Chromatographic Purification and Polishing of AAV Particles.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.

Isolation and Characterization of Exosomes from Ocular Samples.

Exosomes are microsize vesicles secreted by nearly all cells to the extracellular space. The vesicles transport cell signaling and communicate with other cells. Ultracentrifugation is the standard method to isolate exosomes from culture media or body fluid. Without ultracentrifuge, exosomes can be precipitated by polyethylene glycol or separated by size exclusion chromatography. After isolation, nanoparticle tracking analysis can help to estimate the size and concentration of exosome samples. Transmission electron microscopy can directly show the size and morphology of exosomes. Moreover, the sample should be characterized by the expression of several exosome biomarker proteins. Exosomal contents such as proteins and miRNAs could be profiled using appropriate technologies.

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Optimizing Conditions of Polyethylene Glycol Precipitation for Exosomes Isolation From MSCs Culture Media for Regenerative Treatment.

Mesenchymal stem cell (MSC)-derived exosomes, as a cell-free alternative to MSCs, offer enhanced safety and significant potential in regenerative medicine. However, isolating these exosomes poses a challenge, complicating their broader application. Commonly used methods like ultracentrifugation (UC) and tangential flow filtration are often impractical due to the requirement for costly instruments and ultrafiltration membranes. Additionally, the high cost of commercial kits limits their use in processing large sample volumes. Polyethylene glycol (PEG) precipitation offers a more convenient and cost-effective alternative, but there is a critical need for optimized and standardized isolation protocols using PEG precipitation across different cell types and fluids to ensure consistent quality and yield. In this work, we optimized the PEG precipitation method for exosomes isolation and compared its effectiveness to two commonly used methods: UC and commercial exosome isolation kits (ExoQuick). The recovery rate of the optimized PEG method (about 61.74%) was comparable to that of the commercial ExoQuick kit (about 62.19%), which was significantly higher than UC (about 45.80%). Exosome cargo analysis validated no significant differences in miRNA and protein profiles associated with the proliferation and migration of exosomes isolated by UC and PEG precipitation, which was confirmed by gap closure and CCK8 assays. These findings suggest that PEG-based exosomes isolation could be a highly efficient and high-quality method and may facilitate the development of exosome-based therapies for regenerative medicine.

Protocol for isolating leukemia-derived extracellular vesicles from the spleen of preclinical models of leukemia using ultracentrifugation.

Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers. For complete details on the use and execution of this protocol, please refer to Gargiulo et al.1.

Improving the Purity of Extracellular Vesicles by Removal of Lipoproteins from Size Exclusion Chromatography- and Ultracentrifugation-Processed Samples Using Glycosaminoglycan-Functionalized Magnetic Beads.

Extracellular vesicles (EVs) are present in blood at much lower concentrations (5-6 orders of magnitude) compared to lipoprotein particles (LP). Because LP and EV overlap in size and density, isolating high-purity EVs is a significant challenge. While the current two-step sequential EV isolation process using size-expression chromatography (SEC) followed by a density gradient (DG) achieves high purity, the time-consuming ultracentrifugation (UC) step in DG hinders workflow efficiency. This paper introduces an optimized magnetic bead reagent, LipoMin, functionalized with glycosaminoglycans (GAGs), as a rapid alternative for LP removal during the second-step process in about 10 minutes. We evaluated LipoMin's efficacy on two sample types: (a) EV fractions isolated by size exclusion chromatography (SEC + LipoMin) and (b) the pellet obtained from ultracentrifugation (UC + LipoMin). The workflow is remarkably simple, involving a 10 min incubation with LipoMin followed by magnetic separation of the LP-depleted EV-containing supernatant. Results from enzyme-linked immunosorbent assay (ELISA) revealed that LipoMin removes 98.2% ApoB from SEC EV fractions, comparable to the LP removal ability of DG in the SEC + DG two-step process. Importantly, the EV yield (CD81 ELISA) remained at 93.0% and Western blot analysis confirmed that key EV markers, flotillin and CD81, were not compromised. Recombinant EV (rEV), an EV reference standard, was spiked into SEC EV fractions and recovered 89% of CD81 protein. For UC + LipoMin, ApoA1 decreased by 76.5% while retaining 90.7% of CD81. Notably, both colorectal cancer (CRC) and Alzheimer's disease (AD) samples processed by SEC + LipoMin and UC + LipoMin displayed clear expression of relevant EV and clinical markers. With a 10 min workflow (resulting in a 96% time saving compared to the traditional method), the LipoMin reagent offers a rapid and efficient alternative to DG for LP depletion, paving the way for a streamlined SEC + LipoMin two-step EV isolation process.

Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Exosomes Isolation, Purification, and Characterization.

