Abstract: Characterization of the size and material properties of particles in liquid suspensions is in very high demand, for example, in the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, existing methods are limited in their ability to decipher the constituents of realistic samples. Here we introduce iNTA as a new method that combines interferometric detection of scattering with nanoparticle tracking analysis to reach unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multicomponent and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Abstract: Extracellular vesicles (EVs) have attracted much attention as potential diagnostic biomarkers for human diseases. Although both plasma and serum are utilized as a source of blood EVs, it remains unclear whether, how and to what extent the choice of plasma and serum affects the experimental results. To address this issue, in this study, we performed comprehensive characterization of EV fractions derived from plasma and serum, and investigated the differences between these blood EVs. We demonstrated by nanoparticle tracking analysis that EV fractions derived from serum contain more particles than those from plasma of mice. Proteomic analysis demonstrated that platelet-associated proteins are selectively enriched in serum EV fractions from both mice and humans. A literature review of proteomic data of human blood EVs reported by other groups further confirmed that selective enrichment of platelet-associated proteins is commonly observed in serum EVs, and confers different proteome profiles to plasma EVs. Our data provide experimental evidence that EV fractions derived from serum generally contain additional EVs that are released from platelets, which may qualitatively and quantitatively alter EV profiles when using serum as a source of blood EVs.

Abstract: In recent years silver nanoparticles (Ag NPs) gained increased and widespread applications in various fields of industry, technology, and medicine. This study describes the green synthesis of silver nanoparticles (Ag NPs) applying a low-molecular-weight fraction (LMF) of Royal Jelly, the nanoparticle characterization, and particularly their antibacterial activity. The optical properties of NPs, characterized by UV-Vis absorption spectroscopy, showed a peak at ~ 430 nm. The hydrodynamic radius and concentration were determined by complementary dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The particle morphology was investigated using transmission electron microscopy (TEM), and the crystallinity of the silver was confirmed by X-ray diffraction (XRD). The antibacterial activities were evaluated utilizing Gram-negative and Gram-positive bacteria and colony counting assays. The growth inhibition curve method was applied to obtain information about the corresponding minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) required. Obtained results showed that (i) the sizes of Ag NPs are increasing within the increase of silver ion precursor concentration, (ii) DLS, in agreement with NTA, showed that most particles have dimensions in the range of 50-100 nm; (iii) E. coli was more susceptible to all Ag NP samples compared to B. subtilis.

Abstract: Ovarian cancer (OC) is one of the serious threats to the health of women worldwide, and accurate biomarkers are urgently demanded for early diagnosis of OC. We have previously confirmed that miR-205 promotes the invasion and metastasis of OC cells by inhibiting the expression of the tumor suppressor gene TCF21. In this study, we used liquid biopsy technology to detect the expression levels of the four genes, miR-205, CA125, HE4 and TCF21, in the exosomes of plasma of OC patients. Combined with analysis of clinicopathological parameters of OC patients, we aimed to provide efficient and non-invasive laboratory biomarkers for early diagnosis of OC.36 OC patients who were diagnosed in local hospitals from September 2020 to July 2021 were selected as OC group, 31 cases of surgically diagnosed with ovarian benign lesions were selected as benign group, and 32 healthy people who underwent physical examination during the same period were selected as a control group. We employed transmission electron microscope (TEM), Western blotting (WB), and nanoparticle tracking analysis (NTA) to identify biomarkers in the exosomes extracted from plasma of the three groups. The RNA levels of miR-205, CA125, HE4 and TCF21 genes in plasma exosomes were detected by real-time quantitative PCR (qRT-PCR) method. We used clinical pathological parameters and the Receiver Operating Characteristic (ROC) curves to evaluate the diagnostic efficacy for the genes detected in plasma exosomes.We found that the expression level of miR-205 in plasma exosomes of the OC group was significantly higher than that of the benign and control groups (P < 0.05), and the level of miR-205 was elevated during the III-IV periods of OC and lymph node metastasis.The level of miR-205 in plasma exosomes is a valuable tumor biomarker to improve OC diagnosis.

Abstract: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH.

