REVIEW
Introduction of cellulose nanofiber-based EV sheets for novel exosome analyses
2025 Volume 7 Issue 1 Pages 26-29
Details
2025 Volume 7 Issue 1 Pages 26-29
My research group recently developed a novel method for isolating extracellular vesicles (EVs) using cellulose nanofiber (CNF) sheets, termed as EV sheets, which are designed with tunable pore sizes. EVs, including exosomes, play a crucial role in cellular communication and are key targets of disease mechanisms. EV sheets capture intact EVs from minimal volumes of biofluid, such as the surfaces of organs, and store them under dry conditions until analysis. These EV sheets addressed the current limitations of EV research. CNF Sheets provide a breakthrough by effectively isolating and preserving EVs from as little as 10 µL of biofluid. In ovarian cancer models and patient samples, EV sheets revealed spatial heterogeneity in the EV profiles and identified unique miRNA signatures based on their location. This technique detects cancer-associated miRNAs at an early stage before visible symptoms such as ascites develop. Importantly, the miRNA profiles obtained from tumor surfaces differed from those obtained from the surrounding fluids, providing a better understanding of tumor-derived EVs. The EV sheet isolation method has a high EV recovery efficiency and compatibility with small RNA sequencing, demonstrating its potential to advance cancer diagnosis, staging, and treatment planning. EV sheets also offer advantages in EV preservation and transport, making them practical for clinical and research applications. In addition, this method is promising for elucidating EV biology, particularly its role in cancer progression and intercellular communication. Future studies will aim to refine this method for wider clinical use and maximize its potential.
-Cellulose nanofiber-based extracellular vesicles (EVs) sheets efficiently captured and preserved EVs from minimal volumes of biofluid, enabling high-purity isolation and stable storage for advanced molecular analyses.
-Spatial analysis using EV sheets revealed the unique location-based miRNA profiles of EVs in ovarian cancer, aiding early detection and improving our understanding of tumor-derived vesicle biology.
-This innovative EV sheet technology holds potential clinical applications, including cancer diagnosis, staging, and treatment planning, and advances EV research through noninvasive and precise molecular profiling methods.
Extracellular vesicles (EVs), including exosomes, are lipid bilayer-bound particles actively secreted by cells that play essential roles in intercellular communication [1]. They transport bioactive molecules, such as proteins, lipids, and small RNAs, to recipient cells, thereby influencing diverse biological processes, including immune modulation, angiogenesis, and metastasis. EVs are promising biomarkers for disease diagnosis, monitoring, and therapeutic intervention because of their ability to reflect the molecular state of parent cells [2].
Despite the advances in EV research, significant challenges remain with respect to the spatial heterogeneity of EVs in the human body [3]. As shown in Table 1, traditional EV isolation techniques, such as ultracentrifugation, size exclusion chromatography, or immunoaffinity methods, have various advantages and disadvantages and often require large sample volumes [4]. EV isolation using EV sheets is significantly easier than other methods, requires less time, and is less susceptible to individual variations. In addition, this material is composed of pure cellulose and may be available at a lower cost. In addition, important information about location-specific EV characteristics is lost after obtaining body fluids. This is particularly problematic for microenvironments such as ascites, where EVs may exhibit different molecular profiles depending on their proximity to primary tumors or metastatic tissues.