Exosomes are double-layered lipid membranous nanovesicles that are endosomal in origin and secreted by almost all cells. They are 30-130 nm in size and contain various molecular signatures such as miRNAs, mRNAs, DNA, lipids, and proteins. Due to their highly heterogeneous content, exosomes have a major role in influencing cellular physiology and pathology. Although exosome research has been in progress for a long time, its biomedical applications have recently been expanding due to its bio-friendly nature. However, the most challenging part is its isolation to obtain quality exosomes with good yield. Therefore, in this chapter, we have described appropriate protocols for exosome isolation and characterization along with alternative purification methods.

Exosomes: Methods for Isolation and Characterization in Biological Samples.

Exosomes are small lipid bilayer-encapsulated nanosized extracellular vesicles of endosomal origin. Exosomes are secreted by almost all cell types and are a crucial player in intercellular communication. Exosomes transmit cellular information from donor to recipient cells in the form of proteins, lipids, and nucleic acids and influence several physiological and pathological responses. Due to their capacity to carry a variety of cellular cargo, low immunogenicity and cytotoxicity, biocompatibility, and ability to cross the blood-brain barrier, these nanosized vesicles are considered excellent diagnostic tools and drug-delivery vehicles. Despite their tremendous potential, the progress in therapeutic applications of exosomes is hindered by inadequate isolation techniques, poor characterization, and scarcity of specific biomarkers. The current research in the field is focused on overcoming these limitations. In this chapter, we have reviewed conventional exosome isolation and characterization methods and recent advancements, their advantages and limitations, persistent challenges in exosome research, and future directions.

The Method of Lipemia Clearance Should be Based on the Characteristics and the Method of Testing in the Emergency Laboratory.

BACKGROUND: This study aimed to compare the lipemia removal efficiency of highspeed centrifugation, lipid scavengers, and dilution for biochemical analytes. METHODS: We collected 30 cases of lipemic plasma in an emergency laboratory and divided them into 4 aliquots. Lipemia was removed by highspeed centrifugation, lipid scavenger, dilution, and ultracentrifugation, then analytes were measured by an AU5800 analyzer. Taking ultracentrifugation as reference, the efficiencies of the other three methods were evaluated based on the deviation. RESULTS: When highspeed centrifugation was used for lipemia removal, DBIL (18.62%), and Magnesium (6.09%) could not satisfy the criterion. When lipid scavengers were applied to remove lipemia, CRP (-86.70%), TP (-8.29%), CKMB (-44.85%), DBIL (37.96%), Glu (4.20%) and phosphate (14.32%) were not suggested as lipid scavengers. For dilution, nearly half of the analytes could satisfy the criterion, including AMY (2.41%), CRP (5.54%), ALT (2.85%), GGTL (-1.73%), ALP (-0.04%), Glu (-0.84%), LDH (0.06%), CK (0.68%), BUN (3.80%), CREA (-1.54%), UA (5.42%), and magnesium (0.43%). CONCLUSIONS: Neither of the methods for lipid removal could satisfy all emergency department tests for lipid removal. This finding suggests that removing lipemia in the clinical laboratory should be based on the characteristics and the method of testing.

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Flotation Coefficient Distributions of Lipid Nanoparticles by Sedimentation Velocity Analytical Ultracentrifugation.

The robust characterization of lipid nanoparticles (LNPs) encapsulating therapeutics or vaccines is an important and multifaceted translational problem. Sedimentation velocity analytical ultracentrifugation (SV-AUC) has proven to be a powerful approach in the characterization of size-distribution, interactions, and composition of various types of nanoparticles across a large size range, including metal nanoparticles (NPs), polymeric NPs, and also nucleic acid loaded viral capsids. Similar potential of SV-AUC can be expected for the characterization of LNPs, but is hindered by the flotation of LNPs being incompatible with common sedimentation analysis models. To address this gap, we developed a high-resolution, diffusion-deconvoluted sedimentation/flotation distribution analysis approach analogous to the most widely used sedimentation analysis model c(s). The approach takes advantage of independent measurements of the average particle size or diffusion coefficient, which can be conveniently determined, for example, by dynamic light scattering (DLS). We demonstrate the application to an experimental model of extruded liposomes as well as a commercial LNP product and discuss experimental potential and limitations of SV-AUC. The method is implemented analogously to the sedimentation models in the free, widely used SEDFIT software.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

Quality and efficiency assessment of five extracellular vesicle isolation methods using the resistive pulse sensing strategy.

