Microgravity-driven Rab27B activation amplifies mesenchymal stem cell-derived extracellular vesicle production and functions

Abstract

Background

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are emerging as promising treatments for immunomodulation and tissue regeneration. However, the scalable production of functionally enhanced EVs remains a critical challenge. This study introduces microgravity culture using a three-dimensional (3D) rotating cell culture system as a novel strategy to optimize MSC-EVs yield and bioactivity.

Methods

We first investigated the effects of microgravity on the proliferation and stemness of human umbilical cord-derived MSCs (UCMSCs). The yield of microgravity-derived EVs (µg-EVs) was quantified by nanoparticle tracking analysis. The function of µg-EVs was analyzed by proteomic profiling and further assessed by macrophage polarization and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Proteomic analysis of UCMSCs was performed to further explore the underlying mechanisms of EVs biogenesis and functional activity under microgravity condition.

Results

Microgravity culture significantly enhanced UCMSCs proliferation and stemness. Compared with conventional static culture, EVs production increased by 7.7-fold under microgravity. Functionally, µg-EVs more effectively promoted macrophage polarization toward the anti-inflammatory M2 phenotype and significantly enhanced the osteogenic differentiation capacity of PDLSCs. Mechanistically, Rab27B upregulation in microgravity-cultured UCMSCs was associated with increased EVs secretion and enhanced therapeutic efficacy.

Conclusions

This study identifies microgravity as an effective platform for the large-scale production of high-quality UCMSC-EVs, addressing key manufacturing barriers and accelerating the clinical translation of EVs-based therapies.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04711-w.

Introduction

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine due to their immunomodulatory properties and tissue repair capabilities [1]. Recent studies suggest that the therapeutic effects of MSCs are primarily mediated through paracrine secretion of bioactive molecules rather than direct cell differentiation [2]. Extracellular vehicles (EVs) derived from MSCs (MSC-EVs) have emerged as key mediators of these paracrine functions, carrying proteins, nucleic acids, and lipids that mimic the therapeutic potential of parental cells [3]. Preclinical evidence supports the efficacy of MSC-EVs in treating neurological disorders [4–6], autoimmune diseases [7–9], and organ injuries [10–12], positioning them as promising cell-free therapeutics.

Despite their potential, the clinical translation of MSC-EVs faces a major challenge due to the low production yields of conventional two-dimensional (2D) monolayer cultures [13]. In standard tissue culture flasks, adherent MSCs are restricted to rigid plastic surfaces, disrupting natural cell-cell interactions and extracellular matrix (ECM) signaling. This artificial environment accelerates cellular senescence, leading to EVs with reduced regenerative capacity [14–16]. Moreover, 2D systems fail to meet the large-scale EVs quantities required for clinical applications. Three-dimensional (3D) culture strategies have been proposed to address these limitations. Current 3D approaches include scaffold-based systems that mimic ECM structures and scaffold-free systems relying on cell self-assembly (e.g., spheroids). However, scaffold-based methods face challenges in material optimization, while static scaffold-free models often exhibit uneven nutrient distribution and waste accumulation [17]. These limitations highlight the need for dynamic 3D culture systems that better support cell viability and function.

Microgravity culture based on Rotary Cell Culture System (RCCS), a form of dynamic 3D culture, provides unique advantages for cell expansion [18, 19]. This system maintains cells in free-floating conditions by balancing gravitational and hydrodynamic forces during rotation, thereby simulating a microgravity-like state [20, 21]. By minimizing fluid shear forces and enhancing mass transfer, microgravity promotes the formation of tissue-like cell aggregates while maintaining stem cell properties [22, 23]. Previous studies demonstrate that microgravity enhances stemness markers in MSCs and improves their secretory functions [15]. However, its specific effects on MSC-EVs production and therapeutic efficacy remain poorly understood. To solve the question, we cultured umbilical cord mesenchymal stem cells (UCMSCs) under conventional 2D (g-cells) and microgravity 3D (µg-cells) conditions to systematically evaluate their EVs production and biological functions. Through comparative analysis of EVs from both groups (g-EVs vs. µg-EVs), we identified microgravity-induced upregulation of Rab27B as a critical mechanism enhancing EVs yield and therapeutic potency. Our findings provide new insights into efficient UCMSC-EVs production strategies with improved clinical applicability.

Results

Microgravity condition promotes EVs yield.

