Establishment of the collection, storage and preservation methods and their influence on stability of human salivary exosome

Abstract

The functions displayed by exosomes derived from saliva and other body fluids have been established. This paper studied the stability of human salivary exosome beginning from the collection mode, storage, and its preservation methods. Unstimulated saliva samples were collected from healthy subjects. Protease inhibitor was added into each samples and stored under different temperatures and at varying periods of time. The exosomes were isolated by ultracentrifugation and confirmed by using Western Blot. Exosome morphology was inspected by Scanning Electron Microscope (SEM) and the protein concentration was determined using the Protein (Bradford) Assay. The exosome particle size distribution and concentration were calculated using Nanoparticle Tracking Analysis (NTA). The protein assay showed no significant differences in the exosome protein concentration values for all conditions. Western Blot analysis also showed no differences in the presence of exosome and all the samples were positive for protein CD63. SEM analysis showed the fine shape of exosome which is round, in vesicle form with the size ranging between 10 nm and 100 nm. NTA determined the individual mean and the clumping exosome size was 203 nm. Human salivary exosomes remained intact in the absence of protease inhibitor and in different storage temperatures.