Production Of Extracellular Vesicles Research Articles

Role of Hypoxia in Mesenchymal Stem Cells from Dental Pulp: Influence, Mechanism and Application.

Mesenchymal stem cells (MSCs) from dental pulp (DP-MSCs), which include dental pulp stem cells (DPSCs) isolated from permanent teeth and stem cells from human exfoliated deciduous teeth (SHED), have emerged as highly promising cell sources for tissue regeneration, due to their high proliferative rate, multi-lineage differentiation capability and non-invasive accessibility. DP-MSCs also exert extensive paracrine effects through the release of extracellular vesicles (EVs) and multiple trophic factors. To be noted, the microenvironment, commonly referred to as the stem cell niche, plays a crucial role in shaping the functionality and therapeutic effects of DP-MSCs, within which hypoxia has garnered considerable attention. Extensive research has demonstrated that hypoxic conditions profoundly impact DP-MSCs. Specifically, hypoxia promotes DP-MSC proliferation, survival, stemness, migration, and pro-angiogenic potential while modulating their multi-lineage differentiation capacity. Furthermore, hypoxia stimulates the paracrine activities of DP-MSCs, leading to an increased production of EVs and soluble factors. Considering these findings, hypoxia preconditioning has emerged as a promising approach to enhance the therapeutic potential of DP-MSCs. In this comprehensive review, we provide a systematic overview of the influence of hypoxia on DP-MSCs, shedding light on the underlying mechanisms involved. Moreover, we also discuss the potential applications of hypoxia-preconditioned DP-MSCs or their secretome in tissue regeneration. Additionally, we delve into the methodologies employed to simulate hypoxic environments. This review aims to promote a comprehensive and systematic understanding of the hypoxia-induced effects on DP-MSCs and facilitate the refinement of regenerative therapeutic strategies based on DP-MSCs.

Carbon Monoxide-induced Autophagy Enhances Human Mesenchymal Stromal Cell Function via Paracrine Actions in Murine Polymicrobial Sepsis

Sepsis is a life-threatening process due to organ dysfunction resulting from severe infections. Mesenchymal stromal cells (MSCs) are being investigated as therapy for sepsis, along with conditioning regimens to improve their function. Carbon monoxide (CO) gas, which is cytoprotective at low doses, induces autophagy and is a mediator of inflammation. We evaluated CO-induced autophagy in human(h) MSCs, and its impact on cell function in murine cecal ligation and puncture. Conditioning of hMSCs with CO ex vivo resulted in enhanced survival and bacterial clearance in vivo, and neutrophil phagocytosis of bacteria in vitro. Decreased neutrophil infiltration and less parenchymal cell death in organs were associated with increased macrophage efferocytosis of apoptotic neutrophils, promoting resolution of inflammation. These CO effects were lost when the cells were exposed to autophagy inhibition prior to gas exposure. When assessing paracrine actions of CO-induced autophagy, extracellular vesicles (EVs) were predominantly responsible. CO had no effect on EV production, but altered their miRNA cargo. Increased expression of miR-145-3p and miR-193a-3p by CO was blunted with disruption of autophagy, and inhibitors of these miRNAs led to a loss of neutrophil phagocytosis and macrophage efferocytosis. Collectively, CO-induced autophagy enhanced hMSC function during sepsis via paracrine actions of MSC-derived EVs.

Aspects of conceptualization, development, and simulation of an extracellular vesicles' bulk isolation device for cosmetic and therapeutic applications

This paper discusses the concept and model to develop a prototype extracellular vesicle bulk-isolation device (EBID). It proposes an engineering design and potential ways to mitigate limitations in the currently available extracellular vesicle (EV) isolation techniques. A more convenient isolation technique is developed and presented to ensure production-scale isolation and purification of the specific size of EV (approximately 0.1 to 0.34 µm) from a large quantity of cell-culture media or biological fluid sources. A more appropriate design concept was established based on the chitosan-assisted precipitation of EV techniques and the membrane filtration technology-based prototype. After that, a novel theoretical model for bulk isolation of EVs was developed and presented using pressure-driven circulation and particulate flow in the EBID loop, where the EV production results from the biofluid flowing through the EBID membrane filter module (MFM). Governing equations are coupled with Darcy's law to investigate the biofluid flow behavior in the EBID MFM, therefore simulating the isolation process of EVs from their biofluids. The flow characteristics of the EV isolation process within the EBID MFM are theoretically simulated and discussed for improved isolation yield with a high degree of quality and purity.