We successfully isolated UCMSCs which were capable of multi-directional differentiation including osteogenesis, adipogenesis, and chondrogenesis (Fig. S1A-C). Flow cytometric assay showed that the majority of UCMSCs expressed CD90, CD105, CD44, and CD73 while lacking expression of CD34 and CD45 (Fig. S1D). To investigate the effects of microgravity on UCMSCs, the cells were cultured in the conventional condition and the microgravity condition respectively. The optical microscope images showed that g-cells had typically elongated spindle-like morphology, while µg-cells exhibited round and spherical microtissue-like cell mass (Fig. S2A). Compared to g-cells, µg-cells showed faster cell proliferation about 2.5 folds on day 5 (Fig. S2B). Furthermore, the mRNA level of cell stemness markers (Sox2 and Nanog) was significantly increased in µg-cells (Fig. S3), indicating that the microgravity condition enhanced the pluripotency and self-renewal potential of UCMSCs [24, 25]. We collected the supernatants from g-cells and µg-cells and isolated g-EVs and µg-EVs via gradient ultracentrifugation. (Fig. 1A). Transmission electron microscopy (TEM) presented that both EVs displayed typical cup-shaped morphology (Fig. 1B). Western blotting confirmed that both EVs expressed CD63, CD81, and CD9, and were negative for calnexin (Fig. 1C), which well conformed to the identification criteria for EVs [26]. Nanoparticle tracking analysis (NTA) showed that the particle diameter of µg-EVs was significantly larger than that of g-EVs (163.0 ± 0.8 nm in g-EVs vs. 175.8 ± 1.2 nm in µg-EVs) (Fig. 1D-E). The larger size of EVs would offer greater capacity for storing proteins and other cargoes. Additionally, zeta potential analysis suggested no significant difference in the surface charge between the two groups (Fig. S4), indicating similar colloidal stability in both EVs [27]. To compare the yields of g-EVs and µg-EVs, NTA assay showed that the number of nanoparticles derived from the equal volumes of supernatants in µg-EVs group was about 7.7-fold higher than that in g-EVs group (3.07 × 10¹¹ ± 0.50 particles/ml in g-EVs vs. 23.77 × 10¹¹ ± 3.36 particles/ml in µg-EVs) (Fig. 1F). Moreover, compared to the traditional culture, microgravity culture increased EVs secretion per cell by about 3.4 folds (4.12 × 10⁴ ± 0.71 particles/cell in g-EVs and 13.90 × 10⁴ ± 1.32 particles/cell in µg-EVs) (Fig. 1G). Immunoblotting further demonstrated elevated expression of the exosomal marker CD63 in µg-cells compared to g-cells (Fig. 1H-I), suggesting enhanced multivesicular bodies (MVBs) formation in µg-cells [28, 29]. Taken together, the microgravity condition remarkably increased the EVs yield, indicating the microgravity culture could be a potential application for high-efficiency EVs yield.

Fig. 1.

Characterization of g-EVs and µg-EVs. A Schematic diagram of EVs isolation. B Representative TEM images of EVs. Scale bar: 100 μm. C Western blotting of exosomal marker proteins. D NTA analysis of EVs. E Average diameter of EVs. F The total output of EVs. G The yield of EVs per cell. H Representative immunofluorescence images of CD63 in cells. Scale bar: 50 μm. In zoom-in view, scale bar: 20 μm. I Quantitative analysis of CD63 fluorescence intensity from 40 single cells in each group in 5 randomly chosen fields. NTA data are presented as mean ± SD from three independent experiments

Microgravity condition affects the protein profile of EVs

To further characterize the protein cargo of g-EVs and µg-EVs, LC-MS/MS analysis was performed. A total of 2358 proteins were identified in both groups, of which 1956 proteins were commonly expressed. 326 proteins were uniquely detected in µg-EVs, while 76 proteins were specific to g-EVs (Fig. 2B). The hierarchical clustering analysis demonstrated both the homogeneity and heterogeneity in the protein profiles of g-EVs and µg-EVs (Fig. 2C). The volcano plot (Fig. 2D) illustrated the distribution and statistical significance of differentially expressed proteins between the two groups. In addition, as shown in Fig. 2E-F, the proteins upregulated in µg-EVs were primarily associated with immune regulation and tissue repair, suggesting enhanced functional potential of EVs.

Fig. 2.

Bioinformatics analysis of EVs differential proteins. A Schematic diagram of EVs proteomic analysis. B Venn plot for EVs proteins. C Heatmap of the expression of shared proteins from g-EVs and µg-EVs. Red and blue represent up-regulated and down-regulated expression respectively. D Volcano plot of differentially expressed proteins from g-EVs and µg-EVs. E Relative protein levels of top 20 up-regulated proteins in µg-EVs. F Gene Ontology (GO) analysis of significantly up-regulated proteins in µg-EVs

