Oxidative stress-induced death of ovarian granulosa cells (GCs) is a major driver of ovarian functional disorders associated with follicular atresia. Ferroptosis is a key factor in the onset and progression of various ovarian oxidative stress-related diseases, making it a potential target for enhancing reproductive health. Recently, 3D cultured human umbilical cord mesenchymal stem cells (3D hUCMSCs) spheroids have exhibited promising advantages in protecting GCs from oxidative damage. However, it is unclear whether they represent a viable therapeutic strategy for mitigating reproductive failure associated with abnormal follicular atresia by modulating ferroptosis. This study demonstrated that 3D hUCMSC spheroids can effectively protect GCs from hydrogen peroxide (H2O2)-induced oxidative stress and ferroptosis. Additionally, iron overload and lipid peroxidation are two essential features of ferroptosis. 3D hUCMSC spheroids effectively regulate iron uptake and storage to mitigate H2O2-induced iron overload. Furthermore, 3D hUCMSC spheroids mitigate lipid peroxidation induced by H2O2 by restoring GSH metabolic balance and preventing GPX4 inactivation. Mechanistically, the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway was significantly activated by 3D hUCMSCs spheroid treatment. Our findings reveal that Nrf2 knockdown inhibited the 3D hUCMSC spheroids-mediated resistance of GCs to H2O2-induced ferroptosis, and Nrf2 knockdown led to increased iron uptake, resulting in substantial lipid peroxidation through the Fenton reaction, thereby making GCs more susceptible to ferroptosis. This process may involve the ROS-Nrf2-Fe2+ cycle. Significantly, 3D hUCMSC spheroids can mitigate H2O2-induced ferroptosis in GCs by regulating the ROS-Nrf2-Fe2+ cycle. Finally, we confirmed the above results that 3D hUCMSC spheroids ameliorate ovarian oxidative damage in premature ovarian failure (POF) rats. In conclusion, we demonstrated that 3D hUCMSC spheroids regulate oxidative stress and iron homeostasis through the Nrf2 pathway, thereby providing a potential therapeutic target for anovulatory disorders.

Extracellular vesicles (EVs) hold significant promise for the prevention and treatment of various diseases. However, the translation of EV-based therapies into clinical practice faces considerable challenges, particularly in terms of production yield and therapeutic efficacy. Recent studies have emphasized the heterogeneity of EVs and the influence of parental cell microenvironmental signals on their biogenesis, cargo composition, and therapeutic outcomes. This review offers a comprehensive overview of strategies to optimize the therapeutic efficacy of EVs through physical, biochemical, and mechanical modulation. Additionally, it explores how microenvironmental signals affect EV cargoes and the mechanisms by which these signals can improve therapeutic efficacy. The review also addresses current challenges and potential solutions to accelerate the clinical translation of EV therapies. Ultimately, it highlights the potential of microenvironmental modulation in unlocking the full therapeutic capacity of EVs, providing key insights into their production and clinical use for treating various diseases.

Obstructive sleep apnea (OSA) in children is linked to cognitive impairments, potentially due to blood-brain barrier (BBB) dysfunction. Exosomes, small vesicles released by most cells, reflect cellular changes. This study examined the effects of exosomes from children with OSA, with or without cognitive deficits, on neurovascular unit (NVU) models. Twenty-six children were categorized into three groups: healthy controls (Cont, n = 6), OSA without cognitive deficits (OSA-NG, n = 10), and OSA with neurocognitive deficits (OSA-POS, n = 10). Plasma exosomes were characterized and applied to human 3D NVU spheroids for 24 h. Barrier integrity, permeability, and angiogenesis were assessed using trans-endothelial electrical resistance (TEER), tight junction integrity, and tube formation assays. Single-nucleus RNA sequencing (snRNA-seq) and bioinformatics, including CellChat analysis, identified intercellular signaling pathways. Results showed that exosomes from OSA-POS children disrupted TEER, increased permeability, and impaired ZO1 staining in spheroids, compared to the other groups. Both OSA-POS and OSA-NG exosomes increased permeability in NVU cells in monolayer and microfluidic BBB models. snRNA-seq analysis further revealed distinct cell clusters and pathways associated with the different groups. This 3D NVU spheroid model provides a robust platform to study BBB properties and the role of exosomes in OSA. These findings suggest that integrating snRNA-seq with exosome studies can uncover mechanisms underlying neurocognitive dysfunction in pediatric OSA, potentially leading to personalized therapeutic approaches.

Small extracellular vesicle (sEV)-based therapies have gained widespread interest, but challenges persist to ensure standardization and high-scale production. Implementing upstream processes in a chemically defined media in stirred-tank bioreactors (STBr) is mandatory to closely control the cell environment, and to scale-up production, but it remains a significant challenge for anchorage-dependent cells.

We used a human β cell line, grown as monolayer or in suspension as spheroid in stirred systems. We assessed the consequences of culturing these cells in 3D with, or without fetal bovine serum in a chemically defined medium, for cell growth, viability and metabolism. We next explored how different scale-up strategies might influence cell and spheroid formation in spinner flask, with the aim to transfer the process in instrumented Ambr®250 STBr. Lastly, we analyzed and characterized sEV production in monolayer, spinner flask and STBr.

Generation of spheroids in a chemically defined medium allowed the culture of highly viable cells in suspension in stirred systems. Spheroid size depended on the system's volumetric power input (P/V), and maintaining this parameter constant during scale-up proved to be the optimal strategy for standardizing the process. However, transferring the spinner flask (SpF) process to the Ambr®250 STBr at constant P/V modified spheroid size, due to important geometric differences and impeller design. Compared to a monolayer reference process, sEV yield decreased two-fold in SpF, but increased two-fold in STBr. Additionally, a lower expression of the CD63 tetraspanin was observed in sEV produced in both stirred systems, suggesting a reduced release of exosomes compared to ectosomes. This study addresses the main issues encountered in spheroid culture scale-up in stirred systems, rather conducive for the production of ectosomes.