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity

Background: Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. Methods: The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Results: Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. Conclusions: The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Unlocking the Potential of Extracellular Vesicles as the Next Generation Therapy: Challenges and Opportunities.

Mesenchymal stem cells (MSCs) have undergone extensive investigation for their potential therapeutic applications, primarily attributed to their paracrine activity. Recently, researchers have been exploring the therapeutic potential of extracellular vesicles (EVs) released by MSCs. MEDLINE/PubMed and Google scholar databases were used for the selection of literature. The keywords used were mesenchymal stem cells, extracellular vesicles, clinical application of EVs and challenges EVs production. These EVs have demonstrated robust capabilities in transporting intracellular cargo, playing a critical role in facilitating cell-to-cell communication by carrying functional molecules, including proteins, RNA species, DNAs, and lipids. Utilizing EVs as an alternative to stem cells offers several benefits, such as improved safety, reduced immunogenicity, and the ability to traverse biological barriers. Consequently, EVs have emerged as an increasingly attractive option for clinical use. From this perspective, this review delves into the advantages and challenges associated with employing MSC-EVs in clinical settings, with a specific focus on their potential in treating conditions like lung diseases, cancer, and autoimmune disorders.

Process development for the production of mesenchymal stromal cell-derived extracellular vesicles in conventional 2D systems

In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm−2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases.

Pheromone-based communication influences the production of somatic extracellular vesicles in C. elegans

Extracellular vesicles (EVs) are integral to numerous biological processes, yet it is unclear how environmental factors or interactions among individuals within a population affect EV-regulated systems. In Caenorhabditis elegans, the evolutionarily conserved large EVs, known as exophers, are part of a maternal somatic tissue resource management system. Consequently, the offspring of individuals exhibiting active exopher biogenesis (exophergenesis) develop faster. Our research focuses on unraveling the complex inter-tissue and social dynamics that govern exophergenesis. We found that ascr#10, the primary male pheromone, enhances exopher production in hermaphrodites, mediated by the G-protein-coupled receptor STR-173 in ASK sensory neurons. In contrast, pheromone produced by other hermaphrodites, ascr#3, diminishes exophergenesis within the population. This process is regulated via the neuropeptides FLP-8 and FLP-21, which originate from the URX and AQR/PQR/URX neurons, respectively. Our results reveal a regulatory network that controls the production of somatic EV by the nervous system in response to social signals.

Exploring the role of production and release of proteins for microbial interactions in kombucha

Driving kombucha fermentation through microbial consortia remains based on scarce scientific elements, especially regarding the consumption of nitrogenous nutrients. This study aimed at analyzing quantitatively proteins and free amino nitrogen (FAN) concentrations, and qualitatively the soluble proteins profiles during a 12-day production process. The effect of microbial interactions was evaluated by comparing the growth of previously isolated major microorganisms in monoculture and cocultures to original kombucha. Protein and FAN relative concentrations kinetics depended on microbial compositions. These parameters were associated to less variations and a positive effect on total microbial population in original kombucha, underlining a complex yet better management of the nutrients. Analysis of extracellular proteins by electrophoresis showed the presence of a microbial 15 kDa fraction common to all cultures. In contrast, a 21 kDa protein fraction was associated with the presence of Acetobacter indonesiensis. Exploratory proteomics analysis revealed the presence of characteristic extracellular moonlighting proteins, originating from this acetic bacteria. Production of extracellular vesicles was successfully confirmed with the isolation and observation of vesicles in A. indonesiensis culture, as well as in kombucha. The results characterize the central role of microbial interactions on FAN consumption and open new perspectives on the function of the bacterial vesicles observed.

Glyphosate and aminomethylphosphonic acid metabolite (AMPA) modulate the phenotype of murine melanoma B16-F1 cells

Pesticides are contaminants run-offs from agricultural areas with a global concern due to their toxicity for non-target organisms. The Brazilian Health Surveillance Agency reported about 63% of the food contain pesticide residues. Glyphosate is a herbicide used worldwide but its toxicity is not a consensus among specialists around the world. AMPA (aminomethylphosphonic acid) is a glyphosate metabolite that can be more toxic than the parental molecule. Melanoma murine B16-F1 cells were exposed to glyphosate and AMPA to investigate the cell profile and possible induction to a more malignant phenotype. Glyphosate modulated the multi-drug resistance mechanisms by ABCB5 gene expression, decreasing cell attachment, increasing cell migration and inducing extracellular vesicles production, and the cells exposed to AMPA revealed potential damages to DNA. The present study observed that AMPA exhibits high cytotoxicity, which suggests a potential impact on non-tumor cells, which are, in general, more susceptible to chemical exposure. Conversely, glyphosate favored a more metastatic and chemoresistant behavior in cancer cells, highlighting the importance of additional research in this area.

