The effect of the 13C abundance of soil microbial DNA on identifying labelled fractions after ultracentrifugation.

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP. KEY POINTS: • Appropriate 13C-DNA amount was needed for DNA-SIP. • Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions. • Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled.

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.

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.

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.

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.

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.

Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

Using molecular fine structure to identify optimal methods of extracting fungal glycogen.

Glycogen is a highly branched glucose polymer that is an energy storage material in fungi and animals. Extraction of glycogen from its source in a way that minimizes its molecular degradation is essential to investigate its native structure. In this study, the following extraction methods were compared: sucrose gradient density ultracentrifugation, thermal alkali, hot alcohol and hot water extractions. Molecular-size and chain-length distributions of glycogen were measured by size-exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis, respectively. These two fine-structure features are the most likely structural characteristics to be degraded during extraction. The results show that the thermal alkali, hot alcohol and hot water extractions degrade glycogen molecular size and/or chain-length distributions, and that sucrose gradient density ultracentrifugation with neither high temperature nor alkaline treatment is the most suitable method for fungal glycogen extraction.

Isolation of Extracellular Outer Membrane Vesicles (OMVs) from Escherichia coli Using EVscore47 Beads.

Outer membrane vesicles (OMVs) are attractive for biomedical applications based on their intrinsic properties in relation to bacteria and vesicles. However, their widespread use is hampered by low yields and purities. In this study, EVscore47 multifunctional chromatography microspheres were synthesized and used to efficiently isolate functional OMVs from Escherichia coli. Through this technology, OMV loss can be kept to a minimum, and OMVs can be harvested using EVscore47 at 11-fold higher yields and ~13-fold higher purity than those achieved by means of ultracentrifugation. Based on the results presented here, we propose a novel EVscore47-based isolation of OMVs that is fast and scalable.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Quantitation of Enterovirus A71 Empty and Full Particles by Sedimentation Velocity Analytical Ultracentrifugation.

The enterovirus A71 (EV71) inactivated vaccine is an effective intervention to control the spread of the virus and prevent EV71-associated hand, foot, and mouth disease (HFMD). It is widely administered to infants and children in China. The empty particles (EPs) and full particles (FPs) generated during production have different antigenic and immunogenic properties. However, the antigen detection methods currently used were established without considering the differences in antigenicity between EPs and FPs. There is also a lack of other effective analytical methods for detecting the different particle forms, which hinders the consistency between batches of products. In this study, we analyzed the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) in characterizing the EPs and FPs of EV71. Our results showed that the proportions of the two forms could be quantified simultaneously by SV-AUC. We also determined the repeatability and accuracy of this method and found that both parameters were satisfactory. We assessed SV-AUC for bulk vaccine quality control, and our findings indicated that SV-AUC can be used effectively to analyze the percentage of EPs and FPs and monitor the consistency of the process to ensure the quality of the vaccine.

A review on separation and application of plant-derived exosome-like nanoparticles.

Exosomes-like nanoparticles (ELNs) (exosomes or extracellular vesicles) are vesicle-like bodies secreted by cells. Plant ELNs (PENs) are membrane vesicles secreted by plant cells, with a lipid bilayer as the basic skeleton, enclosing various active substances such as proteins and nucleic acids, which have many physiological and pathological functions. Recent studies have found that the PENs are widespread within different plant species and their biological functions are increasingly recognized. The effective separation method is also necessary for its function and application. Ultracentrifugation, sucrose density gradient ultracentrifugation, ultrafiltration, polymer-based precipitation methods, etc., are commonly used methods for plant exosome-like nanoparticle extraction. In recent years, emerging methods such as size exclusion chromatography, immunoaffinity capture-based technique, and microfluidic technology have shown advancements compared to traditional methods. The standardized separation process for PENs continues to evolve. In this review, we summarized the recent progress in the biogenesis, components, separation methods, and some functions of PENs. When the research on the separation method of PENs and their unique biological structure is further studied. A brand-new idea for the efficient separation and utilization of PENs can be provided in the future, which has a very broad prospect.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scattering, and Fluorescence Spectroscopy Methods.

Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.

Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis.

