Hello Xiaoqin Li

Cells secrete many types of membranous vesicles differing in size and molecular weight. There are three major types of extracellular vesicles namely apoptotic bodies, shedding vesicles and exosomes. Apoptotic bodies are the largest with the diameter of 700nm to 5000nm. Shedding vesicles also termed as micro vesicles have a range of sizes from 50nm to 1000nm. The exosomes which you are interested in are smaller in size with diameter ranging from 40nm to 120nm. In order to separate the exosomes from other extracellular vesicles it is important to carry out successive rounds of centrifugation to pellet down the non-required population like the apoptotic bodies and shedding vesicles along with cells and cell debris. If you skip the first two centrifugation steps 1) 300g for 10 mins meant to pellet dead cells and other debris and 2) 2000g for 10 mins meant to separate larger vesicles you will be increasing your chances of getting a lower yield of your final product because of co-sedimentation. Moreover, the final product may not be pure as chances of contamination with other fractions may increase.

Thank you.

Dear Xiaoqin Li. First spin is to sediment cellular debris.

I wonder if it could be substituted with filtration.

Hi Xiaoqin Li several studies have used 2000g to remove cell debris, and 10000g to remove large vesicles ~1000nm. After this step, an addition use of 0.22 nm filtration right before ultracentrifugation. That would make sure there is no large particle in your exosomal samples.

Nguyen Tien Cuong Yeah, I've seen the use of 0.22nm filtration in many papers. I began to focus on the field of MSC-derived exosomes this year and want to know more about them. Understanding the meaning of each step is important, I think. Thanks for your sharing.

Hi Xiaoqin Li, That would be an interesting topic. If you have functional experiments, please consider the time and temperature for storing exosome.

The first two steps are very important to remove the cell debris, especially our lab students must do the first two steps if they have to store the exosome-containing media for later Exo-isolation.

Generally the more steps included the better the purity of the final product.

The advantage of gradually increasing the speed of the centrifugal steps is to try and minimise breakageof various larger cell organelles to produce small fragments and release fragments which can end up in your final product.

Also as it can be difficult to completely prevent material being transferred over to the next centrufation step due to the stability of the pellet, it is advantageous to have several steps to be sure.

Filtration can be included in the purification method. But be sure to use filters of the appropriate pore size.Depth/pre filters can be useful. Filters tend to clog so a gradient of filters starting with those of a large pore size etc.

In this case, your isolated extracellular vesicles and consequently exosomes will be contaminated with cellular debris such as mitochondria, nucleus, .....