Purification Of Vesicles Research Articles

Snorkel-tag based affinity chromatography for recombinant extracellular vesicle purification.

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present the Snorkel-tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non-destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel-tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Analysis of the utility of a rapid vesicle isolation method for clinical strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a pathogen capable of causing diseases ranging from mild to life-threatening, has a large arsenal of virulence factors. Notably, extracellular vesicles have emerged as significant players in the pathogenesis of this organism. However, the full range of their functions is still being studied, and difficulties related to vesicle purification (long protocols, low yields, and specialized instruments) have become a major obstacle for their characterization. In this context, the utility of rapid new methods of vesicle isolation from clinical strains is still unknown. Here, we analyze the utility of the ExoBacteria OMV isolation kit for a collection of clinical strains of P. aeruginosa. We first phenotypically characterized 15 P. aeruginosa strains to ensure that our samples were heterogeneous. We then determined the best conditions for purifying vesicles from P. aeruginosa PAO1 reference strain by the rapid method and used them to isolate vesicles from clinical strains. Our results indicated that M9 minimal medium is the best for obtaining high purity with the rapid isolation kit. Although we were able to isolate vesicles from at least four strains, the low yield and the large number of strains with unpurifiable vesicles showed that the kit was not practical or convenient for clinical strains. Our findings suggest that although fast procedures for vesicle purification can be of great utility for Escherichia coli, the more complex phenotypes of clinical isolates of P. aeruginosa are a challenge for these protocols and new alternatives/optimizations need to be developed.IMPORTANCEPseudomonas aeruginosa is recognized as an opportunistic pathogen in humans and animals. It can effectively colonize various environments thanks to a large set of virulence factors that include extracellular vesicles. Different methods were recently developed to reduce the time and effort associated with vesicle purification. However, the utility of rapid vesicle isolation methods for clinical strains of P. aeruginosa (which are recognized as being highly diverse) is not yet known. In this context, we analyzed the utility of the ExoBacteria OMV Isolation kit for vesicle purification in P. aeruginosa clinical strains. Our findings showed that the kit does not seem to be convenient for research on clinical strains due to low vesicle recovery. Our results underscore the importance of developing new rapid vesicle purification protocols/techniques for specific clinical phenotypes.

Inline Raman Spectroscopy Provides Versatile Molecular Monitoring for Platelet Extracellular Vesicle Purification with Anion-Exchange Chromatography.

Extracellular vesicles (EVs) are relatively recently discovered biological nanoparticles that mediate intercellular communication. The development of new methods for the isolation and characterization of EVs is crucial to support further studies on these small and structurally heterogenous vesicles. New scalable production methods are also needed to meet the needs of future therapeutic applications. A reliable inline detection method for the EV manufacturing process is needed to ensure reproducibility and to identify any possible variations in real time. Here, we demonstrate the use of an inline Raman detector in conjunction with anion exchange chromatography for the isolation of EVs from human platelets. Anion-exchange chromatography can be easily coupled with multiple inline detectors and provides an alternative to size-based methods for separating EVs from similar-sized impurities, such as lipoprotein particles. Raman spectroscopy enabled us to identify functional groups in EV samples and trace EVs and impurities in different stages of the process. Our results show a notable separation of impurities from the EVs during anion-exchange chromatography and demonstrate the power of inline Raman spectroscopy. Compared to conventional EV analysis methods, the inline Raman approach does not require hands-on work and can provide detailed, real-time information about the sample and the purification process.

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Scalable purification of extracellular vesicles with high yield and purity using multimodal flowthrough chromatography

AbstractExtracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC.

Extraction and Purification of Outer Membrane Vesicles and Their Associated RNAs.