Acute myeloid leukemia (AML) is a highly heterogeneous disease with poor therapeutic outcomes and overall prognosis, particularly in c-Mpl+ AML. c-Mpl, a proto-oncogene, is expressed at significantly higher levels in AML compared to normal human tissue cells. This study aimed to develop a type of targeted exosomes (Exos) capable of delivering anticancer drugs directly to c-Mpl+ AML cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) were isolated as the source of Exos. Fusion CD63 proteins with varying numbers of thrombopoietin (TPO)-mimic peptides, designed to target c-Mpl, were bioengineered to be expressed on the membranes of hUCMSCs and their derived Exos. The targeting capability of the fusion proteins was assessed using the DUAL membrane system, fluorescence resonance energy transfer efficiency, and endocytosis assays. After encapsulating the anticancer drug daunorubicin (DNR), these targeted Exos were evaluated for their ability to eliminate c-Mpl+ AML cells. Safety and efficacy were further tested in a mouse AML model.

Our findings showed that the engineered hUCMSCs-derived Exos demonstrated excellent targeting ability to c-Mpl and a strong propensity for endocytic uptake by c-Mpl+ AML cells. Among the engineered Exos, those with the fusion protein containing three TPO-mimic peptides (CD63-mTPO3), named as m3Exos, exhibited the highest binding affinity for c-Mpl. When loaded with DNR, these engineered Exos (m3Exos@DNR) effectively eliminated c-Mpl+ AML cells in both in vitro and in vivo experiments. Furthermore, safety assessments revealed that therapy-related toxicities were within acceptable limits and associated with manageable side effects.

In summary, our results suggest engineered Exos as a highly effective targeted drug delivery vehicle for eliminating c-Mpl+ AML cells while maintaining a favorable safety profile. These findings also provide valuable insights for developing therapeutic strategies for AML and other tumors characterized by specific membrane protein expression.

Wound defects pose a substantial challenge in clinical practice, often resulting in prolonged healing times and an elevated risk of infection. Insufficient vascularization is a critical factor that adversely affects wound healing. Exosomes obtained from bone mesenchymal stem cells (BMSC-exos) have demonstrated significant promise in accelerating tissue repair by promoting angiogenesis. However, their limited yield and suboptimal biological functions impede widespread clinical application in enhancing wound healing. Prior research has indicated that 3D cultures can boost exosome secretion when compared to conventional 2D cultures. However, the currently prevalent 3D culture methods often necessitate expensive equipment or cumbersome procedures. This study investigates a cost-effective and user-friendly 3D culture system developed using gelatin methacrylate (GelMA). Our findings indicate that a 5% concentration of GelMA provides an optimal environment for the 3D culture of BMSCs. Furthermore, we observed that 3D culture significantly delays the senescence of BMSCs, thereby creating favorable conditions for the sustained production of exosomes. Additionally, 3D cultivation has the potential to boost exosome secretion and enhance their angiogenic capabilities. In vivo experiments further confirmed that BMSC-exos from a 3D environment exhibit enhanced capabilities to promote wound healing. These results suggest that GelMA-based 3D cultures offer a novel strategy for both industrial production and clinical application of exosomes.

Viruses may cause a wide range of renal problems. Furthermore, many kidney diseases may be brought on by viral infections. Both the primary cause and a contributing factor of acute kidney injury (AKI) may be viral infections. As an example, it is recommended that patients with dengue virus (DENV) infections undergo careful monitoring of their AKI levels. Also, researchers' data so far lend credence to the several hypothesized pathophysiological mechanisms via which AKI can develop in SARS-CoV- 2 infection. Thus, it is critical to comprehend how viral infections cause AKI. Finding an effective method of treating AKI caused by viruses is also vital. Thus, a potential cell-free method for treating AKI that uses regenerative and anti-inflammatory processes is mesenchymal stem cells (MSCs) and their exosomes (MSC-EXOs). MSCs alleviate tissue damage and enhance protective effects on damaged kidneys in AKI. Furthermore, MSC-EXOs have exhibited substantial regulatory impact on a range of immune cells and exhibit robust immune regulation in the therapy of AKI. Thus, in models of AKI caused by ischemia-reperfusion damage, nephrotoxins, or sepsis, MSCs and MSC-EXOs improved renal function, decreased inflammation, and improved healing. Therefore, MSCs and MSC-EXOs may help treat AKI caused by different viruses. Consequently, we have explored several innovative and significant processes in this work that pertain to the role of viruses in AKI and the significance of viral illness in the onset of AKI. After that, we assessed the key aspects of MSCs and MSC-EXOs for AKI therapy. We have concluded by outlining the current state of and plans for future research into MSC- and EXO-based therapeutic approaches for the treatment of AKI brought on by viruses.

Spinal cord injury (SCI) is a devastating event for the central nervous system (CNS), often resulting in the loss of sensory and motor functions. It profoundly affects both the physiological and psychological well-being of patients, reducing their quality of life while also imposing significant economic pressure on families and the healthcare system. Due to the complex pathophysiology of SCI, effective treatments for promoting recovery remain scarce. Mesenchymal stem cell-derived exosomes (MSC-Exos) offer advantages such as low immunogenicity, good biocompatibility, and the ability to cross the blood-spinal cord barrier (BSCB). In preclinical studies, they have progressively shown efficacy in promoting SCI repair and functional recovery. However, the low yield and insufficient targeting of MSC-Exos limit their therapeutic efficacy. Currently, genetic engineering and other preprocessing techniques are being employed to optimize both the yield and functional properties of exosomes, thereby enhancing their therapeutic potential. Therefore, this paper provides an overview of the pathophysiology of SCI and the biogenesis of exosomes. It also summarizes current approaches to optimizing exosome performance. Additionally, it details the mechanisms through which optimized exosomes provide neuroprotection and explores the potential of combined treatments involving MSC-Exos and hydrogels.