Abstract: Extracellular vesicles (EVs) are nanoparticles (30 to 1000 nm in diameter) surrounded by a lipid-bilayer which carry bioactive molecules between local and distal cells and participate in intercellular communication. Because of their small size and heterogenous nature they are challenging to characterize. Here, we discuss commonly used techniques that have been employed to yield information about EV size, concentration, mechanical properties, and protein content. These include dynamic light scattering, nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, atomic force microscopy, western blotting, and optical methods including super-resolution microscopy. We also introduce an innovative technique for EV characterization which involves immobilizing EVs on a microscope slide before staining them with antibodies targeting EV proteins, then using the reflectance mode on a confocal microscope to locate the EV plane. By then switching to the microscope's fluorescence mode, immunostained EVs bearing specific proteins can be identified and the heterogeneity of an EV preparation can be determined. This approach does not require specialist equipment beyond the confocal microscopes that are available in many cell biology laboratories, and because of this, it could become a complementary approach alongside the aforementioned techniques to identify molecular heterogeneity in an EV preparation before subsequent analysis requiring specialist apparatus.

Abstract: Abstract Purpose Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems. Method Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for β3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites. Results The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (β3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 μm vs. 1860.5 μm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs ( P < 0.05). Conclusion This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

Abstract: Extracellular vesicles (EVs) are cell-derived membrane-bound particles, including exosomes and microvesicles that differ in cellular origin, content, and lipid composition. This study reports that exosomes and microvesicles can be simultaneously separated by size using flow field-flow fractionation (FlFFF) employed with field programming and that the detection of low-concentration EV species can be significantly improved using multiangle light scattering (MALS). The efficiency of ultracentrifugation (UC) and ultrafiltration (UF) in isolating EVs from the culture media of DU145 cells was compared, and the results showed that UF retrieves more EVs than UC. Two size fractions (small and large) of both exosomes and microvesicles were collected during the FlFFF runs and examined using Western blotting to confirm each EV, and transmission electron microscopy was performed for size analysis. Sizes were compared using the root-mean-square radius obtained from the MALS calculation. The collected fractions were further examined using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the size-dependent lipidomic profiles of exosomes and microvesicles, showing that lipids were more enriched in the fraction containing large exosomes than in that containing small exosomes; however, an opposite trend was observed with microvesicles. The present study demonstrated that UF followed by FlFFF-MALS can be utilized for the size separation of exosomes and microvesicles without sequential centrifugation, which is useful for monitoring the changes in the size distribution of EVs depending on the biological status along with generating size-dependent lipidomic profiles.

Abstract: Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women.Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography-tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting.Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers.EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

Abstract: Abstract Extracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation [UC], precipitation [EQ‐O, EQ‐TC], size exclusion chromatography [SEC], and ultrafiltration [UF]) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non‐vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool‐derived EVs as a source for novel biomarkers for early CRC detection.

Abstract: Extracellular vesicles (EVs) are a group of communication organelles enclosed by a phospholipid bilayer, secreted by all types of cells. The size of these vesicles ranges from 30 to 1000 nm, and they contain a myriad of compounds such as RNA, DNA, proteins, and lipids from their origin cells, offering a good source of biomarkers. Exosomes (30 to 100 nm) are a subset of EVs, and their importance in future medicine is beyond any doubt. However, the lack of efficient isolation and detection techniques hinders their practical applications as biomarkers. Versatile and cutting-edge platforms are required to detect and isolate exosomes selectively for further clinical analysis. This review paper focuses on lab-on-chip devices for capturing, detecting, and isolating extracellular vesicles. The first part of the paper discusses the main characteristics of different cell-derived vesicles, EV functions, and their clinical applications. In the second part, various microfluidic platforms suitable for the isolation and detection of exosomes are described, and their performance in terms of yield, sensitivity, and time of analysis is discussed.

Abstract: Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease-specific biomolecular cargoes and importance in cell-cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50-200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (μSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young's modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on-chip nanoliter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50 nm), albumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, EVs are directly isolated from undiluted human platelet-poor plasma using μSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent μSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics.