µg-EVs exhibit enhanced Immunomodulatory and osteogenic effects in vitro

MSC-EVs have been demonstrated to play a critical role in modulating immune responses, including regulating macrophage polarization [30]. To compare the effects of g-EVs and µg-EVs on macrophage inflammation, Raw264.7 cells were first stimulated with lipopolysaccharide (LPS) to induce M0-to-M1 activation, followed by incubation with EVs. After EVs treatment, the gene expression of M1 markers (Nos2, CD86) was significantly reduced, whereas the gene expression of M2 markers (Arg1, CD206) was notably increased. The M1-M2 transformation potency of µg-EVs was superior to that of g-EVs (Fig. 3A). The flow cytometry results (Fig. 3B-C) also showed that µg-EVs more effectively reduced the protein levels of iNOS and CD86, while enhancing the protein levels of Arg1 and CD206. These results suggest that µg-EVs possess superior immunomodulatory potential compared to g-EVs, particularly in promoting macrophage polarization toward the anti-inflammatory M2 phenotype. Periodontal ligament stem cells (PDLSCs), as odontogenic mesenchymal stem cells, are characterized by proliferation, self-renewal, and multidirectional differentiation potential, which are the important seed cells for periodontal regeneration. It has been proved that MSC-EVs can usefully induce the osteogenic differentiation of PDLSCs to facilitate periodontal tissue regeneration [31]. To study the effect of g-EVs and µg-EVs on the osteogenesis of PDLSCs, we performed RT‒qPCR, alkaline phosphatase (ALP) staining, and alizarin red (ARS) staining. Figure 3D showed that the gene expression of osteogenic markers (ALP, OSX, and RUNX2) was significantly higher in the µg-EVs group than in the g-EVs group. Similarly, ALP staining revealed increased ALP activity in PDLSCs following µg-EVs treatment (Fig. 3E). Alizarin red staining (ARS) showed enhanced mineralization in µg-EVs treatment (Fig. 3F). Together, these findings demonstrated that µg-EVs are more effective than g-EVs in enhancing the osteogenic differentiation of PDLSCs.

Fig. 3.

The effects of µg-EVs on macrophage polarization regulation and osteogenic differentiation of PDLSCs. A Gene expression of M1 markers (Nos2, CD86) and M2 markers (Arg1, CD206) after treatment with EVs. B Protein expression of M1 markers (iNOS, CD86) and M2 markers (Arg1, CD206) after treatment with EVs. C Quantitative analysis of flow cytometry. D Gene expression of osteogenic markers (RUNX2, OSX, and ALP) after treatment with EVs. The above data are presented as mean ± SD from three independent experiments. E ALP staining. Scale bar: 100 μm. F ARS staining. Scale bar: 100 μm

Microgravity condition changes the protein profile of UCMSCs

To elucidate the molecular mechanisms underlying the microgravity-induced enhancement in EVs yield and function, we performed proteomic profiling of g-cells and µg-cells using LC-MS/MS analysis (Fig. 4A). A total of 5003 proteins were detected, and hierarchical clustering of differentially expressed proteins between the two groups was shown in Fig. 4B. GO enrichment analysis (Fig. 4C) indicated that proteins upregulated in µg-cells were predominantly associated with “extracellular exosome” and “lysosomal lumen”, suggesting activation of pathways related to EVs biogenesis and intracellular trafficking. Then, we focused on the proteins that were critical for EVs biogenesis and release [32]. As shown in the volcano plot (Fig. 4D), Rab27B was significantly upregulated in µg-cells. Western blotting further confirmed that the expression of Rab27B in µg-cells was significantly upregulated compared with that in g-cells (Fig. 4E-F). Rab27B, a member of the small GTPase Rab family, plays a key role in regulating exosome biogenesis and release [33–36]. Taken together, microgravity may enhance the production and functions of EVs by activating Rab27B.

Fig. 4.

Bioinformatics analysis of differential proteins between g-cells and µg-cells. A Schematic diagram of cellular proteomic analysis. B Heatmap of shared protein expression between g-cells and µg-cells. Red and blue represent up-regulated and down-regulated expression, respectively. C GO analysis of significantly up-regulated proteins in µg-cells. D Volcano plot of differentially expressed proteins between g-cells and µg-cells. E-F Western blotting analysis of Rab27B in g-cells and µg-cells. Data are presented as mean ± SD from three independent experiments

RAB27B knockdown decreases the yield and functions of µg-EVs

To evaluate the role of Rab27B in microgravity-induced changes of MSC-EVs, shRNA was used to downregulate RAB27B expression in UCMSCs (UCMSCs ^shRAB27B). Compared to the control group (UCMSCs ^shControl), both RT-qPCR and western blotting confirmed a significant reduction in Rab27B expression at the mRNA and protein levels in UCMSCs ^shRAB27B (Fig. S6A-C). Next, we evaluated whether RAB27B knockdown affected EVs secretion. NTA assay showed that the number of EVs secreted from UCMSCs ^shRAB27B (sh-µgEVs) was significantly lower than that of µg-EVs (3.73 ± 0.26 × 10¹¹ particles/ml in sh-µg EVs vs. 20.9 ± 2.11 × 10¹¹ particles/ml in µg-EVs) (Fig. 5A). The EVs concentration per cell in sh-µgEVs group was also significantly lower than that in µg-EVs group (Fig. 5B). As shown in Fig. 5D, RAB27B knockdown reduced CD63 expression in sh-µgcells, and quantitative analysis of fluorescence intensity further confirmed this outcome (Fig. 5C). Taken together, these results suggest that microgravity promotes EVs secretion by upregulating Rab27B expression.