Extracellular vesicles (EVs) have been explored as promising vehicles for drug delivery. One of the most valuable features of EVs is their ability to cross physiological barriers, particularly the blood-brain barrier (BBB). This significantly enhances the development of EV-based drug delivery systems for the treatment of CNS disorders. The present review focuses on the factors and techniques that contribute to the successful delivery of EV-based therapeutics to the brain. Here, we discuss the major methods of brain targeting which includes the utilization of different administration routes, capitalizing on the biological origins of EVs, and the modification of EVs through the addition of specific ligands on to the surface of EVs. Finally, we discuss the current challenges in large-scale EV production and drug loading while highlighting future perspectives regarding the application of EV-based therapeutics for brain delivery.

Cancer continues to pose a global threat despite potent anticancer drugs, often accompanied by undesired side effects. To enhance patient outcomes, sophisticated multifunctional approaches are imperative. Small extracellular vesicles (EVs), a diverse family of naturally occurring vesicles derived from cells, offer advantages over synthetic carriers. Among the EVs, the exosomes are facilitating intercellular communication with minimal toxicity, high biocompatibility, and low immunogenicity. Their tissue-specific targeting ability, mediated by surface molecules, enables precise transport of biomolecules to cancer cells. Here, we explore the potential of exosomes as innovative therapeutic agents, including cancer vaccines, and their clinical relevance as biomarkers for clinical diagnosis. We highlight the cargo possibilities, including nucleic acids and drugs, which make them a good delivery system for targeted cancer treatment and contrast agents for disease monitoring. Other general aspects, sources, and the methodology associated with therapeutic cancer applications are also reviewed. Additionally, the challenges associated with translating exosome-based therapies into clinical practice are discussed, together with the future prospects for this innovative approach.

The treatment of congenital deformities, traumatic injuries, infectious diseases, and tumors in the craniomaxillofacial (CMF) region is complex due to the intricate nature of the tissues involved. Conventional treatments such as bone grafts and cell transplantation face limitations, including the need for multiple surgeries, complications, and safety concerns.

This paper aims to provide a comprehensive analysis of the role of exosomes (EXOs) in CMF and dental tissue regeneration and to explore their potential applications in regenerative dental medicine.

An extensive review of advancements in tissue engineering, materials sciences, and nanotechnology was conducted to evaluate the development of delivery systems for EXOs-based therapies. The analysis included how EXOs, as nanovesicles released by cells, can be modified to target specific cells or loaded with functional molecules for drug or gene delivery.

EXOs have emerged as a promising alternative to cell transplant therapy, offering a safer method for cell communication and epigenetic control. EXOs transport important proteins and genetic materials, facilitating intercellular communication and delivering therapeutics effectively. The potential of EXOs in personalized medicine, particularly in diagnosing, customizing treatment, and predicting patient responses, is highlighted.

EXO-mediated therapy holds significant potential for advancing tissue regeneration, offering targeted, personalized treatment options with reduced side effects. However, challenges in purification, production, and standardized protocols need to be addressed before its clinical application can be fully realized.

Human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) have shown great potential in tissue repair and regeneration. However, their scalable production and functional quality are still limited by current expansion technologies. In this study, we propose a production technology for hMSC-EVs based on three-dimensional (3D) microbead culture, which enhances the secretory behaviour of hMSC. Fixed number of MSCs were encapsulated in Matrigel at appropriate densities and printed into 3D microbeads by the custom automated microfluidic bead-jet printing technique. Compared with 2D culture group, EVs derived from 3D hMSC microbead had smaller size and increased yield by 20-fold, and the actin depolymerisation of the cell may be an important mechanism for enhancing EV secretion. Further analysis confirmed that the EVs derived from 3D hMSC microbead exhibited enhanced angiogenic and proliferative capabilities, which promoted the viability and tube-forming capacity of human umbilical vein endothelial cells (HUVEC). In conclusion, this automated microfluidic microbead encapsulation technology increased the yield and therapeutic effect of hMSC-EVs and provides a platform for scalable EV production of regenerative therapies.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as a promising approach in regenerative therapy. However, the clinical application of MSC-EVs is hindered by the presence of xenogenic components, such as fetal bovine serum (FBS), which is the most used culture supplement for MSCs. Human platelet lysate (HPL) has been proposed as an alternative to FBS, but whether MSC-EVs derived from HPL-cultured MSCs are suitable for clinical translation remains unclear. In this study, we comprehensively compared the characterization of EVs derived from MSCs cultured in the medium with FBS (F-EVs) and HPL (H-EVs). Our study showed that HPL promoted MSC-EV production without compromising EVs critical quality attributes. Multiomics sequencing revealed the stability of H-EVs from different umbilical cord donors and global functional alterations for MSC-EVs under different culture conditions. In comparison to F-EVs, H-EVs enriched more angiogenesis-related molecules and exhibited enhanced angiogenesis, which were further confirmed by in vivo and in vitro studies. H-EVs significantly reduced renal microvascular rarefaction and promoted the regeneration of umbilical vein endothelial cells to hypoxia stimulation compared to that of F-EVs. In conclusion, our findings demonstrated that HPL as culture supplements did not alter the critical quality attributes of MSC-EVs, specifically holding a higher yield and quality of MSC-EVs with enhanced angiogenic potential.

