Optimized Wound Healing Assay to Study Extracellular Vesicle-Driven Glioblastoma Cell Migration
Open accessAbstract
Cell migration is a fundamental process in cancer progression, playing a central role in tumor invasion and metastasis. This highly coordinated behavior is regulated by dynamic interactions between cancer cells and extracellular environment. Among the different tumor types, glioblastoma (GB) represents a particularly aggressive form of cancer in which enhanced migratory capacity is a key determinant of diffuse brain infiltration, tumor recurrence, and poor prognosis. In this context, extracellular vesicles (EVs) have emerged as important mediators, regulating cell migration in several cancer types, including GB. EVs are lipid bilayer-enclosed nano- and micro-sized particles, containing various bioactive molecules that can target specific recipient cells, thereby modulating cellular properties, including the migratory behavior. Among the available methods for studying cell migration, the wound healing assay is the most widely used. Although simple, cost-effective and not requiring sophisticated equipment, its reliability and reproducibility can be affected by technical variability and the diversity of existing protocols. Here, we present an optimized protocol for executing and analyzing a cellular wound healing assay designed to assess EV-mediated migration in GB cells. The protocol incorporates the use of silicone culture inserts to enhance wound homogeneity and reproducibility, together with continuous Mitomycin C incubation to inhibit cell proliferation without inducing cytotoxicity, enabling specific assessment of cell migration. We outline a step-by-step description of the procedure, detailing all required materials and equipment and highlighting critical steps, checkpoints, and key parameters. This method provides a robust framework for reproducible wound healing assays to investigate EV effects on GB cell migration.
AI snapshot
Written from the paper’s full text.
TL;DR
This protocol paper presents an optimized insert-based wound healing assay for studying extracellular vesicle (EV)-driven glioblastoma (GB) cell migration, using continuous 2 µg/mL Mitomycin C to block proliferation without cytotoxicity. Applied to U87 GB cells and NTERA2-derived EVs, it reports significantly reduced migration at 24 and 42 h and provides detailed checkpoints for reproducibility.
Key findings
- Continuous 2 µg/mL Mitomycin C inhibited cell proliferation without significant toxicity up to 42 h; 4 and 6 µg/mL caused pronounced cytotoxicity, with most cells detached and floating, preventing measurement.
- NTERA2 EV treatment significantly reduced U87 GB cell migration compared with untreated cells: 62.1% at 24 h (p = 0.0023) and 68.9% at 42 h (p = 0.0042), relative to 100.0% untreated.
- Three NTERA2 EV batches showed an average particle size of 176.19 ± 7.37 nm and a particle-to-protein ratio of 1.92 × 10^8 ± 4.35 × 10^7 particles/μg; 10 µg/mL protein corresponded to an estimated 1.92 × 10^9 ± 4.35 × 10^8 particles/mL.
- The optimized protocol uses ibidi silicone culture inserts to create a 500 μm wound, recommends 18,000 U87 cells per insert, and supports imaging at t0, 16, 24, and 42 h.
Methods
- U87 GB cells were seeded at 18,000 cells per ibidi two-well silicone insert in 24-well plates; after 24 h the insert was removed to create a 500 μm wound, and cells were treated with GB Wound Healing Experimental Medium containing 2% FBS and 2 μg/mL Mitomycin C plus EV samples. Images were acquired at t0, 16, 24, and 42 h at 5× magnification, two fields per well, and analyzed in ImageJ for percentage wound healing and absolute rate of gap closure.
- NTERA2-derived EVs were isolated by differential centrifugation at 10,000× g and characterized by Micro BCA protein assay, interferometric light microscopy (Videodrop), and previously by NTA, cryo-EM, and MISEV markers; EV treatments were normalized to 10 µg/mL protein, using three batches (two independent preparations and one pooled reference).
- The assay was repeated at least three times with different cell and EV batches; statistical analysis included Student’s t-test, one-way ANOVA, and Tukey’s HSD test.
Limitations
- Paper-stated: the protocol was designed for NTERA2-derived EVs and U87 GB cells; Mitomycin C concentration may differ by cell line or batch and should be determined in advance; U87’s highly adhesive phenotype and the small insert area can amplify early seeding asymmetries; the EV dose reference is approximate because not all preparations were individually quantified.
- Paper-stated: the particle-to-protein ratio as a purity indicator requires careful evaluation and can vary with EV origin and isolation method.
- Evident from the text: validation is limited to one GB cell line and one EV source, with no independent head-to-head comparison against scratch or other migration assays reported in this paper.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
22 of 43 cited sources checked · 11 supported · 0 flagged · accuracy not yet measured
- Premisecitation
Extracellular vesicles regulate cell migration in several cancer types, including glioblastoma.
“In this context, extracellular vesicles (EVs) have emerged as important mediators, regulating cell migration in several cancer types, including GB.”
- Also stated in: § 1. Introduction · plain
“EVs are key players in regulating GB pathological features, including cell invasiveness and migration [30,31,32].”
Quote in the paperSubstantial claim2 of 3 sources checked- cites [30] Liguori, G.L. (2024). Challenges and Promise for Glioblastoma Treatment through Extracellular Vesicle Inquiry. Cells, 13. source
Section 3.2 reports that extracellular vesicles can alter glioblastoma cell migration in vitro, and Section 3.1 links EVs to invasiveness in tumors generally. The copy does not specifically establish regulation of cell migration in several cancer types beyond glioblastoma. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [31] Colangelo, T., Saponaro, A.A., Mazzoccoli, G., Serviddio, G., and Villani, R. (2025). Emerging Frontiers in Neuro-Oncology: Insights into Extracellular Vesicle-Driven Tumor Mechanisms and Nanotherapeutic Strategies. Int. J. Mol. Sci., 26. source
Section 4.1 (page unavailable) says GBM-derived EVs enhance invasiveness, supporting a role in glioblastoma cell migration. The passages found do not establish that EVs regulate cell migration in several cancer types; that broader part of the claim is unsupported by our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [32] Tatode, 2026, Glioblastoma Exosomes Reprogramming the Tumor Microenvironment and Evading Therapeutic Challenges, Mol. Biol. Rep. source
- Also stated in: § 1. Introduction · plain
- Study design and conductcitation
Continuous Mitomycin C incubation does not induce cytotoxicity.
“without inducing cytotoxicity, enabling specific assessment of cell migration.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [43] Mantile, F., Kisovec, M., Adamo, G., Romancino, D.P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M.P., and Kisslinger, A. (2022). A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications. Cancers, 14. source
The Wound Healing Assay methods (§2.9; page unavailable) say that 2 μg/mL mitomycin C was present to inhibit proliferation while cell migration was assessed. They do not report whether that incubation was cytotoxic; the viability results (§3.2; page unavailable) concern extracellular vesicles, not mitomycin C. Searched for mitomycin C and cell viability or cytotoxicity. (LLM, conf=0.76)
Experimental — accuracy not yet measured
- Premisecitation
The reliability and reproducibility of the wound healing assay can be severely impacted by many factors if they are not adequately controlled.
“several articles highlight an increasing number of factors that can severely impact the reliability and reproducibility of the assay, if they are not adequately controlled [38,39,40,41,42]”
Quote in the paperSubstantial claim3 of 5 sources checked- cites [38] Yang, C., Yin, D., Zhang, H., Badea, I., Yang, S.M., and Zhang, W. (2024). Cell Migration Assays and Their Application to Wound Healing Assays—A Critical Review. Micromachines, 15. source
- cites [39] Oldenburg, J., Maletzki, L., Strohbach, A., Bellé, P., Siewert, S., Busch, R., Felix, S.B., Schmitz, K., and Stiehm, M. (2021). Methodology for Comprehensive Cell-Level Analysis of Wound Healing Experiments Using Deep Learning in MATLAB. BMC Mol. Cell Biol., 22. source
- cites [40] Planz, 2018, Establishment of a Cell-Based Wound Healing Assay for Bio-Relevant Testing of Wound Therapeutics, J. Pharmacol. Toxicol. Methods source
- cites [41] Sarro, 2019, In Vitro Cell Migration Quantification Method for Scratch Assays, J. R. Soc. Interface source
- cites [42] Potaliya, 2024, In-Vitro Wound Healing Assays: A Comprehensive Review, Mymensingh Med. J.
