A Review of Microfluidic Chip-Based Strategies for Extracellular Vesicle Isolation: Bridging Traditional Methods and Clinical Translation

Development of a microfluidic droplet platform with an antibody-free magnetic-bead-based strategy for high through-put and efficient EVs isolation

Extracellular vesicles (EVs) are lipid bilayer particles released by all cell types, carrying cargos that reflect the cellular states of their origin. Recently, EVs are increasingly recognized as valuable biomarkers and therapeutic vectors in oncology, but their clinical translation is limited by variability in isolation methods and uncertainty regarding long-term storage physical stability. We systematically compared human plasma EVs isolated by ultracentrifugation (UC) or magnetic bead (MB)-based methods under immediate analysis, stable freezing storage, and repeated freeze-thaw conditions. The morphology and protein profiling of EVs were characterized by transmission electron microscopy (TEM) and Western blotting (WB), respectively. EV concentration, particle size, and zeta potential were quantified by particle size analyzer. TEM and WB analyses of human plasma EVs confirmed the efficacy of both the UC and MB isolation methods. UC-isolated EVs are of high yield but low physical stability, featuring size reduction and a shift toward more negative zeta potential values after freeze-thaw cycles. Fresh UC-EVs displayed heterogeneous size profiles, whereas freeze-thawed samples shifted to a dominant peak, consisting of small particles with increased counts. Although lower in yield, MB-isolated EVs retained their physical stability across all conditions. MB-based EV isolation offers physical stability for standardized diagnostic workflows, whereas UC-based EV isolation provides high yield for discovery studies but vulnerable to freeze-thaw stress. These findings provide an evidence-based framework for selecting EV isolation and storage methods to match downstream applications, guiding the standardization of EVs workflows for future precision oncology and personalized medicine.

Neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and multiple sclerosis (MS), are characterized by progressive neuronal dysfunction and limited therapeutic options, largely due to the restrictive nature of the blood-brain barrier (BBB). Exosomes, naturally occurring extracellular vesicles (EVs), have gained attention as innovative drug delivery vehicles owing to their intrinsic ability to cross the BBB, minimal immunogenicity, high biocompatibility, and capability to carry diverse therapeutic cargos such as proteins, nucleic acids, and small molecules. Furthermore, exosomes can be bioengineered to enhance drug-loading efficiency and targeting specificity, positioning them as a versatile and effective platform for treating NDDs. In this review, we summarize recent advances in exosome biogenesis, secretion, and engineering, with an emphasis on innovative strategies for exosome isolation, drug loading, and surface modification. We further explore their roles in modulating neuroinflammation, promoting neural regeneration, and enabling precise therapeutic delivery. Critical challenges associated with large-scale production, quality control, and regulatory compliance under Good Manufacturing Practices (GMP) are also discussed. Collectively, these developments underscore the transformative potential of engineered exosomes in advancing precision therapies for neurodegenerative disorders and offer strategic insights into their clinical translation.

Systematic characterization of mammalian extracellular vesicles using nano-flow cytometry

Extracellular vesicles (EVs) carry a variety of bioactive molecules and are becoming a promising alternative to cell therapy. Scale-up EV isolation is necessary for their functional studies and biological applications, while the traditional methods are challenged by low throughput, low yield, and potential damage. Herein, we developed an ion osmolarity-driven sequential concentration-enrichment strategy (IOSCE) for the EV isolation. IOSCE is composed of a novel superabsorbent polymers (SAPs) for EV concentration and a charged polymer for EV enrichment. Based on the driving force of ionic osmotic pressure, IOSCE can isolate EVs on a large scale from cell culture medium. The saturated water absorption capacity of IOSCE is 13.62 times higher than that of commercial SAPs. Compared with the ultracentrifugation method, IOSCE exhibited a 2.64 times higher yield (6.33 × 108 particles/mL). Moreover, the mesenchymal stem cell-derived EVs isolated using IOSCE demonstrate strong biological activity and can reduce neuroinflammation by affecting RNA metabolism and translation processes. IOSCE provides a cost-effective, high-throughput, and low-damage method for the scale up EV isolation, which is promising for disease diagnosis and treatment.Graphical

