A Holistic Review of the State‐of‐the‐Art Microfluidics for Exosome Separation: An Overview of the Current Status, Existing Obstacles, and Future Outlook

Abstract

Exosomes, which form a class of extracellular vesicles (EVs), are membrane-bound lipid nanovesicles with sizes typically in the Exosomes, a class of small extracellular vesicles (30-150 nm), are secreted by almost all types of cells into virtually all body fluids. These small vesicles are attracting increasing research attention owing to their potential for disease diagnosis and therapy. However, their inherent heterogeneity and the complexity of bio-fluids pose significant challenges for their isolation. Even the "gold standard," differential centrifugation, suffers from poor yields and is time-consuming. In this context, recent developments in microfluidic technologies have provided an ideal system for exosome extraction and these devices exhibit some fascinating properties such as high speeds, good portability, and low sample volumes. In this review, the focus is on the state-ofthe-art microfluidic technologies for exosome isolation and highlight potential directions for future research and development by analyzing the challenges faced by the current strategies. range of 30-150 nm. [1] They are secreted by almost all cells into diverse bio-fluids, including blood, urine, breast milk, saliva, lymph, and cerebrospinal fluid. [2] The discovery of exosomes dates back to the 1980s, but for many years after their discovery, they were regarded as "dust". [3,4] However, recent studies have shown that they play a crucial role in intercellular communication. [5,6] The biogenesis of exosomes includes double invagination of membranes, formation of intracellular multivesicular bodies (MVBs), and the release of exosomes (Figure 1). The first invagination process (plasma-membrane budding) generates early-sorting endosomes (ESEs) that can develop into late-sorting endosomes (LSEs). The LSEs are invaginated once again to form MVBs containing intraluminal vesicles (ILVs). Finally, the MVBs fuse with the plasma membrane to release ILVs (exosomes). [7] After release, these exosomes are taken up by recipient cells via multiple processes, such as macropinocytosis, fusion with the cell membrane, clathrin-dependent endocytosis, and phagocytosis. [8] The exosomes uptaken can act either at the surface of the recipient cells or deliver functional cargo in their bulk, thus affecting recipient-cell behavior. [9] Thus, exosomes play a key role in various physiological and pathological processes, including mammalian reproduction and development, immune responses, and disease progression. [10] Exosomes are reported to exhibit many excellent characteristics for clinical applications (Figure 2). 1) They can reflect the real-time state of the original cell, as they are actively secreted by living cells. 2) They are abundant in virtually all biological fluids (up to 10 10 vesicles per mL). [11] 3) Exosomes have enriched contents, including cell-surface substances and cytoplasmic constituents (including proteins, nucleic acids, lipids, and metabolites). Furthermore, exosomes can not only protect enzyme-sensitive cargos from degradation, but also ...

The significance of exosomal RNAs in the development, diagnosis, and treatment of pancreatic cancer

“…As critical mediators of intercellular crosstalk, exosomes exist in virtually all body fluids, and highly purified exosomes are indispensable for further structural and functional study [44]. However, the acquirement of high-quality exosomes is still challenging due to exosome heterogeneity in the source, size, and content [45]. The International Society for Extracellular Vesicles has suggested that differential ultracentrifugation (DC) is the most frequently used method, with several other methods, such as density gradient ultracentrifugation (DGC), ultrafiltration (UF), precipitation, size-exclusion chromatography (SEC), and immunoaffinity capture [46].…”

Emerging Potential of Exosomal Non-coding RNA in Parkinson’s Disease: A Review

“…However, the high cost of the instrument, the long-time consumption of several hours, the low yield and high protein pollutants caused by the destruction of the integrity of the exosomes limit the popularization and application of DC. In order to improve the shortcomings of DC and improve the yield and purity of exosomes, density gradient ultrasound (DGC) strategies such as isopycnic UC and moving zone UC came into being ( Gupta et al, 2018 ; Ding et al, 2021 ). Ultrafiltration based on nano membrane has the characteristics of simple operation, but there are high abundance of microbubbles, apoptotic bodies and protein contamination.…”

“…Jalaludin et al summarized the currently commercially available size exclusion chromatography kits including qEV (iZON), PURE-EVs (Hansa Biomed), and ExoLutE exosome isolation kit (Rosetta Exosome), precipitation kits including Exoquick (System Biosciences) and Total Exosome Isolation Kit (Thermo Fisher Scientific), and immunoaffinity capture kits including ExoQuant (Biovision), ExoCap Streptavidin Kit (MBL International), and Exosome Isolation Kit Pan (Miltenyi Biotec) ( Jalaludin et al, 2021 ). In order to effectively separate exosomes and minimize time, equipment and cost, microfluidics strategies based on electrophoresis, dielectrophoresis, acoustics, magnetism and immune affinity came into being ( Ding et al, 2021 ). However, the standardization, purity, retention of good biological function and related clinical validation of extracted exosomes remain to be studied.…”

Small Extracellular Vesicles in the Development, Diagnosis, and Possible Therapeutic Application of Esophageal Squamous Cell Carcinoma

“…Also, research must guarantee the homogeneity of exosomes by standardizing protocols for exosome isolation, preparation, and route of administration (8). Currently, inefficient isolation methods of exosomes cannot provide sufficient exosomes to meet cancer therapy requirements (142). Identifying ways to prevent exosomes from being taken up by other cells and to drive the engineered vehicles to targeted cells or organs remains a considerable challenge (143)(144)(145).…”