For studying cellular communicationsex-vivo, a two-dimensional (2D) cell culture model is currently used as the “gold standard”. 2D culture models are also widely used in the study of RNA expression profiles from tumor cells secreted extracellular vesicles (EVs) for tumor biomarker discovery. Although the 2D culture system is simple and easily accessible, the culture environment is unable to representin vivoextracellular matrix (ECM) microenvironment. Our study observed that 2D- culture derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs and DNAs), when compared to the three-dimensional (3D) culture derived EVs. By performing small RNA next-generation sequencing (NGS) analysis of cervical cancer cells and their EVs compared with cervical cancer patient plasma EV-derived small RNAs, we observed that 3D- culture derived EV small RNAs differ from their parent cell small RNA profile which may indicate a specific sorting process. Most importantly, the 3D- culture derived EV small RNA profile exhibited a much higher similarity (~96%) toin vivocirculating EVs derived from cervical cancer patient plasma. However, 2D- culture derived EV small RNA profile correlated better with only their parent cells cultured in 2D. On the other hand, DNA sequencing analysis suggests that culture and growth conditions do not affect the genomic information carried by EV secretion. This work also suggests that tackling EV molecular alterations secreted into interstitial fluids can provide an alternative, non-invasive approach for investigating 3D tissue behaviors at the molecular precision. This work could serve as a foundation for building precise models employed in mimickingin vivotissue system with EVs as the molecular indicators or transporters. Such models could be used for investigating tumor biomarkers, drug screening, and understanding tumor progression and metastasis.

Since the 2013 Nobel Prize was awarded for the discovery of vesicle trafficking, a subgroup of nanovesicles called exosomes has been driving the research field to a new regime for understanding cellular communication. This exosome-dominated traffic control system has increased understanding of many diseases, including cancer metastasis, diabetes, and HIV. In addition to the important diagnostic role, exosomes are particularly attractive for drug delivery, due to their distinctive properties in cellular information transfer and uptake. Compared to viral and non-viral synthetic systems, the natural, cell-derived exosomes exhibit intrinsic payload and bioavailability. Most importantly, exosomes easily cross biological barriers, obstacles that continue to challenge other drug delivery nanoparticle systems. Recent emerging studies have shown numerous critical roles of exosomes in many biological barriers, including the blood–brain barrier (BBB), blood–cerebrospinal fluid barrier (BCSFB), blood–lymph barrier (BlyB), blood–air barrier (BAB), stromal barrier (SB), blood–labyrinth barrier (BLaB), blood–retinal barrier (BRB), and placental barrier (PB), which opens exciting new possibilities for using exosomes as the delivery platform. However, the systematic reviews summarizing such discoveries are still limited. This review covers state-of-the-art exosome research on crossing several important biological barriers with a focus on the current, accepted models used to explain the mechanisms of barrier crossing, including tight junctions. The potential to design and engineer exosomes to enhance delivery efficacy, leading to future applications in precision medicine and immunotherapy, is discussed.

Study of extracellular vesicles (EVs), particularly exosomes, holds significant promise; however, it is technically challenging to define these small and molecularly diverse nanovesicles. With intrinsic molecular payload and biodegradability, molecular engineering of exosomes opens new avenues for mediating cellular responses and developing novel nano-delivery systems in precision therapeutics. Microfluidic lab-on-chip technology is playing pivotal roles in this emerging field. In this review, we have examined scientific advancements of microfluidic technology for engineering exosomes and assessed future applications and perspectives in developing precision therapeutics; this can serve the community via identification of potential new research areas or technologies that are urgently needed in precision therapeutics.

Extracellular vesicles (EVs) play a crucial role in intercellular communication, signaling pathways, and disease pathogenesis by transporting biomolecules such as DNA, RNA, proteins, and lipids derived from their cells of origin, and they have demonstrated substantial potential in clinical applications. Their clinical significance underscores the need for sensitive methods to fully harness their diagnostic potential. In this comprehensive review, we explore EV heterogeneity related to biogenesis, structure, content, origin, sample type, and function roles; the use of EVs as disease biomarkers; and the evolving landscape of EV measurement for clinical diagnostics, highlighting the progression from bulk measurement to single vesicle analysis. This review covers emerging technologies such as single-particle tracking microscopy, single-vesicle RNA sequencing, and various nanopore-, nanoplasmonic-, immuno-digital droplet-, microfluidic-, and nanomaterial-based techniques. Unlike traditional bulk analysis methods, these methods contribute uniquely to EV characterization. Techniques like droplet-based single EV-counting enzyme-linked immunosorbent assays (ELISA), proximity-dependent barcoding assays, and surface-enhanced Raman spectroscopy further enhance our ability to precisely identify biomarkers, detect diseases earlier, and significantly improve clinical outcomes. These innovations provide access to intricate molecular details that expand our understanding of EV composition, with profound diagnostic implications. This review also examines key research challenges in the field, including the complexities of sample analysis, technique sensitivity and specificity, the level of detail provided by analytical methods, and practical applications, and we identify directions for future research. This review underscores the value of advanced EV analysis methods, which contribute to deep insights into EV-mediated pathological diversity and enhanced clinical diagnostics.

Extracellular vesicles (EVs), particularly exosomes (30–150 nm), are an emerging delivery system in mediating cellular communications, which have been observed for priming immune responses by presenting parent cell signaling proteins or tumor antigens to immune cells. Therefore, preparation of antigenic exosomes that can play therapeutic roles, particularly in cancer immunotherapy, is emerging. However, standard benchtop methods (e.g., ultracentrifugation and filtration) lack the ability to purify antigenic exosomes specifically among other microvesicle subtypes, due to the non-selective and time-consuming (>10 h) isolation protocols. Exosome engineering approaches, such as the transfection of parent cells, also suffer from poor yields, low purity, and time-consuming operations. In this paper, we introduce a streamlined microfluidic cell culture platform for integration of harvesting, antigenic modification, and photo-release of surface engineered exosomes in one workflow, which enables the production of intact, MHC peptide surface engineered exosomes for cytolysis activation. A PDMS microfluidic cell culture chip is simply cast from a 3D-printed mold. This proof-of-concept study demonstrated the enhanced ability of harvested exosomes in antigen presentation and T cell activation, by decorating melanoma tumor peptides on the exosome surface (e.g., gp-100, MART-1, and MAGE-A3). Such surface engineered antigenic exosomes were harvested in real-time from the on-chip culture of leukocytes isolated from human blood, leading to much faster cellular uptake. The activation of gp100-specific CD8 T cells which were purified from the spleen of 2 Pmel1 transgenic mice was evaluated using surface engineered exosomes prepared from murine antigen presenting cells. Antigen-specific CD8 T cell proliferation was significantly induced by the engineered exosomes compared to that by native, non-engineered exosomes. This microfluidic platform serves as an automated and highly integrated cell culture device for rapid and real-time production of therapeutic exosomes that could advance cancer immunotherapy.