Outer membrane vesicles (OMVs), produced by Gram negative-bacteria and sRNAs, are key players in cell-to-cell communication and interactions of bacteria with the environment. OMVs act as information carriers and encapsulate various molecules such as proteins, lipids, metabolites, and RNAs. OMVs and sRNAs play a broad range of functions from pathogenesis to stress resistance, to biofilm formation and both mediate interkingdom signaling. Various studies indicate that there is a mechanism of intercellular communication mediated by OMV-derived bacterial RNAs that is conserved among certain bacterial species. Here we describe methods for the extraction and purification of vesicles produced by Gram-negative bacteria, such as Pseudomonas brassicacearum and Escherichia coli, and address methods for the extraction of OMVs-derived sRNA and techniques for the analysis of sRNAs.

Economical large-scale purification of extracellular vesicles from urine

Research on extracellular vesicles (EVs), both in general laboratory methods and in novel therapeutic approaches, is limited by the difficulty of obtaining large quantities of EVs. This is due to the fact that EVs are only present at low concentrations in most starting samples and common methods for concentrating EVs from large volumes have decisive disadvantages. In particular, ultracentrifugation (UC) and tangential flow filtration (TFF) are used in EV bulk production. However, while UC leads to low EV-yield with high levels of impurities and damaged EVs, TFF is suitable for enriching EVs from large volumes, but requires expensive equipment. In addition, both methods require pre-purification of the EV material and downstream EV isolation steps. To circumvent all these obstacles, we evaluated various methods for their applicability to EV mass purification, optimized both their implementation and the workflow, and thus developed the following sequential protocol for EV bulk production: First, a preclearing step is performed by 1 µm filtration, followed by PEG precipitation to reduce total volume. Larger debris and apoptotic bodies are removed by centrifugation and filtration prior to enrichment and pre-purification of EVs by ultrafiltration or using the ExoEasyMaxi kit. Finally, EVs are purified by size exclusion chromatography. Since the availability of suitable starting material to obtain pure EVs is also a challenge, we compared different biological fluids, with urine proving to be the most suitable source. We determined the purity and quantity of the EV samples using nanotracking analysis, dynamic light scattering, phospholipid-, RNA- and protein-concentration measurement, electron microscopy and western blot.In summary, we have developed procedures to obtain about 1012 pure EVs in less than a day, with a handling time under 2 h, and at a cost of approximately €24. This method should facilitate further scientific and technical developments in the field of EV biology.

Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Guidelines for the purification and characterization of extracellular vesicles of parasites

AbstractParasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells

Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer.

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flow-through pores of the monolithic skeleton (median pore size; 5.21 μm) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBS-derived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

Exploiting Sound for Emerging Applications of Extracellular Vesicles.

Extracellular vesicles are nano- to microscale, membrane-bound particles released by cells into extracellular space, and act as carriers of biomarkers and therapeutics, holding promising potential in translational medicine. However, the challenges remain in handling and detecting extracellular vesicles for disease diagnosis as well as exploring their therapeutic capability for disease treatment. Here, we review the recent engineering and technology advances by leveraging the power of sound waves to address the challenges in diagnostic and therapeutic applications of extracellular vesicles and biomimetic nanovesicles. We first introduce the fundamental principles of sound waves for understanding different acoustic-assisted extracellular vesicle technologies. We discuss the acoustic-assisted diagnostic methods including the purification, manipulation, biosensing, and bioimaging of extracellular vesicles. Then, we summarize the recent advances in acoustically enhanced therapeutics using extracellular vesicles and biomimetic nanovesicles. Finally, we provide perspectives into current challenges and future clinical applications of the promising extracellular vesicles and biomimetic nanovesicles powered by sound.