- Premisecitation
Protocols involving extracellular vesicles and glioblastoma cells are particularly poorly validated.
“with protocols involving EVs and GB cells being particularly poorly validated.”
Quote in the paperSubstantial claim3 of 5 sources checked- cites [38] Yang, C., Yin, D., Zhang, H., Badea, I., Yang, S.M., and Zhang, W. (2024). Cell Migration Assays and Their Application to Wound Healing Assays—A Critical Review. Micromachines, 15. source
The Discussion, §3.1, describes consistency problems in cell-migration assays generally, but does not assess validation of protocols involving extracellular vesicles or glioblastoma cells. Searches for those terms and for protocol validation found only an extracellular-vesicles title in the References, not the claimed comparison. (LLM, conf=0.74)
Experimental — accuracy not yet measured - cites [39] Oldenburg, J., Maletzki, L., Strohbach, A., Bellé, P., Siewert, S., Busch, R., Felix, S.B., Schmitz, K., and Stiehm, M. (2021). Methodology for Comprehensive Cell-Level Analysis of Wound Healing Experiments Using Deep Learning in MATLAB. BMC Mol. Cell Biol., 22. source
The Methods (Wound healing experiments; page unavailable) describe assays using human coronary artery endothelial cells, while the Conclusions discuss limitations of wound-healing image analysis. A Discussion passage mentions glioblastoma cells only as a segmentation benchmark. Searches for extracellular vesicles and poorly validated EV or glioblastoma protocols found no passage addressing the claim. (LLM, conf=0.76)
Experimental — accuracy not yet measured - cites [40] Planz, 2018, Establishment of a Cell-Based Wound Healing Assay for Bio-Relevant Testing of Wound Therapeutics, J. Pharmacol. Toxicol. Methods source
- cites [41] Sarro, 2019, In Vitro Cell Migration Quantification Method for Scratch Assays, J. R. Soc. Interface source
The Introduction (p. 1) says scratch assays lack a standard method for quantifying cell migration, but it does not single out extracellular-vesicle or glioblastoma-cell protocols. Searches for those terms and for their abbreviations found no passage addressing whether such protocols are particularly poorly validated. (LLM, conf=0.76)
Experimental — accuracy not yet measured - cites [42] Potaliya, 2024, In-Vitro Wound Healing Assays: A Comprehensive Review, Mymensingh Med. J.
- Study design and conductcitation
Continuous Mitomycin C incubation inhibits cell proliferation.
“continuous Mitomycin C incubation to inhibit cell proliferation without inducing”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [43] Mantile, F., Kisovec, M., Adamo, G., Romancino, D.P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M.P., and Kisslinger, A. (2022). A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications. Cancers, 14. source
- Study design and conductcitation
The optimized protocol uses silicone culture inserts to enhance wound homogeneity and reproducibility.
“The protocol incorporates the use of silicone culture inserts to enhance wound homogeneity and reproducibility”
- Also stated in: § 1. Introduction · plain
“The protocol uses silicone culture inserts to enhance the reproducibility of the wound”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [43] Mantile, F., Kisovec, M., Adamo, G., Romancino, D.P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M.P., and Kisslinger, A. (2022). A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications. Cancers, 14. source
Section 2.9 (Wound Healing Assay; page unavailable) says the researchers used Ibidi inserts in a wound-healing assay. It does not identify the inserts as silicone or say they were used to improve wound homogeneity or reproducibility. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [44] Mantile, 2025, Standard Operating Procedure for Wound Healing Cell Migration Assay within the Nanostructurome Pipeline, Proc. Socrat. Lect.
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Also stated in: § 1. Introduction · plain
- Findingcitation
The particle-to-protein ratio of NTERA2-derived EV preparations is consistent with values reported for large EV fractions of similar size, isolated from other cancer cell lines (e.g., breast) either after 16,500× g sedimentation alone or followed by density cushioning.
“Notably, the particle-to-protein ratio of NTERA2-derived EV preparations is consistent with values reported for large EV fractions of similar size, isolated from other cancer cell lines (e.g., breast) either after 16,500× g sedimentation alone or followed by density cushioning [53].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [53] Lischnig, 2022, Quantitative Proteomics Identifies Proteins Enriched in Large and Small Extracellular Vesicles, Mol. Cell. Proteom. source
The Experimental Procedures describe large EVs from breast cancer cell lines collected at 16,500g, with subsequent density-cushion purification. The Results report particle-to-protein ratios for crude and cushion-purified large EVs (Supplemental Fig. S1C and Fig. 3C). Our copy does not give numerical ratios in the text, so it cannot establish whether the NTERA2 preparations have consistent values or similar sizes. (LLM, conf=0.79)
Experimental — accuracy not yet measured
- Findingown data
Qualitative observations reflected the proliferation block induced by 2 μg/mL Mitomycin C.
“Although quantitative wound closure analysis showed no difference between 2 µg/mL Mitomycin C and the control group, qualitative observations reflected the proliferation block induced by 2 µg/mL Mitomycin C.”
Quote in the paperSubstantial claim - Findingown data
Cryo-electron microscopy confirmed the physical structure of both medium/large and small extracellular vesicle fractions, highlighting their delimitation by a lipid bilayer.
“Cryo-electron microscopy (Thermo Fisher Scientific, Waltham, MA, USA) confirmed the physical structure of both the medium/large and small EV fractions, highlighting their delimitation by a lipid bilayer.”
Quote in the paperNot a substantial claim - Findingown data
At 42 h, no significant difference was observed between the control group (2% FBS without Mitomycin C) and the group treated with 2 μg/mL Mitomycin C.
“At 42 h, no significant difference was observed between the control group (2% FBS without Mitomycin C) and the one treated with 2 µg/mL Mitomycin C (Figure 4A).”
Quote in the paperSubstantial claim - Findingown data
Trypan Blue staining at the end of treatment confirmed that 4 and 6 μg/mL Mitomycin C were highly cytotoxic.
“Trypan Blue staining at the end of the treatment confirmed that 4 and 6 µg/mL Mitomycin C were highly cytotoxic, whereas 2 µg/mL effectively blocked cell division without causing significant toxicity (Figure 4B).”
Quote in the paperSubstantial claim - Findingown data
At 24 h, ANOVA revealed a significant overall difference among the four tested conditions (2% FBS without Mitomycin C and Mitomycin C at 2, 4, and 6 μg/mL).
“At 24 h, ANOVA revealed a significant overall difference among the four tested conditions (2% FBS without Mitomycin C and Mitomycin C, at concentrations of 2, 4, and 6 µg/mL).”
Quote in the paperNot a substantial claim - Findingown data
The three extracellular vesicle batches showed an average particle size of 176.19 ± 7.37 nm.
“The three batches showed an average particle size of 176.19 ± 7.37 nm”
Quote in the paperNot a substantial claim - Findingown data
Quantitative wound closure analysis showed no difference between 2 μg/mL Mitomycin C and the control group.
“Although quantitative wound closure analysis showed no difference between 2 µg/mL Mitomycin C and the control group, qualitative observations reflected the proliferation block induced by 2 µg/mL Mitomycin C.”
Quote in the paperSubstantial claim - Findingown data
The three extracellular vesicle batches showed a particle-to-protein ratio of 1.92 × 108 ± 4.35 × 107 particles/μg.
“and a particle-to-protein ratio of 1.92 × 108 ± 4.35 × 107 particles/μg”
Quote in the paperNot a substantial claim - Findingown data
At 24 h, post hoc pairwise comparisons did not detect any statistically significant difference between individual groups.
“However, post hoc pairwise comparisons did not detect any statistically significant difference between individual groups (Figure 4A).”
Quote in the paperSubstantial claim - Findingown data
At 42 h, 4 and 6 μg/mL Mitomycin C caused pronounced cytotoxicity, with most cells detached and floating, preventing measurement.
