I am trying to extract exosomes from mcf7 cell line. After the sequential centrifugation step I calculated the numberof exosomes per cell based on the nanosight data. It comes to be very low (of the order of 10 exosomes / cell/hour). In the literature I have seen it should be of the order of hunderds. I was wondering if anyone has an estimate on the number of exosomes per cell, based on the seeding density and the time of incubation in the conditioned media.
Thanks
Deepa
Dear Deepa,
we are working with a slow-growing, well-differentiated pancreatic beta cell line. A typical exosome isolation by ultrafiltration and differential centrifugation yields approximatedly 10µg of exosome protein for a T175 culture flask seeded with 27E6 cells. Nanosight analysis shows 1E+11particles/ml (1E+8 - 1E+9 particles/µg). Since exosomes are produced over 4 days, this represents an equivalent of
The yield/cell or the total number may depend on several factors, including the culture conditions (growth factors etc) and the uniformity in size of exosomes. Based on the information you provided, it is likely that you may be loosing a fraction of exosomes during sequential centrifugation. Inclusion of other method may improve the yield.
So you have an approximate idea, we seed 2.5M of HUVEC in a T75 flask and three days later we change the medium to complete-exofree. Three days later we purify the exosomes, and by day 6 from the beggining cells are at congfuence (~6M/flask). Usually we consider a good exosome purification 50ng of protein. Also consider than endothelial cells are some of the cells that produce more exosomes...!
How do you quantify the exact number of exosomes??
it depends... which kind of cell are under consideration. it may vary from grade to grade, source to source, type to type. e.g. if I am using grade III and grade IV glioma cell lines, or different cell lines of prostate cancer, definitely each grade will offer exosme yield at varying numbers depending upon behavior of a particular tumor grade. source may be another factor, e.g. if I am using blood -serum/plasma, urine, or saliva... again the yield per cell from each source might differ. further, I assume that pre-treatment could be another factor i.e. if a patient was on a treatment, therapy, or radiation when you collected samples? VS a patient without having a treatment. same could be applied to cell cultures. which kind of media was in use. Handling practices/protocols may add further variations. Therefore, one cannot fix a number of exosome yield per cell, keeping in view the number of factors. However, we can meet up a range... x - to - z.
Thanks for your answers. I quantify the number of exosomes using the NTA nanosight instrument. Jennifer, from your experience, can you give me some example cell lines that produces the most exosomes?
Dear Deepa,
we are working with a slow-growing, well-differentiated pancreatic beta cell line. A typical exosome isolation by ultrafiltration and differential centrifugation yields approximatedly 10µg of exosome protein for a T175 culture flask seeded with 27E6 cells. Nanosight analysis shows 1E+11particles/ml (1E+8 - 1E+9 particles/µg). Since exosomes are produced over 4 days, this represents an equivalent of
Well Deepa, I have not experience with many cell types, I know that HUVEC cells produce more exosomes than several cancer lines (glioblastoma, breast , pulmonary and colon cancer), but I cannot tell you about any others...
It depends on several factors like cell line, medium/biofluid conditions etc. For sure, the quantity you're getting is too little, proprably due to an incorrect sequential centrifugation protocol.
Can anyone suggest a way to increase exosome production by cancer cells in order to improve their yield with ultracentrifugation?
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