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Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils.

ABSTRACT: OBJECTIVE:Currently, the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of the cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have a relatively equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 h of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry. RESULTS:Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.

SUBMITTER: Alipour R

PROVIDER: S-EPMC6977324 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Mesenchymal stromal cells in platelet lysate versus fetal calf serum

Project description:This SuperSeries is composed of the SubSeries listed below.

2017-08-11 | GSE87798 | GEO

Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.

Project description:Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPAR? activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

| S-EPMC7830683 | biostudies-literature

DNA methylation of mesenchymal stromal cells in platelet lysate versus fetal calf serum

Project description:Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology is influenced by these additives – hence they may favor outgrowth of specific subpopulations, thereby affecting the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow derived MSCs in parallel with HPL or FCS for two passages. In HPL the proliferation was significantly higher and cells reflected more spindle-shaped morphology. In contrast, global DNA-methylation profiles did not reveal any significant differences. None of the CpGs revealed significant differences between MSCs that were cultured in HPL versus FCS if the analysis was corrected for multiple testing (limma-adjusted P-value of <0.05). These results indicate that there is no systematic bias for specific subpopulations of MSCs by using either HPL or FCS.

2017-08-11 | GSE87797 | GEO

Gene expression of mesenchymal stromal cells in platelet lysate versus fetal calf serum

Project description:Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology is influenced by these additives – hence they may favor outgrowth of specific subpopulations, thereby affecting the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow derived MSCs in parallel with HPL or FCS for two passages. In HPL the proliferation was significantly higher and cells reflected more spindle-shaped morphology. Pairwise comparisons of gene expression profiles (Affymetrix HTA 2.0) revealed only moderate differences. When we apply a fold change >1.5 and limma-adjusted P-value of <0.05, only 69 transcripts were differentially expressed. These results indicate that there is no systematic bias for specific subpopulations of MSCs by using either HPL or FCS.

2017-08-11 | GSE87796 | GEO

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

Project description:Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

| S-EPMC5506010 | biostudies-literature

Autologous serum supplement favours in vitro regenerative paracrine factors synthesis.

Project description:OBJECTIVES:Foetal bovine serum (FBS) is often the serum supplement of choice for in vitro human cell culture. This study compares the effect of FBS and autologous human serum (AuHS) supplement in human peripheral blood mononuclear cell (PBMC) culture to prepare secretome. MATERIALS AND METHODS:The PBMC (n = 7) were cultured either in RPMI-1640 containing L-glutamine and 50 units/ml Penicillin-Streptomycin (BM) or in BM with either AuHS or FBS. Viability, proliferation and differentiation of PBMC were evaluated. Paracrine factors present in the secretomes (n = 6) were analysed using ProcartaPlex Human Cytokine panel (17 plex). Ingenuity Pathway Analysis (IPA) was performed to predict activation or inhibition of biological functions related to tissue regeneration. RESULTS:The viability of PBMC that were cultured with FBS supplement was significantly reduced at 96 h compared to those at 0 and 24 h (P < .05). While the reduction of the viability of PBMC that were cultured with AuHS supplement was not significantly different compared to those at 0 and 24 h. The FBS secretomes prepared at 24 h was found to contain significantly higher amount of EGF (P < .05) compared to that in AuHS or BM secretome. The AuHS secretomes contained significantly higher amount of HGF at 24 (P < .05) and 96 h (P < .01), and VEGF-A at 24 h (P < .05) compared to those in the FBS secretomes. SDF-1 was not detected in the FBS secretomes prepared at either 24 or 96 hours. Double immunocytochemical staining revealed a marked increase in co-localization of SDF-1 and its receptor in PBMC that were cultured with AuHS supplement compared to that cultured with FBS supplement. CONCLUSION:In secretome preparation, AuHS supplement favours synthesis of paracrine factors that are needed for regenerative therapy.

| S-EPMC6529128 | biostudies-literature

Addressing reproducibility challenges in cell culture: the impact of oxylipins contained in fetal calf serum

Project description:Reproducibility issues regarding complex in vitro cell culture experiments have been mainly attributed to genetic variations within cell lines. Batch-dependent fetal calf serum variations are well known and also represent relevant influencing factors. However, the molecular constituents mediating such effects remained largely unknown. High resolution mass spectrometry is a powerful tool to identify molecular FCS constituents and investigate their effect on cultured cells. Using a differentiation and inflammatory stimulation protocol on U937 cells we observed FCS batch-dependent variations of secreted cytokines, oxylipins and other inflammatory mediators. In order to detect candidate bioactive molecules contained in FCS, the protein and oxylipin composition of FCS batches was investigated. Remarkably, the protein composition showed some, but much less batch-dependent variation when compared to the oxylipin composition. Efficient uptake of C13-labelled arachidonic acid from medium by U937 macrophages and inflammation-induced release thereof including oxylipin products was evidenced. Balancing out FCS batch-dependent nanomolar concentration differences of two selected oxylipins, 5-HETE and 15-HETE, by spiking experiments, resulted in significant proteome alterations of cultured U937 macrophages indicating PPAR activation. High-resolution microscopy demonstrated HETE-induced formation of peroxisomes, thus independently corroborating the proteome profiling results. In conclusion, the present data demonstrate a strong and previously underappreciated influence of FCS-contained oxylipins on cellular effector functions, thus representing a plausible cause for disturbing reproducibility issues.

2021-06-01 | PXD020617 | Pride

Mesenchymal stromal cells in platelet lysate versus fetal calf serum

Project description:Mesenchymal stromal cells in platelet lysate versus fetal calf serum

| PRJNA347630 | ENA

A human plasma derived supplement preserves function of human vascular cells in absence of fetal bovine serum.

Project description:Current techniques for cell culture routinely use animal-derived components. Fetal bovine serum (FBS) is the most widely applied supplement, but it often displays significant batch-to-batch variations and is generally not suitable for clinical applications in humans. A robust and xeno-free alternative to FBS is of high interest for cellular therapies, from early in vitro testing to clinical trials in human subjects. In the current work, a highly consistent human plasma derived supplement (SCC) has been tested, as a potential substitute of FBS in primary human vascular cells culture. Our results show that SCC is able to support proliferation, preserve cellular morphology and potentiate functionality analogously to FBS. We conclude that SCC is a viable substitute of FBS for culture and expansion of cells in advanced therapies using human vascular cells and fibroblasts.

| S-EPMC5554976 | biostudies-other

Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf Serum.

Project description:Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.

| S-EPMC8622701 | biostudies-literature

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