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  • Direct counting of exosomes in a cell culture medium using neither isolation nor preconcentration.

Direct counting of exosomes in a cell culture medium using neither isolation nor preconcentration.

Analytica chimica acta (2020-05-23)
Tatsuya Fujii, Takashi Kaneta
摘要

Exosomes are expected to be biomarkers of cancer since they contain information about the cells that excrete them. In this study we developed a method to count the exosomes secreted from cancer cells in a culture medium without the need for isolation and/or preconcentration. This detection system consists of a square capillary on which a laser beam is focused in a sheet shape via the use of two cylindrical lenses. A fluorescently labeled anti-CD63 antibody is used to mark the exosomes that are then flowed into the square capillary. In this study, individual exosomes were observed on a trajectory when passing through the laser beam sheet and were counted for 10 min at a constant flow velocity. The total analysis time was less than 1.5 h including the steps required to remove large particles and allow reaction with the antibody. The results for two samples prepared with and without the isolation of exosomes showed a loss of exosomes in the isolation step. We also determined the number of the exosomes secreted by the cells to a culture medium during cultivation. As expected, the total number of exosomes in a culture medium increased with an increase in the cultivation time, and the number of exosomes released every 12 h either remained constant or showed no more than a slight increase for as long as 72 h. It was unclear whether the number exosomes was dependent on the cell population at confluences of 10-60%.

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