scholarly journals Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3600
Author(s):  
Filip Larsberg ◽  
Maximilian Sprechert ◽  
Deike Hesse ◽  
Gudrun A. Brockmann ◽  
Susanne Kreuzer-Redmer
Keyword(s):  
Cell Culture ◽  
Immune Cell ◽  
Calf Serum ◽  
Feed Additives ◽  
Blood Sampling ◽  
Thrombocyte Count ◽  
Modes Of Action ◽  
Optimized Protocol ◽  
Chicken Blood

Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

scholarly journals Eicosanoid content in fetal calf serum accounts for reproducibility challenges in cell culture

2020 ◽  
Author(s):  
Laura Niederstaetter ◽  
Benjamin Neuditschko ◽  
Julia Brunmair ◽  
Lukas Janker ◽  
Andrea Bileck ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Resolution ◽  
Fetal Calf Serum ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Calf Serum ◽  
Protein Composition ◽  
Efficient Uptake ◽  
U937 Macrophage

AbstractReproducibility 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 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 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 in. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ 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.

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro

2011 ◽  
Vol 31 (3) ◽  
pp. 199-210 ◽  
Author(s):  
Nicholas Bryan ◽  
Kirstie D. Andrews ◽  
Michael J. Loughran ◽  
Nicholas P. Rhodes ◽  
John A. Hunt
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Elemental Composition ◽  
Fetal Calf Serum ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Human Umbilical Vein ◽  
Antigenic Expression ◽  
Umbilical Vein Endothelial Cells

One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.

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

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 113
Author(s):  
Laura Niederstaetter ◽  
Benjamin Neuditschko ◽  
Julia Brunmair ◽  
Lukas Janker ◽  
Andrea Bileck ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Resolution ◽  
Fetal Calf Serum ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Calf Serum ◽  
Protein Composition ◽  
Efficient Uptake ◽  
U937 Macrophage

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 PPARγ 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.

scholarly journals New Born Calf Serum Can Induce Spheroid Formation in Breast Cancer KAIMRC1 Cell Line

2021 ◽  
Vol 8 ◽  
Author(s):  
Rizwan Ali ◽  
Sarah Huwaizi ◽  
Alshaimaa Alhallaj ◽  
Arwa Al Subait ◽  
Tlili Barhoumi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Calf Serum ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Cell Junction ◽  
Growth Media ◽  
Easy Method ◽  
New Born

Three-dimensional (3D) cell culture systems have become very popular in the field of drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. We have developed a novel and easy method to produce spheroids from the newly isolated KAIMRC1 cell line in vitro. It can be used as a 3D model to study proliferation, differentiation, cell death, and drug response of cancer cells. Our procedure requires growth media supplemented with 10% new born calf serum (NBCS) and regular cell culture plates to generate KAIMRC1 spheroids without the need for any specialized 3D cell culture system. This procedure generates multiple spheroids within a 12–24-h culture. KAIMRC1 spheroids are compact, homogeneous in size and morphology with a mean size of 55.8 µm (±3.5). High content imaging (HCI) of KAIMRC1 spheroids treated with a panel of 240 compounds resulted in the identification of several highly specific compounds towards spheroids. Immunophenotyping of KAIMRC1 spheroids revealed phosphorylation of FAK, cJUN, and E-cadherin, which suggests the involvement of JNK/JUN pathway in the KAIMRC1 spheroids formation. Gene expression analysis showed upregulation of cell junction genes, GJB3, DSC1, CLDN5, CLDN8, and PLAU. Furthermore, co-culture of KAIMRC1 cells with primary cancer-associated-fibroblasts (CAFs) showcased the potential of these cells in drug discovery application.

scholarly journals Cultivation of hMSCs in Human Plasma Prevents the Cytotoxic and Genotoxic Potential of ZnO-NP In Vitro

2019 ◽  
Vol 9 (23) ◽  
pp. 4994
Author(s):  
Scherzad ◽  
Meyer ◽  
Ickrath ◽  
Gehrke ◽  
Bregenzer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Plasma ◽  
Calf Serum ◽  
Human Mesenchymal Stem Cells ◽  
Industrial Applications ◽  
Culture Conditions ◽  
Zno Nps ◽  
Cytotoxic Effects ◽  
Differentiation Capacity