Fig. 5.

Rab27B tunes the yield of µg-EVs. A Total output of µg-EVs after knockdown of RAB27B. B Yield of µg-EVs per cell after knockdown of RAB27B. C Quantification of CD63 fluorescence intensity was performed on 40 single cells per group, selected from five randomly chosen fields. D Representative immunofluorescence images of CD63 in RAB27B-knockdown cells cultured in microgravity condition. Scale bar: 50 μm. In zoom-in view, scale bar: 20 μm

We further investigated whether Rab27B regulated immunoregulation and tissue repair of µg-EVs. Compared with the µg-EVs group, the gene expression of M1-associated markers (Nos2 and CD86) was greatly higher in the sh-µgEVs group, whereas the gene expression of M2-associated markers (Arg1 and CD206) was significantly lower (Fig. 6A). Consistent with these findings, the flow cytometric analysis showed (Fig. 6B-C) that, compared to the µg-EVs group, more CD86⁺ cells and iNOS⁺ cells were present while CD206⁺ cells and Arg1⁺ cells were reduced in the sh-µgEVs group. Moreover, osteogenic gene markers (ALP, OSX, and RUNX2) were down-regulated in the sh-µgEVs group compared to the µg-EVs group (Fig. 6D). ALP and ARS staining showed consistent experimental results with RT-qPCR, showing a downward trend of ALP activity and mineralized nodule formation capacity in the sh-µgEVs group (Fig. 6E-F). These results confirmed that knockdown of RAB27B adversely affected the immune regulation and tissue regeneration functions of µg-EVs, highlighting the key role of Rab27B in mediating the therapeutic potential of EVs under microgravity condition.

Fig. 6.

Rab27B tunes the functions of µg-EVs. A The gene expression of M1 markers (Nos2, CD86) and M2 markers (Arg1, CD206) after treatment with sh-µgEVs. B The protein expression of M1 markers (iNOS, CD86) and M2 markers (Arg1, CD206) after treatment with sh-µgEVs. C Quantitative analysis of flow cytometry. D Gene expression of osteogenic markers (RUNX2, OSX, and ALP) after treatment with sh-µgEVs. The above data are presented as mean ± SD from three independent experiments. E ALP staining. Scale bar: 100 μm. F ARS staining. Scale bar: 100 μm

µg-EVs inhibited periodontal bone loss in vivo

The µg-EVs facilitated macrophage polarization toward the anti-inflammatory phenotype and enhanced the osteogenic differentiation of PDLSCs, suggesting that µg-EVs could be potentially useful in regulating inflammatory periodontal bone loss. To investigate the hypothesis, mouse periodontitis was induced by ligature and treated with g-EVs or µg-EVs once every two days (Fig. 7A). After ligation for 5 weeks, contrasting with control, obvious alveolar bone resorption was observed surrounding the ligated molar in the periodontitis (PD) group. In comparison, local injection of EVs into the gingiva remarkably reduced the ligature-induced bone loss. Among the treated groups, µg-EVs showed the greatest therapeutic effect, as evidenced by the shortest distance between the alveolar bone crest (ABC) and the cementoenamel junction (CEJ), and the lowest volume of total bone loss (Fig. 7B-D), compared to both g-EVs and sh-µgEVs. Furthermore, hematoxylin-eosin (H&E) staining results revealed that µg-EVs exhibited the least attachment loss (AL) and the lowest infiltration of inflammatory cells, compared to g-EVs and sh-µgEVs groups (Fig. 7E).

Fig. 7.

The therapeutic effect of µg-EVs in periodontitis mice. A Schematic diagram of ligature-induced periodontitis (PD) model and EVs injection. B Representative sagittal 3D and 2D views of the maxillary molar produced by micro-CT. Scale bar: 1 mm. C Average ABC-CEJ distance on buccal and palatal sides. D Bone volume fraction (BV/TV) in each group. E H&E staining (M1 first molar; M2 second molar; black arrows: ABC yellow arrows: CEJ AL attachment loss). Scale bars: 200 μm in low magnification and 100 μm in high magnification. Data are presented as mean ± SD from at least three independent experiments

µg-EVs promote osteogenic differentiation and reduce inflammatory responses in periodontitis mice