Blood-derived extracellular vesicles (EVs) are a popular source of biomarkers for central nervous system (CNS) diseases, but inconsistencies in isolation and analysis hinder their clinical translation. This review summarizes recent studies that investigate the impact of different anticoagulated plasma and serum on the yield, purity, and molecular content of EVs. Specifically, the studies compare ethylenediaminetetraacetic acid (EDTA), citrate, heparin plasma, and serum and highlight the risk of contamination from platelet-derived EVs. Here, I offer practical guidelines for standardizing EV isolation and analysis, recommending the use of plasma anticoagulated with acid-citrate-dextrose (ACD) or citrate followed by EDTA and heparin, subgroup analyses for samples from different biobank repositories, and avoiding serum and plasma-to-serum transformation. Other factors like illness, age, gender, meal timing, exercise, circadian timing, and arm pressure during blood draw can alter EV signatures. Yet, how these variables interact with different anticoagulated plasma or serum samples is unclear, necessitating further research. Furthermore, whether the changes are dependent on the isolation or quantification methodology remains an area of investigation. Importantly, the perspective emphasizes the need for consistency in experimental methodologies to improve the reproducibility and clinical applicability of CNS-originating EV biomarker studies. The proposed guidelines, along with ongoing efforts to standardize blood sample handling and collection, may facilitate the development of more reliable and informative CNS-originating EV biomarkers for diagnosis, prognosis, and treatment monitoring of CNS diseases.

Extracellular vesicles (EVs) hold promise for liquid biopsy and drug delivery applications. However, their heterogeneous nature poses challenges for efficient and selective isolation from complex biofluids. Here, an isolation method based on phase-separated zwitterionic (ZW) coacervates is developed. These coacervates form over a wide range of pH values and ionic strengths, ensuring compatibility with all biofluids. They exhibit antifouling properties that minimize nonspecific binding, allowing for the selective isolation of EVs from biofluids upon functionalization of the polymer with a suitable affinity probe, as proved here with a membrane-sensing peptide. This strategy is applied to pull down, concentrate, and release EVs from urine samples with high yields while retaining their structural integrity. This approach effectively separates EVs from lipoproteins, a challenging task for conventional separation techniques. The power of the approach is demonstrated as a preparative step for downstream analysis and as a one-pot assay to profile EV biomarkers in complex fluids. The latter application, implemented here with flow cytometry, significantly streamlines pre-analytical workflows. Thus, functionalized ZW coacervates represent an effective strategy for the selective isolation and direct analysis of EVs from complex mixtures, paving the way for advances in large-scale manufacturing and diagnostics.

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

Efficient purification of extracellular vesicles via circular multicavity electrophoresis coupled with ultrafiltration.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Small extracellular vesicles (EVs) are nanosized vesicles (< 200nm) secreted from various tissues, including the central nervous system (CNS), into diverse biofluids. Due to their ability to carry molecular cargo that reflects the physiological state of their parental cells, small EVs represent promising diagnostic carriers for neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). While biofluid-derived small EVs offer a "liquid biopsy" solution, their clinical translation is severely hindered by the limitations of conventional isolation methods, which are often time-consuming, costly, or yield low purity. Building upon the "ExoPRISM" (Exosome Precipitation by Ionic Strength Modulation) framework, we developed and validated an optimized, cost-effective ammonium sulfate (AS)-based pipeline for small EVs isolation. We have refined and established the optimal AS ratio specifically for plasma and further demonstrated its feasibility for isolating small EVs from saliva. Subsequently, we introduced a targeted purification step using glutamate aspartate transporter (GLAST) antibodies to specifically isolate central nervous system (CNS)-derived EVs. This step robustly demonstrated that our AS-based approach preserves small EVs integrity and enables the effective isolation of Astrocyte-Derived Extracellular Vesicles (ADEVs) for downstream applications. Finally, the practical utility of this optimized protocol was validated in a clinical cohort. Our findings highlight the robustness, high efficiency, and significant translational potential of this AS-based method for the early and accurate diagnosis of neurodegenerative diseases. We optimized the AS concentration for small EVs precipitation, ultimately identifying 2.66M as the optimal working concentration. This method demonstrated remarkable time efficiency, completing the entire isolation process in approximately 90min. Furthermore, it only requires centrifugation at 12,000 xg, significantly enhancing its practicality in clinical diagnostic scenarios. To expand its potential application scope, we also tested the method's efficacy on other bodily fluids and found that the concentration of 2.66M was equally effective in isolating small EVs from saliva. Comparative analyzes against the commercially available ExoQuick kit demonstrated that our AS-based precipitation method achieved comparable efficacy in isolating small EVs from the plasma of AD patients and in purifying ADEVs. Furthermore, comprehensive validation using nanoparticle tracking analysis (NTA), electron microscopy, and detection of canonical small EVs markers (CD63, CD9) confirmed the functional equivalence of the two isolation methods. The cumulative evidence from this study firmly establishes the AS-based small EVs isolation protocol as a viable and robust efficacy comparable to commercially available kits. This method confers three distinct advantages: enhanced temporal efficiency, reduced cost burden, and minimal interference with downstream assays, rendering it highly amenable to clinical implementation. Notably, it enables the effective isolation of small EVs from plasma, a conventional biofluid widely utilized in clinical diagnostics, as well as from saliva. These attributes underscore the method's potential in advancing the early detection of neurodegenerative disorders. Looking ahead, the versatility of this approach suggests its applicability across a diverse array of biological specimens, thereby facilitating the expansion of small EVs-based diagnostic research and clinical practice.

