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: 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.

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.

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.

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 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.

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.

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.

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.

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.

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.

Isolation of Extracellular Vesicles from Cell Culture and Blood Through Nano-Targeted DLD Microfluidic Device.

Extracellular vesicles (EVs) are secreted by cells and found in biological fluids such as blood, with concentration correlated with oncogenic signals, making them attractive biomarkers for liquid biopsy. The current gold-standard method for EVs isolation requires an ultracentrifugation (UC) step among others. The cost and complexity of this technique are forbiddingly high for many researchers, as well as for routine use in biological laboratories and hospitals. This chapter reports on a simple microfluidic method for EVs isolation, based on a microfluidic size sorting technique named Deterministic Lateral Displacement (DLD). With the design of micrometric DLD array, we demonstrated the potential of our DLD devices for the isolation of nano-biological objects such as EVs, with main population size distribution consistent with UC technique.

A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles.

Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications.

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.

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Recent research on material-based methods for isolation of extracellular vesicles.

Extracellular vesicles (EVs) are nanoparticles secreted by cells with a closed phospholipid bilayer structure, which can participate in various physiological and pathological processes and have significant clinical value in disease diagnosis, targeted therapy and prognosis assessment. EV isolation methods currently include differential ultracentrifugation, ultrafiltration, size exclusion chromatography, immunoaffinity, polymer co-precipitation and microfluidics. In addition, material-based biochemical or biophysical approaches relying on intrinsic properties of the material or its surface-modified functionalized monomers, demonstrated unique advantages in the efficient isolation of EVs. In order to provide new ideas for the subsequent development of material-based EV isolation methods, this review will focus on the principle, research status and application prospects of material-based EV isolation methods based on different material carriers and functional monomers.

Hybrid modeling of an ultracentrifugation process for separation of full and empty adeno-associated virus particles.

Ultracentrifugation is an attractive method for separating full and empty capsids, exploiting their density difference. Changes of the serotype/capsid, density of loading material, or the genetic information contained in the adeno-associated viruses (AAVs) require the adaptation of the harvesting parameters and the density gradient loaded onto the centrifuge. To streamline these adaptations, a mathematical model could support the design and testing of operating conditions.Here, hybrid models, which combine empirical functions with artificial neural networks, are proposed to describe the separation of full and empty capsids as a function of material and operational parameters, i.e., the harvest model. In addition, critical quality attributes are estimated by a quality model which is operating on top of the harvest model. The performance of these models was evaluated using test data and two additional blind runs. Also, a "what-if" analysis was conducted to investigate whether the models' predictions align with expectations.It is concluded that the models are sufficiently accurate to support the design of operating conditions, though the accuracy and applicability of the models can further be increased by training them on more specific data with higher variability.

Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Protocol for separation of fungal extracellular vesicles using ultracentrifugation from solid medium cultures.

Extracellular vesicles (EVs) have been identified in diverse fungi, including human pathogens. In this protocol, we present two techniques for isolating and analyzing fungal EVs. The first is for high-throughput screening, and the second is for yielding concentrated samples suitable for centrifugation-based density gradients. We describe steps for analytical assays such as nano-flow cytometry and nanoparticle tracking analysis to measure EV dimensions and concentration. EV suspensions can serve diverse assays, including electron microscopy, compositional determination, and cell-to-cell communication assays. For complete details on the use and execution of this protocol, please refer to Rizzo et al.,1 Rizzo et al.,2 Reis et al.,3 and Reis et al.4.