Mesenchymal stem cell-derived exosomes exhibit promising potential in tissue regeneration. Recent studies highlight its significant therapeutic potential in various stages of wound healing. However, the clinical translation of exosome-based therapy was hindered due to issues regarding low productivity and the lack of efficient production protocol to obtain a clinically relevant exosome quantity. Therefore, this study established a potential upscaling protocol to produce exosomes derived from canine adipose-derived mesenchymal stem cells (cAD-MSCs) and explored its potential for wound treatment. The potential upscaling protocol, termed VSCBIC-3-3D, was carried out using VSCBIC-3 in-house serum-free exosome-collecting solution in a three-dimensional (3D) culture system followed by the tangential flow filtration (TFF) isolation. Our findings suggest that culturing cAD-MSCs with VSCBIC-3 maintained cell morphology and viability. Compared to conventional two-dimensional (2D) protocols, The potential upscaling protocol increased exosome yield and concentration in conditioned medium by 2.4-fold and 3.2-fold, respectively. The quality assessment revealed enhanced purity and bioactivity of exosomes produced using the VSCBIC-3-3D protocol. In addition, the cAD-MSCs-derived exosomes were shown to significantly improve fibroblast migration, proliferation, and wound healing-related gene expression in vitro. This study collectively demonstrates that potential upscaling protocol establishment allowed robust production of exosomes from cAD-MSCs, which exhibit therapeutic potential for wound healing in vitro.

The therapeutic potential of extracellular vesicles (EVs) in reducing oral inflammation is thoroughly examined in this review, with an emphasis on gingivitis, periodontitis, and oral mucositis. It explains the complex relationship between microbial dysbiosis and host immune responses in the aetiology of oral inflammation. Pathophysiological mechanisms of periodontitis are examined, emphasising the roles played by periodontal pathogens and inflammatory mediators in the disease's chronic course and systemic effects. Preclinical research is providing new evidence that EVs originating from various cellular sources control immune cell dynamics towards a pro-healing phenotype, promote tissue regeneration, and have immunomodulatory qualities. EV-based therapies appear to be a promising new therapeutic technique with potential benefits over traditional methods for the treatment of oral inflammatory illnesses by specifically altering inflammatory signalling pathways. This review highlights the potential of EVs to improve patient outcomes in oral health and emphasises the need for additional clinical research to clarify the therapeutic efficacy and underlying mechanisms of EVs in periodontal therapy.

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) are nanoscale lipid bilayer vesicles secreted by mesenchymal stem cells. They inherit the parent cell's attributes, facilitating tissue repair and regeneration, promoting angiogenesis, and modulating the immune response, while offering advantages like reduced immunogenicity, straightforward administration, and enhanced stability for long-term storage. These characteristics elevate MSC-EVs as highly promising in cell-free therapy with notable clinical potential. It is critical to delve into their pharmacokinetics and thoroughly elucidate their intracellular and in vivo trajectories. A detailed summary and evaluation of existing tracing strategies are needed to establish standardized protocols. Here, we have summarized and anticipated the research progress of MSC-EVs in various biomedical imaging techniques, including fluorescence imaging, bioluminescence imaging, nuclear imaging (PET, SPECT), tomographic imaging (CT, MRI), and photoacoustic imaging. The challenges and prospects of MSC-EV tracing strategies, with particular emphasis on clinical translation, have been analyzed, with promising solutions proposed.

Interest in Current Good Manufacturing Practices (cGMP)-grade extracellular vesicles (EVs) is expanding. Some obstacles in this new but rapidly growing field include a lack of standardization and scalability. This review focuses on automated biomanufacturing of EVs in conditioned media collected from cultured mesenchymal stromal cells (MSCs). Different automated cell culture systems are discussed, including factors affecting EV quantity and quality, isolating EVs manufactured in an automated system, and validations needed. The ultimate goal when manufacturing cGMP-grade EVs is to identify a specific application and characterize the EV population in detail. This is achieved by validating every step of the process, choosing appropriate release criteria, and assuring batch-to-batch consistency. Due to the lack of standards in the field, it is critical to ensure that the cGMP-grade EVs meet FDA standards pertaining to identity, reproducibility, sterility, safety, purity, and potency. A closed-system automated bioreactor can be a valuable tool to generate cGMP-EVs in a scalable, economical, and reproducible manner.

Stromal cell-derived exosomes have demonstrated their value in the field of biomedical engineering. However, the low production and specific requirements of different diseases limited the practical efficacy of these exosomes and restricted their wider applications. Here, we presented a method to culture genetically engineered mesenchymal stromal cells (MSC) that overexpressed the hepatocyte growth factor (HGF) in microfluidics and harvest mass HGF overexpressed exosomes for wound healing. The microfluidic chips were featured with herringbone grooves and micropillar arrays, where sufficient fluidic mechanical stimuli and efficient nutrient delivery were promoted by a turbulent vortex. It was demonstrated that the production of exosomes was much higher than by the traditional flask cell culture, along with higher HGF content. In addition, the MSCHGF-secreted exosomes were applied for wound healing in diabetic rat model, showing superior angiogenesis, cell migration, and immune modulation capabilities. These features indicated that the genetically engineered MSCHGF exosomes from high-throughput herringbone microfluidics possess great potential for wound healing and related biomedical applications.

Mesenchymal stem/stromal cells (MSCs) are a valuable source of paracrine factors, as they have a remarkable secretory capacity, and there is a sizeable knowledge base to develop industrial and clinical production protocols. Promising cell-free approaches for tissue regeneration and immunomodulation are driving research towards secretome applications, among which extracellular vesicles (EVs) are steadily gaining attention. However, the manufacturing and application of EVs is limited by insufficient yields, knowledge gaps, and low standardization. Facing these limitations, hydrogels represent a versatile three-dimensional (3D) culture platform that can incorporate extracellular matrix (ECM) components to mimic the natural stem cell environment in vitro; via these niche-mimicking properties, hydrogels can regulate MSCs' morphology, adhesion, proliferation, differentiation and secretion capacities. However, the impact of the hydrogel's architectural, biochemical and biomechanical properties on the production of EVs remains poorly understood, as the field is still in its infancy and the interdependency of culture parameters compromises the comparability of the studies. Therefore, this review summarizes and discusses the reported effects of hydrogel encapsulation and culture on the secretion of MSC-EVs. Considering the effects of cell-material interactions on the overall paracrine activity of MSCs, we identify persistent challenges from low standardization and process control, and outline future paths of research, such as the synergic use of hydrogels and bioreactors to enhance MSC-EV generation.

