I don't under stand why are you dissolving your drugs in 100% ethanol when they have been completely homogenized in DMSO (100%). Moreover there is no need to again make a dilution in PBS too. While testing them in Cell lines you just dilute them in the culture media and use it directly but remember that the DMSO final concentration should never be increased more than 0.3% (v/v) . Use a separate control well with 0.3% (v/v) DMSO alone which will serve as Vehicle treated. However, if your extracts are highly hydrophobic (aromatic chemicals) then they would probably make crystal like structures in the culture well with media after some hours but that would happen at a very high concentration. Try to go for serial dilutions in media only without the cells so that u would come to know that after some time which concentration range is having the precipitates. Avoid them and use the lower concentrations. Good luck

I confirm the answer of Vivek, the maximum final dose of DMSO in an in vitro system does not exceed 0.1-0.3 %. so you must try to prepare a started solution of foreign compound in order to avoid any precipitate when you add it to culture medium. i

Thanks,

Yes at first I dissolved in DMSO and then diluted in PBS. But they formed crystals. I did not try in media. Thought it wont be different. Later found in some discussion (in some webpage), some one suggesting to use ethanol, hence tried that step. At lower dilution, I did not find any crystals, thought it might be because, number of crystals will be very low so could not find. However, at higher concentration (>50ug/ml) crystals formed but virus did not grow. I am new to this anti-viral drug testing. Might be my methods or questions are amateurish.

I agree with Vivek Vaish. All you need do is make your dilutions in the media. You may need to also include the DMSO alone as well as drugs in DMSO in order to eliminate interference if any arise due to the hydrophobic nature of drugs...Also remeber that your diluted conc in the media will be 2x of desired. Good luck

I agree too with Vivek. According to my experience some types of cells (Caco-2; HT-29; U2Os or HepG2) are viable also with a concentration of 1% DMSO in cell culture medium. But in most cases I use 0.2-0,1% DMSO in control cells for my experiments.

You can never exceed the solubility of your compound in the assay medium. If you do the assay will read out the activity of only the compound soluble concentration. The remaining drops out and equivalent to stone. if you add solvent you reach a point where the solvent disrupts the medium and test becomes invalid. Test the compound at it's saturation point or find a compound that is more water soluble.

Programs die as a result of faulty data generated by weakly soluble compounds. The activity reported is at the saturation concentration and that level is not recorded and different for each compound.

The literature has many examples and if data doesn't correlate that is the first place to start.

Based upon my own expertise, loss of cell-viability can be observed at DMSO concentrations as low as 1% V/V, including for colorectal cells like the ones cited above by Maria - I guess that there is some interlab variability. Normally I don't exceed 0.1% - 0.2% of DMSO when conducting experiments, and adding the same in control wells is an absolute requirement indeed, as underlined by others. Having said that, poor druggability is a a common concern when working on news chemical entities - and the junk yards of pharmaceutical companies are full of highly active compounds just unable to be properly solubilized in aqueous media, despite promising QSAR evidenced in silico. I mean that if does not dissolve properly in saline or in culture media to reach a minimal effective concentration in vitro, well then just kill it and make another lead identification: sooner or later you will have to face serious similar issues when using your drug in vivo. If you can't get rid of the problem without using high levels of solvent, and if you don't want to No Go your drug, the other option is to develop some smart biopharmaceutics such as liposomes - they are not that tricky to develop (basic apparatus can do the job), you could then bypass the poor solubility profile by incorporating your drug into the lipidic membrane of the liposome and use them to deliver your drug to the cells. Standard bench-liposomes are not that difficult to prepare, but it's time consuming and not very stable throughout time. So basically, it all depends to what extent you want to keep this particurlar drug, or not.

According to literature, less than 0.1%

we have been maintaining various flavonoids preparastions of various concenrations all dissolved in 0.5% DMSO for long and have found no wrong using them in microbiological studies as of now.....even with the control of 0.5%DMSO there was a good growth of culture of E.coli appreciable to that with PBS or Distilled water....think u must once go for a trial work with ur culture with ur necessary growth conditions.....also check for pH coz any change in pH with the same dilutions of ur sample will certainly hamper ur results....good luck...

