Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles (EVs)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA553766 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9667740.fastq.gz | Fastqsanger.gz | |||
| SRR9667741.fastq.gz | Fastqsanger.gz | |||
| SRR9667742.fastq.gz | Fastqsanger.gz | |||
| SRR9667737.fastq.gz | Fastqsanger.gz | |||
| SRR9667738.fastq.gz | Fastqsanger.gz |
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Project description:With the goal to identify miRNAs and other small RNAs involved in EV-mediated oligodendrocyte-neuron communication, we performed deep sequencing (RNA-seq) of small RNAs derived from EVs isolated from culture supernatants of primary mouse oligodendrocytes by differential centrifugation. Oligodendrocytes were cultured and EVs harvested under serum-free conditions in presence of B27 supplement. We found that RNA isolated from EVs harvested from cells under serum-replacement conditions contains miRNA contaminants carried into the sample by defined media components.
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Project description:Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background.
| S-EPMC6746277 | biostudies-literature
Project description:Dietary supplements Metagenome
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Project description:Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential.
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Project description:Analysis of gene expression in skin of rats subjected to electron irradiation and the effect of vitamin A supplements. Keywords: other
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Project description:Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential. Microarray hybridizations (Agilent: Whole Human Genome Oligo Microarray; 41k unique probe) were carried out with 5 µg Cy3 labeled cRNA and 5 µg Cy5 labeled cRNA, both prepared from each sample.
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Project description:The cell seretome is diverse and contains extracellular secreted vesicles (EVs) as well as non-vesicular extracellular nanoparticles (NVEPs) called exomeres and supermeres, all of which carry different kinds of cell signaling cargos. We have developed new techniques to purify exomeres and supermeres by ultracentrifugation (UC) and by fast protein liquid chromatography (FPLC) from cell conditioned media and from blood plasma. We utilized a proteomics approach to compare UC and FPLC methods with standard UC and density gradient methods of isolating EVs and new methods of isolating NVEPs. We acquired LC-MS/MS data from conditioned media using standard 2D plastic and hollow fiber 3D bioreactor production from a CRC cell line, DiFi, using all these methods and growth culture conditions.
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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.
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