Zinc oxide nanoparticles (ZnO-NPs) are commonly used for industrial applications. Consequently, there is increasing exposure of humans to them. The in vitro analysis of cytotoxicity and genotoxicity is commonly performed under standard cell culture conditions. Thus, the question arises of how the results of genotoxicity and cytotoxicity experiments would alter if human plasma was used instead of cell culture medium containing of fetal calf serum (FCS). Human mesenchymal stem cells (hMSCs) were cultured in human plasma and exposed to ZnO-NPs. A cultivation in expansion medium made of DMEM consisting 10% FCS (DMEM-EM) served as control. Genotoxic and cytotoxic effects were evaluated with the comet and MTT assay, respectively. hMSC differentiation capacity and ZnO-NP disposition were evaluated by histology and transmission electron microscopy (TEM). The protein concentration and the amount of soluble Zn2+ were measured. The cultivation of hMSCs in plasma leads to an attenuation of genotoxic and cytotoxic effects of ZnO-NPs compared to control. The differentiation capacity of hMSCs was not altered. The TEM showed ZnO-NP persistence in cytoplasm in both groups. The concentrations of protein and Zn2+ were higher in plasma than in DMEM-EM. In conclusion, the cultivation of hMSCs in plasma compared to DMEM-EM leads to an attenuation of cytotoxicity and genotoxicity in vitro.

1120: Citrate and Vitamin E Blunt the SWL-Induced Free Radical Surge in an In-Vitro MDCK Cell Culture Model

2004 ◽  
Vol 171 (4S) ◽  
pp. 295-295
Author(s):  
Fernando C. Delvecchio ◽  
Ricardo M. Brizuela ◽  
Karen J. Byer ◽  
W. Patrick Springhart ◽  
Saeed R. Khan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Free Radical ◽  
Vitamin E ◽  
Mdck Cell ◽  
Cell Culture Model ◽  
Culture Model

3D in vitro reconstruction of synthetic liver sinusoids in a microfluidic cell culture device

2013 ◽  
Vol 51 (01) ◽  
Author(s):  
J Böttger ◽  
J Schütte ◽  
K Benz ◽  
C Freudigmann ◽  
B Hagmeyer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Liver Sinusoids ◽  
Microfluidic Cell Culture

The Natural Course of Defibrination Syndrome Caused by Echis Colorata Venom in Man

1974 ◽  
Vol 31 (03) ◽  
pp. 420-428 ◽  
Author(s):  
M Fainaru ◽  
S Eisenberg ◽  
N Manny ◽  
C Hershko
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Major Bleeding ◽  
Renal Damage ◽  
Specific Treatment ◽  
Natural Course ◽  
Factor V ◽  
Thrombocyte Count

SummaryThe natural course of defibrination syndrome caused by Echis colorata venom (ECV) in five patients is reported. All patients developed afibrinogenemia within six hours after the bite. Concomitantly a depression in factor V was recorded. Factor VIII and thrombocyte count in blood were normal in most patients. In the light of the known effects of ECV on blood coagulation in vivo and in vitro it is concluded that the afibrinogenemia is due to intravascular clotting.Four patients had transient renal damage, manifested by oliguria, azotemia, albuminuria and cylindruria, ascribed to microthrombi in the renal glomeruli.After the bite, the natural course was benign, no major bleeding was observed, and all signs of coagulopathy reverted to normal within 7 days. Therefore we recommend no specific treatment for this condition. In the case of heavily bleeding patients, administration of antiserum against ECV and/or heparin should be considered.

Influence of Certain Drugs on the Adhesiveness of Human, Rat, and Rabbit Blood Platelets in Vitro

1965 ◽  
Vol 13 (02) ◽  
pp. 428-438 ◽  
Author(s):  
K Reber ◽  
A Studer
Keyword(s):  
Sodium Citrate ◽  
Blood Platelets ◽  
Drug Application ◽  
Normal Range ◽  
Blood Samples ◽  
Serotonin Antagonist ◽  
Thrombocyte Count ◽  
Particle Count ◽  
Rabbit Blood

SummaryThis is a comparative study of the methods described by H. P. Wright and O’Brien for determining the adhesiveness of thrombocytes. An attempt is made to characterize and statistically correlate both techniques. With the aid of a Coulter Counter for thrombocyte counts, a normal range is presented for human, rat, and rabbit blood. Anticoagulants used are sodium citrate and Heparin.The influence of Cocaine and the Serotonin antagonist Ro 3-0837 was studied on these same substrates, to determine a pharmacological interference with results of either Wright’s test or O’Brien’s. Both drugs are found to induce a statistically significant increase in the “thrombocyte count” as compared to the corresponding controls. These effects are not real but to be attributed to an increase in particle count due to thrombocyte fragmentation as a consequence of drug application. There is no evidence for the claim that these drugs decrease the adhesiveness of thrombocytes.Numerical results of both tests often show a high and statistically significant correlation, especially following the addition of Ro 3-0837. Such is not true of individual blood samples to which no drug has been added. Evidentally, both tests are not specific for the same characteristic of normal blood platelets. But, when Ro 3-0837 is added, the breakdown of unstable platelets is induced; and the corresponding increase in count of thrombocyte fragments is expressed by both tests in the same fashion.

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