| Method | Description | Advantages | Disadvantages |
|---|---|---|---|
| Ultracentrifugation | High-speed centrifugation separates EVs based on their density and size. | Widely used, reliable, cost-effective. | Time-consuming, purity of EVs requires specialized equipment. |
| Density gradient centrifugation | Separation using a density gradient medium, such as sucrose or iodixanol. | High purity of isolated EVs. | Limited scalability, labor-intensive, time-consuming. |
| Size-exclusion chromatography | Separation based on size by passing through a porous matrix. | High purity of isolated EVs, simple operation. | Limited scalability, potential contamination with similarly sized particles. |
| Ultrafiltration | Membrane filteration to isolate EVs based on size. | Fast, cost-effective, scalable. | Risk of clogging or damaging EV structure. |
| Immunoaffinity capture | Antibodies specific capturing to surface markers. | High specificity, ability to isolate sub-populations of EVs. | Expensive, isolate specific EVs, requires characterized markers, limited scalability. |
| Precipitation-based methods | Using polymers to precipitate EVs from solution. | Simple, does not require specialized equipment. | Low purity of EVs. |
| Microfluidics-based methods | Utilizes microchips with specific designs for size or marker-based isolation. | High precision, small sample volume requirement. | Expensive, requires specialized devices, limited throughput. |
Understanding the spatial dynamics of EVs is critical for cancer biology. For example, in ovarian cancer, EVs within the ascites fluid play critical roles in tumor progression, immune evasion, and metastasis [5, 6]. However, the heterogeneity of EVs in different regions of ascites, such as near the tumor surface, liver, or pelvic peritoneum, remains unexplored. This limitation is due to the lack of technology for capturing EVs directly from organ surfaces or trace amounts of biofluids during surgery. However, no tools are currently available to address these issues.
To address these limitations, my research group has developed and introduced cellulose nanofiber (CNF) EV sheets as a breakthrough platform for EV isolation and analysis [7]. EV sheets are biocompatible, highly absorbent, and capable of capturing EVs from the microvolumes of biofluids or moist tissue surfaces. By tailoring the nanostructure of the EV sheets (Fig. 1), a novel method for capturing, preserving, and analyzing EVs, including their small RNA cargo, with high efficiency and purity was developed. The utility of EV sheets has been demonstrated in murine ovarian cancer models and clinical samples, revealing site-specific EV heterogeneity and its implications in cancer progression and biomarker discovery.
Conceptual diagram of extracellular vesicle (EV) isolation by the EV sheet.
The EV sheets developed in this study were fabricated using tailored porous nanostructures that enable efficient EV capture and storage. The process includes three main steps: (1) EV capture by capillary uptake into the nanopores of the sheet; (2) EV preservation by drying, which closes the nanopores to protect EV integrity; and (3) EV release in phosphate-buffered saline (PBS) for downstream analysis. This method allows the isolation of EVs from as little as 10 µl of biofluid, overcoming the limitations of conventional ultracentrifugation-based techniques.
The EV sheets outperformed conventional methods in terms of EV recovery efficiency and purity. Compared to ultracentrifugation, EV Sheets recovered higher concentrations of intact EVs from smaller volumes of biofluid with minimal contamination from free miRNAs or proteins. Cryoelectron microscopy confirmed the structural integrity of EVs captured by EV Sheets, whereas immunoblotting and nanoparticle tracking analysis validated the presence of EV markers, such as CD63 and CD81. The size of EVs captured by EV sheets was approximately 100 nm, indicating that most EVs were exosomes and not other larger vesicles. Regarding purity, owing to the low-volume requirement of the EV sheet, it is difficult to directly compare the performance of the EV sheet with other methods. However, the calculated purity, determined by dividing the concentration by the protein content, was even higher with the EV sheets than with the conventional serial centrifugation method in serum.
Using mouse ovarian cancer models, this study demonstrated the efficacy of EV sheets in capturing EVs directly from organ surfaces. Mice injected with ID8 ovarian cancer cells showed tumor progression in the peritoneal cavity without apparent ascites in the early stages. EV sheets attached to various organs, including the pelvic peritoneum, liver surface, and omentum, successfully captured EVs. Small-RNA sequencing revealed distinct miRNA profiles in EVs from different anatomical locations. For example, mmu-miR-615-3p and mmu-miR-196b-5p are enriched in pelvic peritoneal EVs, whereas liver surface EVs exhibit high levels of mmu-miR-122-3p and mmu-miR-335-5p. These findings highlight the spatial heterogeneity of EVs and suggest that their profiles are influenced by their microenvironments.