Human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEV) have recently garnered attention as a potential therapeutic approach for kidney diseases with anti-inflammatory effects. Infiltrated macrophages play an important role in facilitating tissue regeneration. However, the intricate regulatory effects of hucMSC-sEV on macrophages during cisplatin-induced acute kidney injury (AKI) remain unknown. In this study, we uncovered that hucMSC-sEV exhibited potent anti-inflammation and effectively inhibited the polarization of M1 phenotype macrophages. Mechanically, miRNA sequencing analysis and qRT-PCR indicated that a novel miRNA, named miR-13896, was enriched in hucMSC-sEV. When transfected with miR-13896 mimic, macrophages displayed M2 phenotype with elevated levels of Arg1 and IL-10, while miR-13896 inhibitor promoted M1 phenotype. Furthermore, we firstly established that miR-13896 repressed Tradd expression by targeting its 3' untranslated region and subsequently inhibited NF-κB signaling pathway in macrophages. Additionally, to improve therapeutic effects, hucMSC-sEV were engineered with elevated levels of miR-13896 through electroporation, which resulted in promoting M2 phenotype macrophages, inhibiting inflammatory factors, and enhancing kidney repair. Conclusively, our findings provide novel insights into the mechanisms underlying the effects of hucMSC-sEV on macrophages and AKI, while also highlighting electroporation as a promising strategy for treating cisplatin-induced AKI.

The therapeutic potential of exosomes derived from mesenchymal stem cells (MSCs) is increasingly recognized in veterinary medicine. This study explored the feasibility of a microcarrier-based three-dimensional (3D) culture system for producing the exosomes (cEXO). Investigations were conducted to enhance production efficiency, ensure stability, and evaluate the therapeutic potential of cEXO for anti-inflammatory applications while assessing their safety profile.

The microcarrier-based 3D culture system improved efficient production of cEXO, yielding exosomes with acceptable profiles, including a size of approximately 81.22 nm, negative surface charge, and high particle concentration (1.32 × 109 particles/mL). Confocal imaging proved dynamic changes in cell viability across culture phases, highlighting the challenges of maintaining cell viability during repeated exosome collection cycles. Characterization via transmission electron microscopy, nanoparticle tracking analysis, and zeta-potential measurements confirmed the stability and functionality of cEXO, particularly when stored at -20 °C. Functional assays showed that cEXO exerted significant anti-inflammatory activity in RAW264.7 macrophages in an inverse dose-dependent manner, with no observed cytotoxicity to fibroblasts or macrophages. Acute toxicity testing in rats revealed no adverse effects on clinical parameters, organ health, or body weight, supporting the safety of cEXO for therapeutic use.

This study highlights the potential of a microcarrier-based 3D culture system for scalable cEXO production with robust anti-inflammatory activity, stability, and safety profiles. These findings advance the development of cEXO-based therapies and support their application in veterinary regenerative medicine.

Among the most challenging diseases to treat is lung cancer (LC). While immunotherapy has a checkered history, it has lately shown great promise in the treatment of LC, and interest in this promising new approach is on the rise around the globe. Immunotherapy using mesenchymal stem cells (MSCs) is gaining popularity. Regenerative medicine, cell therapy, and immune modulation are three areas that have shown significant interest in MSCs. More than that, MSCs have recently attracted attention as potential anti-cancer drug delivery vehicles due to their inherent ability to go home to tumor locations. Making MSCs a double-edged sword in the fight against neoplastic illnesses, they are also known to impart pro-oncogenic properties. Additionally, multiple studies have proposed extracellular vesicles (EVs) secreted by MSCs as a potential therapeutic agent or method for delivering anti-cancer drugs. However, there has been conflicting evidence regarding the impact of MSCs or MSC-EV on the behavior of cancer cells, and the exact mechanism for this effect is still unknown. Our research has focused on MSCs and their key characteristics, such as their immunomodulatory capabilities for cancer therapy. Our research has also explored the potential of MSCs and their derivatives to treat small-cell and non-small-cell lung cancers (NSCLC and SCLC, respectively) by leveraging MSCs' immunomodulatory characteristics. At the end of this article, we covered the pros and cons of this therapy procedure, as well as what researchers want to do in the future to make it more suitable for clinical application in LC treatment.

Extracellular vesicles (EVs) are secreted from biological cells and contain many molecules with diagnostic values or therapeutic functions. There has been great interest in academic and industrial communities to utilize EVs as tools for diagnosis or therapeutics. In addition, EVs can also serve as delivery vehicles for therapeutic molecules. An indicator of the enormous interest in EVs is the large number of review articles published on EVs, with the focus ranging from their biology to their applications. An emerging trend in EV research is to produce and utilize "engineered EVs", which are essentially the enhanced version of EVs. EV engineering can be conducted by cell culture condition control, genetic engineering, or chemical engineering. Given their nanometer-scale sizes and therapeutic potentials, engineered EVs are an emerging class of nanomedicines. So far, an overwhelming majority of the research on engineered EVs is preclinical studies; there are only a very small number of reported clinical trials. This Review focuses on engineered EVs, with a more specific focus being their applications in therapeutics. The various approaches to producing engineered EVs and their applications in various diseases are reviewed. Furthermore, in vivo imaging of EVs, the mechanistic understandings, and the clinical translation aspects are discussed. The discussion is primarily on preclinical studies while briefly mentioning the clinical trials. With continued interdisciplinary research efforts from biologists, pharmacists, physicians, bioengineers, and chemical engineers, engineered EVs could become a powerful solution for many major diseases such as neurological, immunological, and cardiovascular diseases.

