Exosome Isolation Methods
There are various methods for isolating exosomes with varying degrees of complexity and contamination with non-exosomal vesicles and/or non-vesicular products. The prevalent method for exosome isolation is differential ultracentrifugation. It involves sequential spinning of a fluid at increasing speed and duration to pellet dead cells and cellular debris, microvesicles and eventually, exosomes (75). This method is not suitable for fluids with high viscosity such as blood since higher speeds and longer duration of centrifugation are required (76). Due to similarity of sedimentation properties of various types of extracellular vesicles the yield and purity using this method may be low (77,78)
Density gradient ultracentrifugation uses a concept similar to differential ultracentrifugation. The initial steps are similar to DUC but sucrose is added at a later point. Due to the buoyant density of exosomes (1.15 to 1.19 g/mL) in sucrose, it sediments separately from other denser extracellular vesicles during centrifugation (79,80). This method yields purer fractions compared with differential ultracentrifugation (81), but has same disadvantage of need for long periods of processing and requirement of costly equipment.
Size based isolation techniques make use of the smaller size of exosomes compared with microvesicles, apoptotic bodies and other molecules to separate them in a fluid sample. Ultrafiltration, a size based technique uses membrane filters with decreasing pore sizes to isolate exosomes (82). It is a faster method compared with ultracentrifugation and does not require costly equipment. While this method yields pure vesicles, there is difficulty in removing contaminating proteins but can be combined with ultrcentrifugation for removal of proteins (83). Other size based techniques include size exclusion chromatography which involves differential passage of various vesicles and particles through a gel column and their recovery in different elution fractions (84). SEC allows for pure exosomal isolates with intact physical characteristics (85). The run time is however long and limits its use in multiple biological specimens.
In polymer-based precipitation, a polymer, e.g. polyethylene glycol (PEG) precipitates exosomes in a biofluid and the exosomes recovered by low speed centrifugation to pellet down the precipitate (86).
Immunoaffinity enrichment methods use exosomal membrane markers, typically the tetraspanins CD9, CD63 and CD81, to isolate exosomes from biofluids (87,88). The antibodies are immobilized on varying media including magnetic beads, microfluidic devices and chromatography matrices (89,90).
Newer methods of exosome isolation include contact-free sorting which uses ultrasound waves to exert differential acoustic forces on vesicles based on their size and density (91), microfluidics, which utilizes size dependent position of flow of various vesicles in a biofluid in a channel to isolate exosomes (92) and nanoplasmon-enhanced scatter which uses immobilized exosomal antibodies and gold plated nanoparticles to capture and detect exosomes based on scatter patterns (93). Confirmation of exosome isolation can be done using multiple techniques including electron microscopy which identifies the typical cup shaped morphology and vesicle size of 30-120nm, western blotting for exosome markers such as tetraspanins (CD63/CD9/CD81), ALIX, flotillin, hsp70, TSG101, and also nanoparticle tracking analysis for vesicle size and concentration determination (94).
Exosome Use in DLBCL
Due to their relative abundance, longer half-life, presence in multiple body fluids, and a payload that includes nucleic acids, proteins, and small molecules (95) exosomes are poised to contain biomarkers that may be informative in the prognostication and management of DLBCL patients at multiple stages. The nucleic acid and/or protein cargo in exosomes can be used for molecular analyses in DLBCL. Considering the relative abundance of exosomes in body fluids, especially serum where there is a concentration of about 3x106 exosomes/microliter, this presents a readily available source of biologic material for multiparametric studies leading to more exhaustive characterization of DLBCL types, prognostication, surveillance during and post treatment, and understanding of mechanisms of resistance to therapy. Rutherford et al characterized mutations in cell lines and their exosomes using RNA sequencing and found that nearly one third of mutations in the cell lines were detected in the exosomes (96). This was surprising as exosomes contained a smaller subset of RNAs than the cell lines, a finding that also suggests a likely enrichment of mutated RNA in exosomes.