Oxysterols in Cell Viability, Phospholipidosis and Extracellular Vesicles Production in a Lung Cancer Model.

The study carried out systematic research on the influence of selected oxysterols on cells viability, phospholipidosis and the level of secreted extracellular vesicles. Three oxidized cholesterol derivatives, namely 7α-hydroxycholesterol (7α-OH), 7- ketocholesterol (7-K) and 24(S)-hydroxycholesterol (24(S)-OH) were tested in three different concentrations: 50 μM, 100 μM and 200 μM for 24 h incubation with A549 lung cancer cell line. All the studied oxysterols were found to alter cells viability. The lowest survival rate of the cells was observed after 24 h of 7-K treatment, slightly better for 7α-OH while cells incubated with 24(S)-OH had the best survival rate among the oxysterols used. 7-K increased phospholipids accumulation in cells, however, most noticeable effect was noticed for 24(S)-OH. Changes in the level of extracellular vesicles secreted in cells culture after the treatment with oxysterols were also observed. It was found that all oxysterols used increased the level of secreted vesicles, both exosomes and ectosomes. The strongest effect was noticed for 24(S)-OH. Taken together, these results suggest that 7-K is the most potent inducer of cancer cell death, while 7α-OH is slightly less potent in this respect. The lower cytotoxic effect of 24(S)-OH correlates with greater phospholipids accumulation, extracellular vesicles production and better cells survival.

Dendritic cell-targeted delivery of antigens using extracellular vesicles for anti-cancer immunotherapy.

Neoantigen delivery using extracellular vesicles (EVs) has gained extensive interest in recent years. EVs derived from tumour cells or immune cells have been used to deliver tumour antigens or antitumor stimulation signals. However, potential DNA contamination from the host cell and the cost of large-scale EV production hinder their therapeutic applications in clinical settings. Here, we develop an antigen delivery platform for cancer vaccines from red blood cell-derived EVs (RBCEVs) targeting splenic DEC-205+ dendritic cells (DCs) to boost the antitumor effect. By loading ovalbumin (OVA) protein onto RBCEVs and delivering the protein to DCs, we were able to stimulate and present antigenic OVA peptide onto major histocompatibility complex (MHC) class I, subsequently priming activated antigen-reactive T cells. Importantly, targeted delivery of OVA using RBCEVs engineered with anti-DEC-205 antibody robustly enhanced antigen presentation of DCs and T cell activation. This platform is potentially useful for producing personalised cancer vaccines in clinical settings.

Mitochondrial extracellular vesicles, autoimmunity and myocarditis.

For many decades viral infections have been suspected as 'triggers' of autoimmune disease, but mechanisms for how this could occur have been difficult to establish. Recent studies have shown that viral infections that are commonly associated with viral myocarditis and other autoimmune diseases such as coxsackievirus B3 (CVB3) and SARS-CoV-2 target mitochondria and are released from cells in mitochondrial vesicles that are able to activate the innate immune response. Studies have shown that Toll-like receptor (TLR)4 and the inflammasome pathway are activated by mitochondrial components. Autoreactivity against cardiac myosin and heart-specific immune responses that occur after infection with viruses where the heart is not the primary site of infection (e.g., CVB3, SARS-CoV-2) may occur because the heart has the highest density of mitochondria in the body. Evidence exists for autoantibodies against mitochondrial antigens in patients with myocarditis and dilated cardiomyopathy. Defects in tolerance mechanisms like autoimmune regulator gene (AIRE) may further increase the likelihood of autoreactivity against mitochondrial antigens leading to autoimmune disease. The focus of this review is to summarize current literature regarding the role of viral infection in the production of extracellular vesicles containing mitochondria and virus and the development of myocarditis.

Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome

BackgroundHematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow causing cytopenias, increasing susceptibility to infections and death. We and others have shown that cellular therapies like human mesenchymal stromal cells (MSCs), or monocytes/macrophages educated ex-vivo with extracellular vesicles (EVs) from MSCs were effective in a lethal H-ARS mouse model. However, given the complexity of generating cellular therapies and the potential risks of using allogeneic products, development of an “off-the-shelf” cell-free alternative like EVs may have utility in conditions like H-ARS that require rapid deployment of available therapeutics. The purpose of this study was to determine the feasibility of producing MSC-derived EVs at large scale using a bioreactor and assess critical quality control attributes like identity, sterility, and potency in educating monocytes and promoting survival in a lethal H-ARS mouse model.MethodsEVs were isolated by ultracentrifugation from unprimed and lipopolysaccharide (LPS)-primed MSCs grown at large scale using a hollow fiber bioreactor and compared to a small scale system using flasks. The physical identity of EVs included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow-cytometry. Comparison of the RNA cargo in EVs was determined by RNA-seq. Capacity of EVs to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed in vivo using a lethal ARS model with NSG mice.ResultsPhysical identity of EVs at both scales were similar but yields by volume were up to 38-fold more using a large-scale bioreactor system. RNA-seq indicated that flask EVs showed upregulated let-7 family and miR-143 micro-RNAs. EEMos educated with LPS-EVs at each scale were similar, showing increased gene expression of IL-6, IDO, FGF-2, IL-7, IL-10, and IL-15 and immunophenotyping consistent with a PD-L1 high, CD16 low, and CD86 low cell surface expression. Treatment with LPS-EVs manufactured at both scales were effective in the ARS model, improving survival and clinical scores through improved hematopoietic recovery. EVs from unprimed MSCs were less effective than LPS-EVs, with flask EVs providing some improved survival while bioreactor EVs provide no survival benefit.ConclusionsLPS-EVs as an effective treatment for H-ARS can be produced using a scale-up development manufacturing process, representing an attractive off-the-shelf, cell-free therapy.

Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

RAS protein activator-like 2 (RASAL2) initiates peritubular capillary rarefaction in hypoxic renal interstitial fibrosis

The progression of chronic kidney disease (CKD) often involves renal interstitial fibrosis (RIF) and subsequent loss of peritubular capillaries (PTCs), which enhances disease severity. Despite advancements in our understanding of fibrosis, effective interventions for reversing capillary loss remain elusive. Notably, RIF exhibits reduced capillary density, whereas renal cell carcinoma (RCC) shows robust angiogenesis under hypoxic conditions. Using RNA sequencing and bioinformatics, we identified differentially expressed genes (DEGs) in hypoxic human renal tubular epithelial cells (HK-2) and renal cancer cells (786-0). Analysis of altered Ras and PI3K/Akt pathways coupled with hub gene investigation revealed RAS protein activator-like 2 (RASAL2) as a key candidate. Subsequent in vitro and in vivo studies confirmed RASAL2′s early-stage response in RIF, which reduced with fibrosis progression. RASAL2 suppression in HK-2 cells enhanced angiogenesis, as evidenced by increased proliferation, migration, and branching of human umbilical vein endothelial cells (HUVECs) co-cultured with HK-2 cells. In mice, RASAL2 knockdown improved Vascular endothelial growth factor A (VEGFA) and Proliferating cell nuclear antigen (PCNA) levels in unilateral ureteral occlusion (UUO)-induced fibrosis (compared to wild type). Hypoxia-inducible factor 1 alpha (HIF-1α) emerged as a pivotal mediator, substantiated by chromatin immunoprecipitation (ChIP) sequencing, with its induction linked to activation. Hypoxia increased the production of RASAL2-enriched extracellular vesicles (EVs) derived from tubular cells, which were internalized by endothelial cells, contributing to the exacerbation of PTC loss. These findings underscore RASAL2′s role in mediating reduced angiogenesis in RIF and reveal a novel EV-mediated communication between hypoxic tubular- and endothelial cells, demonstrating a complex interplay between angiogenesis and fibrosis in CKD pathogenesis.

Culture of Hoffa fat pad mesenchymal stem/stromal cells on microcarrier suspension in vertical wheel bioreactor for extracellular vesicle production