Calcium Triggered Compound Fusion at Ribbon Synapses During Exocytosis

AbstractBackgroundExocytosis in photoreceptors involves fusion of vesicles with the plasma membrane at the base of a specialized proteinaceous electron‐dense structure known as a synaptic ribbon. The molecular mechanisms underlying exocytosis at ribbon synapses are not well understood. Research suggests a compound fusion model where vesicles at ribbon synapses undergo homotypic fusion forming larger vesicles prior to fusion with the plasma membrane. But how this process is mediated remains unclear. Ca2+ triggers several forms of exocytosis in different types of eukaryotic cells through activation of the SNARE complex. SNARE‐mediated fusion of membranes is triggered by the calcium sensor Synaptotagmin, but not much is known about whether this applies to compound fusion. Here, we report that, like typical synaptic exocytosis, compound fusion is mediated by Ca2+ influx.MethodTo examine the role of calcium in compound fusion, we performed vesicle purification experiments from Mus musculus (C57BL/6 WT) retina. Brain tissue was also used as a control where we expected no compound fusion. Retina and brain tissue were dissected, homogenized, and ultracentrifuged. The supernatant was incubated in 2mM Ca2+ for 5 minutes then vesicles from the Ca2+samples and controls containing no Ca2 were isolated using magnetic beads coupled with rho1D4 monoclonal antibody. Vesicles were negatively stained and viewed under TEM. Vesicle sizes of the Ca2+ and control samples were determined via blind analysis using ImageJ.ResultsIn retina samples, exogenous Ca2+ caused a significant increase in vesicle size when viewed under TEM. In the retina Ca2+ samples, vesicle diameter ranged from 30nm to 250nm. This effect was not seen in retina controls where vesicle diameter ranged from 30nm to 60nm. No large vesicles were seen in any of the brain samples.ConclusionOur research supports the compound fusion model of vesicle exocytosis at ribbon synapses. We suggest that in retina, the larger vesicles formed during exocytosis are a result of smaller vesicles fusing with each other and that Ca2+ has a significant role in this process. Future studies investigating the role of calcium sensors such as synaptotagmin will help to further elucidate the mechanisms underlying compound fusion and its therapeutic potential in neurological disorders.

A microfluidic device for extracellular vesicle purification: an in-vitro study on mammary gland tumours

A microfluidic device for extracellular vesicle purification: an in-vitro study on mammary gland tumours

Interplay of electric field and pressure-driven flow inducing microfluidic particle migration

The lateral migration of colloidal particles inside a microfluidic channel has gained attention due to being both fundamentally intriguing and applicable for particle separation, such as cancer cell isolation or extracellular vesicle purification. Applying an external electric field combined with a pressure-driven flow induces such lateral migrations. In this study, new modes of lateral particle migration have been found by experimentally investigating 6 µm particles in the co-presence of electric field and pressure-driven flow. The experiments revealed the importance of the relative strengths of electric field and pressure gradient in determining particle lateral positioning. We hypothesize that the nonuniformity of the polarization caused by the external electric field and the rotation of the particle due to the background pressure-driven flow result in these modes of transverse migration. These new migration patterns are further utilized to perform microparticle separation and, more importantly, present a novel separation modality.

Methods of Isolation and Purification of Extracellular Vesicles from Different Biological Matrixes: Special Issue at a Glance

ARTICLES DISCUSSED: Hanine El Itawi, H., et al. Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection. J Vis Exp. 186, DOI: 10.3791/63480 (2022) Jones, M.T., Manioci S.W., S1, Russell, A.E. Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media. J Vis Exp. 186, DOI: 10.3791/63614 (2022) Ryan, J.M., Dobos, K.M., Kruh-Garcia, N.A. Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography. J Vis Exp. 186, DOI: 10.3791/63895 (2022) Valle-Tamayo, N., et al. Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma J Vis Exp. 186, DOI: 10.3791/64107 (2022) Koh, B., et al. A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells J Vis Exp. 186, DOI: 10.3791/64106 (2022)

Anion exchange chromatography-based platform for the scalable purification of extracellular vesicles derived from human mesenchymal stromal cells