“In contrast, 4 and 6 µg/mL Mitomycin C caused pronounced cytotoxicity, with most cells detached and floating, thereby preventing measurement (Figure 3A).”
Quote in the paperSubstantial claim - Findingnone given
The described protocol was successfully applied to study the impact of NTERA2-derived EVs on the migration of U87 GB cells.
“The described protocol was successfully applied to study the impact of NTERA2-derived EVs on the migration of U87 GB cells.”
Quote in the paperSubstantial claim - Findingown data
For NTERA2 EV batches, a protein concentration of 10 μg/mL corresponds to an estimated assay concentration of 1.92 × 10^9 ± 4.35 × 10^8 particles/mL.
“For NTERA2 EV batches, this corresponds to an estimated assay concentration of 1.92 × 109 ± 4.35 × 108 particles/mL.”
Quote in the paperNot a substantial claim - Findingown data
For our EV preparations, a protein concentration range of 5-20 μg/mL corresponds to approximately (9.60 ± 2.18) × 10^8 to (3.84 ± 0.87) × 10^9 particles/mL.
“For dose–response evaluation, we suggest beginning with EV concentrations within the range of 5–20 µg/mL of protein, which for our EV preparations corresponds to approximately (9.60 ± 2.18) × 108 to (3.84 ± 0.87) × 109 particles/mL.”
Quote in the paperSubstantial claim - Findingown data
At 24 h, EV treatment significantly reduced cell migration to 62.1% compared with untreated cells (100.0%), p = 0.0023.
“Overall, results at both 24 and 42 h (Figure 5B) show a statistically significant reduction in cell migration in the presence of EVs (62.1% at 24 h, p = 0.0023 and 68.9% at 42 h, p = 0.0042) compared with untreated cells (100.0%).”
Quote in the paperSubstantial claim - Findingown data
Batch 1 exhibited the lowest particle-to-protein ratio (1.44 × 108 ± 3.20 × 107).
“Batch 1 exhibited the lowest particle-to-µg ratio (1.44 × 108 ± 3.20 × 107), whereas Batch 3 showed the highest ratio (2.29 × 108 ± 2.76 × 107), allowing a broader range of particle concentrations to be tested”
Quote in the paperSubstantial claim - Findingnone given
A concentration of 2 μg/mL Mitomycin C has been confirmed across different batches of Mitomycin C and GB U87 cells.
“A concentration of 2 μg/mL has been confirmed across different batches of Mitomycin C and GB U87 cells.”
Quote in the paperSubstantial claim - Findingown data
At 42 h, EV treatment significantly reduced cell migration to 68.9% compared with untreated cells (100.0%), p = 0.0042.
“Overall, results at both 24 and 42 h (Figure 5B) show a statistically significant reduction in cell migration in the presence of EVs (62.1% at 24 h, p = 0.0023 and 68.9% at 42 h, p = 0.0042) compared with untreated cells (100.0%).”
Quote in the paperSubstantial claim - Findingown data
Batch 3 showed the highest particle-to-protein ratio (2.29 × 108 ± 2.76 × 107).
“Batch 1 exhibited the lowest particle-to-µg ratio (1.44 × 108 ± 3.20 × 107), whereas Batch 3 showed the highest ratio (2.29 × 108 ± 2.76 × 107), allowing a broader range of particle concentrations to be tested”
Quote in the paperSubstantial claim - Interpretationcitation
Glioblastoma cells are responsive to signals emitted by NTERA2 cells.
“suggesting that GB cells are responsive to signals emitted by NTERA2 cells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [47] Attia, 2019, Cationic Niosome-Based HBMP7 Gene Transfection of Neuronal Precursor NT2 Cells to Reduce the Migration of Glioma Cells in Vitro, J. Drug Deliv. Sci. Technol. source
- Premisecitation
Conditioned medium from NTERA2 cells affects glioblastoma cell behavior.
“it has been found that conditioned medium from NTERA2 cells affects GB cell behavior [47]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [47] Attia, 2019, Cationic Niosome-Based HBMP7 Gene Transfection of Neuronal Precursor NT2 Cells to Reduce the Migration of Glioma Cells in Vitro, J. Drug Deliv. Sci. Technol. source
- Interpretationreasoning
Using individual EV preparations enables precise assessment of batch-specific properties.
“Using individual EV preparations enables precise assessment of batch-specific properties, whereas pooling multiple EV preparations provides a more representative sample of the overall population.”
Quote in the paperSubstantial claim - Interpretationown data
In samples treated with 2 μg/mL Mitomycin C, wound closure was primarily driven by migration.
“By contrast, fewer and more elongated cells were observed in samples treated with 2 µg/mL Mitomycin C, suggesting that closure was primarily driven by migration (Figure 3B).”
Quote in the paperSubstantial claim - Interpretationreasoning
The optimized protocol provides a robust framework for reproducible wound healing assays to investigate extracellular vesicle effects on glioblastoma cell migration.
“This method provides a robust framework for reproducible wound healing assays to investigate EV effects on GB cell migration.”
Quote in the paperSubstantial claim - Interpretationown data
In the control group, wound closure involved both proliferation and migration.
“In the control group, wound areas exhibited a higher cell density with numerous rounded cells, indicating that closure involved both proliferation and migration (Figure 3B).”
Quote in the paperSubstantial claim - Interpretationown data
The three extracellular vesicle batches indicated uniformity and consistency across preparations.
“indicating uniformity and consistency across preparations.”
Quote in the paperSubstantial claim - Premisecitation
NTERA2 teratocarcinoma cells closely resemble neural stem/progenitor cells.
“NTERA2 teratocarcinoma cells closely resemble neural stem/progenitor cells, as they are capable of differentiating into glia and neurons, and of locating and targeting glioma neoplasms in mouse models, particularly following treatment with retinoic acid [45,46,47,48].”
Quote in the paperNot a substantial claim0 of 4 sources checked- cites [45] Andrews, 1998, Teratocarcinomas and Human Embryology: Pluripotent Human EC Cell Lines. Review Article, Apmis source
- cites [46] Ahmed, 2011, The Use of Neural Stem Cells in Cancer Gene Therapy: Predicting the Path to the Clinic, Curr. Opin. Mol. Ther.
Not checked in the free run.
- cites [47] Attia, 2019, Cationic Niosome-Based HBMP7 Gene Transfection of Neuronal Precursor NT2 Cells to Reduce the Migration of Glioma Cells in Vitro, J. Drug Deliv. Sci. Technol. source
- cites [48] Zhao, 2010, Human NT2 Neural Precursor-Derived Tumor-Infiltrating Cells as Delivery Vehicles for Treatment of Glioblastoma, Hum. Gene Ther. source
- Interpretationreasoning
Continuous Mitomycin C incubation enables specific assessment of cell migration.
“enabling specific assessment of cell migration.”
Quote in the paperSubstantial claim - Study design and conductown data
Mycoplasma absence was verified in the cell cultures using the Mycoplasma PCR detection kit.
“we verified its absence in the cell cultures using the Mycoplasma PCR detection kit”
Quote in the paperSubstantial claim - Interpretationown data
The 2 μg/mL Mitomycin C condition is suitable for wound healing assays of at least 42 h duration.
“Overall, continuous incubation with 2 µg/mL Mitomycin C for up to 42 h effectively inhibits proliferation without affecting viability or migration, making this condition suitable for wound healing assays of at least 42 h duration.”
Quote in the paperSubstantial claim - Study design and conductreasoning
Seeding 18,000 GB cells/insert in the presence of Mitomycin C creates a defined but not-overgrown cell monolayer suitable for assays up to 42 h without changing the culture medium.
“We set up the GB cell number (18,000 cells/insert) to seed in the presence of an antimitotic treatment (Mitomycin C) to create a defined but not-overgrown cell monolayer, suitable for assays up to 42 h, without changing the Culture Medium.”
Quote in the paperSubstantial claim - Interpretationreasoning
The smaller size of inserts reduces amounts of cells, medium, reagents and especially EVs needed for the assay.