For validation in clinical settings, EV sheets were used during surgery in patients with ovarian cancer. The EV sheets were sterilized with ethylene oxide gas and sterility was ensured by the Japanese Society of Medical Instrumentation [8]. After sterilization, the EV isolation performance of the sheets did not change. EVs from tumor surfaces, ascites, and adjacent tissues were obtained using a sterilized EV sheet. Small RNA sequencing confirmed that tumor surface EVs had miRNA profiles closely resembling those of tumor tissues, whereas whole ascites EVs showed distinct patterns. For example, miRNAs such as hsa-miR-200b-3p and hsa-miR-429, which are known to be associated with tumor progression [9], were significantly enriched in tumor-derived EVs, but were less abundant in whole ascites EVs. These results indicated that EV sheets can reveal previously unrecognized location-dependent EV heterogeneity, thereby providing valuable insights into tumor biology.
Furthermore, the utility of EV sheets for monitoring disease progression and therapeutic response has been demonstrated. In a patient with metastatic ovarian cancer, EV sheets revealed that the EV miRNA profiles from different anatomical sites reflected the extent of tumor dissemination. miRNA expression levels were the highest at tumor rupture sites and decreased with increasing distance from the tumor, demonstrating the dynamic nature of EV-mediated communication. Importantly, miRNA levels such as those of hsa-miR-200c-3p and hsa-miR-429 decreased after surgery, highlighting their potential as biomarkers for treatment monitoring.
The findings of this study open numerous possibilities for advancing EV research and its clinical applications. First, their ability to capture EVs directly from microenvironments, such as organ surfaces or localized biofluids, provides a new dimension to EV biology. As demonstrated in the present study, the spatial heterogeneity of EVs suggests that they carry microenvironment-specific molecular signatures that can be exploited for diagnostic and therapeutic purposes [10].
Clinically, EV sheets can be used for real-time EV analysis during surgery, allowing surgeons to assess tumor biology and disease progression based on EV miRNA profiles. This could improve decisionmaking in cancer staging, treatment planning, and recurrence monitoring. For example, miRNAs, such as hsa-miR-200b-3p and hsa-miR-429, identified in tumor-derived EVs, could serve as biomarkers for detecting residual disease or early metastasis.
Further research is required to expand the utility of EV sheets beyond small RNA profiling. EVs carry diverse cargo, including proteins, lipids, and DNA, which can provide additional insights into disease mechanisms [1]. Integrating proteomics, lipidomics, and genomic analyses with EV isolation using EV sheets could enhance our understanding of EV biology and its role in diseases such as cancer, cardiovascular disease, and neurodegeneration.
The scalability and biocompatibility of the EV sheets make them promising candidates for clinical applications. Large-scale production and regulatory approval are critical for its widespread adoption in clinical practice. In addition, EVs in the sheet could be stored under dry conditions for one week, as the quality of the EVs did not change. Therefore, this method is suitable for the direct transport of EV samples and is well-suited for clinical use. Validation in larger patient cohorts is necessary to confirm the reliability of EV sheet-based biomarkers for cancer diagnosis and monitoring.
In conclusion, this study demonstrated the power of EV sheets as a next-generation EV isolation platform capable of capturing, preserving, and analyzing EVs with spatial resolution. By uncovering the location-specific heterogeneity of EVs, EV sheets provide new opportunities to understand disease mechanisms, identify biomarkers, and improve patient care. This technology represents a significant advancement in basic EV research and translational medicine.
There is no COI to disclose.
The authors deeply appreciate Dr. Takao Yasui (Department of Life Science and Technology, Institute of Science, Tokyo) and Dr. Hirotaka Koga (Institute of Scientific and Industrial Research, Osaka University) for the co-invention of the CNF sheets. This work was financially supported by the following grants: Fusion Oriented Research for Disruptive Science and Technology (FOREST; JPMJFR204J, to A.Y.) and Project for Cancer Research and Therapeutic Evolution (P-PROMOTE) grant number 22ama221407h0001 from the Japan Agency for Medical Research and Development (AMED).