Abstract: Lipid-shelled nanobubbles (NBs) are emerging as potential dual diagnostic and therapeutic agents. Similar to their micron-scale counterparts, microbubbles (1-10 μm), they can act as ultrasound contrast agents as well as locally enhance therapeutic uptake. Recently, it has been shown that the reduced size of NBs (<1 μm) promotes increased uptake and accumulation in tumor interstitial space, which can enhance their diagnostic and therapeutic performance. However, accurate characterization of NB size and concentration is challenging and may limit their translation into clinical use. Their submicron nature limits accuracy of conventional microscopy techniques, while common light scattering techniques fail to distinguish between subpopulations present in NB samples (i.e., bubbles and liposomes). Due to the difficulty in the characterization of NBs, relatively little is known about the influence of size on their therapeutic performance. In this study, we describe a novel method of using a commercially available nanoparticle tracking analysis system, to distinguish between NBs and liposomes based on their differing optical properties. We used this technique to characterize three NB populations of varying size, isolated via centrifugation, and subsequently used this to assess their potential for enhancing localized delivery. Confocal fluorescence microscopy and image analysis were used to quantify the ultrasound enhanced uptake of fluorescent dextran into live colorectal cancer cells. Our results showed that the amount of localized uptake did not follow the expected trends, in which larger NB populations out-perform smaller NBs, at matched concentration. To understand this observed behavior, the stability of each NB population was assessed. It was found that dilution of the NB samples from their stock concentration influences their stability, and it is hypothesized that both the total free lipid and interbubble distance play a role in NB lifetime, in agreement with previously proposed theories and models.

Abstract: Abstract Extracellular vesicles (EVs) are complex biological nanoparticles endogenously secreted by all eukaryotic cells. EVs carry a specific molecular cargo of proteins, lipids, and nucleic acids derived from cells of origin and play a significant role in the physiology and pathology of cells, organs, and organisms. Upon release, they may be found in different body fluids that can be easily accessed via noninvasive methodologies. Due to the unique information encoded in their molecular cargo, they may reflect the state of the parent cell and therefore EVs are recognized as a rich source of biomarkers for early diagnostics involving liquid biopsy. However, body fluids contain a mixture of EVs released by different types of healthy and diseased cells, making the detection of the EVs of interest very challenging. Recent research efforts have been focused on the detection and characterization of diagnostically relevant subpopulations of EVs, with emphasis on label-free methods that simplify sample preparation and are free of interfering signals. Therefore, in this paper, we review the recent progress of the label-free optical methods employed for the detection, counting, and morphological and chemical characterization of EVs. We will first briefly discuss the biology and functions of EVs, and then introduce different optical label-free techniques for rapid, precise, and nondestructive characterization of EVs such as nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy, surface plasmon resonance spectroscopy, Raman spectroscopy, and SERS spectroscopy. In the end, we will discuss their applications in the detection of neurodegenerative diseases and cancer and provide an outlook on the future impact and challenges of these technologies to the field of liquid biopsy via EVs.

Abstract: Chronic kidney disease is a progressive, incurable condition that involves a gradual loss of kidney function. While there are no non-invasive biomarkers available to determine whether individuals are susceptible to developing chronic kidney disease, small RNAs within urinary exosomes have recently emerged as a potential candidate to use for assessing renal function. Ultracentrifugation is the gold standard for urinary exosome isolation. However, extravesicular small RNA contamination can occur when isolating exosomes from biological fluids using ultracentrifugation, which may lead to misidentifying the presence of certain small RNA species in human urinary exosomes. Therefore, we characterized human urinary exosomal preparations isolated by ultracentrifugation alone, or via ultracentrifugation followed by size exclusion chromatography (SEC) column-purification. Using nanoparticle tracking analysis, we identified SEC fractions containing robust amounts of exosome-sized particles, that we further characterized using immunoblotting. When compared to exosomal preparations isolated by ultracentrifugation only, SEC fractionated exosomal preparations showed higher levels of the exosome-positive marker CD81. Moreover, while the exosome-negative marker calnexin was undetectable in SEC fractionated exosomal preparations, we did observe calnexin detection in the exosomal preparations isolated by ultracentrifugation alone, which implies contamination in these preparations. Lastly, we imaged SEC fractionated exosomal preparations using transmission electron microscopy to confirm these preparations contained human urinary exosomes. Our results indicate that combining ultracentrifugation and SEC column-purification exosome isolation strategies is a powerful approach for collecting contaminant-free human urinary exosomes and should be considered when exosomes devoid of contamination are needed for downstream applications.