Thank you all for your suggestions.

I had dissolved the extract in 100% DMSO but it precipitate when added to the media. In such case will it dissolve in 10% DMSO? I am more concerned to test with different DMSO % (V/V) because I have very less quantity of the extracts and not possible to take fractions of it.

I feel Mr. Joseph Ciccolini 's point would be the case in my experiments, with their dissolving property. I will try to get more information about the properties of the extract from the provider which may solve the problem.

I get the impression that you are dealing with a solubility issue regarding your drug. I am assuming that you are trying to use a hydrophobic(ish) drug and so are disolving the drug first in DMSO before you add it to the culture media. My first reaction would be to test what concentration of DMSO your cells can tolerate without suffering any side effects first, so you can identify the upper limits of how much DMSO you can use. I would then test what the maximum concentration of drug you can then put into that amount of DMSO without it precipitating, and then test it on your cells. However I suspect that if you see the drug precipitate at high concentration it will still be precipitating at lower concentrations as well, just not as visibly.

If you have lots of the drug and you can easily measure the drug in the tissue culture media (e.g. HPLC) you could do some formulation experiments to see how much of the compound you can get to go into aqueous solution and identify the saturation point.

If you are part of a drug discovery programme, there are chemical tricks used by synthetic chemists to increase solubility which may or may not have an effect on the efficacy of the compound, sometimes there has to be a trade off between deliverability and potency, especially in vivo. Dr Cicciolini does make a good point about liposomes, and they are worth considering.

Good luck

Thank you Dr. Andrew Sunters,

I will check the soluble concentration, as you have mentioned. At present, my work is in preliminary level, and continuing even though some of the extracts precipitate. They were obtained from our project partners, so will be getting about the nature of the compounds first before checking other options. However, your points are very useful in designing the next experiments.

Thank you.

I agree with Joseph. In my experiments I use 0.1-0.2% as maximum concentration of DMSO when I work with HT-29 and HCT116. But as the tolerance to DMSO dipends on the cell line, I found that RKO are more sensitive and, in that case, I use a concentration lower than 0.1%. So, as Andrew said, it's important that you know which is the best concentration of DMSO you have to use on your cells before setting up experiments with your drug

Thanks Dr. Katia Rieta,

I had checked for DMSO toxicity and 0.1% is quite OK. However, problem with my work is precipitation of the extracts hence wished to know some easy methods to improve dissolution rate.

Have you tried adding the drug directly to cell culture media at 1 microlit per ml i.e 0.1% and then checking the precipitation via turbidimetry.

I did not try, since they precipitate in media itself, there wont be any difference in directly adding to cells. Moreover, it will not be good to add 1 ul of highly concentrated drug to the cell culture because (1) cannot mix well in the 96 well plate (2) pipetting error will be high.

If you are looking for accurate assay results test the compound at its saturation concentration, and do a dilution assay for a dose response. Determine the concentration at saturation and you will have a meaningful number. Assaying at higher concentrations will be fantasy.

Thanks, I will check the precipitation inhibitors. And true, concentration at saturation would be very important. My work got diverted from last 2 weeks. Will check all the possibilities mentioned.

Thank you all.

Cyclodextrin, liposomes, vessel walls, assay media provide depots for excess compound and do not give you higher concentration of compound in solution. Depots add additional kinetics to the equilibrium and not higher concentrations. Magic is not part of reliable results.

i also want to ask a question that my plant extract is soluble in methanol so i am using methanol as a control is it safe? I am using 100ul of methanol in 2 ml of cell culture media

Hi Satheesh,

About DMSO;

1. In order to dissolve some the proteins/ or pool of synthetic peptides we use upto 90% of DMSO and 10% of ultrapure water! There are several instances we do use 100% DMSO to completely dissovle proteins, peptides...