“The smaller size of the inserts compared to 6- or 12-well plates normally used for making the scratch, offers the significant advantage of reducing the amounts of cells, medium, reagents and especially of EVs needed for the assay.”
Quote in the paperSubstantial claim- Rests on an assumption: The scratch assay has low reproducibility of the scratch, resulting in heterogeneous wound areas and affecting reliability and reproducibility.
- Interpretationreasoning
Small differences in initial cell distribution within the insert can be amplified over time.
“As a consequence, small differences in the initial cell distribution within the insert, potentially arising from microflows during the addition of the cell suspension and/or movements of the well plate during adhesion, can be amplified over time.”
Quote in the paperSubstantial claim- Rests on an assumption: U87 GB cells have a highly adhesive phenotype, making them especially sensitive to minor variations during seeding.
- Interpretationreasoning
Continuous incubation with Mitomycin C for 42 h avoids confounding effects on evaluation of cell migration.
“We set up continuous incubation with an antimitotic drug such as Mitomycin C to inhibit cell proliferation for 42 h, avoiding confounding effects on the evaluation of cell migration.”
Quote in the paperSubstantial claim- Rests on an assumption: The wound healing assay can be inherently heterogeneous, potentially concealing complex underlying dynamics.
- Interpretationreasoning
Early asymmetries due to the reduced surface area of the insert may become increasingly evident during wound closure, particularly under treatment conditions.
“to the reduced surface area of the insert, these early asymmetries may become increasingly evident during wound closure, particularly under treatment conditions, and should therefore be carefully considered when interpreting migration data.”
Quote in the paperSubstantial claim - Study design and conductcitation
Taking two images of each technical replicate at 5× magnification and measuring the entire gap area corresponding to the sum of the two imaging fields provides an accurate representation of cell migratory behavior inside the well.
“Taking two images of each technical replicate at 5× magnification and measuring the entire gap area corresponding to the sum of the two imaging fields can provide an accurate representation of cell migratory behavior inside the well.”
Quote in the paperSubstantial claim - Interpretationreasoning
The approach combining pooled and individual EVs enhances the reliability of the experiments.
“This approach combines the consistency of pooled EVs with the specificity of individual batches, thereby enhancing the reliability of the experiments.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 24
- May combine two claims. 10
Probably not a substantial claim: may not pass S3 (load-bearing).
FindingcitationThe ratio of our extracellular vesicle batches aligns with the ratio reported for large extracellular vesicle fractions of similar size isolated from breast cancer cell lines.
“The ratio of our EV batches aligns with the ratio reported for large EV fractions of a similar size isolated from breast cancer cell lines, either after 16,500× g sedimentation alone or followed by density cushion centrifugation [53].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [53] Lischnig, 2022, Quantitative Proteomics Identifies Proteins Enriched in Large and Small Extracellular Vesicles, Mol. Cell. Proteom. source
May combine two claims.
FindingcitationThe identity of the extracellular vesicles was confirmed by showing they possessed specific positive markers CD63 and HSP70, while lacking the negative marker Calnexin.
“The identity of the EVs was confirmed by showing they possessed specific positive markers, such as the CD63 membrane-bound marker and the HSP70 luminal marker, while lacking negative markers specific of intracellular compartments (Calnexin) [43].”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [43] Mantile, F., Kisovec, M., Adamo, G., Romancino, D.P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M.P., and Kisslinger, A. (2022). A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications. Cancers, 14. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe Videodrop system detects nanoparticles in solution by analyzing the interference pattern generated by light scattered by each particle, enabling real-time tracking of their Brownian motion.
“The Videodrop system detects nanoparticles in solution by analyzing the interference pattern generated by light scattered by each particle, enabling real-time tracking of their Brownian motion.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [52] Romolo, A., Jan, Z., Zavec, A.B., Kisovec, M., Arrigler, V., Spasovski, V., Pocsfalvi, G., Kogej, K., and Kralj-Iglič, V. (2022). Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy. Int. J. Mol. Sci., 23. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe core methodology was first established in a previous study on the effect of NTERA2 teratocarcinoma-derived extracellular vesicles on U87 glioblastoma cell migration.
“The core methodology was first established in our previous study on the effect of NTERA2 teratocarcinoma-derived EVs on the U87 GB cell migration [43]”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [43] Mantile, F., Kisovec, M., Adamo, G., Romancino, D.P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M.P., and Kisslinger, A. (2022). A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications. Cancers, 14. source
Classified as probably not a substantial claim.
Study design and conductcitationExtracellular vesicle isolation and characterization were performed according to MISEV 2018 and 2023.
“EV isolation and characterization were performed according to MISEV 2018 and 2023 [49,50], as previously reported [43].”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [49] Witwer, 2018, Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines, J. Extracell. Vesicles source
- cites [50] Welsh, 2023, Minimal Information for Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced Approaches, J. Extracell. Vesicles source
- cites [43] Mantile, F., Kisovec, M., Adamo, G., Romancino, D.P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M.P., and Kisslinger, A. (2022). A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications. Cancers, 14. source
Materials and Methods §2.2 describes EV isolation by differential centrifugation, and §2.11 says the study followed MISEV2018. Results §3.1 describes EV characterization using markers recommended by MISEV2018. The source does not support the attributed reference to MISEV 2023. (LLM, conf=0.90)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationThe particle-to-protein ratio has been proposed as an indicator for estimating the purity of EV preparations, although its applicability requires careful evaluation since expected ratios can vary depending on factors such as EV origin and isolation method.
“The particle-to-protein ratio has been proposed as an indicator for estimating the purity of EV preparations [60], although its applicability requires careful evaluation [49,50], since expected ratios can vary depending on multiple factors, such as EV origin and isolation method.”
Quote in the paperNot a substantial claim3 of 3 sources checked- cites [60] Webber, 2013, How Pure Are Your Vesicles?, J. Extracell. Vesicles source
- cites [49] Witwer, 2018, Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines, J. Extracell. Vesicles source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured - cites [50] Welsh, 2023, Minimal Information for Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced Approaches, J. Extracell. Vesicles source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataTrypan Blue staining at the end of treatment confirmed that 2 μg/mL Mitomycin C effectively blocked cell division without causing significant toxicity.
“Trypan Blue staining at the end of the treatment confirmed that 4 and 6 µg/mL Mitomycin C were highly cytotoxic, whereas 2 µg/mL effectively blocked cell division without causing significant toxicity (Figure 4B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingnone givenU87 cells do not migrate as a compact front but rather independently, so the distance between the two fronts can be extremely heterogeneous.
“This is particularly true when cells do not migrate as a compact front but rather independently, as in the case with U87 cells, meaning that the distance between the two fronts can be extremely heterogeneous.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBatch 2 had a higher relative standard deviation (37%) compared to Batch 1 (22%) and Batch 3 (12%), indicating greater uncertainty in particle concentration per μg.
“Batch 2 had a higher relative standard deviation (37%) compared to Batch 1 (22%) and Batch 3 (12%), indicating greater uncertainty in particle concentration per µg.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).
Interpretationnone givenThe protocol presented here has been further refined to ensure higher reproducibility and detailed quantification.
“The protocol presented here has been further refined to ensure higher reproducibility and detailed quantification.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationNTERA2 cells can locate and target glioma neoplasms in mouse models, particularly following retinoic acid treatment.
“NTERA2 teratocarcinoma cells closely resemble neural stem/progenitor cells, as they are capable of differentiating into glia and neurons, and of locating and targeting glioma neoplasms in mouse models, particularly following treatment with retinoic acid [45,46,47,48].”
Quote in the paperSubstantial claim0 of 4 sources checked- cites [45] Andrews, 1998, Teratocarcinomas and Human Embryology: Pluripotent Human EC Cell Lines. Review Article, Apmis source
- cites [46] Ahmed, 2011, The Use of Neural Stem Cells in Cancer Gene Therapy: Predicting the Path to the Clinic, Curr. Opin. Mol. Ther.
Not checked in the free run.
