ИСТИНА

Introduction: Differential centrifugation is going to be one of the most widely used methods for exosomes isolation, even though it is labour intensive and in most cases require large amount of source material. In the present work, we illustrate the theoretical consideration of microparticle sedimentation process with the results of the analysis of morphology, size distribution and amount of the objects sedimented at different centrifugation steps. Methods: HT29 cell culture supernatant was subjected to centrifugation at 500 g for 5 minutes, 2,000 g for 10 minutes, 10,000 g for 30 minutes and 100,000 g for 1 h. Measurements of particle size and concentration were done by NTA. TEM was used to determine the morphology of the particles. Results: According to our data, the essential part of the population of 70–100 nm spheric particles is sedimented at 10,000 g and such a loss is obviously to increase in case of using smaller tubes or rotors with smaller k-factors. On the contrary, the increase of rotor k-factor or of the particle path length (tube volume) leads to contamination of exosomes with population of bigger particles. To demonstrate the fact that exosomes themselves can be partly sedimented at 10,000 g, the population of exosomes non-contaminated with bigger particles was subjected to centrifugation at 10,000 g for 30 minutes. The analysis revealed the presence of identical particles in both the pellet and supernatant. To increase the yield and the purity of exosome population, we eliminated the 10,000 g centrifugation step and used non-equilibrium zonal centrifugation in sucrose. Summary/conclusion: Differential centrifugation fails to well separate the objects having similar sedimentation velocities as exosomes and microvesicles. We demonstrated that in accordance with theoretical predictions, the essential part of the population of 70–100 nm particles is sedimented at 10,000 g. We are discussing the possibility of increasing the yield and the purity of exosome population by using non-equilibrium zonal centrifugation.