Augmented extracellular matrix (ECM) stiffness is a mechanical hallmark of cancer. Mechanotransduction studies have extensively probed the mechanisms by which ECM stiffness regulates intracellular communication. However, the influence of stiffness on intercellular communication aiding tumor progression in three-dimensional microenvironments remains unknown. Small extracellular vesicles (EVs) are communicators of altered biophysical cues to distant sites through EV-ECM interactions and EV-mediated recipient cell-ECM interactions. Here we demonstrate stiffness-mediated modulation of small EVs secretion and cargo from three-dimensional oral squamous cell carcinoma spheroids. Using a spheroid culture platform with varying matrix stiffness properties, we show that small EVs carry parental biomolecular cargo, including mechanosensitive Piezo1 ion channel and adhesion molecule CD44. We comprehensively validate the presence of both markers in our EV populations using proteomic and genetic analysis. Transcriptomic analysis of microRNA and long non-coding RNA cargo of small EVs released from soft and stiff ECM spheroids revealed enrichment of tumorigenic and metastatic profiles in EVs from stiff ECM cultures compared to that of soft ones. Gene set enrichment analysis of a comparative dataset obtained by overlaying spheroid mRNA and EV miRNA profiles identified key oncogenic pathways involved in cell-EV crosstalk in the spheroid model.

Intervertebral disc degeneration is the leading cause of low back pain, imposing significant burdens on patients, societies, and economies. Advancements in regenerative medicine have spotlighted extracellular vesicles as promising nanoparticles for intervertebral disc degeneration treatment. Extracellular vesicles retain the potential of cell therapy and serve as carriers to deliver their cargo to target cells, thereby regulating cell activity. This review summarizes the biogenesis and molecular composition of extracellular vesicles and explores their therapeutic roles in intervertebral disc degeneration treatment through various mechanisms. These mechanisms include mitigating cell loss and senescence, delaying extracellular matrix degeneration, and modulating the inflammatory microenvironment. Additionally, it highlights recent efforts in engineering extracellular vesicles to enhance their targeting and therapeutic efficacy. The integration of extracellular vesicle-based acellular therapy is anticipated to drive significant advancements in disc regenerative medicine.

Existing clinical treatment strategies often fail to effectively address the challenges associated with regenerating degenerated intervertebral discs. As a new regenerative medicine strategy, the extracellular vesicle strategy avoids the risks associated with cell transplantation and shows great promise in treating intervertebral disc degeneration by carrying therapeutic cargo. This review comprehensively examines the latest research, underlying mechanisms, and therapeutic potential of extracellular vesicles, offering a promising new strategy for intervertebral disc degeneration treatment.

Extracellular vesicles (EVs) have emerged as a promising strategy for treating a wide spectrum of pathologies, as they can deliver their cargo to recipient cells and regulate the signaling pathway of these cells to modulate their fate. Despite the great potential of EVs in clinical applications, their low yield and the challenges of cargo loading remain significant obstacles, hindering their transition from experimental research to clinical practice. Therefore, promoting EV release and enhancing EV cargo-loading are promising fields with substantial research potential and broad application prospects. In this review, we summarize the clinical applications of EVs, the methods and technologies for their large-scale production, engineering, and modification, as well as the challenges that must be addressed during their development. We also discuss the future perspectives of this exciting field of research to facilitate its transformation from bench to clinical reality.

Kidney diseases are characterized by their intricate nature and complexity, posing significant challenges in their treatment and diagnosis. Nanoparticles (NPs), which can be further classified as synthetic and biomimetic NPs, have emerged as promising candidates for treating various diseases. In recent years, the development of engineered nanotherapeutics has focused on targeting damaged tissues and serving as drug delivery vehicles. Additionally, these NPs have shown superior sensitivity and specificity in diagnosis and imaging, thus providing valuable insights for the early detection of diseases. This review aims to focus on the application of engineered synthetic and biomimetic NPs in kidney diseases in the aspects of treatment, diagnosis, and imaging. Notably, the current perspectives and challenges are evaluated, which provide inspiration for future research directions, and encourage the clinical application of NPs in this field.

The global issue of aging populations has become increasingly prominent, thus the research and development for anti-aging therapies to assure longevity as well as to ameliorate age-related complications is put high on the agenda. The young humoral milieu has been substantiated to impart youthful characteristics to aged cells or organs. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membrane-limited structures that serve as couriers of proteins and genetic material to regulate intercellular communication. Of note, EVs appeared to be an indispensable component of young blood in prolonging lifespans, and circulating EVs have been indicated to mediate the beneficial effect of a young milieu on aging. Human umbilical cord mesenchymal stem cell-derived EVs (HUCMSC-EVs), isolated from the youngest adult stem cell source, are speculated to reproduce the function of circulating EVs in young blood and partially revitalize numerous organs in old animals. Robust evidence has suggested HUCMSC-EVs as muti-target therapeutic agents in combating aging and alleviating age-related degenerative disorders. Here, we provide a comprehensive overview of the anti-aging effects of HUCMSC-EVs in brain, heart, vasculature, kidney, muscle, bone, and other organs. Furthermore, we critically discuss the current investigation on engineering strategies of HUCMSC-EVs, intending to unveil their full potential in the field of anti-aging research.

Exosomes, small extracellular vesicles secreted by cells, have emerged as focal mediators in intercellular communication and therapeutic interventions across diverse biomedical fields. Inflammatory disorders, including inflammatory bowel disease, acute liver injury, lung injury, neuroinflammation, and myocardial infarction, are complex conditions that require innovative therapeutic approaches. This review summarizes recent advances in exosome-based therapies for inflammatory disorders, highlighting their potential as diagnostic biomarkers and therapeutic agents. Exosomes have shown promise in reducing inflammation, promoting tissue repair, and improving functional outcomes in preclinical models of inflammatory disorders. However, further research is needed to overcome the challenges associated with exosome isolation, characterization, and delivery, as well as to fully understand their mechanisms of action. Current limitations and future directions in exosome research underscore the need for enhanced isolation techniques and deeper mechanistic insights to harness exosomes' full therapeutic potential in clinical applications. Despite these challenges, exosome-based therapies hold great potential for the treatment of inflammatory disorders and may offer a new paradigm for personalized medication.