Extracellular vesicles (EVs) have emerged as a novel cell-free strategy with broad biomedical applications in disease diagnosis and prognosis as well as therapy. Moving EVs to the clinics will require the development of cost-effective manufacturing processes capable of delivering a high-quality product with high yield. In this work, we explore the purification of EVs from conditioned media derived from human mesenchymal stromal cell bioreactor cultures using a nuclease digestion followed by anion exchange (AEX) chromatography as a scalable purification platform. The performance of different nucleases was evaluated and optimized, with MNase being the top performer. Concerning the chromatographic step, all anion-exchange resins successfully isolated EVs, being Capto™ Q ImpRes the lead resin with recovery yields exceeding 85% concomitant with removal of more than 98% of proteins and host cell DNA. Overall, our results show that the purification platform proposed in this work is a viable alternative to the existing methods, capable of delivering highly purified EVs and able to meet the requirements imposed by the regulatory agencies for clinical trial testing, and that can contribute for the integration of EVs-based therapies in the clinic.

In vitro reconstitution of COPII vesicles from Arabidopsis thaliana suspension-cultured cells.

Transport vesicles mediate protein traffic between endomembrane organelles in a highly selective and efficient manner. In vitro reconstitution systems have been widely used for studying mechanisms of vesicle formation, polar trafficking, and cargo specificity in mammals and yeast. However, this technique has not yet been applied to plants because of the large lytic vacuoles and rigid cell walls. Here, we describe an Arabidopsis-derived in vitro vesicle formation system to reconstitute, purify and characterize plant-derived coat protein complex II (COPII) vesicles. In this protocol, we provide a detailed method for the isolation of microsomes and cytosol from Arabidopsis thaliana suspension-cultured cells (7-8 h), in vitro COPII vesicle reconstitution and purification (4-5 h) and biochemical and microscopic analysis using specific antibodies against COPII cargo molecules for reconstitution efficiency evaluation (2 h). We also include detailed sample-preparation steps for analyzing vesicle morphology by cryogenic electron microscopy (1 h) and vesicle cargoes by quantitative proteomics (4 h). Routinely, the whole procedure takes ~18-20 h of operation time and enables plant researchers without specific expertise to achieve organelle purification or vesicle reconstitution for further characterization.

Filtration-based technologies for isolation, purification and analysis of extracellular vesicles.

The involvement of extracellular vesicles (EVs) in cellular communication with multifactorial and multifaceted biological activity has generated significant interest, highlighting their potential diagnostic and therapeutic applications. EVs are found in nearly all biological fluids creating a broad spectrum of where potential disease markers can be found for liquid biopsy development and what subtypes can be used for treatment of diseases. Complexity of biological fluids has generated a variety of different approaches for EV isolation and identification that may in one way or another be most optimal for research studies or clinical use. Each approach has its own advantages and disadvantages, significance of which can be evaluated depending on the end goal of the study. One of the methods is based on filtration which has received attention in the past years due its versatility, low cost and other advantages. Introduction of different approaches for EV capture and analysis that are based on filtration gave rise to new subcategories of filtration techniques which are presented in this overview. Miniaturization and combination of filtration-based approaches with microfluidics is also highlighted due its future prospects in healthcare, especially point-of-need technologies.

Advances in Purification, Modification, and Application of Extracellular Vesicles for Novel Clinical Treatments.

Extracellular vesicles (EV) are membrane vesicles surrounded by a lipid bilayer membrane and include microvesicles, apoptotic bodies, exosomes, and exomeres. Exosome-encapsulated microRNAs (miRNAs) released from cancer cells are involved in the proliferation and metastasis of tumor cells via angiogenesis. On the other hand, mesenchymal stem cell (MSC) therapy, which is being employed in regenerative medicine owing to the ability of MSCs to differentiate into various cells, is due to humoral factors, including messenger RNA (mRNA), miRNAs, proteins, and lipids, which are encapsulated in exosomes derived from transplanted cells. New treatments that advocate cell-free therapy using MSC-derived exosomes will significantly improve clinical practice. Therefore, using highly purified exosomes that perform their original functions is desirable. In this review, we summarized advances in the purification, modification, and application of EVs as novel strategies to treat some diseases.