Increased Yield of Extracellular Vesicles after Cytochalasin B Treatment and Vortexing

Abstract

Extracellular vesicles (EVs) are promising therapeutic instruments and vectors for therapeutics delivery. In order to increase the yield of EVs, a method of inducing EVs release using cytochalasin B is being actively developed. In this work, we compared the yield of naturally occurring extracellular vesicles and cytochalasin B-induced membrane vesicles (CIMVs) from mesenchymal stem cells (MSCs). In order to maintain accuracy in the comparative analysis, the same culture was used for the isolation of EVs and CIMVs: conditioned medium was used for EVs isolation and cells were harvested for CIMVs production. The pellets obtained after centrifugation 2300× g, 10,000× g and 100,000× g were analyzed using scanning electron microscopy analysis (SEM), flow cytometry, the bicinchoninic acid assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). We found that the use of cytochalasin B treatment and vortexing resulted in the production of a more homogeneous population of membrane vesicles with a median diameter greater than that of EVs. We found that EVs-like particles remained in the FBS, despite overnight ultracentrifugation, which introduced a significant inaccuracy in the calculation of the EVs yield. Therefore, we cultivated cells in a serum-free medium for the subsequent isolation of EVs. We observed that the number of CIMVs significantly exceeded the number of EVs after each step of centrifugation (2300× g, 10,000× g and 100,000× g) by up to 5, 9, and 20 times, respectively.

Investigation of the Affinity of Aptamers for Bacteria by Surface Plasmon Resonance Imaging Using Nanosomes

“…In a sample containing both small and large particles, small particles will hardly be visualized and the average size will be overestimated. 24 In contrast, NTA is appropriate for a polydisperse sample because it tracks the Brownian motion of each particle in the suspension. 25 The nanosome concentration was obtained by NTA with settings optimized for quantification of B. cereus nanosomes.…”

“…DLS measures particle size according to changes in the scattered intensity of particles in suspension. In a sample containing both small and large particles, small particles will hardly be visualized and the average size will be overestimated . In contrast, NTA is appropriate for a polydisperse sample because it tracks the Brownian motion of each particle in the suspension .…”

Using Targeted Nano-Antibiotics to Improve Antibiotic Efficacy against Staphylococcus aureus Infections

“…While we have observed differences in the drug release profiles, we cannot exclude the possibility of variations in the number of NP-LEV@Staph and NP-CIP@Staph particles adhering to bacteria after the incubation period. Quantifying these targeted nano-antibiotics on the bacterial surface in future experiments is necessary to investigate this aspect further [ 53 , 54 ]. Moreover, conducting in vivo animal studies is essential to gain a more comprehensive understanding of the therapeutic effects of nano-antibiotics.…”

Synergistic vesicle-vector systems for targeted delivery

“…Chemical stimulation methods involve the simple addition of specific chemicals to the growth medium. For instance, cytochalasin-B treatment resulted in the production of at least five times more extracellular vesicle (EV) particles compared to conventional isolation methods [ 120 ]. Moreover, EVs treated with cytochalasin exhibited an enhanced capacity for drug loading [ 121 ].…”