Adsorption of extracellular vesicles onto the tube walls during storage in solution

Abstract

Short term storage of extracellular vesicle (EV) solutions at +4°C is a common practice, but the stability of EVs during this procedure has not been fully understood yet. Using nanoparticle tracking analysis, we have shown that EVs isolated from the conditioned medium of HT-29 cells exhibit a pronounced concentration decrease when stored in PBS in ordinary polypropylene tubes within the range of (0.5–2.1) × 1010 particles/ml. EV losses reach 51±3% for 0.5 ml of EVs in Eppendorf 2 ml tube at 48 hours of storage at +4°C. Around 2/3 of the observed losses have been attributed to the adsorption of vesicles onto tube walls. This result shows that the lower part (up to at least 2 × 1010 particles/ml) of the practically relevant concentration range for purified EVs is prone to adsorption losses at +4°C. Total particle losses could be reduced to 18–21% at 48 hours by using either Eppendorf Protein LoBind tubes or ordinary tubes with the surface blocked with bovine serum albumin or EVs. Reduction of losses to 15% has been shown for isolated EVs dissolved in the supernatant after 100 000 g centrifugation as a model of conditioned medium. Also, a previously unknown feature of diffusion-controlled adsorption was revealed for EVs. In addition to the decrease in particle count, this process causes the predominant losses of smaller particles.

Analysis of MicroRNA Profile Alterations in Extracellular Vesicles From Mesenchymal Stromal Cells Overexpressing Stem Cell Factor

“…Configuration included 405-nm, 65-mW laser unit with passive temperature readout and high sensitivity camera of EMCCD type. All measurements were performed according to ASTM E2834 ( ASTM, 2018 ), using previously reported camera- and video-processing setups, optimized for EVs ( Livshits et al, 2015 ; Silachev et al, 2019 ; Evtushenko et al, 2020 ). Briefly, samples were diluted by particle-free PBS down to the optimal concentration around 1.5 × 10 8 particles/ml.…”

Impact of Storage Conditions on EV Integrity/Surface Markers and Cargos

“…Some researchers warned that isolated EVs could be adsorb onto tube walls, which would decrease EV concentration. Using Eppendorf Protein LoBind tubes or adding bovine serum albumin or Tween-20 to block the wall can decrease the loss [ 104 , 105 ]. Hermida-Nogueira et al (2020) found that seven days following a pathogen reduction technology (PRT) + Mirasol ® (vitamin B2 with ultraviolet B light), 151 proteins, including EV markers and regulatory proteins released by platelets, were upregulated compared to their levels after two days of storage [ 106 ].…”

Classification of Extracellular Vesicles based on Surface Glycan Structures by Spongy-like Separation Media

“…These are more likely to occur for purified vesicles. [50][51][52] However, there is not a consensus on a method to suppress nonspecific adsorption of EVs. Here, we consider a blocking treatment with excess protein or hydrophilic polymers as a practical solution to prevent nonspecific binding in various bioanalytical methods.…”