To further confirm the therapeutic effect of µg-EVs on experimental periodontitis in mice, immunohistochemical staining for the osteogenic marker ALP was performed. Semi-quantitative analysis revealed that ALP expression in the µg-EVs-treated group was significantly higher than that in the g-EVs and sh-µgEVs groups (Fig. 8A-B), indicating enhanced osteogenic activity. In addition, we examined the phenotype of macrophages in the periodontium after µg-EVs treatment. F4/80⁺ cells were regarded as a general marker for macrophages. iNOS⁺ cells represent pro-inflammatory macrophages and CD206⁺ cells indicate anti-inflammatory macrophages. As shown in Fig. 8C-F, the µg-EVs group exhibited a significantly higher number of CD206⁺ cells and fewer iNOS⁺ cells compared to the g-EVs and sh-µgEVs groups, suggesting that µg-EVs promoted macrophage polarization toward the anti-inflammatory phenotype in vivo. Furthermore, Fig. S7 showed that no significant pathologic changes were observed in the main organs including the heart, liver, spleen, lungs, and kidneys in all groups, indicating no evident systemic toxicity of EVs treatment. Taken together, these findings demonstrated that µg-EVs not only promoted local osteogenic activity but also facilitated the conversion of macrophages to the anti-inflammatory phenotype, thereby alleviating alveolar bone loss in periodontitis.

Fig. 8.

µg-EVs promote osteogenic differentiation and reduce inflammatory responses in periodontitis mice. A Representative images of ALP immunohistochemical staining. Scale bars: 200 μm in low magnification and 100 μm in high magnification. B Semi-quantitative analysis of immunohistochemistry. C Representative immunofluorescence images of iNOS in macrophages in periodontium. Scale bar: 100 μm. D Semi-quantitative analysis of immunofluorescence. E Representative immunofluorescence images of CD206 in macrophages in periodontium. Scale bar: 100 μm. F Semi-quantitative analysis of immunofluorescence

Discussions

MSC-EVs have demonstrated great immunoregulatory and tissue regenerative functions, holding considerable promise for treating diverse diseases. However, the scalable production of high-functional MSC-EVs remains a major challenge, limiting their clinical translation. In this study, we demonstrated that microgravity culture using the rotary cell culture system significantly promoted the proliferation and self-renewal capacity of UCMSCs. Furthermore, the microgravity environment markedly increased EVs secretion by up to 7.7-fold, along with improved immunomodulatory and osteogenic functions. Mechanistically, we identified Rab27B as a key regulatory molecule upregulated under microgravity condition, contributing to both enhanced EVs biogenesis and bioactivity. Collectively, these findings establish microgravity culture as a robust and promising platform to improve the production efficiency and functional activity of MSC-EVs.

The efficient production and functional quality of MSC-EVs are closely influenced by the physiological state of their parental MSCs [15, 37]. In this study, we first evaluated the biological behavior of UCMSCs cultured under microgravity condition using RCCS. The RCCS, widely adopted as a standard platform to simulate microgravity, rotates the culture vessel on a horizontal axis to balance gravitational and hydrodynamic forces, thereby maintaining cells in continuous suspension. This low-shear, three-dimensional environment promotes the formation of spheroidal microtissues [20, 21]. The results showed that microgravity culture significantly promoted UCMSCs proliferation and enhanced their stemness. These findings are consistent with previous studies in other stem cell types, including bone marrow stem cells (BMSCs) [38, 39], adipose-derived stem cells (ADSCs) [40], dental pulp stem cells (DPSCs) [41], and periodontal ligament stem cells (PDLSCs) [42], suggesting a common regulatory effect of microgravity on stem cells. Gene ontology (GO) enrichment analysis (Fig. 4C) further revealed that upregulated proteins under microgravity culture were mainly enriched in terms such as “extracellular matrix” and “cell adhesion”. This suggests enhanced extracellular matrix remodeling and stronger cell–niche interactions in µg-cells, which are critical for maintaining stemness and supporting continuous proliferation [43, 44]. It should be noted that the RCCS inherently combines simulated microgravity, spheroid formation, and dynamic fluid forces. Future work should aim to disentangle the individual contributions of these factors. Nevertheless, our findings demonstrate that RCCS-based microgravity culture provides a favorable microenvironment for UCMSC stemness maintenance and large-scale expansion.

Furthermore, we observed a substantial increase in EVs yield under microgravity condition. Specifically, the total EVs particle production per milliliter increased by 7.7-fold, and the per-cell EVs yield was 3.4 times higher than that observed under conventional static culture. These results indicated that the enhanced EVs particle production induced by microgravity was attributed to increased cell numbers and upregulation of vesicle secretion capacity at the single-cell level. Furthermore, we found that microgravity significantly upregulated the expression of Rab27B to promote EVs secretion. Rab27B is a key small GTPase critically involved in the fusion of multivesicular bodies (MVBs) with the plasma membrane [45, 46]. This suggests that microgravity may enhance EVs release by promoting Rab27B-mediated vesicle trafficking, thereby markedly increasing per-cell EVs output. In addition, the enhanced activation of Rab27B under microgravity may be attributed to cytoskeleton-driven regulation of guanine nucleotide exchange factors (GEFs), which facilitate its conversion to the active GTP-bound form and promote EVs secretion [35, 45, 47].

