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Extracellular vesicles (EVs) include a variety of nanosized vesicles released to the extracellular microenvironment by the vast majority of cells transferring bioactive lipids, proteins, mRNA, miRNA or non-coding RNA, as means of intercellular communication. Remarkably, among other fields of research, their use has become promising for immunomodulation, tissue repair and as source for novel disease-specific molecular signatures or biomarkers. However, a major challenge is to define accurate, reliable and easily implemented techniques for EV isolation due to their nanoscale size and high heterogeneity. In this context, differential ultracentrifugation (dUC) has been the most widely used laboratory methodology, but alternative procedures have emerged to allow purer EV preparations with easy implementation. Here, we present and discuss the most used of the different EV isolation methods, focusing on the increasing impact of size exclusion chromatography (SEC) on the resulting EV preparations from in vitro cultured cells-conditioned medium and biological fluids. Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, which are not discarded using dUC or precipitating agents, while being more user friendly and less time-consuming than gradient-based EV isolation. Also, SEC highly maintains the major EVs’ characteristics, including vesicular structure and content, which guarantee forthcoming applications. In sum, together with scaling-up possibilities to increase EV recovery and manufacturing following high-quality standards, SEC could be easily adapted to most laboratories to assist EV-associated biomarker discovery and to deliver innovative cell-free immunomodulatory and pro-regenerative therapies.

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria during growth, mainly under stress conditions. OMV-based vaccines have played an important role in vaccination against Neisseria meningitidis serogroup B (MenB), stimulating research into novel approaches for developing more effective vaccines. OMVs released by the bacterium Neisseria lactamica have emerged as a promising platform for new vaccine development, especially as carriers in subunit vaccines. Despite their importance, some challenges remain in obtaining and purifying OMVs. The most commonly employed methods for OMV isolation and purification are ultracentrifugation (UC) and size exclusion chromatography (SEC). However, these techniques could present limitations for large-scale production and often result in low yields. This study investigated techniques such as tangential flow filtration (TFF), membrane chromatography, and mixed-mode (multimodal) chromatography as potential replacements for UC and SEC. Among the TFF methods evaluated, the sample obtained on the membrane with a 300-kDa cutoff showed a profile more similar to UC but with more than double the total protein recovery. Sartobind® Q membrane chromatography was ineffective for OMV purification, in the conditions evaluated, with a recovery of 8.7%. Conversely, multimodal Capto™ Adhere chromatography recovered 59.0%, while Capto™ Core 400 yielded a recovery of 72.0%, proving to be more effective for purification when analyzed by high-performance liquid chromatography (HPLC). Thus, combining TFF with a 300-kDa membrane followed by Capto™ Core 400 chromatography can be applied as strategy for large-scale applications offering high recovery and purity. Key points • Evaluation of TFF, membrane and multimodal chromatography techniques for OMV purification. • Improved Neisseria lactamica OMV yields combining TFF and multimodal chromatography. • A process for OMV purification from a non-pathogenic organism feasible to scale up.

Bio-based and biodegradable materials play a vital role in a sustainable and green economy. These materials must exhibit properties that are similar to or better than the properties of oil- or coal-based materials and require sophisticated synthesis technologies and detailed knowledge of structure–property correlations. For comprehensive molecular structure elucidation, advanced analytical methods, including coupled and hyphenated techniques that combine advanced fractionation and information-rich spectroscopic detectors, are an indispensable tool. One important tool for fractionating complex polymers regarding molecular size is size exclusion chromatography. For fractionating polymers with regard to chemical composition, solvent (or temperature) gradient HPLC has been developed. The combination of different liquid chromatography methods in comprehensive two-dimensional HPLC setups is another important tool. Today, a toolbox of HPLC methods is in place that enables the fractionation of complex bio-based and biodegradable polymers according to the most important molecular parameters including molecular size, composition, functionality, and branching. Here, an overview of the different techniques and some major applications is presented. Some representative developments in the field are discussed, and different techniques, experimental protocols, and applications are highlighted.

