Getting Started with Extracellular Vesicles

Extracellular vesicles (EVs) are produced by cells from all three domains of life (Archaea, Bacteria, and Eukarya) and are an essential part of intercellular communication and signaling(1)

Due to this, the production of EVs can be conveniently and straightforwardly accomplished using conditioned cell cultures grown in defined media. Both adherent and suspension cultures in a multitude of culture vessels ranging from microplates to baffled flasks to bioreactors can be employed for EV production.(2-4) 

Generally speaking, most cultured cell lines will produce EVs constitutively under all conditions.(1) However, the quality and yield of EVs can be impacted by numerous factors. 

  • It is well documented that EV populations vary greatly from cell line to cell line.(1, 4-11) Not only is there variability with respect to size distribution and molecular contents, but the relative concentration of EVs expressed is also cell-line dependent.(12, 13)

EV Image from BioLector PPT

  • Although the choice of cell line and the efficiency of the EV isolation step are critical, EV yield is also limited in the upstream process by the cellular capacity of standard shake flasks.(12) Notably, novel culturing approaches including continuous, semi-continuous, and three-dimensional are being explored to improve EV yield. 
  • In addition, cells used for EV production are usually not supplemented with standard serum (i.e. fetal bovine serum). Instead, these cultures are grown in a serum-free environment or the serum is clarified prior to its use through ultracentrifugation. This is done to reduce or eliminate the presence of contaminating EVs that are found in serum while still ensuring cell proliferation and integrity.(14)
  • Although standard serum can still be used, its removal from the culture media prior to EV isolation is required and this process stresses the cells and, consequently, influences the integrity of the resulting EVs. 

EVs can also be isolated noninvasively from most human biological fluids including blood, urine, amniotic fluid, and saliva.

References

  1. Van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213-28.
  2. Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 2015;4:27031.
  3. Xu R, Greening DW, Zhu HJ, Takahashi N, Simpson RJ. Extracellular vesicle isolation and characterization: toward clinical application. J Clin Invest. 2016;126(4):1152-62.
  4. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373-83.
  5. Bruno S, Porta S, Bussolati B. Extracellular vesicles in renal tissue damage and regeneration. Eur J Pharmacol. 2016;790:83-91.
  6. Forsberg MH, Kink JA, Hematti P, Capitini CM. Mesenchymal Stromal Cells and Exosomes: Progress and Challenges. Front Cell Dev Biol. 2020;8:665.
  7. Gimona M, Pachler K, Laner-Plamberger S, Schallmoser K, Rohde E. Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use. Int J Mol Sci. 2017;18(6).
  8. Andaloussi SEL, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12(5):347-57.
  9. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2(8):569-79.
  10. Zhang X, Yuan X, Shi H, Wu L, Qian H, Xu W. Exosomes in cancer: small particle, big player. J Hematol Oncol. 2015;8:83.
  11. Zhang ZG, Chopp M. Exosomes in stroke pathogenesis and therapy. J Clin Invest. 2016;126(4):1190-7.
  12. Salem I, Naranjo NM, Singh A, DeRita R, Krishn SR, Sirman LS, et al. Methods for extracellular vesicle isolation from cancer cells. Cancer Drug Resist. 2020;3:371-84.
  13. San Lucas FA, Allenson K, Bernard V, Castillo J, Kim DU, Ellis K, et al. Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes. Ann Oncol. 2016;27(4):635-41.
  14. De la Torre Gomez C, Goreham RV, Bech Serra JJ, Nann T, Kussmann M. "Exosomics"-A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk. Front Genet. 2018;9:92.

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