Hi all,

I am dissolving some cell culture drugs in 100% DMSO to make stock solutions of 10 mM. Im then adding 5 ul of this stock to 1 ml of media in 12 well plates to make a final concentration of 50 uM. This constitutes a 0.5% DMSO percentage which the cells are exposed to which should be safe for them. However at 37C some of the drugs come out of solution. I was going to then make a 1mM solution and add 25 ul of this to 500 ul of media to make a 50uM final concentration. This would constitute a 5% DMSO exposure.

Could anyone advise me if 5%DMSO is safe for Caco-2 cells to be exposed to for 24 h at 37C? What is the highest % of DMSO that the cells could withstand? I am intending on measuring amounts of cytokines and MMP-9 in the supernatants.

I get around 20% “loss” of CACO cells with 1% DMSO over 24hrs.

Your cells will be affected down to at least 0.1% DMSO. 5% is very high and will be dissolving the cell membranes. I would not advise using it at this concentration without appropriate controls for your experiments, and very careful analysis to ensure that any effects you are seeing are not due to the DMSO.

bob1 on May 27 2009, 02:13 AM said:

Bob1 is completely correct, DMSO for cell line experiments should always be below 0.1%, as it is cytotoxic at higher concentrations.
Primary cell cultures are far more sensitive, so if it is primaries you are using then do a further dose/response curve (viability) at concentrations below 0.1%.

Hope this is useful

Kindest regards

Rhombus

Hi Rhombus,

Thanks for the advice. I read a paper in the Biological and Pharmacological Bulletin from 2002 (Da Violente et al) whoch found that Caco-2s could withstand up to 10% DMSO when using an LDH assay to investigate cytotoxic effects. Tomorrow I will try the 5% approach and see what happens to my cells. Im not comfortable using this high a percentage of DMSO as it is a nasty chemical, but unfortunately my drugs are very hydrophobic and polar and will only dissolve readily in DMSO/acetonitrile. Im just hoping that increasing the % of DMSO in the plate wells will help ensure the drugs don't precipitate and do not kill the cells.

jakatta70 on May 28 2009, 06:54 AM said:

Do you mean nasty as in dangerous?
I’d be more concerned with what might be dissolved in the DMSO rather than the DMSO itself (LD50 in the g/kg). Mind you, it would take an awfully good curry to get that taste out of your mouth if you were to try some .

No I meant nasty as in the cells don't like being exposed to it; although it is dangerous in that it can be absorbed through the skin.

5% is quite high - most people try to avoid concentrations above 1-2% DMSO for cells.
My personal experience is usually up to 1% does not cause severe cytotoxicity in most cell lines.

Anyway: don't forget the vehicle control in your experiments

I found that I could go up to 3% DMSO final concentration when using Caco-2 cells and the monolayer remained viable and intact. Surprising!

jakatta70 on Jun 4 2009, 12:57 PM said:

yah..but it's what is going on inside your cells that you cannot see that you should be concerned about..because DMSO dissolves the endomembrane system at variable rates in different cell types from different species with greater effects on golgi stacks in some lines, ER in other etc.

most people will not use greater than 1% DMSO. sure the caco cells might appear to tolerate more DMSO...but what good is it if your studies cannot extend into other cell types etc. because you were measuring effects of high DMSO that was masking or potentiating the drug effect (which already will have a number of "off-targets" that you need to control for...e.g., testing LiCl inhibition of GSK should be done in parallel with a NaCl control AND roscovitine since too high LiCl in some cell lines will hit cdks as well as GSK)...same deal for your experiments

if you cannot get around using high concentrations of DMSO then you should be also doing MTT assay to calculate cell death and maybe blot for activation of caspase-3 in case your cells look "OK" to your eye, but are undergoing apoptosis.