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Purification and in vitro analysis of exosomes secreted by malignantly transformed human cells
Authors:T A Shtam  S N Naryzhny  S B Landa  V S Burdakov  T O Artamonova  M V Filatov
Institution:(1) Krefting Research Centre, Sahlgrenska Academy, University of Gothenburg, Box 424, 405 30 Gothenburg, Sweden;(2) Department of Medicine, Clinical Allergy Research Unit, Karolinska University Hospital Solna, Stockholm, Sweden;(3) Dept. of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Gothenburg, Guldhedsgatan 10A, 413 46 Gothenburg, Sweden
Abstract:Exosomes are natural nanoparticles secreted by different cells and capable of carrying protein markers and genetic information, thus participating in cellular communication. There is good reason to think that quantitative and qualitative characterization of these microparticles produced by different tissues in normal and pathological states can give valuable diagnostic and prognostic information and be a biomarker of different diseases, including oncological ones. Elaboration of the purification of exosomes and their proteome analysis was the aim of the present work. An original approach to enhancing exosome production in cultured transformed human cells was developed. The data obtained allowed us to detect exosomes in cultural conditioned samples and control the quality of produced exosomes at all stages of their purification. Electrophoretic analysis of proteins obtained from exosomes of different origins shows differences in protein profiles. Proteins from exosomes of glioblastoma cell lines were separated by two-dimensional electrophoresis. Protein profiles were further analyzed by densitometry and mass spectrometry, which allowed more than 30 proteins, including specific tumor markers, to be identified.
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