Project description:We apply 3D directed induced transfer culture system alongsideand ultra-high speed centrifugation technology to break through the mass production bottleneck of ht-SKPs-EV (human transformed skin-derived precursors). We hope to compare its protein expression with that of fibroblasts extracellular vesicles (FBs-EV) in traditional adherent culture mode.The experimental procedure for analyzing ht-SKPs-EV and FBs-EV proteins using proteomics involves extracting proteins from ht-SKPs-EV and FBs-EV, subjecting them to gel electrophoresis, transferring the proteins onto a membrane, probing with specific antibodies, and detecting the protein bands to assess expression levels.
Project description:In the research field of extracellular vesicles (EVs), the use of EV-depleted fetal bovine serum (FBS) for in vitro studies is highly recommended to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or bought commercially, nevertheless these depletion methods do not guarantee an RNA-free preparation. In this study we have addressed the RNA contamination issue in FBS, ultracentrifuged EV-depleted FBS, commercially available EV-depleted FBS, and also from our recently developed filtration based EV depleted FBS. Commercially available serum-free, xeno-free defined media were also screened for RNA contamination.
Project description:The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is therefore to always perform HI of FBS prior to EV depletion.
Project description:Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades, and it represents one of the most commonly used reagents in life science research. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here we report that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, likely due to the confounding effect of the FBS. Furthermore, although the absolute amount of individual RNA species in FBS is low, they can be taken up by cultured cells and affect the results and interpretations of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA.
Project description:We used male C57BL/6 mice (Charles River Laboratories, Wilmington, MA), housed under SPF conditions. Primary adipocytes from the inguinal fat depot at postnatal day (P6, young mice) and postnatal day 56 (P56, adult mice) were isolated by collagenase digestion and separation of cell fractions and subsequently cultured in EV-free media (FBS was EV-depleted). Article: Röszer T., MicroRNA Profile of Mouse Adipocyte-Derived Extracellular Vesicles
Project description:Human mesenchymal stem/stromal cells (hMSCs) hold significant potential for regenerative medicine due to their anti-inflammatory and pro-angiogenic secretome. In particular, 3D hMSC aggregates secrete EVs that have enhanced anti-inflammatory and immunomodulatory properties. However, safety and efficacy concerns associated with stem cell therapy have led to the discovery of cell-free approaches utilizing hMSC-derived extracellular vesicles (EVs). Despite their therapeutic promise, the clinical application of hMSC-EVs is limited by low production yield. This study investigates the scalable generation of hMSC-EVs from 3D aggregates in a novel Vertical-Wheel Bioreactor (VWBR) through shear stress-mediated biochemical cues, promoting cargo expression relevant to nerve regeneration and neuropathic treatment. Bone marrow-derived hMSCs were cultured as 3D aggregates in VWBRs and exposed to two different culture media—αMEM/FBS (serum-containing) and DMEM/F12/B27 (serum-free)—under three agitation speeds (25, 40, and 64 rpm). Metabolite and gene analyses were performed to assess EV biogenesis, focusing on ESCRT machinery markers. Results showed that hMSCs cultured in VWBR exhibited higher expression of EV biogenesis genes and glycolytic genes compared to static culture. αMEM/FBS (serum-containing) condition was more robust than DMEM/F12/B27 (serum-free) condition. EV yield (EV number per cell) increased by 3-10 fold (in serum-containing medium) in VWBR compared to static culture, with size of 120-180 nm and EV marker expression. microRNA-sequencing of the EVs shows the upregulation of miR-29a-3p, miR-451a, miR-224-5p, miR-16-5p, miR-133a-3p, and miR-143-3p, etc., reflecting a combination that enhances EV biogenesis, promotes metabolic reprogramming, and supports immunomodulatory behavior characteristics. Functional assays demonstrated that the generated EVs effectively modulated Schwann cell responses under neural inflammation, supporting their therapeutic potential. These findings highlight the role of VWBR-driven hydrodynamics in promoting EV production from 3D hMSC aggregates. This study advances the knowledge of dynamic aggregation and metabolic influence on 3D hMSC-EV production and fundamentally scale up the preconditioned techniques used to promote hMSC aggregation and the consequent EV secretion.
Project description:Extracellular Vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, we isolated EV from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV were characterized in terms of expression of specific markers, yield, and size. Bioactivity was assessed in human umbilical vein endothelial cells (HUVEC) and hiPSC-CM. Small RNA-Seq was performed to identify the differentially expressed miRNA in the four EV groups. Bioactivity assays showed increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increased hiPSC-CM proliferation. Global miRNA expression profiles corroborated an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification. A stemness maintenance miRNA cluster upregulated in hiPSC-EV was found to target the PTEN/PI3K/AKT pathway. Moreover, hiPSC-EV treatment mediated PTEN suppression and increased AKT phosphorylation. Overall, our findings validate hiPSC as suitable cell biofactories for EV production for cardiac regenerative applications.
Project description:To screen for potential miRNA that may contribute to the etiopathogenesis of EDS-HT/JHS miRNA expression profiling was performed using the Affymetrix GeneChip® miRNA 3.0 Array and comparing the miRNA expression changes of skin fibroblasts of five EDS-HT/JHS patients with those of six healthy individuals
Project description:COmparison of the MCF7 proteomes after being cultured for 3 d in either 10% FBS (standard, commercial) or in 10% EV-depleted FBS (EDS, commercial) to validate the absence of EV specific proteins in EDS to exclude potential cross-contamination of xeno-EVs