Pin Li, Melisa Kaslan, Sze Han Lee, Justin Yao, and Zhiqiang Gao
Exosomes are extracellular vesicles that are predominately involved in cell to cell communication, along with a wide range of other biological processes not greatly understood. Evidence suggests that the genetic material within exosomes can play a major role in the development of tumors.
Exosomes are now being examined as biomarkers to be used to aid in the diagnosis of disease. This recent examination has created a demand for inexpensive and effective methods for isolating exosomes.
Exosomes consist of a lipid bilayer consisting of “ceramide (sometimes used to differentiate exosomes from lysosomes), cholesterol, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains.” The outer surface consists of proteins and is dense with saccharide chains.
The outer surface proteins provide insight into the quality of the parental cells the exosomes were derived from. “Proteins typically found in exosomes include platelet derived growth factor receptor, lactadherin, transmembrane proteins and lysosome associated membrane protein-2B, membrane transport and fusion proteins like annexins, flotillins, GTPases, heat shock proteins, tetraspanins, proteins involved in multivesicular body biogenesis, as well as lipid-related proteins and phospholipases.” The physiology of an exosomes is almost identical to the parental cell The DNA inside of exosomes is needed after the isolation process for proper quantification and accurate diagnosis of disease.
Exosomes can be isolated from blood, urine, and cerebrospinal fluid. Five different isolation techniques are currently used; differential ultracentrifugation-based techniques, size-based techniques, immunoaffinity capture-based techniques, exosome precipitation, and microfluidic-based techniques.
Ultracentrifugation-based Isolation Techniques
A centrifugation process that generates forces up to 1,000,000 x g. There are two types of ultracentrifugation, analytical and preparative. The analytic approach examines the “the physiochemical properties of particulate materials and molecular interactions of polymeric materials.” The preparative approach fractionates “small bioparticles such as viruses, bacteria, subcellular organelles, and extracellular vesicles.”Ultracentrifugation is the most common isolation technique used currently.
Size-based Isolation Techniques
Ultrafiltration is a separation method that depends on the size or molecular weight of the cell. It is faster than centrifugation and does not require special equipment. A disadvantage of ultrafiltarion is the potential for damage of large vesicles due to the use of force.
Exclusion chromatography isolates exosomes by sorting macromolecules and bioparticles by size using the porous stationary phase of the cell. Larger molecules are unable to pass through the pores.
Flow field-flow fractionation utilizes a porous rectangular channel. “While a parabolic flow runs along the channel axis carries a sample towards the end of the channel, a crossflow across the channel controls the retention of the sample.65 The crossflow distributes particulate components against the channel wall based on the diffusivity of the components. Smaller particles diffuse further from the accumulation wall and are eluted earlier than larger ones.” Similar to exclusion chromatography, larger molecules are separated from smaller particles. This method enables the characterization of exosomes and is can be done rapidly to increase efficiency.
Immunoaffinity Capture-based Techniques
Immunoisolation focuses on the “immunoaffinitive interactions between those proteins (antigens) and their antibodies, and specific interactions between the receptors and ligands,” and is a highly specific isolation method. Biomarkers typically need to be membrane bound, lack soluble counter parts, and be concentrated on the surface of the exosome.
Isolation is done by altering the solubility or dispersibility of the exosomes. An easy method to use that does not require special equipment.
Microfluidics-based Isolation Techniques
Isolation is completed by utilizing alternative sorting methods such as acoustic, electrophoretic, and electromagnetic manipulations. A common effect of this method is a diminished sample volume,reagent consumption, and isolation time.