Extracellular vesicle Characterization: Quantitative Analysis of Cells and Cell-Derived Vesicles at the Single Cell Level Using NTA and SC-ICP-MS

Abstract

Extracellular vesicles (EVs) are important mediators of cell signaling pathways that contain diverse biological information such as DNAs, RNAs, lipids, and proteins. It is attracting attention in the diagnosis, treatment, basic research, and clinical applications of various diseases. Therefore, accurate and robust quantitation methods for size, concentration, and other properties such as protein quantification are essential. However, extracellular vesicle (EV) is secreted from all cells, so it is very heterogeneous, and it is divided into microvesicles (MVs) and exosomes according to the production process. And, it is difficult to distinguish and measure EV subgroups even if the generation process and size are different. In addition, existing general analytical methods have limited information on a single EV due to bulk analysis of isolated EV samples. Therefore, a study on the characterization method of a specific EV should be conducted. In the Chapter 1, NTA and mass cytometry are used to evaluate the characterization method of exosomes in a single cell unit. These two measuring instruments perform immunostaining using CD63, a biomarker of exosome, to phenotyping only exosomes in EVs and to analyze single exosome units. Measurement of single exosomes is important as it provides information about the heterogeneity and subgroups of exosomes. However, exosomes have a detection limit due to their very small size and low refractive index. To solve this problem, Qdot-conjugated antibody was introduced instead of a traditional fluorescence antibody or metal conjugated antibody. Nanoparticle tracking analysis (NTA) is one of the widely used exosome characterization methods. However, size-dependent sensitivity and detection limits result in biased measurements of the size distribution and number concentration of exosomes. In this study, the size distribution and number concentration of exosomes derived from A549 cells were compared and analyzed using the conventional NTA (C-NTA) and fluorescence NTA (F-NTA) methods. And mass cytometry, which combines single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) and immunostaining method that uses rare-earth metal ion-tagged antibodies, can compare the expression and phenotype of cells into various subtypes by biosignals such as proteins and DNAs. However, there are no prior research results on exosome protein expression analysis using mass cytometry. In this paper, a method for quantifying the surface protein of exosomes was studied using single cell-based mass spectrometry technology. In the Chapter 2, To analyze the platelet division mechanism, platelets were isolated from platelet rich plasma and labeled using the surface antigens of platelets, CD41a and CD61. The platelet count is very important because when a blood vessel is damaged, it attaches to the blood vessel wall and is formed by a hemostatic plug. Recently, evidence of platelet division has been presented, but the mechanism of how many and at what proportion platelets divide to be studied. In this study, the metal ions mass per antibody of a metal tagged antibody reagent was quantified to quantitatively measure and analyze platelet division using Single Cell Inductively Coupled Plasma Mass Spectrometry (SC-ICP-MS). And to observe the platelet division mechanism, the number of platelet surface antigens of CD41a and CD61 according to time of 0h, 6h, and 20h was counted.