Mesenchymal stem cells (MSCs) are highly valued in regenerative medicine due to their ability to self-renew and differentiate into various cell types. Their therapeutic benefits are primarily due to their paracrine effects, in particular through extracellular vesicles (EVs), which are related to intercellular communication. Recent advances in EV production and extraction technologies highlight the potential of MSC-derived EVs (MSC-EVs) in tissue engineering and regenerative medicine. MSC-EVs offer several advantages over traditional cell therapies, including reduced toxicity and immunogenicity compared with whole MSCs. EVs carrying functional molecules such as growth factors, cytokines, and miRNAs play beneficial roles in tissue repair, fibrosis treatment, and scar prevention by promoting angiogenesis, skin cell migration, proliferation, extracellular matrix remodeling, and reducing inflammation. Despite the potential of MSC-EVs, there are several limitations to their use, including variability in quality, the need for standardized methods, low yield, and concerns about the composition of EVs and the potential risks. Overall, MSC-EVs are a promising alternative to cell-based therapies, and ongoing studies aim to understand their actions and optimize their use for better clinical outcomes in wound healing and skin regeneration.

Fungi-derived exosome-like nanovesicles (ENs) are emerging as a highly promising class of nanoparticles, particularly noted for their cost-effective production. However, their impact on immune regulation and their potential as anti-tumor agents need further exploration. Our study specifically focused on the investigation of the immunomodulatory and anti-tumor properties of ENs derived from Cordyceps militaris, an edible fungus that had achieved large-scale commercial production, referred to as CMDENs.

The ENs of C. militaris were collected through ultra-high-speed centrifugation, followed by characterization of their physicochemical properties and contents. Subsequently, the biological distribution of these vesicles was investigated using in vivo fluorescence imaging experiments. Finally, the immune activation and polarization of macrophages were examined through both in vitro and in vivo experiments.

Herein, we presented the discovery of CMDENs that were rich in proteins, lipids, flavonoids and alkaloids. Immunomodulatory experiments conducted in vivo demonstrated that CMDENs exhibited protective effects against cyclophosphamide-induced immunosuppression in mice by significantly enhancing macrophage phagocytosis and peripheral blood immune cell counts. Moreover, CMDENs effectively induced the polarization of M0- and M2-like macrophages toward M1-like phenotype by activating MAPKs signaling pathway. Notably, CMDENs exhibited remarkable capabilities in inhibiting tumor growth by reprogramming tumor-associated macrophages and activating tumor-infiltrating T lymphocytes, without any observed toxicity in mice bearing tumors.

Our research suggested that CMDENs possessed the potential to be explored as a nano-immunomodulatory agent for cancer.

Since the discovery that exosomes can exchange genes, their potential use as tools for tissue regeneration, disease diagnosis, and therapeutic applications has drawn significant attention. Emerging three-dimensional (3D) printing technologies, such as bioprinting, which allows the printing of cells, proteins, DNA, and other biological materials, have demonstrated the potential to create complex body tissues or personalized 3D models. The use of 3D spheroids in bioprinting facilitates volumetric tissue reconstruction and accelerates tissue regeneration via exosome secretion. In this review, we discussed a convergence approach between two promising technologies for bioprinting and exosomes in regenerative medicine. Among the various 3D cell culture methods used for exosome production, we focused on spheroids, which are suitable for mass production by bioprinting. We then summarized the research results on cases of bioprinting applications using the spheroids and exosomes produced. If a large number of spheroids can be supplied through bioprinting, the spheroid-exosome-based bioprinting technology will provide new possibilities for application in tissue regeneration, disease diagnosis, and treatment.

Small extracellular vesicles (sEVs) have great promise as effective carriers for drug delivery. However, the challenges associated with the efficient production of sEVs hinder their clinical applications. Herein, we report a stimulative 3D culture platform for enhanced sEV production. The proposed platform consists of a piezoelectric nanofibrous scaffold (PES) coupled with acoustic stimulation to enhance sEV production of cells in a 3D biomimetic microenvironment. Combining cell stimulation with a 3D culture platform in this stimulative PES enables a 15.7-fold increase in the production rate per cell with minimal deviations in particle size and protein composition compared with standard 2D cultures. We find that the enhanced sEV production is attributable to the activation and upregulation of crucial sEV production steps through the synergistic effect of stimulation and the 3D microenvironment. Moreover, changes in cell morphology lead to cytoskeleton redistribution through cell-matrix interactions in the 3D cultures. This in turn facilitates intracellular EV trafficking, which impacts the production rate. Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials for enhanced sEV production. This platform is expected to accelerate the potential use of sEVs for drug delivery and broad biomedical applications.

Renal fibrosis is a complex and multifactorial process that involves inflammation, cell proliferation, collagen, and fibronectin deposition in the kidney, ultimately leading to chronic kidney disease and even end-stage renal disease. The main goal of treatment is to slow down or halt the progression of fibrosis and to improve or preserve kidney function. Despite significant progress made in understanding the underlying mechanisms of renal fibrosis, current therapies have limited renal protection as the disease progresses. Exosomes derived from stem cells are a newer area of research for the treatment of renal fibrosis. Exosomes as nano-sized extracellular vesicles carry proteins, lipids, and nucleic acids, which can be taken up by local or distant cells, serving as mediators of intercellular communication and as drug delivery vehicles. Exosomes deliver molecules that reduce inflammation, renal fibrosis and extracellular matrix protein production, and promote tissue regeneration in animal models of kidney disease. Additionally, they have several advantages over stem cells, such as being non-immunogenic, having low risk of tumor formation, and being easier to produce and store. This review describes the use of natural and engineered exosomes containing therapeutic agents capable of mediating anti-inflammatory and anti-fibrotic processes during both acute kidney injury and chronic kidney disease. Exosome-based therapies will be compared with stem cell-based treatments for tissue regeneration, with a focus on renal protection. Finally, future directions and strategies for improving the therapeutic efficacy of exosomes are discussed.

