6 Steps for Successful in vitro Drug Treatment

You might have seen one of the many anti-drug ads the 80s had to offer (including this delightful message from Robocop) and rightfully steered clear of drugs. But when it comes to biology, we use in vitro drug treatment for many experimental purposes, including testing anti-cancer treatments or synchronizing the cell cycle.

If you are facing your first in vitro drug treatment and don’t know where to start, don’t worry, we’ve put together 6 steps to get your experiments off to a flying start.

Step 1: Know Your Drug

Start by learning as much as possible about how the drug works and any properties that are relevant to your experimental plans. This knowledge will be essential for understanding the question being addressed by the experiment, for designing the experiment, and for understanding how your cells will likely react to the treatment. The key information you will want to know about the mode of action of the drug is:

• its cellular target;
• the effect that it has on cellular processes.

From this information, you should be able to make predictions about the experimental outcomes. You also want to know some of the key properties of the drug including:

• reversibility: drugs can reversibly (non-covalent interaction) or irreversibly (covalent bond formation) bind their target. If you have plans to wash out the drug for a pulse-chase or to track recovery, make sure your drug is reversible.
• permeability: this refers to the ability of the drug to cross the cell membrane. There are instances when you will want a permeable drug and instances when you will want it to be impermeable. It all comes down to your purpose. In many cases, alternative drugs can be used if some property is found to be incompatible with experimental plans.

Step 2: Know the Solubility and Storage Conditions

Drug Solubility

There isn’t a single universal solvent in which all drugs are soluble, nor is there a standard concentration at which all drugs are soluble. The most common solvents in my experience are water (or PBS, or media), dimethylsulfoxide (DMSO), and ethanol. For a particular solvent, a drug will only be soluble up to a certain concentration (e.g. 50mM), which will be relevant when making stock solutions.

So where can you find information on your drugs solubility? The product data sheet! This sheet should come with the drug, but it can also be found online through the manufacturer’s website.

Drug Stability and Storage

Stability will also differ from drug to drug and this will affect storage. You will want to follow the recommended storage conditions as you would with any reagent to maintain the drug’s activity and to avoid waste or inconsistent experimental results. Keep in mind that storage conditions for a drug in powder form versus one in solution may vary. For example, storing a powder at room temperature might be appropriate, but the solution may require colder temperatures.

You should also know how long a solution of your drug will be stable. This will tell you if you can make a stock solution that can be aliquoted and kept for a while or if you will have to make a fresh solution for each experiment.

Again, information regarding storage should be available from the product data sheet for the drug. This may also include information on the stability and storage of a working solution of the drug. Don’t forget to look over the Material Safety Data Sheet (MSDS), so you understand any potential hazards before handling. Your lab should have a binder that contains the MSDS (assuming the drug is already in your possession), but I often find it quicker and easier to simply google the drug’s MSDS.

Step 3: Find a Working Concentration

Now it is time to use the drug, but what concentration should you use? Not only can there be a wide range of potentially appropriate concentrations, but this can vary between cell types, so proceed with caution. I always start with a literature search, looking for concentrations as well as treatment times (the next step). Ideally, you will find that someone has already found an appropriate concentration range in the same cell type, but finding concentrations for other cells can still be helpful.

Even if you find an exact concentration or range, I recommend doing some serial dilutions as a quick way to test several concentrations. Serial dilutions can be done in many ways to quickly and simultaneously test narrow or wide ranges of drug concentrations. Check out, “Three Steps for Setting up a Drug Screening Assay” for an explanation of serial dilutions.

Throughout initial tests, check for unhealthy, rounded up cells which can indicate that a concentration is too high. Additionally, some drugs can have off-target effects at higher concentrations which could make your results difficult to interpret. Ultimately, you will want to find the lowest concentration with the desired effect without causing undue stress to the cells.

Step 4: Find the Optimal Time for in vitro Drug Treatment

Finding a treatment time is very similar to finding a working concentration. You should have a good idea of where to start from your literature search. However, plan on testing out a series of time points while maintaining the drug concentration from Step 3.

Start with the longest time point and work backwards to the 0-hour condition. Think strategically about your range of time points and when to begin so that you won’t find yourself in the lab during the middle of the night.

When doing Steps 3 and 4, always remember your intended application. For example, if doing anything involving dose-dependency or calculating rates, you will be finding a range instead of a single concentration or time point.

Step 5: Determine Appropriate Controls

Hopefully, you were already planning on including a no drug treatment control, where you have cells that have not been treated with your drug.

Another important control is the vehicle control. The vehicle is the solvent used to dissolve the drug (e.g. DMSO). Unless the solvent is media, this type of control is necessary so that you can be confident that any observed effects are due to the drug alone and not the solvent. The same volume of vehicle and vehicle + drug should be added to their respective wells since the only difference between these conditions is the inclusion of the drug.

Finally, depending on your downstream application, there may be additional experiment-specific controls to include. For example, if the next step involves immunofluorescence, you will need to factor in typical controls for immunofluorescence. Take care to ensure that all conditions, including controls, are treated the same way through all other steps.

Step 6: Put Everything Together and Run Your Experiment!

Your experiments may require additional steps or considerations before you run any experiments, such as cell density, but I hope this guide serves as a good starting point for your in vitro drug treatment endeavours. If you are already familiar with drug treatment experiments and have any other tips, please comment below!