Zen-Bio
Executive Director, Regnerative Medicine
The term exosome is generally understood to reference a specific class of lipid-membrane bound extracellular vesicle (EV) characterized by a diameter of 40 nm to 150 nm and a density of 1.09 g/ml to 1.18 g/ml. Exosomes participate in a variety of cellular activities and have been shown to be isolatable from multiple body fluids, including saliva, urine, plasma, serum, breast milk and amniotic fluid, as well as from the conditioned media of cultured cells [1]. In fact, exosomes can be isolated from any cell type that can be cultured. Purified exosomes have been demonstrated to have clinically relevant therapeutic bioactivity across multiple in vitro and in vivo models [2,3]. In the diagnostic arena, exosomes isolated from bodily fluids, most notably blood, have proven their worth as biomarkers for specific diseases. In this article, you will find tips for optimizing isolation of exosomes from cells of your choice.
Culturing without FBS
Bovine serum contains an abundance of exosomes. To optimize analysis of the exosome targets of interest, it is best to remove serum from the culture medium that is to be harvested. Most cultured cells can tolerate 24 or maybe 48 hours of growth in serum-free media (testing the cells’ tolerance is relatively simple—check for cell viability after incubation in a serum-free medium). After your cultures are established with a serum-containing medium, wash the attached cells at least three times with an abundance of serum-free medium, then add a minimum amount of serum-free medium to the culture and return to the incubator for 24 to 48 hours. The resulting conditioned-medium will contain an abundance of exosomes from your cells of interest. Should your cells not tolerate 24 hours without serum, there are commercially available exosome-reduced serum options.
Phenol red-free media
Ultracentrifugation of a conditioned medium is one of the more popular methods to isolate exosomes, especially if you have larger quantities of medium (>100 ml). Following ultracentrifugation to pellet exosomes, in spite of the most rigorous methods to rid the centrifuge tube of excess culture medium, there will often be some medium carryover when resuspending the pellet in phosphate-buffered saline (PBS) or another clear buffer. For some downstream applications, particularly direct spectrophometric analysis, the presence of even small amounts of phenol red can affect your readings. Phenol red-containing media for the growth of your cells are acceptable, as long as you wash and incubate the cultures with a phenol red-free medium for conditioning and subsequent exosome isolation.
Approximating protein and RNA amounts
You’ve isolated your exosomes and want to determine approximately how much protein and RNA you have. Standard measurements typically require a lysis step and may require 10 ml to 100 ml of sample. Examples include a standard Bradford assay to measure proteins or a spectrophotometric reading to record exosomal RNA levels. In many cases, your exosomes may be in short supply or in a minimum volume of less than 100 ml. As these methods are only an approximation of the amount, you may be better off using an instrument such as a NanoDrop ND-1000 spectrophotometer that can take a direct measurement from 1 ml to 1.5 ml of sample. Then you will have more precious exosome material on which to perform either functional or biochemical analysis.
Correlations can be tricky
Although it is true that having more cells will result in the isolation of more exosomes, a specific exosome number does not seem to have a consistent total protein or RNA content. This lack of predictability, despite controlling cell number and passage, media volume and incubation time, appears to extend within and across the same and different cell types. Even with the experience of hundreds of exosome isolations from conditioned culture media from at least 20 primary and line cells, a predictable correlation among these parameters has been elusive. This can pose a challenge when comparing different batches of exosomes within the same lab as well as comparing results to those from other labs. Keep this in mind when comparing results from different preparations by different researchers and in different labs.
Conclusions
Although many methods are available, a uniform consensus on how best to isolate, quantitate, size and phenotypically characterize exosomes is still lacking. Remember this when reviewing results from other labs and comparing them to your data. Different technologies designed to measure the same parameters, such as EV size and concentration, often produce somewhat different results. Whatever methods and technologies you use to obtain the results you are looking for, follow these suggested tips and stay consistent with your approach.
References
[1] Thery, C, et al., “Isolation and characterization of exosomes from cell culture supernatants and biological fluids,” Curr Prot Cell Biol, 3.22.1-3.22.29, 2006. DOI:10.1002/0471143030.cb0322s30.
[2] Basu, J, Ludlow, JW, “Exosomes for repair, regeneration and rejuvenation,” Expert Opin Biol Ther, 16(4):489-506, 2016. [PMID: 26817494]
[3] Whitford, W, Ludlow, JW, Cadwell, JJS, “Continuous production of exosomes,” Gen Engin and Biotechnol News, 35(16):34, 2015.
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