Restoring the function of the ovary is important for chemotherapy-induced ovarian failure (COF) patients. Stem cell and extracellular vesicles (EVs) therapy show promise but need further improvement.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) were primarily cultured and further three-dimensional (3D) cultured using an ultra-low attachment surface method. The expression levels of nutritional cytokines and immunomodulatory and stemness-related genes of 3D-cultured hUC-MSCs were analyzed. EVs were isolated by ultracentrifugation and characterized. Ovaries were decellularized with sodium dodecyl sulfate to obtain extracellular matrix (ECM). Lyophilized EVs from three-dimensional (2D) or 3D hUC-MSCs were mixed with ECM to prepare the 2D/3D-MSC-EVs-ECM gels. The therapeutic effect of the MSC-EVs-ECM gel on cyclophosphamide (CTX) -treated rats was analyzed through various tests. RNA sequencing was used to analyze the expression changes of genes before and after treatment.

After culturing in ultra-low attachment dishes, hUC-MSCs aggregated into spheroids and significantly upregulated the expression levels of immunomodulatory and stemness-related genes. The total EVs yield was also upregulated (5.6-fold) after 3D culture. The cell viability of CTX-treated ovarian granulosa cells (OGCs) was significantly rescued by coculture with the 3D-MSC-EVs-ECM gel. Hormones indicative of ovarian function, AMH, E2, and FSH, were recovered in both the CTX + 2D-MSC-EVs-ECM gel group and the CTX + 3D-MSC-EVs-ECM gel group, while the apoptosis-related protein Bax was significantly downregulated. The 3D-MSC-EVs-ECM gel was more effective than the 2D-MSC-EVs-ECM gel. Significantly differentially expressed genes, such as Hbb-b1, Gpd1, and Sirpa, were detected by RNA sequencing. Hbb-b1 was increased in the ovaries of CTX-treated rats, and this increase was attenuated by injecting the 2D/3D-MSC-EVs-ECM gel. Gpd1 was increased after CTX treatment, and this increase was reversed by the 3D-MSC-EVs-ECM gel. Sirpa was decreased in the ovaries of CTX-treated rats, and this decrease was attenuated by injecting the 3D-MSC-EVs-ECM gel.

Our study demonstrated that the 3D-MSC-EVs-ECM gel is an efficient strategy for the recovery of ovarian function in CTX-induced ovarian failure.

Extracellular vesicles (EVs) are natural carriers of biomolecules that play a crucial role in cell-to-cell communication and tissue homeostasis under normal and pathological conditions, including inflammatory diseases and cancer. Since the discovery of the pro-regenerative and immune-modulating properties of EVs, EV-based therapeutics have entered clinical trials for conditions such as myocardial infarction and autoimmune diseases, among others. Due to their unique advantages-such as superior bioavailability, substantial packaging capacity, and the ability to traverse biological barriers-EVs are regarded as a promising platform for targeted drug delivery. However, achieving a sufficient accumulation of therapeutic agents at the target site necessitates a larger quantity of EVs per dose compared to using EVs as standalone drugs. This challenge can be addressed by administering larger doses of EVs, increasing the drug dosage per administration, or enhancing the selective accumulation of EVs at target cells. In this review, we will discuss methods to improve the isolation and purification of EVs, approaches to enhance cargo packaging-including proteins, RNAs, and small-molecule drugs-and technologies for displaying targeting ligands on the surface of EVs to facilitate improved targeting. Ultimately, this guide can be applied to the development of novel classes of EV-based therapeutics and to overcoming existing technological challenges.

Extracellular vesicles (EV) are a heterogeneous group of lipid particles excreted by cells. They play an important role in regeneration, development, inflammation, and cancer progression, together with the extracellular matrix (ECM), which they constantly interact with. In this review, we discuss direct and indirect interactions of EVs and the ECM and their impact on different physiological processes. The ECM affects the secretion of EVs, and the properties of the ECM and EVs modulate EVs' diffusion and adhesion. On the other hand, EVs can affect the ECM both directly through enzymes and indirectly through the modulation of the ECM synthesis and remodeling by cells. This review emphasizes recently discovered types of EVs bound to the ECM and isolated by enzymatic digestion, including matrix-bound nanovesicles (MBV) and tissue-derived EV (TiEV). In addition to the experimental studies, computer models of the EV-ECM-cell interactions, from all-atom models to quantitative pharmacology models aiming to improve our understanding of the interaction mechanisms, are also considered. STATEMENT OF SIGNIFICANCE: Application of extracellular vesicles in tissue engineering is an actively developing area. Vesicles not only affect cells themselves but also interact with the matrix and change it. The matrix also influences both cells and vesicles. In this review, different possible types of interactions between vesicles, matrix, and cells are discussed. Furthermore, the united EV-ECM system and its regulation through the cellular activity are presented.

Extracellular vesicles (EVs) are minute sacs released by cells into the extracellular milieu, harboring an array of biomolecules including proteins, nucleic acids, and lipids. Notably, a large number of studies have demonstrated the important involvement of EVs in both physiological and pathological aspects of renal function. EVs can facilitate communication between different renal cells, but it is important to recognize their dual role: they can either transmit beneficial information or lead to renal damage and worsening of existing conditions. The composition of EVs in the context of the kidneys offers valuable insights into the intricate mechanisms underlying specific renal functions or disease states. In addition, mesenchymal stem cell-derived EVs have the potential to alleviate acute and chronic kidney diseases. More importantly, the innate nanoparticle properties of EVs, coupled with their engineering potential, make them effective tools for drug delivery and therapeutic intervention. In this review, we focus on the intricate biological functions of EVs in the kidney. In addition, we explore the emerging role of EVs as diagnostic tools and innovative therapeutic agents in a range of renal diseases.