Notably, EVs derived from microgravity condition exhibited enhanced biological activity. Specifically, µg-EVs effectively promoted macrophage polarization toward the anti-inflammatory M2 phenotype and significantly enhanced the osteogenic differentiation of PDLSCs. We further observed that microgravity culture significantly upregulated Rab27B expression in UCMSCs. Importantly, knockdown of RAB27B markedly attenuated the immunomodulatory and pro-osteogenic effects of µg-EVs, suggesting microgravity enhanced EVs function by upregulating the expression of Rab27B. Our findings are supported by previous work showing that Rab27B plays a critical role in EVs function. Cheng et al. reported that lower Rab27B expression in MSCs from older donors was associated with diminished anti-inflammatory capacity [48]. These observations suggested that Rab27B may contribute to cargo selection and functional output. Future studies should explore its potential as a biomarker to predict EVs potency and as a molecular target to enhance EVs-based therapeutic applications.

Conclusions

In summary, this study suggests that microgravity is an effective strategy to enhance EVs yield and augment EVs functions for macrophage immunomodulation and PDLSCs osteogenesis, exhibiting greater treatment efficacy in periodontitis. Mechanistically, the upregulation of Rab27B in microgravity-cultured UCMSCs was linked to increased EVs secretion and enhanced therapeutic efficacy. These results indicated microgravity culture was a potential platform to produce EVs with augmented immunomodulatory and regenerative properties, allowing to optimize manufacturing process of UCMSC-EVs for mass production.

Experimental Section

MSC isolation

Human UCMSCs were isolated from fresh umbilical cords and human PDLSCs were isolated from healthy premolars extracted for orthodontic purposes from donors. The detailed information of donors was provided in Table S3. UCMSCs and PDLSCs were obtained using the standard plastic adherence method following enzymatic digestion according to previous studies [49, 50]. Briefly, for UCMSCs isolation, the umbilical cord was cut into pieces, treated with collagenase type I (Sigma, GER), and incubated in Minimum Essential Medium α (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Procell, CHN). For PDLSCs isolation, the periodontal ligament tissue was scraped, cut into pieces, and cultured in α-MEM supplemented with 10% FBS. Both cells were passaged when the density reached 90% confluence. All experiments were performed using cells between passages 3–6. Cells were cryopreserved in liquid nitrogen with standard freezing medium (90% fetal bovine serum (Procell, CHN) + 10% Dimethyl sulfoxide (D8371, Solarbio, CHN).

UCMSCs identification

To examine osteogenic differentiation, UCMSCs were cultured in osteogenesis-induced medium (α-MEM supplemented by 10% FBS, 0.1 µM dexamethasone, 50 µM ascorbate-2-phosphate and 10 mM β-glycerophosphate) and detected by ARS staining. To examine adipogenic differentiation, UCMSCs were cultured using MesenCultTM Adipogenic Differentiation Kit (Stemcell, CAN) and detected by Oil Red O staining. To examine chondrogenic differentiation, UCMSCs were cultured using MesenCultTM-ACF Chondrogenic Differentiation Kit (Stemcell, CAN) and detected by Alcian Blue (Sigma, USA) staining. Positive (CD44, CD73, CD90, and CD105) and negative (CD45, CD34) surface markers were detected by flow cytometry.

Cell culture

To imitate microgravity conditions, UCMSCs were cultured in RCCS (Synthecon, US, RCCS-4SQ)rotating at a speed of 27 rpm. UCMSCs were seeded in conventional flasks and RCCS at a density of 10⁶ cells/mL with exosome-free medium. The same UC-MSCs cell line was used for all experiments, with EVs isolated from generation 3 to 6 (P3-P6) cells to ensure generational consistency.

Isolation of MSC-EVs

Three independent UCMSC lines were used for all experiments, and EVs were isolated from cells at passages 3 to 6 (P3–P6). The cell supernatants were harvested every 48 h. The supernatants were centrifuged at 300 × g for 10 min at 4 °C to remove residual cells, further centrifugated at 3,000 × g for 10 min at 4 °C to isolate dead cells, and subsequently centrifuged at 10, 000 × g for 30 min at 4 °C to remove residual apoptotic bodies and cellular debris. Finally, the supernatants were ultracentrifuged at 120 000 × g for 70 min at 4 °C (Beckman Coulter, USA) to obtain EVs. All EVs were preserved in PBS and stored at − 80 °C. All in vitro EV treatments were administered at an equal protein concentration of 20 µg/mL.

