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The optimal way to isolate Exosomes

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Posted May 7, 2019

There are few topics that have gained enough attention to be considered the next big thing. Exosome is one of those topics. They were initially considered waste products and were ignored as such. Nowadays it’s well-established that they play key role in cell communication and they have linked to several diseases including cancer.

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At a glance, this exosome celebrity appears well deserved. However, there is too much mystery around these tiny extracellular vesicles. One of the main reasons why the questions regarding exosomes remain unanswered is mainly to the lack of optimized isolation techniques. But before going into details, let’s explain what makes them important.

What are exosomes

Exosomes are membrane vesicles of endocytic origin whose size varies between 30 and 150 nm. They are found in extracellular medium and in bodily fluids such as blood, urine, saliva etc. These extracellular vesicles and contain proteins, lipids, nucleic acids and other metabolites. These macromolecules are transmitted from one cell to another resulting in the modulation of the behavior of the recipient cell.

They are key players in inter-cell communication. The latter is an important mechanism for information exchange, promoting cell survival and proliferation. Hence, it is no wonder that research data has linked exosomes to both normal and pathological processes. Their involvement in cancer has particularly attracted attention.

Exosome and cancer

There are increasing evidence to suggest that exosomes are key players in almost all the steps that eventually lead to cancer progression. For instance, research data suggests that exosomes participate in inflammatory response, angiogenesis, lymphogenesis, cell migration, cell proliferation, immune suppression, invasion, epithelial-to-mesenchymal transition and even metastasis.

It is believed that exosomes act as signal amplifier for cancerous cells[1]. For example, higher-grade cells secrete a higher number of exosomes than low grade cancerous cells. The exosome content that derived from cancerous cells is also different from that of normal cells. It has been shown that exosome issued from cancerous cells influence non-cancer cells to generate tumor microenvironment that is permissive of tumor growth and metastasis1. More precisely it has been demonstrated that exosomes that derive from cancer cells influence endothelial cells to support neo-angiogenesis, fibroblast differentiation into pro-angiogenic and pro-tumoric cancer associated fibroblasts and suppress immune cells to evade immune detection of tumors[2]. All this step clearly indicates exosome implication in cancer.

It has been suggested that they can be potential diagnostic and therapeutic tools. For starters, higher exosome secretion is seen as indication of cancer progression1. Furthermore, exosome are being explored as a new way to deliver anti-cancer drugs. It is believed that this method may have better specificity and sensitivity. Another advantage of exosome-drug loading is that it is better supported by the immune system. Scientists believe that exosome-drug delivery may be the solution to a cancer treatment with less side effects.

Regardless, there is a consensus among exosome-research related community that there is a need for optimization and standardization of methods applied to isolate and purify these extracellular vesicles. So far, the technique of reference is the ultracentrifugation with Optiprep™ procedure. Optimization and standardization of exosome isolation is the first barrier to overcome for further downstream analysis.

What is the optimal exosome isolation method

Following the important role of exosomes, several methods have been developed for optimal exosome separation. Among the different techniques, ultracentrifugation with Optiprep™ outperformed the others in term of the biological purity of the final product[3]. However, there are a few drawbacks to it.

Firstly, ultracentrifugation with Optiprep™ procedure has been criticized for not resulting in the highest protein amount[4]. However, it has been suggested that the higher amount of protein collected from other methods, may be as a result protein co-isolation of contamination ( for example albumin, apoliproteine E or THP).

Secondly, ultracentrifugation with Optiprep™ gradient is also known to be labor-intensive and time-consuming. This may explain why this procedure is not universally used although it may optimize downstream applications.

Thirdly, the procedure is not designed to be applied at industrial scale. The method involves several centrifugation steps which makes the standardization difficult. At the end of the procedure, the result may not be the same. The final yield, ie. the exosome content issued from initial sample, is too small to justify the cost of this method in a large scale.

Conclusion

In conclusion, there are increasing evidence to suggest that exosomes are essential elements for normal and pathological processes. Their involvement in cancer have attracted a lot of attention. It has been suggested that these extracellular vesicles may be an innovative way to detect and/or treat cancer. Prior to confirming these hypotheses, there is a need to optimize their isolation yield. Ultracentrifugation with Optiprep™ procedure is considered to be the golden standard. Regardless, that technique too requires further optimization.

[1] Wu K, Xing F, Wu SY, Watabe K. Extracellular vesicles as emerging targets in cancer: Recent development from bench to bedside. Biochim Biophys Acta Rev Cancer. 2017;1868(2):538–563. doi:10.1016/j.bbcan.2017.10.001

[2] Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ. Extracellular vesicles in cancer – implications for future improvements in cancer care. Nat Rev Clin Oncol. 2018;15:617–638

[3] A. Abramowicz , P. Widlak and M. Pietrowska Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation Mol. BioSyst., 2016, 12 , 1407 -1419

[4] Tang, Y., Huang, Y., Zheng, L., Qin, S., Xu, X., An, T., Xu, Y., Wu, Y., Hu, X., Ping, B., Wang, Q.”Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum”. International Journal of Molecular Medicine 40.3 (2017): 834-844.

Additional references

  • Edgar J. R. Q&A: what are exosomes, exactly? BMC Biology. 2016;14(1):46–47. doi: 10.1186/s12915-016-0268-z
  • György B., Szabó T. G., Pásztói M., et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and Molecular Life Sciences. 2011;68(16):2667–2688. doi: 10.1007/s00018-011-0689-3
  • Hessvik, Nina Pettersen, and Alicia Llorente. “Current knowledge on exosome biogenesis and release.” Cellular and molecular life sciences : CMLS vol. 75,2 (2017): 193-208. doi:10.1007/s00018-017-2595-9
  • Wu AY, Ueda K, Lai CP. Proteomic analysis of extracellular vesicles for cancer diagnostics. Proteomics. 2018;19:e1800162

Source: proteogenix-products.com

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