I'm culturing cells with exosomes from IPF patient serum and it causes our media to turn incredibly vicious - has anyone else noticed this. We want to western our media but I wonder if the thickness will affect it? We are thinking of adding some trypsin but aren't sure if this is the best course of action. Any advice welcome!
I have faced this problem with some of my experiments.
Do you wash your exosome prep with PBS after isolation? How many exosomes do you add to the media? If its a high concentration, then it is going to be very viscous due to the very nature of these vesicles. Do you properly resuspend the exosomes before adding to the media/cell cultures?
I found that resuspending the exosome suspension, at the desired final concentration, in a larger volume of culture media (which you would then add directly to the cell cultures) helps.
Are they, purified exosomes? If yes, how many you have loaded? Becomes it quickly viscous?
if you have loaded purified exosome and low quantity, and it become viscous slowly, there is the possibility that the viscous media is caused by the synthesis of extracellular matrix by the cells treated with exosomes.
Daniele - they become viscous after 24hrs and they are purified from serum. We're presuming that it is the generation of collagen.
How do you prepare your exosomes? Are you using ultracentrifugation or ExoQuick? Are the cells viable? Are there lots of detached cells?
It depends on the method of isolation and sterility. I do not have experience with serum Exosomes but if the isolation was done in non-sterile conditions (is the kit sterile ? isolation done outside the hood, unclean tubes,......), a very big chance of contamination can occur and could cause viscosity and turbidity.
I have isolated Cardiac Exosomes by a kit and used onto Cardiac myocytes for overnight. Didn't really observe any viscosity issues in the media.
I think that your deduction may be correct. In fact, if the viscosity appears after 24 hours is not possible that it is due to the amount of lipid injected with exosomes ...
How was made the purifications? I make two successive centrifugations (5 'to 300 rpm at RT temperatures, and 30' at 3000 rpm at 4 ° C) to remove cells and debris, filtration with a filter to 20 microns, and subsequently a ultracentrifugation (2 h at 35000 rpm at 4 ° C in SW28 rotor). The pellet resuspended in PBS. Everything under a laminar flow hood.
So the sw28 is a swing rotor with metal buckets for each tube. Also the tubes are probably not sterile but easily sterilized. I would make sure the metal buckets and tubes are sterilized (check beckman for recommendations, if alcohol is permitted then its easy, soak then dry in hood, some tubes are not compatible with Alcohol)
When you say 20 micron ? what is that (0.22 um filter you mean ?)
Serum is filled with many molecules and it is challenging to really purify exosomes from serum. It must be done with great care with verification of purity, otherwise you will not know exactly what you are testing. Samples purified only by ultracentrifugation will contain many, many proteins, which are sticky and will adhere to exos. Plus your isolation may contain other microvesicles. Albumin and gamma globulin are abundant in serum samples. Serum also can contain a lot of DNA, which can greatly increase viscosity. Run an SDS PAGE of sonicated exosomes purified with your current approach and evaluate the range of proteins present in your sample. It is critical to be sure your purification is good before investing much time and energy in treatment of other cells with exosomes that might not be pure exosomes.
dear ola Elgamal, the tubes are of the UltraClear TM family of beckman and the little apertures are closed hot.
the used filters are actually of 0.22 micron
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