Hello..

Exosomes are small, membrane-bound vesicles that are released by cells and can be found in various bodily fluids, such as blood, urine, and saliva. They have many potential uses in medicine and research, but it is important to ensure that they are free of contaminants before they are used.

There are several methods that can be used to sterilize exosomes, but the most suitable method will depend on the specific properties of the exosomes and the intended use. Some options to consider include:

Filtration: Exosomes can be sterilized by passing them through a filter with a pore size that is small enough to remove contaminants, but large enough to allow the exosomes to pass through. This method is effective for removing bacteria, fungi, and other particulates, but may not be suitable if the exosomes are prone to aggregation.

Chemical treatment: Exosomes can be sterilized by treating them with a chemical disinfectant, such as ethanol, hydrogen peroxide, or chloroform. This method is effective for killing bacteria and viruses, but may not be suitable if the exosomes are sensitive to the chemical being used.

Virucidal agents: Exosomes can be sterilized by treating them with agents that are specifically designed to kill viruses. These agents may include antiviral drugs, such as ribavirin or interferon, or physical methods, such as high-pressure homogenization or ultrasonic treatment.

It's worth noting that each of these methods has its own advantages and disadvantages, and it may be necessary to use a combination of methods to achieve the desired level of sterilization. If you are uncertain about which method to use, I recommend consulting a scientist or expert in the field to discuss the best options for your specific needs.

Regards.

Dear Nourhan

I have no experience with exosomes. Nevertheless, I can guess that the dilution of exosomes using an appropriate buffer to make the aggregation-free sterile filtration possible, followed by the re-concentration utilizing centrifugal filtration (amicon filters with 10-100 KDa porosity) might be useful.

Hello Nourhan S. Elkholy

If none of the above methods of sterilization works, and you need the exosomes to be sterile, then you are left with no other option, but to limit the potential for contamination. So, it becomes necessary to use sterile equipment and consumables as far as possible.

If sterility is a necessity, sterile centrifuge and ultracentrifuge tubes must be used, and all steps up to the time when CM-containing tubes or tube holders are closed must be performed in a tissue culture hood. To sterilize ultracentrifuge tubes, wash the clean tubes and their lids briefly in 70% ethanol, rinse twice in sterile PBS, discard the PBS, and drain the remaining drops of PBS with a pipette before use. The rotor lid or the lid for each tube holder must also be cleaned with 70% ethanol before closing the rotor.

As far as possible, perform all the steps required for exosome isolation in the biosafety cabinet.

Best.

Do you want to separate aggregated exosomes or sterilize them?

For sterilization: the starting buffer for precipitation is crucial and should be in a sterile condition. Afterward, you can dilute the EVs in sterile PBS or ddH20 and snap-freeze (1-3s) in liquid nitrogen to inactivate any contaminant/bacteria. However, there has been a point of contention as to using fresh EVs or thawed EVs from -80 for functional assays. if you want to just perform cytotoxic tests, then either might be ok but for cargo delivery assays, then fresh EVs might work well.

For separating aggregated EVs: I think this may depend on the downstream application that you want to use them for, for characterization studies you may need not worry about the aggregation as that itself can be a point in your data. However, for single EV analysis- after diluting the EVs in your choice of buffer (you might see some precipitate at the base of the tube, this should in itself give a vague idea of how condensed the exosomes are), carefully passage the EVs either with 20ul tip of syringe for about 10-20 times and then you might want to sonicate them briefly as well.

hope this helps Nourhan S. Elkholy