BackgroundMesenchymal stromal cells (MSCs) are increasingly employed in regenerative medicine approaches for their immunomodulatory and anti-inflammatory properties, which are encoded in their secretome including extracellular vesicles (EVs). The Hoffa fat pad (HFP) located infrapatellarly harbours MSCs that could assist in tissue homeostasis in osteoarthritic joints. Intraarticular injection therapies based on blood products could modulate the populations of released HFP-MSC-EVs in a quantitative manner.MethodsTo obtain amounts of HFP-MSC-derived EVs that allow pre-clinical evaluation, suitable EV production systems need to be developed. This work investigates the release of EVs from primary HFP-MSCs cultivated in a 3D environment using microcarrier suspension culture in a vertical wheel bioreactor in comparison to conventional 2D culture. To simulate an intraarticular blood product therapy, cultures were treated with citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) before EV collection. HFP-MSC-EVs are enriched via ultrafiltration and characterised via Western Blot, nanoparticle tracking analysis in scatter as well as fluorescence mode. EV potency was determined via RT-qPCR analysing the expression of type II and X collagen (COL2 and COL10), as well as inducible nitric oxide synthase (iNOS) in primary OA chondrocytes.ResultsBlood product supplementation elevated HFP-MSC metabolic activity as determined via XTT assay over the course of 14 days. 3D culture resulted in a roughly 100-fold EV yield compared to 2D culture and elevated number of EVs released per cell. Total protein content correlated with the EV concentration. While typical EV marker proteins such as CD9, CD63 or Alix were detected in total protein extracts, CD9 and CD73 colocalised on individual EVs highlighting their cell origin. The type of blood product treatment did not affect the size or concentration of EVs obtained from HFP-MSCs. Assessing potency of 3D culture EVs in comparison to 2D EVs revealed superior biological activity with regard to inhibition of inflammation, inhibition of chondrocyte hypertrophy and induction of cartilage-specific ECM production.ConclusionsHFP-MSCs proliferate in presence of human blood products indicating that animal serum in culture media can be avoided in the future. The culture of HFP-MSCs in the employed bioreactor was successfully used to generate quantities of EVs that could allow evaluation of HFP-MSC-EV-mediated effects in pre-clinical settings. In addition, EV potency of 3D EVs is superior to EVs obtained in conventional 2D culture flasks.

Abstract 1362 Investigating the effects of antibiotics on the production of extracellular vesicles in E. coli

Abstract 1362 Investigating the effects of antibiotics on the production of extracellular vesicles in E. coli

The Chinese herbal prescription JZ-1 promotes extracellular vesicle production and protects against herpes simplex virus type 2 infection in vitro

ObjectiveGenital herpes, primarily caused by HSV-2 infection, remains a widespread sexually transmitted ailment. Extracellular vesicles play a pivotal role in host-virus confrontation. Recent research underscores the influence of Chinese herbal prescriptions on extracellular vesicle production and composition. This study aims to probe the impact of JieZe-1 (JZ-1) on extracellular vesicle components, elucidating its mechanisms against HSV-2 infection via extracellular vesicles. MethodsThe JZ-1's anti-HSV-2 effects were assessed using CCK-8 assay. Extracellular vesicles were precisely isolated utilizing ultracentrifugation and subsequently characterized through TEM, NTA, and Western Blot analyses. The anti-HSV-2 activity of extracellular vesicles was gauged using CCK-8, Western Blot, and immunofluorescence. Additionally, high-throughput sequencing was employed to detect miRNAs from extracellular vesicles, unraveling the potential antiviral mechanisms of JZ-1. ResultsAntiviral efficacy of JZ-1 was shown in VK2/E6E7, HeLa, and Vero cells. The samples extracted from cell supernatant by ultracentrifugation were identified as extracellular vesicles. In VK2/E6E7 cells, extracellular vesicles from JZ-1 group enhanced cell survival rates and diminished the expression of intracellular viral protein gD, contrasting with the inert effect of control group vesicles. Extracellular vesicles from JZ-1 treated Vero cells demonstrated a weaker yet discernible anti-HSV-2 effect. Conversely, extracellular vesicles of HeLa cells exhibited no anti-HSV-2 effect from either group. High-throughput sequencing of VK2/E6E7 cell extracellular vesicles unveiled significant upregulation of miRNA-101, miRNA-29a, miRNA-29b, miRNA-29c, and miRNA-637 in JZ-1 group vesicles. KEGG pathway analysis suggested that these miRNAs may inhibit PI3K/AKT/mTOR signaling pathway and induce autophagy of host cells to protect against HSV-2. Western blot confirmed the induction of autophagy and inhibition of AKT/mTOR in VK2/E6E7 cells with JZ-1 group extracellular vesicles treatment. ConclusionJZ-1 had an anti-HSV-2 efficacy. After JZ-1 stimulation, VK2/E6E7 cells secreted extracellular vesicles which protect host cells from HSV-2 infection. High-throughput sequencing showed that these extracellular vesicles contained a large number of miRNAs targeting PI3K/AKT/mTOR pathway. JZ-1 group extracellular vesicles could inhibit the activation of AKT/mTOR pathway and induce the host cells autophagy.

Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system

Background & aimsCell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. MethodsMSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. ResultsTaking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). ConclusionsTaken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.