Extracellular vesicles (EVs) have attracted great interest due to their great potential in disease diagnosis and therapy. The separation of EVs from complex biofluids with high purity is essential for the accurate analysis of EVs. Despite various methods, there is still no consensus on the best method for high-quality EV isolation and reliable mass production. Therefore, it is important to offer a standardized method for characterizing the properties (size distribution, particle concentration and purity) of EV preparations from different isolation methods. Herein, we employed a NanoCoulter Counter based on the resistive pulse sensing (RPS) strategy that enabled multi-parameter analysis of single EVs to compare the quality and efficiency of different EV isolation techniques including traditional differential ultracentrifugation, ultrafiltration, size exclusion chromatography, membrane affinity binding and polymer precipitation. The data revealed that the NanoCoulter Counter based on the RPS strategy was reliable and effective for the characterization of EVs. The results suggested that although higher particle concentrations were observed in three commercial isolation kits and ultrafiltration, traditional differential ultracentrifugation showed the highest purity. In conclusion, our results from the NanoCoulter Counter provided reliable evidence for the assessment of different EV isolation methods, which contributed to the development of EV-based disease biomarkers and treatments.

Flow-electricity coupling fields enhance microfluidic platforms for efficient exosome isolation.

Recently, exosomes have emerged as important biomarkers for cancer diagnosis, playing a significant role in disease diagnosis. Consequently, efficient isolation of exosomes from complex body fluids is now a critical focus in clinical research. We have designed and fabricated an exosome separation chip, leveraging the synergies of flow and electric fields through 3D printing technology. This approach harnesses the combined strengths of both fields, substantially enhancing separation efficiency and purity. This also effectively reduced the voltage required to form an electric field (from 120 V down to 10 V), minimizing the risk of Joule heating, thereby preserving the structural integrity and biological activity of the exosomes. Compared with the standard exosome separation method of ultracentrifugation (UC), our chip offers numerous benefits: it is cost-effective (under 50 RMB), boasts a high recovery rate (64.8%) and high purity (almost 100%), achieves remarkable separation efficiency (within 30 minutes), and is straightforward to operate. Moreover, since an unmarked separation method is used, the separated exosomes can be directly used for downstream detection and analysis, which has certain practicality for future clinical research and application.

Quality control and validation of extracellular vesicles isolated from cultured human breast cancer cells.

OBJECTIVE: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFß1, ß-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

Using absorbance detection for hs-SV-AUC characterization of adeno-associated virus.

Data are presented demonstrating that absorbance detection can be used during high-speed sedimentation velocity analytical ultracentrifugation (hs-SV-AUC) experiments to characterize the size distribution of adeno-associated virus (AAV) drug products accurately. Advantages and limitations of being able to use this detector in this specific type of SV-AUC experiment are discussed.

Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics.

The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultaneously and without any sample preparation step, the sedimentation coefficients of both the LNP-mRNA formulation and the mRNA molecules. This allows measuring several quality attributes, such as particle size distribution, encapsulation efficiency and density of the formulation. The technique can also be applied to study the stability of the formulation under stress conditions and different buffers.

Comparison of morphology, protein concentration, and size distribution of bone marrow and Wharton's jelly-derived mesenchymal stem cells exosomes isolated by ultracentrifugation and polymer-based precipitation techniques.

Exosomes which are tiny extracellular vesicles (30-150 nm), transport vital proteins and gene materials such as miRNA, mRNA, or DNA, whose role in cell communication and epithelia regulation is critical. Many techniques have been developed as a result of studying exosomes' biochemical and physicochemical properties, although there is still no standard method to isolate exosomes simply with high yield. Commercial kits have gained popularity for exosome extraction despite concerns about their effectiveness in scientific research. On the other hand, ultracentrifugation remains the gold standard isolation method. This study compares these two common exosome isolation methods to determine their impact on the quality and quantity of exosomes isolated from bone marrow (BM) and Wharton's jelly (WJ)-derived mesenchymal stem cells. Isolated exosomes from the two sources of the cell's conditioned medium by two methods (polymer kit and ultracentrifuge) were characterized using western blotting, scanning electron microscopy (SEM), dynamic light scattering (DLS), and the Bradford assay. Western blot analysis confirmed separation efficiency based on CD81 and CD63 markers, with the absence of calnexin serving as a negative control. The Morphology of exosomes studied by SEM image analysis revealed a similar round shape appearance and their sizes (30-150 nm) were the same in both isolation techniques. The DLS analysis of the sample results was consistent with the SEM ones, showing a similar size range and very low disparity. The exosome protein content concentration analysis revealed that exosomes isolated by the polymer-based kits contained higher protein concentration density and purity (p <0.001). In general, though the protein yield was higher when the polymer-based kits were used, there were no significant differences in morphology, or size between WJ-derived and BM-derived exosomes, regardless of the isolation method employed.