Characterization of MSC-EVs

The morphology of EVs was monitored via TEM (Hitachi, Japan). Surface markers of exosome (CD9, CD81, CD63, and Calnexin) were measured by western blotting. Size distribution and concentration of EVs were measured by nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Malvern, UK) equipped with a red laser and an sCMOS camera. Measurements were performed with NTA software version 3.4 Build 3.4.4, using a camera gain of 55. Each sample was recorded for 30s in triplicate. The surface potential of EVs was measured by Zetasizer Nano ZSE (Malvern, UK).

Liquid chromatography with tandem mass spectrometry (LC-MS/MS)

Protein samples were digested enzymatically into peptides and subsequently analyzed by label-free quantitative LC-MS/MS. The resulting peptides were loaded onto a 25 cm column (150 μm inner diameter, packed with ReproSil-Pur C18-AQ 1.9 μm silica beads; Beijing Qinglian Biotech Co., Ltd., Beijing, China). Chromatographic separation was performed using a gradient of 8–12% solvent B over 7 min, 12–30% solvent B over 48 min, followed by an increase to 40% in 10 min and a final wash at 95% solvent B for 15 min, at a flow rate of 600 nL/min. Solvent A consisted of 0.1% formic acid in water, and solvent B was 80% acetonitrile with 0.1% formic acid in water. Mass spectrometry was carried out on a quadrupole-Orbitrap instrument (Q Exactive HF-X, Thermo Fisher Scientific, Bremen, Germany) coupled to an EASY-nLC 1200 ultra-high-pressure liquid chromatography system (Thermo Fisher Scientific) via a nano-electrospray ion source. The instrument was operated in data-dependent acquisition (DDA) “top-40” mode, acquiring full MS spectra in the Orbitrap mass analyzer (resolution 120,000; m/z range 350–1500), with an AGC target of 3 × 10^6 and a maximum ion injection time of 80 ms. DDA MS data from six fractionated pools and DIA MS data from single-shot subject samples were used to construct a DDA-based library and a direct-DIA library, respectively. These libraries were computationally merged into a hybrid library using Spectronaut software (Biognosys, version 15.7.220308.50606). Database searches were performed with carbamidomethylation set as a fixed modification, while protein N-terminal acetylation and methionine oxidation were specified as variable modifications. The |log2(FC)| ≥ 1 and p-value ≤ 0.05 were regarded as the screening criterion for identifying differentially expressed proteins.

Western blotting (WB)

Cells were lysed by Cell Lysis Buffer (NCM Biotech; CHN) to extract proteins. The protein samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore; USA). After being blocked by 5% milk for 1 h at room temperature (RT), the membranes were incubated with primary antibodies at 4 °C overnight. Details of primary antibodies were included in Table S1 in the supporting information. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (Boster, CHN) for 1 h at RT. Finally, the membranes were observed using chemiluminescence (NCM Biotech; CHN).

RT‒qPCR

Total RNA was isolated with Trizol (Accurate Biology, CHN) and reverse-transcribed using the PrimeScript TM RT kit (Accurate Biology, CHN). Quantitative real-time PCR was performed using the PrimeScriptTM RT Reagent Kit (Accurate Biology, CHN). Primer sequences are listed in Table S2 in supporting information.

Immunofluorescence staining (IF)

Briefly, the samples were fixed with 4% paraformaldehyde (PFA) for 10 min at 4 ℃, followed by permeabilization using 0.25% Triton X-100 (Solarbio, CHN) for 5 min at RT. Then, the samples were incubated with primary antibodies at 4 °C overnight and subsequently stained by Alexa 594-labeled fluorescent secondary antibodies (Boster, CHN) for 1 h at RT. Next, the cell nuclei were stained by DAPI (Beyotime, China) (1:1000) and filamentous actin (F-actin) was stained with Multi-rAb CoraLite^® Plus 594-Goat Anti-Rabbit Recombinant Secondary Antibody (H + L) (Proteintech, CHN) (1:1000). Finally, the fluorescence images were obtained using fluorescence microscope (FV3000, Olympus JPN). The primary antibodies for immunofluorescence staining were provided in Table S1 in supporting information.

Flow cytometry (FCM)

Samples were incubated with commercial antibodies (Table S1, Supporting Information) for 1 h at 4 °C in the dark and analyzed by flow cytometer (CytoFLEX SRT, Beckman Coulter, USA). For each sample, 10,000 events were acquired. The data were analyzed using FlowJo software. The gating strategy consisted of FSC-A vs. SSC-A to identify the main cell population and exclude debris, after which marker expression was analyzed.

Cell transfection

For RAB27B knockdown, UCMSCs were transduced with lentiviral vectors encoding shRNA against RAB27B (Shanghai Taiertu Gene Company). UCMSCs transduced with a non-targeting shRNA (UCMSCs^shControl) served as the control, and knockdown efficiency was confirmed by RT-qPCR and western blotting. EVs derived from UCMSCs^shControl were used as controls in all in vitro experiments assessing the yield and function of Rab27B-knockdown EVs to ensure valid comparisons.

