Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS‐HAT). We report that PBS‐HAT buffer facilitates clearly improved short‐term and long‐term EV preservation for samples stored at ‐80°C, stability throughout several freeze‐thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.
Extracellular vesicles (EVs) are nanosized cell-derived vesicles produced by all cells, which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The equilibrium between the EV-producing pathways is heavily influenced by factors in the extracellular environment, in such a way that can be taken advantage of to boost production of engineered EVs. In this study, a quantifiable EV-engineering approach is used to investigate how different cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production at the physical, molecular, and transcriptional levels. Further, it is demonstrated that the ceramide-dependent EV biogenesis route is the major pathway to production of engineered EVs during optimized EV-production. These findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods.
Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Although chemotherapy is the primary means in colorectal cancer treatment, it is burdenerd by adverse drug effects. Drug-resistance is one of the most important challenges for chemotherapy and epithelial-mesenchymal transition (EMT) plays critical role in the development of drug resistance. Aims: The aim of this study was to investigate the mechanisms underlying the effect of astragaloside IV (AS-IV) on miR-134 expression, EMT and chemotherapeutic sensitivity in CRC. Methods: Cell proliferation, transfection assay, western blot, real-time PCR, cell migration and invasion assay and luciferase reporter assay were used to detect the effects of AS-IV on CRC. Results: AS-IV significantly inhibited CRC cell migration and invasion by inducing miR-134 expression. Moreover, AS-IV and miR-134 increased the sensitivity of CRC tumors to oxaliplatin (OXA) chemotherapy. cAMP responsive element-binding protein 1 (CREB1), which was required for CRC cells migration, invasion and drug sensitivity, was significantly down-regulated by AS-IV. Conclusions: Our results indicated that AS-IV inhibited CRC EMT by inducing miR-134 expression which significantly down-regulated the CREB1 signaling pathway, and therefore increased the sensitivity to chemotherapy. Our findings provided new insight into the mechanisms of chemotherapy-resistant CRC, and may open new therapeutic options in the treatment of this devastating disease.
BackgroundConbercept is a novel vascular endothelial growth factor (VEGF) inhibitor for the treatment of wet age-related macular degeneration (AMD). This systematic review aims to assess the efficacy and safety of conbercept in the treatment of wet AMD.MethodsPubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, VIP database, and Wanfang database were searched from their earliest records to June 2017. We included randomized controlled trials (RCTs) evaluating the efficacy and safety of conbercept in wet AMD patients. Outcomes included the mean changes from baseline in best-corrected visual acuity (BCVA) score (primary outcome), central retinal thickness (CRT), plasma level of vascular endothelial growth factor (VEGF) over time, and the incidence of adverse events (AEs).ResultsEighteen RCTs (1285 participants) were included in this systematic review. Conbercept might improve BCVA compared to triamcinolone acetonide [MD = 0.11, 95% CI (0.08, 0.15)], and reduce CRT compared to the other four therapies (conservative treatment, ranibizumab, transpupillary thermotherapy, and triamcinolone acetonide). The incidence of AEs in patients receiving conbercept was significantly lower than those receiving triamcinolone acetonide [RR = 0.25, 95% CI (0.09–0.72)], but was similar to the other therapies. Conbercept seemed to be more effective than ranibizumab in lowering the plasma level of VEGF [MD = − 15.86, 95% CI (− 23.17, − 8.55)].ConclusionsCurrent evidence shows that conbercept is a promising option for the treatment of wet AMD. Nevertheless, further studies are required to compare the efficacy, long-term safety and cost-effectiveness between conbercept and other anti-VEGF agents in different populations.Electronic supplementary materialThe online version of this article (10.1186/s12886-018-0807-1) contains supplementary material, which is available to authorized users.
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