Background: ExoRNAs offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Methods: Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by DLS and TEM, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Results: Extracted exosomes displayed a similar range of size and morphology. We found that PEG-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. Conclusion: We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

BASIS: BioAnalysis SEDFIT integrated software for cGMP analysis of SV-AUC data.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has long been an important method for characterization of antibody therapeutics. Recently, SV-AUC has experienced a wave of new interest and usage from the gene and cell therapy industry, where SV-AUC has proven itself to be the "gold standard" analytical approach for determining capsid loading ratios for adeno-associated virus (AAV) and other viral vectors. While other more common approaches have existed in the realm of cGMP-compliant techniques for years, SV-AUC has long been used strictly for characterization, but not for release testing. This manuscript describes the challenges faced in bringing SV-AUC to a cGMP environment and describes a new program, "BASIS", which allows for 21 CFR Part 11-compliant data handling and data analysis using the well-known and frequently cited SEDFIT analysis software.

Rapid high-resolution size distribution protocol for adeno-associated virus using high speed SV-AUC.

Simulated SV-AUC data for an adeno-associated virus (AAV) sample consisting of four components having closely spaced sedimentation coefficients were used to develop a high-speed protocol that optimized the size distribution analysis resolution. The resulting high speed (45K rpm) SV-AUC (hs-SV-AUC) protocol poses several experimental challenges: 1) the need for rapid data acquisition, 2) increased potential for optical artifacts from steep and fast moving boundaries and 3) the increased potential for convection. To overcome these challenges the protocol uses interference detection at low temperatures and data that are confined to a limited radial-time window. In addition to providing higher resolution AAV SV-AUC data and very short run times (<20 min after temperature equilibration), the need to match the sample and reference solvent composition and meniscus positions is relaxed making interference detection as simple to employ as absorbance detection. Finally, experimental data comparing hs-SV-AUC (at 45K rpm) with standard low-speed (15K rpm) SV-AUC on the same AAV sample demonstrate the size distribution resolution improvement. These experiments also validate the use of a radial-time window and show how quickly data can be acquired using the hs-SV-AUC protocol.

Separation of high-purity plasma extracellular vesicles for investigating proteomic signatures in diabetic retinopathy.

Extracellular vesicles (EVs) play a multifaceted role in intercellular communication and hold significant promise as bio-functional indicators for clinical diagnosis. Although plasma samples represent one of the most critical sources of circulating EVs, the existing technical challenges associated with plasma-EV isolation have restricted their application in disease diagnosis and biomarker discovery. In this study, we introduce a two-step purification method utilizing ultracentrifugation (UC) to isolate crude extracellular vesicle (EV) samples, followed by a phospholipid affinity-based technique for the selective isolation of small EVs, ensuring a high level of purity for downstream proteomic analysis. Our research demonstrates that the UC & TiO2-coated magnetic bead (TiMB) purification system significantly improves the purity of EVs when compared to conventional UC or TiMB along. We further revealed that proteomic alterations in plasma EVs effectively reflect key gene ontology components associated with diabetic retinopathy (DR) pathogenesis, including the VEGF-activated neuropilin pathway, positive regulation of angiogenesis, angiogenesis, cellular response to vascular endothelial growth factor stimulus, and immune response. By employing a comprehensive analytical approach, which incorporates both time-series analysis (cluster analysis) and differential analysis, we have identified three potential protein signatures including LGALS3, MYH10, and CPB2 that closely associated with the retinopathy process. These proteins exhibit promising diagnostic and severity-classification capabilities for DR disease. This adaptable EV isolation system can be regarded as an effective analytical tool for enhancing plasma-based liquid biopsies toward clinical applications.

Methods for the Design and Analysis of Analytical Ultracentrifugation Experiments.

Analytical ultracentrifugation experiments play an integral role in the solution-phase characterization of biological macromolecules and their interactions. This unit discusses the design of sedimentation velocity and sedimentation equilibrium experiments performed with a Beckman Proteomelab XL-A or XL-I analytical ultracentrifuge and with a Beckman Optima AUC. Instrument settings and experimental design considerations are explained, and strategies for the analysis of experimental data with the UltraScan data analysis software package are presented. Special attention is paid to the strengths and weaknesses of the available detectors, and guidance is provided on how to extract maximum information from analytical ultracentrifugation experiments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.