Animal experiment

30 male C57BL/6 mice (6–8 weeks old, 20–30 g) were housed in pathogen-free conditions at a 12-h light/dark cycle. The mice were randomly divided into the following groups using random number generation (n = 6 in each group): (1) healthy group (Control), (2) periodontitis group (PD), (3) PD + g-EVs treatment, (4) PD + µg-EVs treatment, and (5) PD + sh-µgEVs treatment. During the course of the procedures, 5 mice died, leaving 25 animals available for final analysis, with 5 biological samples per group. All animal surgical procedures were performed under inhalation anaesthesia induced with 4% (v/v) isoflurane. Based on previous studies, the periodontitis model in mice was established by tying a 5 − 0 silk ligature (HUAYON, CHN) surrounding the bilateral maxillary second molar. After ligation, 50 µg EVs (1 µg/µL) were injected into the gingiva of the ligated second maxillary molar with a microliter syringe (HUAYON, CHN) once every two days. After 5 weeks, mice were euthanized through CO₂ exposure, and maxillary alveolar bones were dissected and fixed with 4% PFA for further analysis. Multiple experimentalists collaborated on blinded procedures throughout the animal experiment. The work has been reported in line with the ARRIVE guidelines 2.0.

Microcomputed tomography (Micro-CT)

The maxillary bones were examined by a micro-CT scanner (Quantum GX, PerkinElmer, USA). Region of interest (ROI) was defined as a 15 × 15-pixel² area between the first and the second molar on the alveolar bone with the height of 50 slices. The 2D and 3D images, the distance between ABC and CEJ, and BV/TV were analyzed.

Histological analysis

The fixed samples were decalcified using 10% EDTA (AccuRef Scientific, CHN), embedded in paraffin, and cut into slices (thickness: 5 μm). H&E staining was accomplished with commercial kits (Biosharp, BL700B). Immunohistochemical staining was performed as follows. Antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0; Biosharp, BL604A) at 95 °C for 20 min. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide (H₂O₂) for 30 min at room temperature. Sections were then blocked with 5% bovine serum albumin (BSA) for 1 h, followed by incubation with polyclonal anti-rabbit ALP antibodies (1:200; Affinity, DF6225) overnight at 4 °C. After washing, sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (Boster, BA1039) for 1 h and DAB substrate (ZSGB-Bio, ZLI-9018) for 3 min at room temperature.

Statistical analysis

All assays were performed in at least three technical replicates. Statistical analysis was measured by GraphPad Prism software. For data comparisons within two groups, a student’s t-test was used for analysis. For data comparisons among three or more groups, a one-way analysis of variance was used for analysis. p < 0.05 was regarded to indicate statistical significance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

YD: Methodology, Project administration, Writing—original draft, Data curation. YRF: Investigation, Writing—review &editing. MS, YL: Software, Formal analysis. AL, YL, YRF: Funding acquisition, supervision, design, and conceptualization. All authors read and approved the final manuscript.

Funding

This work was supported by Key Project of Shaanxi Provincial Natural Science Foundation (2025 JC-QYXQ-045), Key R&D Program of Shanxi Province (2025YF-04), the National Natural ScienceFoundationofChina(GrantNo. 82370939), the Key International (Regional) Joint Research Program funded by National Natural Science Foundation of China (Grant No. W2411086), the Shaanxi Provincial High-level Talent Program and Young Talent Support Plan of Xi’an Jiaotong University and the Shaanxi Natural Science Basic Research Program (Grant No. 2025JC-YBQN-1059).

Data availability

The data are available from the corresponding author.

Declarations

Ethics approval and consent to participate

Human umbilical cord tissue was obtained at Renmin Hospital of Wuhan University under the project title of “Umbilical Cord Mesenchymal Stem Cell-Based Therapy for Venous Malformations in Mice” by the Clinical Research Ethics Committee of Renmin Hospital of Wuhan University (Ethics No. WDRY2022-K034, 25 February 2022). Written informed consent was obtained from the parents or legal guardians of all donors for the collection and use of umbilical cord samples. Human teeth were obtained at Xi’an Jiaotong University Affiliated Hospital of Stomatology under the project title of “Effect of Kaempferol in Treating Periodontitis-Related Bone Defects via EphrinB2” by the Medical Ethics Committee of the Stomatological Hospital, Xi’an Jiaotong University (Ethics No. 2023-XJKQIEC-KY-GXB-0008-002, 19 December 2023). Written informed consent was obtained from the parents or legal guardians of all donors. All the animal experiments were approved by the Biomedical Ethics Committee of Health Science Center of Xi’an Jiaotong University under the project title of “Mechanistic study on the regulation of stem cell-derived exosome secretion and function under microgravity conditions” (Ethics No. XJTUAE2014-2786, 27 December 2024).

Competing interests

The authors declare no competing interests.