Consequently, the advancement of the field relies on the creation of novel methodologies and instruments that facilitate investigation into the fundamental biology of EVs. The monitoring of EV production and release commonly utilizes methods that employ either antibody-based flow cytometric assays or systems featuring genetically encoded fluorescent proteins. selleck chemical Previously, we had generated artificially barcoded exosomal microRNAs (bEXOmiRs) which were used as high-throughput reporters of EV release. The initial phase of this protocol meticulously outlines the essential steps and factors to consider in the development and replication of bEXOmiRs. The procedure for examining bEXOmiR expression and abundance in both cells and isolated extracellular vesicles is detailed next.
By carrying nucleic acids, proteins, and lipid molecules, extracellular vesicles (EVs) facilitate communication between cells. Exosomes' biomolecular payload can alter the recipient cell's genetic, physiological, and pathological states. Exploiting the innate capability of EVs, the cargo of interest can be directed to a particular cell or organ. Their capability to pass through the blood-brain barrier (BBB) is a key characteristic of extracellular vesicles (EVs), making them ideal for transporting therapeutic drugs and macromolecules to inaccessible organs like the brain. Subsequently, the current chapter describes laboratory procedures and protocols centered on the modification of EVs for neuronal research applications.
Nearly all cells release exosomes, small extracellular vesicles measuring 40 to 150 nanometers in diameter, which are crucial in mediating intercellular and interorgan communication. The vesicles secreted by source cells are packed with diverse biologically active materials such as microRNAs (miRNAs) and proteins, enabling these components to modify the molecular properties of distant target cells. In consequence, microenvironmental niches within tissues experience regulated function through the agency of exosomes. How exosomes selectively adhere to and are directed toward specific organs remained largely a mystery. The recent years have shown integrins, a large family of cell-adhesion molecules, to be critical in the process of directing exosome transport to specific tissues, analogous to their role in controlling the cell's tissue-specific homing process. To this end, a crucial experimental step is to define the roles of integrins on exosomes in their specific tissue localization. This chapter outlines a protocol for investigating the integrin-mediated targeting of exosomes, considering both in vitro and in vivo experimental environments. selleck chemical We are particularly interested in examining the role of integrin 7 in the phenomenon of lymphocyte homing to the gut, which is well-established.
Investigating the intricate molecular mechanisms of extracellular vesicle uptake by target cells is a vital area of focus within the EV community. EVs are crucial for intercellular communication, impacting tissue balance or diverse disease pathways, like cancer or Alzheimer's disease progression. Given the nascent state of the electric vehicle (EV) sector, the standardization of methods for fundamental procedures like isolation and characterization remains a work in progress and a subject of ongoing discussion. The study of electric vehicle adoption also reveals the significant shortcomings inherent in the presently utilized strategies. Novel methods should aim to distinguish surface EV binding from uptake events, or enhance the sensitivity and accuracy of the assays. To analyze and assess EV uptake, we introduce two complementary methods, which we believe will address some existing methodological constraints. The two reporters are sorted into EVs with the help of a mEGFP-Tspn-Rluc construct. Employing bioluminescence signaling for quantifying EV uptake enhances sensitivity, distinguishes EV binding from cellular internalization, permits kinetic analysis within live cells, and remains amenable to high-throughput screening. A flow cytometry assay is utilized in the second approach to stain EVs with a maleimide-fluorophore conjugate. This chemical compound forms a covalent bond with proteins at sulfhydryl sites, offering a viable replacement for lipidic dyes. The technique is compatible with sorting cells that have incorporated the labeled EVs using flow cytometry.
Vesicles, minuscule in size, are secreted by every cellular type, and these exosomes are proposed to be a natural, promising means of intercellular communication. Exosome-mediated intercellular communication may arise from the transport of their endogenous cargo to nearby or distant cells. The recent development of cargo transfer has presented a novel therapeutic strategy, involving the investigation of exosomes as vectors for loaded cargo, particularly nanoparticles (NPs). The procedure for encapsulating NPs involves incubating cells with NPs, and subsequently determining cargo content and minimizing any harmful changes to the loaded exosomes.