- cites [47] Attia, 2019, Cationic Niosome-Based HBMP7 Gene Transfection of Neuronal Precursor NT2 Cells to Reduce the Migration of Glioma Cells in Vitro, J. Drug Deliv. Sci. Technol. source
- cites [48] Zhao, 2010, Human NT2 Neural Precursor-Derived Tumor-Infiltrating Cells as Delivery Vehicles for Treatment of Glioblastoma, Hum. Gene Ther. source
Probably not a substantial claim: may not pass S4 (based).
Study design and conductnone givenQuantifying wound closure based on the total gap, calculated by summing the area of both imaging fields, minimizes potential bias caused by spatial heterogeneity.
“Quantify wound closure based on the total gap, calculated by summing the area of both imaging fields to minimize potential bias caused by spatial heterogeneity.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.
InterpretationreasoningThis formalized protocol provides a valuable tool to support the reproducibility of the experiments, while ensuring quality assurance of procedures and quality control of data.
“This formalized protocol provides a valuable tool to support the reproducibility of the experiments, while ensuring quality assurance of procedures and quality control of data.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenIn our study, cell samples were excluded when the total wound area at t0, calculated across two imaging fields, deviated by more than 10% from the corresponding expected value.
“In our study, cell samples were excluded when the total wound area at t0, calculated across two imaging fields, deviated by more than 10% from the corresponding expected value.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNTERA2 cells are capable of differentiating into glia and neurons.
“NTERA2 teratocarcinoma cells closely resemble neural stem/progenitor cells, as they are capable of differentiating into glia and neurons, and of locating and targeting glioma neoplasms in mouse models, particularly following treatment with retinoic acid [45,46,47,48].”
Quote in the paperNot a substantial claim0 of 4 sources checked- cites [45] Andrews, 1998, Teratocarcinomas and Human Embryology: Pluripotent Human EC Cell Lines. Review Article, Apmis source
- cites [46] Ahmed, 2011, The Use of Neural Stem Cells in Cancer Gene Therapy: Predicting the Path to the Clinic, Curr. Opin. Mol. Ther.
Not checked in the free run.
- cites [47] Attia, 2019, Cationic Niosome-Based HBMP7 Gene Transfection of Neuronal Precursor NT2 Cells to Reduce the Migration of Glioma Cells in Vitro, J. Drug Deliv. Sci. Technol. source
- cites [48] Zhao, 2010, Human NT2 Neural Precursor-Derived Tumor-Infiltrating Cells as Delivery Vehicles for Treatment of Glioblastoma, Hum. Gene Ther. source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe wound healing assay is one of the most frequently used methods for studying cell migration.
“The wound healing assay remains one of the most frequently used methods for studying cell migration [37].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [37] Liang, 2007, In Vitro Scratch Assay: A Convenient and Inexpensive Method for Analysis of Cell Migration in Vitro, Nat. Protoc. source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenCells were maintained at low passage number (<3 passages post-thawing) for all assays.
“Cells were maintained at low passage number (<3 passages post-thawing) for all assays.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThis approach provides a rapid, label-free assessment of extracellular vesicle concentration and size distribution, with high reproducibility and without requiring sample fixation or additional processing.
“This approach provides a rapid, label-free assessment of EV concentration and size distribution, with high reproducibility and without requiring sample fixation or additional processing.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataContinuous incubation with 2 μg/mL Mitomycin C for up to 42 h effectively inhibits proliferation without affecting viability or migration.
“Overall, continuous incubation with 2 µg/mL Mitomycin C for up to 42 h effectively inhibits proliferation without affecting viability or migration, making this condition suitable for wound healing assays of at least 42 h duration.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenFor each acquisition, a minimum of 300 particles were tracked to ensure robust estimation of particle parameters.
“For each acquisition, a minimum of 300 particles were tracked to ensure robust estimation of particle parameters.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningDue to the reduced surface area of the insert, early asymmetries may become increasingly evident during wound closure, particularly under treatment.
“Due to the reduced surface area of the insert, these early asymmetries may become increasingly evident during wound closure, particularly under treatment”
Quote in the paperNot a substantial claim- Rests on an assumption: U87 GB cells have a highly adhesive phenotype, making them especially sensitive to minor variations during seeding.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
Interpretationnone givenThe protocol can be readily adapted to other GB cell lines and/or adherent tumor cell cultures, provided that critical process parameters are appropriately identified and described.
“Furthermore, it can be readily adapted to other GB cell lines and/or adherent tumor cell cultures, provided that critical process parameters are appropriately identified and described.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Interpretationnone givenDifferent EV subtypes and fractions may require specific dose optimization due to heterogeneity in size, particle-to-protein ratio, physicochemical properties, molecular content, and functional activity, as well as in isolation and characterization methods.
“Different EV subtypes and fractions may require specific dose optimization due to heterogeneity in size, particle-to-protein ratio, physicochemical properties, molecular content, and functional activity, as well as in isolation and characterization methods.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Interpretationnone givenEV concentrations above 20 μg/mL protein may lead to EV aggregation, which can affect EV uptake and subsequent phenotypic responses.
“Concentrations below this range may be insufficient to produce a reproducible and stable effect, while concentrations above this range may lead to EV aggregation, which can affect EV uptake and subsequent phenotypic responses.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S4 (based).
Interpretationnone givenPooling multiple EV preparations provides a more representative sample of the overall population.
“Using individual EV preparations enables precise assessment of batch-specific properties, whereas pooling multiple EV preparations provides a more representative sample of the overall population.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductnone givenAll EV treatments were normalized based on protein concentration (10 μg/mL).
“All EV treatments were normalized based on protein concentration (10 µg/mL), with nanoparticle measurements performed for selected batches (Table 1).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenNot all of the EV preparations included in this protocol have been individually quantified.
“This value should be considered an approximate average, since not all of the preparations included in this protocol have been individually quantified.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
InterpretationreasoningEV concentrations below 5 μg/mL protein may be insufficient to produce a reproducible and stable effect.
“Concentrations below this range may be insufficient to produce a reproducible and stable effect, while concentrations above this range may lead to EV aggregation, which can affect EV uptake and subsequent phenotypic responses.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningUsing ibidi silicone culture inserts creates more homogeneous and reproducible gaps without damaging cells or activating a cellular program that could interfere with measurement of cell migration.
“This approach has the important advantage of creating more homogeneous and reproducible gaps, without damaging the cells or activating a cellular program that could interfere with the measurement of cell migration.”
Quote in the paperSubstantial claim- Rests on an assumption: The scratch assay has low reproducibility of the scratch, resulting in heterogeneous wound areas and affecting reliability and reproducibility.
- Rests on an assumption: The scratch may cause damage to cells at the edge of the wound, affecting cell migration and introducing variability.
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningThe reduced insert size and well-defined outer edge facilitate spatial referencing under manual, non-motorized acquisition.
“Last, but not least, the reduced insert size and well-defined outer edge of the insert facilitate spatial referencing, particularly under manual, non-motorized acquisition settings.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based). May combine two claims.
Study design and conductnone givenThe protocol includes all the necessary details for full replication, as well as guidance on identifying and accounting for potential sources of experimental failure.
“The protocol includes all the necessary details for full replication, as well as guidance on identifying and accounting for potential sources of experimental failure.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
Glioblastoma is an aggressive brain tumor with poor prognosis, largely due to highly infiltrative glioblastoma cells that hamper complete surgical removal and increase relapse likelihood.
“Glioblastoma (GB) is an aggressive brain tumor with a poor prognosis, largely due to the highly infiltrative nature of GB cells, which hampers complete surgical removal and increases the likelihood of relapse.”
The paper relies on this without support.
Irregular wounds can affect the reproducibility of the wound healing assay.
“Irregular wounds might affect the reproducibility of the assay.”
The paper relies on this without support.
The optimal Mitomycin C concentration to inhibit cell proliferation without causing cytotoxicity differs for each cell line.
“The concentration of Mitomycin C to be used in the Experimental Medium may differ for each cell line and should be determined in advance to inhibit cell proliferation without causing cytotoxicity.”
The paper relies on this without support.
