Browse
Submit Data
Databases
API
Help

Dataset Information

14 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Effect of fetal bovine serum in culture media on MS analysis of mesenchymal stromal cells secretome.

ABSTRACT: The analysis of mesenchymal stromal cells secretome is fundamental to identify key players of processes involving these cells. Truly secreted proteins may be difficult to detect in MS based analysis of conditioned media (CM) due to proteins supplemented with fetal bovine serum (FBS). We compared different growth conditions to determine the effect of varying FBS concentration on the number and quantity of truly secreted human proteins vs contaminating bovine proteins. The results suggest that to minimize interference cells should be grown in presence of FBS until confluence and transferred into a serum-free medium prior to secretome collection.

SUBMITTER: Nonnis S

PROVIDER: S-EPMC5988613 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Effect of fetal bovine serum in culture media on MS analysis of mesenchymal stromal cells secretome.

Nonnis Simona S Maffioli Elisa E Zanotti Lucia L Santagata Fabiana F Negri Armando A Viola Antonella A Elliman Stephen S Tedeschi Gabriella G

EuPA open proteomics 20160107

The analysis of mesenchymal stromal cells secretome is fundamental to identify key players of processes involving these cells. Truly secreted proteins may be difficult to detect in MS based analysis of conditioned media (CM) due to proteins supplemented with fetal bovine serum (FBS). We compared different growth conditions to determine the effect of varying FBS concentration on the number and quantity of truly secreted human proteins <i>vs</i> contaminating bovine proteins. The results suggest t ...[more]

PMID: 29900097

Similar Datasets

Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media.

Project description:It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 10? ± 1.39 × 10? EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

| S-EPMC6275013 | biostudies-literature

Proteomic Profiling of the Human Fetal Multipotent Mesenchymal Stromal Cells Secretome.

Project description:Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-?) have not been identified in the FetMSCs secretome.

| S-EPMC7716221 | biostudies-literature

A Simple Method for In-Depth Proteome Analysis of Mammalian Cell Culture Conditioned Media Containing Fetal Bovine Serum.

Project description:A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

| S-EPMC7961985 | biostudies-literature

Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media.

Project description:In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA, Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.

| S-EPMC6445286 | biostudies-literature

Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

Project description:Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate 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, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results 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.

| S-EPMC4977539 | biostudies-literature

Feline embryo development in commercially available human media supplemented with fetal bovine serum.

Project description:Feline embryo development was examined for 7 days after fertilization using commercially available human media supplemented with 0.3% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS). Cumulus-oocyte complexes were categorized as Grades 1, 2, and 3 according to morphology. Only-One Medium (OM) was used for in vitro culture (IVC) in OM + BSA, OM + FBS, and OM + BSA/FBS, with BSA supplementation for the first 2 days and FBS for the subsequent 5 days. Embryos cultured in Early Culture Medium (1-2 days) and Blastocyst Medium (3-7 days) were defined as EB + BSA and EB + BSA/FBS. The developmental rate until the blastocyst stage of Grade 1 and 2 oocytes cultured in OM + BSA/FBS was higher than for the other groups and was significantly higher than for the OM + BSA and EB + BSA groups (P<0.01). Grade 3 oocytes cultured in OM + BSA/FBS also showed the greatest proportion of blastocyst formation. However, FBS supplementation throughout the IVC period reduced blastocyst number. The percentage of 2 pronuclei after fertilization as well as blastocyst cell number were significantly higher in Grade 1 and 2 than Grade 3 oocytes when cultured in OM + BSA/FBS (P<0.05). These results indicate that commercially available OM supplemented with BSA for the first 2 days of culture and FBS for the subsequent 5 days is suitable for feline embryo development until the blastocyst stage.

| S-EPMC6483925 | biostudies-literature

Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media.

Project description:INTRODUCTION: Despite having a proven immunosuppressive potential in vitro, human mesenchymal stromal cells (MSCs) are reported to display variable efficacy in vivo and, in fact, their proven benefit in the clinical practice is still limited and controversial. METHODS: The interplay between clinical grade MSCs and pre-activated donor lymphocytes or selected lymphocyte subsets was studied in vitro. The kinetics of MSC growth and viability was evaluated by adhesion-dependent changes of culture plate impedance and biochemically by a colorimetric assay. Activation of natural killer (NK) cells was assessed as well, using a flow cytometry assay. RESULTS: A strong inhibition of MSC growth was rapidly induced by the addition of pre-activated lymphocytes but not of resting lymphocytes. Inhibition seems not to be attributable to a single cell population, as similar results can be obtained by depleting NK cells or by using either selected CD4+ or CD8+ lymphocytes. In addition, conditioned medium (CM) from activated lymphocytes was able to inhibit MSC growth in a dose-dependent manner. Furthermore, licensing with IFN-? partially protected MSCs from pre-activated lymphocytes but not from their CM. These results suggest an inhibitory role of lymphocyte-activation-derived substances. However, the identification of a single molecule responsible for MSC inhibition remained elusive, even if preliminary experiments showed that ATP and, to a lesser extent, TNF-? might play a role. CONCLUSIONS: These results suggest that survival of MSCs can be affected by soluble mediators released by activated lymphocytes. Thus it can be hypothesized that MSC immunosuppressive action in vivo could be impaired by ongoing immune activation through the release of inflammatory mediators.

| S-EPMC4055165 | biostudies-literature

RNA in Fetal Bovine Serum Interferes with Cell Culture Studies

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.

2016-09-19 | GSE78970 | GEO

Questioning the purity of the media - Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media

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.

2019-04-30 | GSE120594 | GEO

Serum-free media for the production of human mesenchymal stromal cells: a review.

Project description:The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large-scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)-supplement for optimal cell proliferation. FBS-based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum-use to serum-free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large-scale production of MSCs.

| S-EPMC6496935 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data