There is a great need for techniques which enable reproducible separation of extracellular vesicles (EVs) from biofluids with high recovery, purity and throughput. The development of new techniques for isolation of EVs from minute sample volumes is instrumental in enabling EV-based biomarker profiling in large biobank cohorts and paves the way to improved diagnostic profiles in precision medicine. Recent advances in microfluidics-based devices offer a toolbox for separating EVs from small sample volumes. Microfluidic devices that have been used in EV isolation utilise different fundamental principles and rely largely on benefits of scaling laws as the biofluid processing is miniaturised to chip level. Here, we review the progress in the practicality and performance of both passive devices (such as mechanical filtering and hydrodynamic focusing) and active devices (using magnetic, electric or acoustic fields). As it stands, many microfluidic devices isolate intact EV populations at higher purities than centrifugation, precipitation or size-exclusion chromatography. However, this comes at a cost. We address challenges (in particular low throughput, clogging risks and ability to process biofluids) and highlight the need for more improvements in microfluidic devices. Finally, we conclude that there is a need to refine and standardise these lab-on-a-chip techniques to meet the growing interest in the diagnostic and therapeutic value of purified EVs. Graphical abstract

Monostructured single-wall carbon nanotubes (SWCNTs) are important in both scientific research and electronic and biomedical applications; however, the bulk separation of SWCNTs into populations of single-chirality nanotubes remains challenging. Here we report a simple and effective method for the large-scale chirality separation of SWCNTs using a single-surfactant multicolumn gel chromatography method utilizing one surfactant and a series of vertically connected gel columns. This method is based on the structure-dependent interaction strength of SWCNTs with an allyl dextran-based gel. Overloading an SWCNT dispersion on the top column results in the adsorption sites of the column becoming fully occupied by the nanotubes that exhibit the strongest interaction with the gel. The unbound nanotubes flow through to the next column, and the nanotubes with the second strongest interaction with the gel are adsorbed in this stage. In this manner, 13 different ( n , m ) species were separated. Metallic SWCNTs were finally collected as unbound nanotubes because they exhibited the lowest interaction with the gel. Large-scale separation of single-wall carbon nanotubes into populations of single chirality is a significant challenge in the practical application of nanotubes. Now, using multicolumn gel chromatography, the large-scale separation of 13 different carbon nanotube species is achieved.

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC–DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.

Butyrylcholinesterase (BChE; EC 3.1.1.8) is an enzyme found in blood plasma and various tissues, playing a key role in metabolizing esters and detoxifying various substances. In this study, we developed a modified purification protocol for BChE from human serum, achieving a higher purification yield (38.3%) and specific activity (15.09 U/mg protein) compared to previous reports. The method employed a single round of acid dialysis, Sephadex G50 gel filtration chromatography, and procainamide Sepharose 4 fast flow affinity chromatography. Our new approach excludes the commonly used DEAE Trisacryl M chromatography. The goal was to compare this method with our previously employed purification protocols. This study demonstrates that optimizing chromatography steps can enhance enzyme recovery and activity, though further refinement may be needed for higher purification folds. This improved methodology offers a valuable approach for efficient BChE purification with potential for broader applications.

The analysis of serum for biomarkers is a standard method in clinical diagnosis and health assessment. The application of Raman spectroscopy to probe biomarkers in serum is increasingly investigated due to its time- and cost-efficiency. However, time-consuming sample preparation is often required to analyze the serum samples. Additionally, hemolyzed samples are commonly discarded due to interference in the measurements. This study focuses on the application of the online coupling of size exclusion chromatography (SEC) to diode array detector (DAD) and capillary-enhanced Raman spectroscopy (CERS) for direct analysis of hemolyzed serum samples. We demonstrate that different protein classes such as serum albumin and immunoglobulin G (IgG) can be identified in hemolyzed serum according to a calculated hit quality index (HQI). Additionally, different oxidation and binding states of the heme prosthetic group are investigated at 532-nm excitation. The online coupling of SEC-DAD-CERS enables the detailed characterization of blood serum proteins, including the differentiation of IgG, serum albumin, and hemoglobin. Graphical Abstract