Performing the wound healing assay for at least 24 h and no more than 42 h preserves the healthy state of the cells.
“We recommend performing the assay for at least 24 h and no more than 42 h to preserve the healthy state of the cells.”
The paper relies on this without support.
The wound healing assay can be inherently heterogeneous, potentially concealing complex underlying dynamics.
“Although the wound healing assay is seemingly straightforward to perform and analyze, it can still be inherently heterogeneous, potentially concealing complex underlying dynamics.”
The paper relies on this without support.
The scratch assay has low reproducibility of the scratch, resulting in heterogeneous wound areas and affecting reliability and reproducibility.
“The main weakness lies in the low reproducibility of the scratch, which can result in the wound areas being very heterogeneous, thereby affecting both reliability and reproducibility of the results.”
The paper relies on this without support.
The scratch may cause damage to cells at the edge of the wound, affecting cell migration and introducing variability.
“Moreover, the scratch may cause damage to cells at the edge of the wound, which could affect cell migration and introduce additional variability to the assay.”
The paper relies on this without support.
U87 GB cells have a highly adhesive phenotype, making them especially sensitive to minor variations during seeding.
“U87 GB cells are characterized by a highly adhesive phenotype, making them especially sensitive to minor variations during the seeding procedure.”
The paper relies on this without support.
10,000× g sedimented EV fractions remain less extensively characterized than 118,000× g isolated EVs.
“In particular, we provide a valuable reference for 10,000× g sedimented EV fractions, which remain less extensively characterized than 118,000× g isolated EVs, despite attracting increasing interest.”
Cites
Théry, C., Witwer, K. W., Aikawa, E., Alcaraz, M. J., Anderson, J. D., Andriantsitohaina, R., Antoniou, A., Arab, T., Archer, F., Atkin‐Smith, G. K., et al. (2018) · Journal of Extracellular Vesicles · doi:10.1080/20013078.2018.1535750 · Cited by 11,099 · see citing works
The biology <b>,</b> function <b>,</b> and biomedical applications of exosomes
Kalluri, R., & LeBleu, V. S. (2020) · Science · doi:10.1126/science.aau6977 · Cited by 10,770 · see citing works
Shedding light on the cell biology of extracellular vesicles
van Niel, G., D'Angelo, G., & Raposo, G. (2018) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm.2017.125 · Cited by 8,369 · see citing works
In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro
Liang, C.-C., Park, A. Y., & Guan, J.-L. (2007) · Nature Protocols · doi:10.1038/nprot.2007.30 · Cited by 4,696 · cited 1× · see citing works
“The wound healing assay remains one of the most frequently used methods for studying cell migration [37].”
Welsh, J. A., Goberdhan, D. C. I., O'Driscoll, L., Buzas, E. I., Blenkiron, C., Bussolati, B., Cai, H., Di Vizio, D., Driedonks, T. A. P., Erdbrügger, U., et al. (2024) · Journal of Extracellular Vesicles · doi:10.1002/jev2.12404 · Cited by 4,663 · see citing works
Extracellular vesicles as tools and targets in therapy for diseases
Kumar, M. A., Baba, S. K., Sadida, H. Q., Al-Marzooqi, S. K., Jerobin, J., Altemani, F. H., Algehainy, N. A., Alanazi, M. A., Abou‐Samra, A., Kumar, R., et al. (2024) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-024-01735-1 · Cited by 1,447 · cited 1× · see citing works
“For this reason, EVs are increasingly acknowledged as vital mediators that coordinate intercellular communication in both health and disease [10–13], including cardiac, metabolic and renal pathologies [14,15], as well as neurological disorders [16–18] and cancers [4,19,20].”
The roles of extracellular vesicles in the immune system
Buzás, E. I. (2022) · Nature reviews. Immunology · doi:10.1038/s41577-022-00763-8 · Cited by 1,236 · see citing works
An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays
(2020) · doi:10.1371/journal.pone.0232565 · Cited by 851 · cited 1× · see citing works
“You can define the area both manually or using the ImageJ Wound Healing Size Tool plugin, described by Suarez-Arnedo and coauthors [55].”
Webber, J., & Clayton, A. (2013) · Journal of Extracellular Vesicles · doi:10.3402/jev.v2i0.19861 · Cited by 817 · see citing works
The principles of directed cell migration
SenGupta, S., Parent, C. A., & Bear, J. E. (2021) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-021-00366-6 · Cited by 654 · cited 1× · see citing works
“Furthermore, cell movement is involved in numerous pathological processes, such as immune activation, tissue repair, and cancer invasion and metastasis [1,2].”
A review of the regulatory mechanisms of extracellular vesicles-mediated intercellular communication
Liu, Y.-J., & Wang, C. (2023) · Cell Communication and Signaling · doi:10.1186/s12964-023-01103-6 · Cited by 362 · see citing works
Quantitative Proteomics Identifies Proteins Enriched in Large and Small Extracellular Vesicles
Lischnig, A., Bergqvist, M., Ochiya, T., & Lässer, C. (2022) · Molecular & Cellular Proteomics · doi:10.1016/j.mcpro.2022.100273 · Cited by 189 · cited 1× · see citing works
“The ratio of our EV batches aligns with the ratio reported for large EV fractions of a similar size isolated from breast cancer cell lines, either after 16,500 × g sedimentation alone or followed by density cushion centrifugation [53].”
<i>In vitro</i> cell migration quantification method for scratch assays
Bobadilla, A. V. P., Arévalo, J., Sarró, E., Byrne, H. M., Maini, P. K., Carraro, T., Balocco, S., Meseguer, A., & Alarcón, T. (2019) · Journal of The Royal Society Interface · doi:10.1098/rsif.2018.0709 · Cited by 179 · see citing works
All Roads Lead to Directional Cell Migration
Shellard, A., & Mayor, R. (2020) · Trends in Cell Biology · doi:10.1016/j.tcb.2020.08.002 · Cited by 172 · cited 1× · see citing works
“Furthermore, cell movement is involved in numerous pathological processes, such as immune activation, tissue repair, and cancer invasion and metastasis [1,2].”
Mycoplasma infection of cultured cells
Hay, R. J., Macy, M. L., & Chen, T. R. (1989) · Nature · doi:10.1038/339487a0 · Cited by 145 · cited 1× · see citing works
“As Mycoplasma is an invisible contaminant that could alter the structure, metabolism and behavior of the cells [54], thereby affecting the reproducibility of results, we verified its absence in the cell cultures using the Mycoplasma PCR detection kit (Applied Biological Materials Inc., Richmond,…”
Teratocarcinomas and human embryology: Pluripotent human EC cell lines
ANDREWS, P. W. (1998) · APMIS · doi:10.1111/j.1699-0463.1998.tb01331.x · Cited by 140 · cited 1× · see citing works
“NTERA2 teratocarcinoma cells closely resemble neural stem/progenitor cells, as they are capable of differentiating into glia and neurons, and of locating and targeting glioma neoplasms in mouse models, particularly following treatment with retinoic acid [45–48].”
InfluentialEXTRACELLULAR VESICLES: CRITICAL PLAYERS DURING CELL MIGRATION
Sung, B., Parent, C., & Weaver, A. M. (2021) · Developmental Cell · doi:10.1016/j.devcel.2021.03.020 · Cited by 133 · cited 1× · see citing works
“They modulate tumor cell traits such as proliferation, epithelial–mesenchymal transition, migration, invasion, dormancy, and chemoresistance, while shaping interactions with the tumor microenvironment and immune system [4,21–27].”
Unravelling cell migration: defining movement from the cell surface
Merino-Casallo, F., Gómez‐Benito, M. J., Hervás-Raluy, S., & García‐Aznar, J. M. (2022) · Cell Adhesion & Migration · doi:10.1080/19336918.2022.2055520 · Cited by 108 · see citing works
Chen, H., Zhang, S., Fang, Q., He, H., Ren, J., Sun, D., Lai, J., Ma, A.-Q., Chen, Z., Liu, L., et al. (2022) · ACS Nano · doi:10.1021/acsnano.2c08861 · Cited by 89 · see citing works
Vang Mouritzen, M., & Jenssen, H. (2018) · Journal of Visualized Experiments · doi:10.3791/57691 · Cited by 83 · see citing works
The use of neural stem cells in cancer gene therapy: predicting the path to the clinic.