Abstract: We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Abstract: The oocyte microenvironment constituted by the follicular fluid (FF) is a key for the optimal development of female gametes. Its composition reflects the physiological state of the ovarian follicle. The particularity of FF is to contain a huge diversity of extracellular vesicles specific to women, in the same way as seminal plasma in men. Here, we described and compared morphological aspects of broad subcategories of human FF-related Extracellular Vesicles (EVs). EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. EVs isolated from FF are involved in different biological functions related to follicular growth, oocyte maturation, and embryo development. However, knowledge on the morphology of FF-derived EVs is limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. The aim of this study was to provide a comprehensive morphological description of EVs from FF of healthy subjects and quantification. EVs separation was realized by centrifugation, with comparison of the EV yield obtained from differential centrifugation and one-step ultracentrifugation. Cryo-Transmission Electron Microscopy was used to reveal the morphology, size, and phenotype of EVs. Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) were used to quantify and analyze the size distribution for each centrifugation step. We performed a comprehensive inventory of human follicular fluid EVs. We show that human FF contains a huge diversity of EVs. This study brings novel insights on EVs from normal FF and provides a reference for further studies of EVs in ovarian diseases.

Abstract: ABSTRACT Purpose: Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Extracellular vesicles (EVs) have been extensively studied as a biomarker to monitor disease in patients. The study of new biomarkers in melanoma patients could prevent metastasis by earlier diagnosis. In this study, we determined the proteomic profile of EVs isolated from aqueous humor (AH), vitreous humor (VH), and plasma from UM patients in comparison with cancer-free control patients. Methods: AH, VH and plasma were collected from seven patients with UM after enucleation; AH and plasma were collected from seven cancer-free patients with cataract (CAT; control group). EVs were isolated using the membrane-based affinity binding column method. Nanoparticle tracking analysis (NTA) was performed to determine the size and concentration of EVs. EV markers, CD63 and TSG101, were assessed by immunoblotting, and the EV proteome was characterized by mass spectrometry. Results: Mean EV concentration was higher in all analytes of UM patients compared to those in the CAT group. In the UM cohort, the mean concentration of EVs was significantly lower in AH and plasma than in VH. In contrast, the mean size and size distribution of EVs was invariably identical in all analyzed analytes and in both studied groups (UM vs. CAT). Mass spectrometry analyses from the different analytes from UM patients showed the presence of EV markers. Conclusion: EVs isolated from AH, VH, and plasma from patients with UM showed consistent profiles and support the use of blood to monitor UM patients as a noninvasive liquid biopsy.

Abstract: Background: Recent studies have reported that the incidence of sensitive skin is increasing. Skin sensitivity and skin barrier functions were related to many skin diseases including atopic dermatitis, psoriasis, rosacea, and so on. Mesenchymal stem cell (MSC)-derived exosomes (hMSC) might be considered as a new effective therapeutic scheme. Aims: This study aims to investigate the safety and efficacy of hMSC exosomes as a novel topical treatment for sensitive skin. Patients/Methods: Exosomes were extracted from primary hMSC via ultracentrifugation method. The morphology of hMSC exosomes was studied via transmission electron microscope. Expression of exosome specific surface marker was detected via Western blot. 22 subjects (female, aged 18–55) diagnosed with sensitive skin were enrolled. Follow-up was conducted before, 7-day, 14-day, and 28-day after hMSC exosomes use. Transepidermal water loss (TEWL), surface hydration, sebum secretion, and L*a*b* value were simultaneously tested at the same time point in an environment-controlled room. Results: Under transmission electron microscopy, the extracted hMSC exosomes were circular or elliptical with intact membrane structure, and their diameters ranged mainly from 40 to 80 nm. Western blot showed that the expression of markers CD63, CD9, and Tsg101 was positive. Brownian motion based nanoparticle trajectory analysis (NTA) showed that the main peak of particle size distribution occurred around 96 nm, the average particle size was 122 nm, and the main peak accounted for 96.7%. All this conformed to the biological characteristics of exosomes standardized by the International Society for Extracellular Vesicles. In the clinical trial, scores of objective symptoms including roughness, scales, erythema, and subjective symptoms including tension, burning, or itching, were improved after 7-, 14-, and 28- day using hMSC-exosomes. TEWL, hydration, sebum, pH, and a* values were tended to return to the level of healthy skin. Conclusion: The hMSC-exosomes, with the advantages of biocompatibility and biodegradability, could improve clinical symptoms and eruptions in sensitive skin patients, and might be as an MSC cell-free novel therapy in sensitive skin-related disease treatment.