“Universal solvent” –DMSO

DMSO(Dimethylsulfoxide) was called a “miracle drug” when it was first discovered because of its widespread use, and then restricted by the FDA. How toxic are solvents that are widely disseminated, and how do we use them in our experiments? Today we will reveal the correct use of DMSO, the “universal solvent”, in cell and animal experiments.

Introduction to DMSO

Dimethylsulfoxide (DMSO) is a sulfur-containing organic compound whose structure is shown below.

Physical properties: colorless odorless transparent liquid at room temperature, with strong hygroscopic, freezing point of 18.55℃, density and water close to 1.100g/mL;

Chemical properties: The molecular formula is (CH3) 2SO, because the structure has a hydrophilic thionyl group and two hydrophobic methyl groups, it can be miscible with aqueous solution and most organic solvents except petroleum ether; It can dissolve about 80% of the compounds, and most of the water-soluble compounds and fat-soluble compounds can be dissolved by it, so DMSO is also known as “universal solvent”.

History of DMSO

DMSO was discovered by Russian physician AlexanderM.Saytzeff in 1866. In the 1950s, British scientists discovered that it could be used as an antifreeze to store bone marrow and blood cells. This unique by-product of the wood pulp industry can also be used as an industrial solvent and paint thinner. Scientists have conducted a large number of studies, and the results show that DMSO can quickly relieve pain, end headaches, and even quickly recover and restore muscle sprains and burns.

In 1963, the FDA approved DMSO for human trials, and some big pharmaceutical companies conducted an unprecedented number of experiments, spending a total of 250 million dollars, testing more than 100,000 people, and each group of experiments showed that DMSO was a very safe and effective substance. But a study by Merck, a drug company, showed some cloudy changes in the lens of the eye in some animals receiving DMSO. On December 25, 1965, based on Merck’s research, the FDA banned all human use of DMSO. As can be seen from the figure below, after 1965, scientists conducted extensive studies on the toxicity of DMSO, but it was not found to have serious toxic side effects on healthy experimental animals and human beings, but it was relatively toxic to cells, so DMSO was restricted by FDA to be used in non-special and non-irreplaceable cases.

DMSO toxicity

1. Cytotoxicity of DMSO: need to control < 0.1% (v/v)

In 2008, QiWeidong et al. ‘s study found that when DMSO was contained in the culture solution at 0.1%-0.25% (v/v), there was no damage or impact on rat dasyic cells within 24h, but when the DMSO content in the culture solution reached 0.5%-6%, the dasyic cells showed damage and dose-dependent cell death.

In 2014, He Bing et al. ‘s study found that different concentrations of DMSO in cell culture medium had certain effects on the growth of rabbit chondrocytes cultured in vitro. When the concentration of DMSO was 2.0% (v/v), it showed a strong inhibitory effect on chondrocytes, but it had little effect on cell growth in the concentration range of 0.1%-1.0%.

2. Enzyme toxicity of DMSO: need to control < 0.1% (v/v)

In 2011, RamakrishnaNirogi et al. found that in the incubation experiment of human liver microsomes, DMSO content above 0.1% in the incubation system could inhibit the activity of CYP1A2 in human liver microsomes. However, 2% concentration of methanol or acetonitrile had no effect on enzyme activity.

In 2003, MasuhiroNISHIMURA et al. found that when a compound is administered to human liver cells, Of DMSO concentration at 0.1% or lower level on the drug metabolic enzymes in a phase of liver cell subtype cytochrome P450 enzyme (CYPs), two-phase drug metabolic enzymes in cell subtype glucuronyl transferase (UGTs) as well as the main transmembrane transporter ABC transporter (ATPBingCasstteeTransporters The expression amount of) had no effect, and higher concentration would change its expression.

From the above toxicological data, it can be seen that compared with other safe and non-toxic solvents, although DMSO has certain toxicity, DMSO also has its own unique properties, before finding a better cell antifreeze and a “universal solvent” with higher flux, Biological experiments and drug preliminary screening stage can also make correct experimental results without reagent interference quickly through reasonable use of DMSO. Here’s a look at how to use DMSO correctly in an experiment.

How to use DMSO correctly in cell experiments?

1. Cell freezing agent

DMSO is the best protective agent for cell cryopreservation at present, but it has certain toxicity. Generally, when used, the concentration is controlled below 10% of the final volume, and can be reduced to less than 10% for cells with weak tolerance, such as 8%. Note that dimethyl sulfoxide should be washed off as soon as possible when the cell recovers, otherwise it will cause serious cell toxicity. The results showed that when the concentration of DMSO in culture medium was 10%, the inhibition rate of cell growth was nearly 100%. At 1% concentration, the inhibition rate was 35%, and even at 0.04% concentration, DMSO had adverse effects on cell growth.

2. Solvent of the compound during cell administration

The dose of DMSO should be strictly controlled when the cell is administered with dissolved compounds: Different cells may be sensitive to different levels of DMSO, but what is more acceptable now is that when DMSO compounds are prepared for cell administration, the final concentration of DMSO is controlled within 0.1%, which is considered to have no interference with cell experiments, that is, the volume of DMSO in 1ml medium cannot exceed 1μl. If the volume is found to exceed this ratio, we can reduce the amount of DMSO by increasing the concentration of the drug, or using aqueous multi-step dilution.

3. What is the grade of DMSO and the way of aseptic filtration?

The DMSO grade used for cryopreservation must be Tissueculturegrade DMSO (such as SigmaD2650), which itself is sterile. After opening the bottle for the first time, a small amount of it should be divided into sterile test tubes and stored at 4℃ to avoid the cracking of DMSO and the release of harmful substances caused by repeated freezing and thawing. And reduce the chance of pollution. If you want to filter DMSO, you must use a DMSO-resistant Nylon filter membrane.

Note in the use of DMSO

1. Avoid skin contact

The study found that DMSO has a small molecular weight and is easily absorbed through the skin. If the skin is exposed to DMSO for a long time, it will show a small local irritation response, such as local skin irritation response, itching and burning sensation. During the experiment, 1%-5% ammonia water can be prepared for backup, and the skin should be washed with a lot of water and dilute ammonia water after it is stained. At the same time, you should also avoid contact with dimethyl sulfoxide solution containing other toxic substances, because dimethyl sulfoxide will penetrate the skin, under certain conditions will bring toxic substances into the skin.

2. Avoid inhaling DMSO

Inhaling DMSO is most commonly associated with nausea, vomiting, and exhaled breath that smells of garlic, onion, and oyster. High concentration of large area use can affect nervous function and lead to hemolysis, when using to avoid its volatilization.

In conclusion, although DMSO has certain toxicity, it is not unavailable; In the absence of a suitable alternative, it is permissible to apply DMSO in moderate amounts to obtain research data.