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Browsing by Subject "Ultracentrifugation"

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  • Maran, Suveda Vignesh (2019)
    Extracellular vesicles (EVs) are a diverse set of cell-derived membranous vesicles which have exosomes or small EVs (40 -200nm) ,large EVs (>200 nm) and larger apoptotic bodies/oncosomes (>1000 nm). They are present in most biological fluids, such as blood, sweat, and urine etc. EVs are expressed by almost all cells.Recent evidence suggests that EVs are a form of cellular communication, resulting in an intensive investigation of their role in different biological processes and also due to their biomarker potential. Ultracentrifugation (UC) is the standard method used for the isolation of EVs from different biological fluids. UC has some limitations related to laborious operation and scalability issues. Hence, there is a need for new methods for the isolation of EVs.Nanocellulose(NC) is a biopolymer obtained from wood biomass and has the ability to be functionalised with various chemical groups on its surface. The chemical groups alter the surface properties of NC for usability in different applications. Currently, It finds extensive use in different biomedical applications due to its unique physicochemical properties. The aim of the thesis is to evaluate the use of functionalised nanocellulose (FNC) as a novel alternative compared to the standard method of ultracentrifugation in the isolation of EVs from conditioned cell culture media. Cancer cell lines and mesenchymal stromal cells (MSCs) were grown in EV-depleted cell culture media after expansion in cell culture media. The EV-depleted media was collected from the cells after 48 hours and further processed by filtration to obtain the necessary conditioned cell culture media for EV isolation. Extracellular vesicles were isolated from the conditioned cell culture media by using UC and FNC. The isolated EVs from both methods were characterised and evaluated based on their different parameters using Bicinchoninic Acid (BCA) assay, Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and Western Blot. The EVs isolated by FNC were smaller in size with few vesicle-like structures around 20-40 nm compared to EVs isolated by UC 60-200nm. Most EVs isolated by UC had a higher particle-to-protein ratio which indicated more purity and reduced levels of non-vesicular contaminants in the EV isolations. The EVs isolated by FNC for many samples had a low particle-to-protein ratio indicating potentially higher levels of non-vesicular contaminants being present. Hsp70, a surface marker of EVs and soluble stress protein was observed in EVs obtained from a primary MSC and another cancer cell line among all the isolation methods. No contamination from Endoplasmic reticulum(ER) components was observed in any of the samples. The EVs isolated by FNC could not be analysed more thoroughly by some of the characterisation methods due to issues such as lack of standardised sample preparation protocols, possible interference from isolation components and sample variations. The isolation method will really benefit from further optimisation of sample preparation steps and the adoption of alternative characterisation methods for evaluation. A narrower research question with focus on FNC-EDA isolation of EVs from HOS-143B and AT-MSC will be an excellent starting point for more detailed investigations.The availability of different surface modifications, faster processing and more research on the isolation mechanism will make this novel method a great alternative to UC for isolation of EVs.