2. while freezing mixture for mouse/rat Hybridoma cells - we use 10% DMSO, 40%RPMI medium,50% Fetal bovine serum (FBS)!

3. There are references, where 10% of DMSO and 90% of FBS is being used to freeze mouse hybridoma cell lines

What matters is concetration of proteins/molecues used to dissolve in DMSO, so that, finally you are only taking couple of micro-liters for your bio-assays, therby reducing the DMSO toxicity to in the culture medium.

Raj

Dear all again,

Usual cut-off with using DMSO in biology is 1% but actually at the bench we mostly use the 0.1% one - based upon my own expertise 1% DMSO can have an impact indeed on some sort of cells, including nasty cancer ones (e.g., HT29, to name but a few). Methanol as proposed above can be quite toxic as well. Can't tell you in virology, whether or not the vehicle/solvent may bias your observations and generate overestimated activity eventually.

There are plenty of smart ways to prevent precipitation of big molecules in biologic media (check above), including developing nanoparticles - it's all a question of time, money and effort and once again if I were you, this solubility issue should be taken as a critical parameter to go/nogo your compounds upon screening. My opinion is still that it may be pointless to further develop compounds if at the very early stages you have to deal with complicated physico-chemical properties. Honestly I can't tell you with antiviral compounds, but in oncology docetaxel and paclitaxel are two major anticancer drugs that have been approved despite crap solubility profiles - consequently, solvents required to administrate them in patients (cremophor, ethanol, castor oil and so on) proved to be highy toxic at the bedside, with several toxic-deaths reported in frail patients, including in children. Poor efficacy/toxicity balance with taxoïds is partly caused by the vehicle, not the drug itself. Twenty years after their first approval, people are still trying to find better way to handle these drugs (nab-conjugates, liposomal formulations, pegylated forms, etc...) in a safer way.

The issue is solubility rather than concentration of DMSO. Can you test the solubility of your extract products? For example, weight some your product and dissolve it in suitable (or minimal) amount of pure DMSO to make xx mg/mL of clear stock, and then test it in PBS or your culture media. There are a number ways to do it, you can use a 96-well clear plate, add DMSO to column 1, add and dilute your stock from well A (2-fold serial dilution) to well H,  add PBS or media to columns 2 to 5. Perform 10-fold dilution from column 1 to column 2 (now 10% DMSO), then column 2 to column 3 (now 1% DMSO), column 3 to column 4 (0.1% DMSO). Check which well is cloudy or clear under microscope. You also can use a few small vials to do it.  For cancer cell lines, 1% of DMSO may inhibit some cell growth, 0.3% of DMSO is much weaker or has no significant effect, 0.1% DMSO in general is safe.  You can check both 1% DMSO (column 3) and 0.1% DMSO (column 4) dilutions and decide the final dilution (solubility) to be used. If your media contains serum, do use the same (warm, if 37C incubator used) media for solubility test, protein-binding also can influence the solubility.

Dear Satheesha,

DMSO concentration 1% is okay and tolerable by any type of human cell line. In order to avoid precipitation, After dissolving the drug in 100% DMSO, use the culture medium of the cell line to dilute the extract further to attain the concentration of 1% for the Assay.

Do not use ethanol or PBS again since that is causing precipitation of your extract.

I hope this helps you because it works for me on several plant crude extract I have been working on for anti-viral and trypanocidal cytotoxicity

Hello,

A quick question.

What solvents did you use in obtaining your extract. I just need to know if those suggestions do not help you. So that I can advice another path.

Pls Im new to MTT assay could you please explain  the stock solution preparation for  drug concentration how it can mix with the media

is your problem resolved?

Why you are dissolving in DMSO? why don't you try another solvent like DMF?

DMF is good choice if you plan to add your drug preparation to an aqueous solution to avoid precipitation.

Hello,

 You try to dissolve the anti viral compounds in minimum DMSO solution so that it wont affect cells when you use it in cell culture.or else u dissolve on 100/DMSO and dillute in such a way that the final concentration is 5-10 percent.

all the best