Effects of heparin-binding substances in fetal bovine serum upon proliferation and differentiation of trophoblast cells in the mouse blastocysts cultured in vitro

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 × 109 ± 1.39 × 108 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.

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Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays

10.1101/2021.07.19.452897 ◽

2021 ◽

Author(s):

Xenia Dolde ◽

Christiaan Karreman ◽

Marianne Wiechers ◽

Stefan Schildknecht ◽

Marcel Leist

Keyword(s):

Neural Crest ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Developmental Toxicity ◽

Video Tracking ◽

Large Panel ◽

Human Proteins ◽

Fetal Bovine ◽

Targeted Approach

Fetal bovine serum (FBS) is the only known stimulus for migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We confirmed here in an optimized transwell assay that FBS triggers directed migration along a concentration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chemotactic for NCCs in animal models (e.g. VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined in a broad screening approach, whether human cells would produce any factor able to trigger NCC migration. We found that HepG2 hepatoma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease-sensitivity suggested a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

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Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽

10.1182/blood.v76.6.1150.1150 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1150-1157 ◽

Cited By ~ 21

Author(s):

AR Migliaccio ◽

G Migliaccio ◽

M Brice ◽

P Constantoulakis ◽

G Stamatoyannopoulos ◽

...

Keyword(s):

Bovine Serum ◽

Fetal Bovine Serum ◽

Mrna Level ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Gm Csf ◽

Gamma Globin ◽

Fetal Bovine ◽

Globin Synthesis

Abstract We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

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Elucidation of the Interaction between Flavan-3-ols and Bovine Serum Albumin and Its Effect on Their In-Vitro Cytotoxicity

Molecules ◽

10.3390/molecules24203667 ◽

2019 ◽

Vol 24 (20) ◽

pp. 3667

Author(s):

Yasuyuki Fujii ◽

Yoshitomo Suhara ◽

Yusuke Sukikara ◽

Tomohiro Teshima ◽

Yoshihisa Hirota ◽

...

Keyword(s):

Cell Culture ◽

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

In Vitro Cytotoxicity ◽

Binding Energies ◽

Docking Simulations ◽

Fetal Bovine

Flavan-3-ols (FLs), specifically catechin and its oligomer B-type procyanidins, are suggested to potently bind to bovine serum albumin (BSA). We examined the interaction between BSA and FLs by fluorescence quenching and found the following order of binding activities to BSA: cinnamtannin A2 (A2; tetramer) > procyanidin C1 (C1; trimer) ≈ procyanidin B2 (B2, dimer) > (−)epicatechin (EC, monomer). Docking simulations between BSA and each compound at the binding site showed that the calculated binding energies were consistent with the results of our experimental assay. FLs exerted cytotoxicity at 1000 μg/mL in F11 cell culture with fetal bovine serum containing BSA. In culture containing serum-free medium, FLs exhibited significant cell proliferation at 10−4 μg/mL and cytotoxicity was observed at concentrations greater than 10 μg/mL. Results of this study suggest that interactions between polyphenols and BSA should be taken into account when evaluating procyanidin in an in vitro cell culture system.

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