Ahmed, A. U., Alexiades, N. G., & Lesniak, M. S. (2010) · PubMed · Cited by 83 · see citing works
Extracellular vesicles: New horizons in neurodegeneration
Chen, J., Tian, C., Xiong, X., Yang, Y., & Zhang, J. (2025) · eBioMedicine · doi:10.1016/j.ebiom.2025.105605 · Cited by 53 · see citing works
Extracellular Vesicles: Recent Advances and Perspectives
Ragni, E. (2025) · Frontiers in Bioscience-Landmark · doi:10.31083/fbl36405 · Cited by 52 · see citing works
Extracellular vesicles in neurodegenerative diseases: A systematic review
Raghav, A., Singh, M., Jeong, G., Giri, R., Agarwal, S., Kala, S., & Gautam, K. (2022) · Frontiers in Molecular Neuroscience · doi:10.3389/fnmol.2022.1061076 · Cited by 49 · see citing works
On the road: extracellular vesicles in intercellular communication
Weßler, S., & Meisner-Kober, N. C. (2025) · Cell Communication and Signaling · doi:10.1186/s12964-024-01999-8 · Cited by 46 · see citing works
Long non‑coding RNA PVT1 promotes glioma cell proliferation and invasion by targeting miR‑200a
Zhang, Y., Yang, G., & Luo, Y. (2018) · Experimental and Therapeutic Medicine · doi:10.3892/etm.2018.7083 · Cited by 43 · see citing works
Recent advances in microfluidics-based cell migration research
Ren, J., Wang, N., Guo, P., Fan, Y., Lin, F., & Wu, J. (2022) · Lab on a Chip · doi:10.1039/d2lc00397j · Cited by 38 · cited 1× · see citing works
“More recent methods rely on transient agarose spots or three-dimensional (3D) microfluidic channels [34–36]; however, these approaches have not yet been widely adopted.”
Exogenous Extracellular Vesicles as Emerging Platforms in Translational Medicine
Bai, M., Li, Z., Shi, T., et al. (2025) · BIO Integration · doi:10.15212/bioi-2025-0122 · Cited by 36 · cited 1× · see citing works
“Thanks to their intrinsic biocompatibility, low immunogenicity, and specific bioactivity, exogenous EVs, whether pristine or engineered for drug delivery, are poised to become the next-generation platforms for precision therapy, including in oncological applications [28,29].”
Extracellular Vesicle-Based Therapeutic Cargo Delivery for Cancer Therapy
Huang, Z., Cheng, J., Deng, Z., Liu, C., Huang, T., & Lin, W. (2025) · International Journal of Nanomedicine · doi:10.2147/ijn.s548006 · Cited by 30 · cited 1× · see citing works
“Thanks to their intrinsic biocompatibility, low immunogenicity, and specific bioactivity, exogenous EVs, whether pristine or engineered for drug delivery, are poised to become the next-generation platforms for precision therapy, including in oncological applications [28,29].”
Zhao, Y., & Wang, S. (2010) · Human Gene Therapy · doi:10.1089/hum.2009.196 · Cited by 25 · cited 1× · see citing works
“NTERA2 teratocarcinoma cells closely resemble neural stem/progenitor cells, as they are capable of differentiating into glia and neurons, and of locating and targeting glioma neoplasms in mouse models, particularly following treatment with retinoic acid [45–48].”
InfluentialHitting the target: cell signaling pathways modulation by extracellular vesicles
Cerrotti, G., Buratta, S., Latella, R., Calzoni, E., Cusumano, G., Bertoldi, A., Porcellati, S., Emiliani, C., & Urbanelli, L. (2024) · Extracellular Vesicles and Circulating Nucleic Acids · doi:10.20517/evcna.2024.16 · Cited by 25 · cited 1× · see citing works
“Interaction between EVs and recipient cells occurs through various mechanisms, including direct membrane receptor activation, or internalization through endocytosis, macropinocytosis or phagocytosis, with subsequent modulation of signaling pathways and gene expression [8,9].”
Sausset, R., Krupova, Z., Guédon, É., Péron, S., Grangier, A., Petit, M., Sordi, L. D., & Paepe, M. D. (2023) · Journal of Extracellular Biology · doi:10.1002/jex2.75 · Cited by 24 · cited 1× · see citing works
“Interferometric light microscopy (ILM) analysis [51] was also performed on random batches to control the number and size distribution of the particles.”
Challenges and Promise for Glioblastoma Treatment through Extracellular Vesicle Inquiry
Liguori, G. L. (2024) · Cells · doi:10.3390/cells13040336 · Cited by 24 · cited 1× · see citing works
“EVs are key players in regulating GB pathological features, including cell invasiveness and migration [30–32].”
Self-citationCirculating Extracellular Vesicles in Cardiovascular Disease
Cappucci, I. P., Tremoli, E., Zavan, B., & Ferroni, L. (2025) · International Journal of Molecular Sciences · doi:10.3390/ijms26146817 · Cited by 24 · cited 1× · see citing works
“For this reason, EVs are increasingly acknowledged as vital mediators that coordinate intercellular communication in both health and disease [10–13], including cardiac, metabolic and renal pathologies [14,15], as well as neurological disorders [16–18] and cancers [4,19,20].”
Quality-based model for Life Sciences research guidelines
Digilio, F. A., Lanati, A., Bongiovanni, A., Mascia, A., Carlo, M. D., Barra, A., Cirafici, A. M., Colotti, G., Kisslinger, A., Lacerra, G., et al. (2016) · Accreditation and Quality Assurance · doi:10.1007/s00769-016-1205-0 · Cited by 23 · see citing works
Self-citationCell Migration Assays and Their Application to Wound Healing Assays—A Critical Review
Yang, C., Yin, D., Zhang, H., Badea, I., Yang, S.-M., & Zhang, W. (2024) · Micromachines · doi:10.3390/mi15060720 · Cited by 22 · cited 1× · see citing works
“Despite its widespread use, several articles highlight an increasing number of factors that can severely impact the reliability and reproducibility of the assay, if they are not adequately controlled [38–42], with protocols involving EVs and GB cells being particularly poorly validated.”
Liguori, G. L., & Kralj‐Iglič, V. (2023) · Cancers · doi:10.3390/cancers15184425 · Cited by 20 · cited 1× · see citing works
“In this context, tumor-derived EVs have attracted considerable attention as cancer-targeted therapeutic agents, both in immunotherapy and as drug delivery systems capable of efficiently transporting cargos to malignant cells [22].”
Self-citationThe uptake of extracellular vesicles: Research progress in cancer drug resistance and beyond
Qian, L., Chen, P., Zhang, S., Wang, Z., Guo, Y., Koutouratsas, V., Fleishman, J. S., Huang, C., & Zhang, S. (2025) · Drug Resistance Updates · doi:10.1016/j.drup.2025.101209 · Cited by 20 · see citing works
Extracellular vesicles and the tumour microenvironment
Belényesi, S.-K., Patmore, S., & O’Driscoll, L. (2025) · Biochimica et Biophysica Acta (BBA) - Reviews on Cancer · doi:10.1016/j.bbcan.2025.189275 · Cited by 20 · see citing works
Establishment of a cell-based wound healing assay for bio-relevant testing of wound therapeutics
Planz, V., Wang, J., & Windbergs, M. (2018) · Journal of Pharmacological and Toxicological Methods · doi:10.1016/j.vascn.2017.10.003 · Cited by 19 · see citing works
Romolo, A., Jan, Z., Bedina Zavec, A., Kisovec, M., Arrigler, V., Spasovski, V., Podobnik, M., Iglič, A., Pocsfalvi, G., Kogej, K., et al. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms232415801 · Cited by 19 · cited 1× · see citing works
“We used the Videodrop instrument (Myriade, Paris, France), according to the manufacturer’s recommendations [52].”
