Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids.Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes.To this end, size exclusion chromatography (SEC) and gradient density ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed.MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different types of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease. Results Protocol comparison: bdEV separationThe tissue processing and bdEV separation protocol previously published by several members of the author team (Vella et al., JEV, 2017) 13 was followed through the 2,000 x g centrifugation step ( Figure 1). After enrichment of large EVs (lEVs) by filtration and 10,000 x g centrifugation, supernatant was subjected to SDGU (as previously published) or SEC. Where indicated, SEC fractions were then concentrated by UC or ultrafiltration (UF) (Figure 1). Comparison of bdEV particle count, morphology, and protein markers in different fractions of SDGU and SEC: human and mouse tissueParticle yield per 100 mg tissue input (human or mouse) was determined by nanoparticle tracking analysis (NTA). Particle yield was highest for F2 from SDGU and F7-10 from SEC+UC. Particle concentration was below the reliable range of meas...

Extracellular Vesicles in neural cell interaction and CNS homeostasis

“…In fact, the largest proportion of neural EVs will be cleared from the interstitial space by surrounding cells and never reach these body fluids. Recent efforts aimed at recovering neural EVs from the brain parenchyma starting with frozen human, macaque or mouse brain tissue (79)(80)(81)(82)(83). The basic idea is to dissociate the tissue under mild conditions with as few as possible damage to the cells and collect EVs present in the interstitial fluid.…”

“…The challenge is to minimize co-isolation of non-EV contaminants with the same physiochemical properties such as intracellular vesicles, intraluminal vesicles or membranous particles released from broken cells during tissue harvest, processing, or storing (79,84). Following gentle tissue disruption (mechanical procedures, enzymatic digestion), EV separation can be performed by ultracentrifugation (85), density-gradient ultracentrifugation (79)(80)(81)83), size exclusion chromatography (82,83), or precipitation (86). However, it is unavoidable that during brain dissociation artificial membrane fragments are generated from thin axons, dendrites (spines), synapses and myelin, which form small vesicles co-purifying with interstitial EVs.…”

“…In addition, the pre-analytical conditions (species, death-to-processing period, storage conditions) greatly impact the yield, purity and stability of the recovered EV fractions and require thorough reporting and standardization as recommended by the MISEV and EV-TRACK initiatives, which impart Accepted Article essential guidelines for increasing transparency and harmonizing EV-research (84,87). A stringent characterization including screening for potential contaminants following EV enrichment from brain tissue is mandatory to meet the standards and requirements for the definition of EVs and subtypes (79,82,88). Until now, an absolute separation of EVs from cellular compartments and a resolution of the existing EV subtypes is unreached.…”

Characterization of the microRNA Transcriptomes and Proteomics of Cochlea Tissue-derived Small Extracellular Vesicles From Different Age of Mice After Birth

“…The cochlea is surrounded by the otic vesicle that gradually ossi es and becomes rigid as the mouse ages, especially after P10, and this makes it di cult to dissect the basilar membrane for extracting cochlear tissue-derived sEVs. Some recent studies have used enzyme digestion for the purpose of maintaining the integrity of the cells as much as possible in order to extract EVs from fat, brain, and tumor tissues [35,[83][84][85][86], while other studies have ground the tissues as a necessary step for extracting EVs [85,[87][88][89]. Crescitelli et al showed that the digestive enzymes in the existing tissue extraction methods are ineffective for bone tissue, and the methods for this type of tissue need further optimization [90].…”

Surface phenotyping and quantitative proteomics reveal differentially enriched proteins of brain-derived extracellular vesicles in Parkinson’s disease

“…Although peripheral biofluid EV molecular profiles have been examined at the RNA and protein levels (Dutta et al, 2021; Jiang et al, 2021; Shi et al, 2014), how these findings compare with EVs present in the brain remains unclear. Brain-derived EVs (bdEVs) can be separated by gentle tissue digestion in a reproducible manner (Huang et al, 2020; Vella et al, 2017). Here, we separated and profiled bdEVs from PD, PSP, and control tissues.…”