Isolation and Detection of Extracellular Vesicles

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http://urn.fi/URN:NBN:fi-fe2017121155628
Julkaisun nimi: Isolation and Detection of Extracellular Vesicles
Tekijä: Tear, Jing Ying Crystal
Muu tekijä: Helsingin yliopisto, Matemaattis-luonnontieteellinen tiedekunta, Kemian laitos
Opinnäytteen taso: pro gradu -tutkielmat
Tiivistelmä: Extracellular vesicles (EVs) are bilayer nanoparticles ranging from 40 nm to 5 μm in size and are mainly categorized as exosomes, microparticles, and apoptotic bodies. Recent studies reveal their involvement in various metabolic mechanisms in the human body. In particular, certain types of platelet-derived EVs found in plasma and serum are related to blood coagulation diseases and strokes. Merely, the diversity and heterogeneity of EVs in cellular systems and the lack of their isolation and detection methods are known about EVs. The fact is that a wide range of nomenclatures and basic questions regarding their morphology, size, and phenotype has remained debatable. As a result, there is a need to discover and standardize a simple and robust protocol for the isolation and detection of EVs. The literature part of the thesis covers an overview of what is defined as EVs and their functions undertaken, followed by a summary of the conventional methods for isolation and detection of EVs. The literature part finally concludes with an insight into upcoming and novel isolation and detection methods. Ultracentrifugation is the “golden” method used in the isolation of EVs. The long extraction times and varying reproducibility makes ultracentrifugation less viable, resulting in the exploration of other methods including size exclusion chromatography and immunoaffinity methods. Currently, none of these techniques are able to effectively distinguish between subtypes of EVs and matrix contaminants such as proteins remaining in the isolates. Each isolation method has its own edging advantages and disadvantages. Nevertheless, immunoaffinity methods showed greater potential in EVs extraction due to their high specificity and selectivity to process purified EVs samples. Detection methods are hindered by the minute sizes of EVs and the presence of contaminants in the isolates. Most detection methods involve the use of antibodies to detect or microscopic imaging to verify their presence based on their morphology. The trend is, however, skewing towards more reliable detection methods such as the use of mass spectrometry and microfluidic devices. The aim of the experimental portion is the ability to isolate purified platelet-derived EVs from complex human plasma samples and enable fast isolation of EVs. The use of immunoaffinity chromatography using antibody immobilized carbonyldiimidazole (CDI) monolithic disk was explored. Studies using specific platelet EV biomarker, anti-human CD61 antibody had successfully isolated platelet-derived EVs from plasma samples. In these studies, diluted plasma in phosphate buffer saline (PBS) solution (1:20 v/v) was injected through the housing setup containing the anti-human CD61 antibody immobilized monolithic disk. The eluate was obtained by injection of carbonate-bicarbonate solution and neutralized with hydrochloric acid. Detection analyses were then performed with nanoparticle tracking analysis (NTA), protein assays, Western blot and transmission electron microscopy (TEM). The average particle counts obtained in plasma isolates were found to achieve 10,000 particles more than isolates obtained after ultra-centrifugation reported in the literature. Positive presence of cytosolic protein TSG101 and transmembrane protein CD9 in Western blots, and transmission electron microscopic images of circular bilayer particles confirmed the isolation of platelet-derived EVs. Optimization studies also showed the inverse relationship between the flow rate in isolation step and the yield of platelet-derived EVs obtained. Affinity chromatography using antibody immobilized monolithic disk proved its success in the quick isolation (≤45 minutes) of platelet-derived EVs from plasma samples. The fast, miniaturized, compact setup, and simple methodology make the method viable for automation such as high throughput screening and suitable for applications of microfluidic microchips and censoring techniques on-line coupled for non-invasive and portable diagnostics.
URI: URN:NBN:fi-fe2017121155628
http://hdl.handle.net/10138/229529
Päiväys: 2017
Oppiaine: Analytical Chemistry
Analyyttinen kemia
Analytisk kemi


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