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.