Mantile, F., Kisovec, M., Adamo, G., Romancino, D. P., Hočevar, M., Božič, D., Bedina Zavec, A., Podobnik, M., Stoppelli, M. P., Kisslinger, A., et al. (2022) · Cancers · doi:10.3390/cancers14153700 · Cited by 18 · cited 4× · see citing works
“[49,50], as previously reported [43].”
InfluentialSelf-citationAttia, N., Mashal, M., Grijalvo, S., Eritja, R., Puras, G., & Pedraz, J. L. (2019) · Journal of Drug Delivery Science and Technology · doi:10.1016/j.jddst.2019.101219 · Cited by 17 · cited 1× · see citing works
“Furthermore, it has been found that conditioned medium from NTERA2 cells affects GB cell behavior [47], suggesting that GB cells are responsive to signals emitted by NTERA2 cells.”
Oldenburg, J., Maletzki, L., Strohbach, A., Bellé, P., Siewert, S., Busch, R., Felix, S. B., Schmitz, K.-P., & Stiehm, M. (2021) · BMC Molecular and Cell Biology · doi:10.1186/s12860-021-00369-3 · Cited by 16 · see citing works
Extracellular Vesicles in Cancer: Mechanistic Insights and Clinical Applications
Alhumaydhi, F. A., & Anwar, S. (2026) · Cancers · doi:10.3390/cancers18030537 · Cited by 16 · cited 1× · see citing works
“For this reason, EVs are increasingly acknowledged as vital mediators that coordinate intercellular communication in both health and disease [10–13], including cardiac, metabolic and renal pathologies [14,15], as well as neurological disorders [16–18] and cancers [4,19,20].”
Mimi, M. A., Sohag, S. M., Toma, S. N., & Hasan, M. M. (2025) · Future Pharmacology · doi:10.3390/futurepharmacol5030048 · Cited by 14 · cited 1× · see citing works
“For this reason, EVs are increasingly acknowledged as vital mediators that coordinate intercellular communication in both health and disease [10–13], including cardiac, metabolic and renal pathologies [14,15], as well as neurological disorders [16–18] and cancers [4,19,20].”
D’Antonio, C., & Liguori, G. L. (2024) · Frontiers in Immunology · doi:10.3389/fimmu.2024.1441914 · Cited by 9 · cited 1× · see citing works
“They modulate tumor cell traits such as proliferation, epithelial–mesenchymal transition, migration, invasion, dormancy, and chemoresistance, while shaping interactions with the tumor microenvironment and immune system [4,21–27].”
Self-citationMethods and computational tools to study eukaryotic cell migration in vitro
Toscano, E., Cimmino, E., Pennacchio, F. A., Riccio, P., Poli, A., Liu, Y., Maiuri, P., Sepe, L., & Paolella, G. (2024) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2024.1385991 · Cited by 8 · cited 1× · see citing works
“More recent methods rely on transient agarose spots or three-dimensional (3D) microfluidic channels [34–36]; however, these approaches have not yet been widely adopted.”
The influence of extracellular vesicles on tumor evolution and resistance to therapy
Baston, E., García-Agulló, J., & Peinado, H. (2025) · Physiological Reviews · doi:10.1152/physrev.00019.2024 · Cited by 8 · see citing works
Transient Agarose Spot (TAS) Assay: A New Method to Investigate Cell Migration
Veres-Székely, A., Pap, D., Szebeni, B., et al. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23042119 · Cited by 7 · see citing works
Modeling Glioblastoma for Translation: Strengths and Pitfalls of Preclinical Studies
D’Antonio, C., & Liguori, G. L. (2025) · Biology · doi:10.3390/biology14111490 · Cited by 7 · cited 1× · see citing works
“Traditionally, two-dimensional (2D) GB monolayer cell cultures have been used for the rapid and easy testing of candidate anti-migratory drugs [33].”
Self-citationChung, W. Y., Lam, S., Pernari, B., Kim, Y., Cho, S., Burger, D. E., & Sweeney, G. (2025) · Mechanisms of Ageing and Development · doi:10.1016/j.mad.2025.112078 · Cited by 6 · cited 1× · see citing works
“For this reason, EVs are increasingly acknowledged as vital mediators that coordinate intercellular communication in both health and disease [10–13], including cardiac, metabolic and renal pathologies [14,15], as well as neurological disorders [16–18] and cancers [4,19,20].”
Extracellular vesicles in tumor microenvironment modulation and clinical diagnosis
Liu, H., Niu, D., Yu, T., Wang, Y.-H., Ran, K.-L., Liu, Y.-H., Yin, D.-C., & Zhang, C. (2025) · Pathology - Research and Practice · doi:10.1016/j.prp.2025.156339 · Cited by 4 · see citing works
Hamdalla, R. H., Bhaskar, V. B., & Tian, C. (2026) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2025.1710150 · Cited by 4 · cited 1× · see citing works
“For this reason, EVs are increasingly acknowledged as vital mediators that coordinate intercellular communication in both health and disease [10–13], including cardiac, metabolic and renal pathologies [14,15], as well as neurological disorders [16–18] and cancers [4,19,20].”
Glioblastoma exosomes reprogramming the tumor microenvironment and evading therapeutic challenges
Tatode, A., Premchandani, T., Chaudhary, A. A., Ali, M. A. M., Qutub, M., Taksande, J., Khan, R., Hussain, U. M., & Umekar, M. (2026) · Molecular Biology Reports · doi:10.1007/s11033-026-11526-2 · Cited by 3 · cited 1× · see citing works
“EVs are key players in regulating GB pathological features, including cell invasiveness and migration [30–32].”
Mimi, M. A., Sohag, S. M., Toma, S. N., et al. (2025) · doi:10.20944/preprints202506.1931.v1 · Cited by 3
(2024) · PubMed · Cited by 2 · see citing works
Hollmann, S., Regierer, B., D’Elia, D., Kisslinger, A., & Liguori, G. L. (2022) · Advances in biomembranes and lipid self-assembly · doi:10.1016/bs.abl.2022.05.001 · Cited by 2 · see citing works
Self-citationColangelo, T., Saponaro, A. A., Mazzoccoli, G., Serviddio, G., & Villani, R. (2025) · International Journal of Molecular Sciences · doi:10.3390/ijms262411826 · Cited by 1 · see citing works
D’Antonio, C., Mantile, F., & Liguori, G. L. (2025) · doi:10.55295/psl.12.2025.iii9
Self-citation
Flagged first, then by citations
Research works printed but not linked: 1
- Mantile, F., Franco, P., Stoppelli, M.P., and Liguori, G.L. (2020). Biological Role and Clinical Relevance of Extracellular Vesicles as Key Mediators of Cell Communication in Cancer. Biological Membrane Vesicles: Scientific, Biotechnological and Clinical Considerations. Advances in Biomembranes and Lipid Self-Assembly, Elsevier.
- Authors
- D’Antonio, C., Mantile, F., Pocsfalvi, G., & Liguori, G. L.
- Affiliations
- Institute of Genetics and Biophysics (IT)
- Institute of Biosciences and Bioresources (IBBR), National Research Council of Italy, 80131 Naples, Italy
- Venue
- Methods and Protocols
- Publisher
- MDPI AG
- Published
- 2026-05-31
- Volume
- 9
- Issue
- 3
- Pages
- 81
- ISSN
- 2409-9279 (ISSN-L)
- PMC
- PMC13305267
- OpenAlex
- W7163367707
- Cited by
- 0
- References
- 53
- FWCI
- 0.00
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Methods and Protocols (publishedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Archivio Istituzionale della Ricerca - Università degli Studi della Campania "Luigi Vanvitelli" (submittedVersion)
- Funders
- European Union—NextGenerationEU (CEVITA)
- Regione Campania (PO FESR 2014–2020 SATIN)
- University of Ljubljana (Nanostructurome)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Keywords
- glioblastoma, extracellular vesicles, cell migration, wound healing assay, mitomycin C, cell proliferation inhibition, tumor invasion, assay optimization
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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