Review on Strategies and Technologies for Exosome Isolation and Purification

Abstract

Exosomes, a nano-sized subtype of extracellular vesicles secreted from almost all living cells, are capable of transferring cell-specific constituents of the source cell to the recipient cell. Cumulative evidence has revealed exosomes play an irreplaceable role in prognostic, diagnostic, and even therapeutic aspects. A method that can efficiently provide intact and pure exosomes samples is the first step to both exosome-based liquid biopsies and therapeutics. Unfortunately, common exosomal separation techniques suffer from operation complexity, time consumption, large sample volumes and low purity, posing significant challenges for exosomal downstream analysis. Efficient, simple, and affordable methods to isolate exosomes are crucial to carrying out relevant researches. In the last decade, emerging technologies, especially microfluidic chips, have proposed superior strategies for exosome isolation and exhibited fascinating performances. While many excellent reviews have overviewed various methods, a compressive review including updated/improved methods for exosomal isolation is indispensable. Herein, we first overview exosomal properties, biogenesis, contents, and functions. Then, we briefly outline the conventional technologies and discuss the challenges of clinical applications of these technologies. Finally, we review emerging exosomal isolation strategies and large-scale GMP production of engineered exosomes to open up future perspectives of next-generation Exo-devices for cancer diagnosis and treatment.

Keywords: cancer; exosome isolation; exosome separation; exosomes; microfluidics.

FIGURE 1

The number of exosomal publications. The graph was generated from Web of Science.

FIGURE 2

Biogenesis of exosomes and other vesicles (Hessvik and Llorente, 2018) (van der Pol et al., 2012) (Gurunathan et al., 2019).

FIGURE 3

Schematic of exosomal molecular composition. Exosomes contain various important biomarkers, such as proteins, lipids, and miRNAs.

FIGURE 4

Schematic representation of common exosomal separation techniques. (A) Ultracentrifugation, (B) Density gradient centrifugation, (C) Dead-end filtration (DEF), (D) Tangential flow filtration (TFF), (E) Size-exclusion chromatography, and (F) Immunoaffinity.

FIGURE 5

Schematic representation of Membrane-based exosome isolation techniques. (A) The phosphate groups on the membrane surface of exosomes can specifically bind to metal oxides (TiO₂). Adapted from (Gao et al., 2019), copyright 2019 Royal Society of Chemistry. (B) The positively charged molecules enrich exosomes. Adapted from (Kim and Shin, 2021), copyright 2021 MDPI. (C) The lipid nanoprobes with lipid tail are capable of inserting into the exosomal membrane structure. The wings modified with lipid nanoprobes can promote the efficiency and speed of exosome binding to nanoprobes. Adapted from (Han et al., 2020), copyright 2020 Elsevier Ltd.

FIGURE 6

Schematic representation of physical property-based microfluidic isolation techniques. (A) An acoustic-based separation microfluidic chip employing acoustic forces and droplet spinning for isolation of exosomes from biofluids. Adapted from (Gu et al., 2021), copyright 2021 American Association for the Advancement of Science. (B) An electrical-based separation device integrated the focusing power of isotachophoresis and paper-based filtering ability. Adapted from (Guo et al., 2020), copyright 2020 Elsevier Ltd. (C) A ZnO nanowires array for exosome capture. Adapted from (Suwatthanarak et al., 2021), copyright 2021 Royal Society of Chemistry. (D) Hydrodynamic-based microfluidic strategy for isolating exosomes from whole blood. Adapted from (Tay et al., 2021), copyright 2021 Royal Society of Chemistry.

FIGURE 7

Scheme of immunoaffinity-based microfluidics for exosome isolation and enrichment. (A) Microfluidic Raman chip for exosome isolation and detection. Adapted from (Wang et al., 2020), copyright 2020 Royal Society of Chemistry. (B) Scheme of lipid membranes microarrays functionalized with antibodies. Adapted from (Liu H. Y. et al., 2021), copyright 2021 Wiley-VCH Verlag GmbH & Co. (C) 3D nanopatterned EV-CLUE chip were manufactured by colloidal inkjet printing. Adapted from (Zhang et al., 2020), copyright 2020 American Association for the Advancement of Science.