Toward the isolation of exosomes by flow cytometry

• Autor(a) principal: Mozes, André Ribeiro da Graça Lima
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.1/13556
• topic: Vesículas extracelulares; Exosomas; FACS; Isolamento; Domínio/Área Científica::Ciências Médicas::Outras Ciências Médicas

In the last two decades the Extracellular Vesicles (EVs) field has attracted a lot of attention from the scientific community, especially after the discovery that EVs can shuttle functional proteins and nucleic acids between cells. Some recent studies have shown an association between tumorigenesis and increased exosomes production. Exosomes and their influence has also been reported in the establishment of new metastatic niches. Besides that, the EV field remains confusing due to numerous and ambiguous definitions, specially caused by the huge heterogeneity of the vesicles, both in composition and function. Extracellular vesicles are divided into microvesicles which are originated from the plasma membrane and exosomes which have an endosomal origin. For now, it is technically challenging to obtain a pure exosome fraction, free from non-vesicular components, due to the fact the extracellular milieu is quite complex and can contain microvesicles or apoptotic bodies similar in size and structure to exosomes. The two most used methods, ultracentrifugation and commercial kits, don’t show a good efficiency when distinguishing the exosomes fraction specifically from the microvesicles fraction. Due to this sub-optimal efficiency demonstrated by these two methods, we have decided to use Flow Cytometry to see if we can achieve better exosome purification. We will use Fluorescence-activated cell sorting (FACS) to purify endogenous exosomes. This would be quite challenging especially due to the exosomes size and heterogeneity but on the other hand, if we have success with our approach, it would be possible to do downstream analysis in order to know their protein composition, functions and elaborate some more studies to try to find some “exosome-specific” marker. This would have a huge impact in the pharmaceutical industry, both for diagnosis and therapy.