Extracellular vesicles (EVs) have emerged as important carriers for intercellular communication and biological sources for diagnosis and therapeutics. Low efficiency in EV isolation from biofluids, however, severely restricts their downstream characterization and analysis. Here, we introduced a novel strategy for EV isolation from urine for prostate cancer diagnosis using bifunctionalized magnetic beads through high affinity Ti(IV) ions and the insertion of a phospholipid derivative, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, into the EV membrane synergistically. We demonstrated its efficient isolation of EVs from urine samples with low contamination, high recovery (>80%), and short separation time (within 1 h), resulting in the identification of 36,262 unique EV peptides corresponding to 3302 unique proteins and 3233 unique phosphopeptides representing 1098 unique phosphoproteins using only 100 μL and 5 mL urine samples, respectively. Coupled with trapped ion mobility spectrometry and parallel accumulation-serial fragmentation for phosphosite-specific resolution, quantitative phosphoproteomics of urine samples from prostate cancer patients and healthy individuals revealed 121 upregulated phosphoproteins in cancer patients in contrast to the healthy group. These particular advantages indicate that the novel bifunctional material enables sensitive EV phosphoproteomic analysis for noninvasive biomarker screening and early cancer diagnosis.

Reliable isolation and quantitation of extracellular vesicles (EVs), which function as natural bioactive nanocarriers in biological processes, are essential to uncovering their underlying mechanisms and applications. To meet these requirements, we present here a peptide-engineered biomimetic nanoplatform featuring cell-membrane camouflage. This biomimetic nanoplatform utilizes specific peptide ligands to facilitate the "capture-release" isolation of EVs while enhancing performance by harnessing the antifouling and fluidity advantages afforded by the camouflage of red blood cell membranes. Furthermore, this nanoplatform streamlines the electrochemical quantitation of EVs via a nondestructive labeling and delabeling process, showing accuracy comparable to that of widely used nanoparticle tracking analysis. Validating with EVs from breast cancer cells and human embryonic stem cells, this nanoplatform is also proved to effectively maintain the biological activities of the isolated EVs, thereby enabling precise regulation of cell migration and antiapoptotic response. As such, this biomimetic nanoplatform stands as a highly effective solution for isolating and quantitatively assessing EVs from diverse sources, thus propelling the potential applications of EVs in biomedical and clinical research.

Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.

Wound healing in adult mammals is a regulated but imperfect process in which fibrotic repair dominates over proper regeneration. Mesenchymal stem/stromal cells (MSCs) are considered promising in regenerative medicine; however, evidence suggests that their benefits are primarily mediated by paracrine signaling, including the secretion of extracellular vesicles (EVs). We conducted a structured review to evaluate the potential of MSC-derived EVs (MSC-EVs) in wound management. PubMed was searched for original studies published between January 2015 and July 2025. Of 171 records, 19 met the inclusion criteria. The data included the MSC source, EV isolation, dosing, and effects on wound repair. The review is organized into six themes. First, MSC-EVs consistently promoted the proliferation and migration of keratinocytes, fibroblasts, and endothelial cells, enhancing wound closure in vitro and in vivo. Second, pro-angiogenic effects were evident, often mediated through the PI3K/AKT/mTOR/HIF-1α signaling pathway, although tissue-specific responses were observed. Third, MSC-EVs exhibit immunomodulatory activity by reducing pro-inflammatory cytokines, inducing M2 macrophage polarization, and improving outcomes in chronic and diabetic wound models. Fourth, in extracellular matrix (ECM) remodeling, MSC-EVs increased collagen synthesis, regulated metalloproteinases, and reduced scar formation. Fifth, strategies such as MSC preconditioning and hydrogel-based delivery enhanced EV stability, prolonged activity, and improved therapeutic efficacy. Finally, we assessed methodological challenges, including heterogeneity in EV isolation and characterization, inconsistent dosing, limited adherence to ISCT and ISEV standards, and a lack of GMP-compliant protocols. In summary, MSC-EVs emerge as multifaceted acellular therapeutics that influence key phases of wound repair, including cell activation, angiogenesis, immune modulation, and ECM remodeling. They hold promise for accelerating healing and reducing fibrosis. However, substantial barriers remain before clinical translation, particularly the standardization of EV preparation and regulatory compliance. Addressing these gaps is essential for advancing MSC-EVs into safe, effective, and scalable therapies for wound healing.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04813-5.