Exosomes have a crucial impact on the regulation of tumor development, progression, and resistance to anti-angiogenesis treatments (AATs). Exosomes can be found emanating from both tumor cells and surrounding endothelial cells (ECs). Our methodology for exploring cargo transfer between tumor cells and endothelial cells (ECs) is described, utilizing a novel four-compartment co-culture system. Furthermore, we detail the investigation of the tumor cell impact on endothelial cell angiogenic ability using Transwell co-culture.
Selective isolation of biomacromolecules from human plasma is achievable through immunoaffinity chromatography (IAC) using antibodies immobilized on polymeric monolithic disk columns, followed by further fractionation of relevant subpopulations, such as small dense low-density lipoproteins, exomeres, and exosomes, using asymmetrical flow field-flow fractionation (AsFlFFF or AF4). An online coupled IAC-AsFlFFF system is utilized to describe the process of isolating and fractionating extracellular vesicle subpopulations without the presence of lipoproteins. The developed methodology allows for a rapid, reliable, and reproducible automated isolation and fractionation of challenging biomacromolecules from human plasma, thereby ensuring high purity and high yields of subpopulations.
Clinical-grade extracellular vesicles (EVs) necessitate reproducible and scalable purification protocols for the development of an EV-based therapeutic product. Frequently employed isolation procedures, such as ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer precipitation, suffered from limitations related to extraction yield, the purity of the vesicles, and the volume of sample available. Through a strategy incorporating tangential flow filtration (TFF), we developed a GMP-compliant methodology for the scalable production, concentration, and isolation of EVs. For the purpose of isolating extracellular vesicles (EVs) from the conditioned medium (CM) of cardiac stromal cells, specifically cardiac progenitor cells (CPCs), a known therapeutic asset in treating heart failure, we utilized this purification technique. Exosome vesicle (EV) isolation, achieved through tangential flow filtration (TFF) from conditioned medium, exhibited a consistent recovery of approximately 10^13 particles per milliliter, predominantly in the 120-140 nanometer size range. EV preparations demonstrated a remarkable 97% decrease in major protein-complex contaminants, maintaining consistent biological activity. Methods for determining EV identity and purity, as well as procedures for downstream applications like functional potency assays and quality control testing, are detailed in the protocol. The production of GMP-quality electric vehicles on a large scale offers a flexible protocol, applicable to various cell types across diverse therapeutic domains.
The release of extracellular vesicles (EVs) and their constituent molecules are sensitive to diverse clinical conditions. Intercellular communication is facilitated by EVs, which are hypothesized to reflect the pathophysiological state of the cells, tissues, organs, or the entire system they interact with. Urinary EVs have been shown to correlate with the pathophysiology of renal system diseases, presenting a supplementary, non-invasively obtainable source of potential biomarkers. selleck chemical Interest in the cargo of electric vehicles has been primarily focused on proteins and nucleic acids, though it has been further diversified to include metabolites more recently. Living organisms' internal processes are mirrored in the downstream alterations of the genome, transcriptome, and proteome, ultimately seen as changes in metabolites. Their research relies heavily on nuclear magnetic resonance (NMR) in conjunction with tandem mass spectrometry, employing liquid chromatography-mass spectrometry (LC-MS/MS). This study demonstrates the reproducibility and non-destructive nature of NMR, outlining the methodological protocols for urinary extracellular vesicle metabolomic analysis. Furthermore, the procedure for a targeted LC-MS/MS analysis is detailed, allowing for a seamless transition to untargeted methodologies.
Extracting extracellular vesicles (EVs) from conditioned cell culture media has been a demanding and often complex procedure. The task of obtaining numerous, completely pure and undamaged EVs proves exceptionally formidable. The diverse benefits and limitations associated with each of the commonly employed methods, including differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification, are evident. A multi-step purification protocol, employing tangential-flow filtration (TFF), is presented here, integrating filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) for high-purity EV isolation from substantial cell culture conditioned medium volumes. Integrating the TFF step ahead of PEG precipitation decreases protein presence, potentially preventing their clumping and co-purification with extracellular vesicles in the next purification stages.