scholarly journals In Vitro Culture of Bovine Fibroblasts using Select Serum-Free Media Supplemented with Chlorella vulgaris Extract

2021 ◽  
Author(s):  
Galileo Defendi-Cho ◽  
Timothy Gould
Keyword(s):  
Cell Culture ◽  
Growth Factors ◽  
Cell Growth ◽  
Culture Media ◽  
Serum Free ◽  
The Media ◽  
Chlorella Algae ◽  
Free Media ◽  
Cells Cultured

Standard cell culture practices require addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5-10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the cost of media while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC-MS, we compared molecular signatures of FBS to Chlorella algae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented with C. vulgaris extract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover, C. vulgaris extract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present in C. vulgaris extract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications.

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

1998 ◽  
Vol 78 (4) ◽  
pp. 587-597 ◽  
Author(s):  
M. Y. Yang ◽  
R. Rajamahendran
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Culture System ◽  
Physiological Role ◽  
Serum Free ◽  
Igf I ◽  
The Media ◽  
Estradiol 17Β ◽  
Insulin Like Growth Factors

The objectives of this study were: 1) to develop a bovine granulosa cell (GC) culture system; and 2) to use this system to evaluate the effects of gonadotropins (FSH and LH) and insulin-like growth factors-I and -II (IGF-I and IGF-II) on steroidogenesis of bovine GC derived from small, medium, and large antral follicles (diameters ≤4, 5–8 and >8 mm, respectively). Granulosa cells were cultured (concentration, 5 × 105 cells per well) in serum-free medium for 48 h with variable doses of hormones and growth factors. Concentrations of progesterone (P4) and estradiol-17β (E2) in the media were determined by radioimmunoassay. Basal E2 production by GC from follicles of all sizes decreased with time of culture (P < 0.01) while basal P4 production increased (P < 0.01). Basal E2 and P4 production increased with increasing size of follicles (P < 0.01). Only very low concentrations of FSH stimulated E2 production from medium and large follicles. Follicle-stimulating hormone stimulated P4 production by GC of follicles of all sizes (P < 0.05). Luteinizing hormone inhibited E2 production by GC in medium and large follicles (P < 0.05), suggesting that LH is responsible for the rise in plasma E2 through effects on both theca cells and GC. A dose of 100 ng mL−1 of IGF-I increased E2 production by GC from medium and large follicles (P < 0.05). Progesterone production by GC from all categories of follicles was also stimulated by IGF-I (P < 0.05). Estradiol-17β production by GC from large follicles decreased in response to IGF-II (P < 0.05). The physiological role of IGF-II on steroidogenesis in the bovine ovary remains to be elucidated. In summary, these results demonstrate the development of a serum-free culture system for bovine GC, and that FSH, LH, IGF-I and IGF-II have different effects on steroidogenesis by bovine GC from different size follicles. Key words: Granulosa cells, gonadotropins, Insulin-like growth factors, progesterone, estradiol-17β, cows

scholarly journals Rapid light-dependent degradation of fluorescent dyes in formulated serum-free media

2019 ◽  
Author(s):  
Peter A. Morawski ◽  
Samantha J. Motley ◽  
Daniel J. Campbell
Keyword(s):  
Cell Culture ◽  
Fluorescent Dyes ◽  
Culture Media ◽  
Dye Degradation ◽  
Fluorescence Signal ◽  
Specific Cell ◽  
Signal Loss ◽  
Serum Free ◽  
Antibody Conjugates ◽  
Free Media

Chemically defined serum-free media is increasingly used as a tool to help standardize experiments by eliminating the potential variability contributed by pooled serum. These media are formulated for the culture and expansion of specific cell types, maintaining cell viability without the need for exogenous animal proteins. Formulated serum-free media could thus help improve viability and reduce variability during sample preparation for flow cytometry, yet a thorough analysis of how such media impact fluorochrome-antibody conjugates has not been performed. In this study, we expose fluorescent antibody-labeled cells or antibody capture beads to white light in the presence of various hematopoietic cell culture media and provide evidence that formulated serum-free media permit rapid light-initiated fluorescent dye degradation in a cell-independent manner. We observed fluorescence signal loss of several dyes, which included fluorescence spillover into adjacent detectors. Finally, photostability of antibody-fluorochrome conjugates in formulated serum-free media is partially restored in the presence of either serum or vitamin C, implicating reactive oxygen species in the observed signal loss. Thus, our data indicate that formulated serum-free media designed to standardize cell culture are not currently optimized for use with fluorochrome-antibody conjugates, and thus extreme caution should be exercised when using these media in cytometric experiments.

scholarly journals The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

2013 ◽  
Vol 2013 ◽  
pp. 1-22 ◽  
Author(s):  
Ana Lúcia Vargas Arigony ◽  
Iuri Marques de Oliveira ◽  
Miriana Machado ◽  
Diana Lilian Bordin ◽  
Lothar Bergter ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Culture Media ◽  
Genomic Stability ◽  
Sole Source ◽  
Cell Culture Media ◽  
The Media ◽  
Fetal Bovine

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic thein vivoenvironment, providingin vitromodels used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previousin vitroexperiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

Isolation, Characterization, and Differentiation of Stem Cells From Various Dental Sources: An In Vitro Study

2021 ◽  
pp. 232020682110107
Author(s):  
Sandeep S. Katti ◽  
Kishore Bhat ◽  
Chetana Bogar
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Statistical Analysis ◽  
Specific Growth ◽  
Culture Media ◽  
In Vitro Study ◽  
Central Research ◽  
Cell Lineages ◽  
Apical Papilla

Aim: The aim of the current study was to isolate stem cells from various dental sources such as dental pulp, periodontal ligament (PDL), and apical papilla, and to characterize stem cells by staining for the presence/absence of specific surface markers and also to differentiate stem cells into osteogenic, chondrogenic, and adipogenic cell lineages by exposing them to specific growth factors under the ideal conditions. Materials and Methods: A total of 117 samples were included in the study, consisting of 30 pulp, 50 gingival, 35 PDL, and 2 apical papilla samples. The pulp was extirpated and transported to the Central Research Laboratory. Gingival connective tissue was collected from the participants undergoing any crown lengthening procedure or any gingivectomy procedure from the Department of Periodontology. A similar procedure was also followed for apical papilla and PDL. Isolation was done followed by the identification of the cells by immunocytochemistry using different markers. Once the identity of cells was confirmed, these cells were treated with different culture media to attain 70% to 100% confluency. Then the medium was replaced with a conditioning medium containing specific growth factors for differentiation into osteogenic, chondrogenic, and adipogenic cell lineages. Result: In our study, the number of samples collected and processed was 117. The isolation rate of stem cells from the above-collected samples was 70%. Statistical analysis—no statistical analysis was done as there was no variability expected. Conclusion: Our study showed that stem cells could be isolated, differentiated, and characterized from different dental sources.

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

2021 ◽  
Author(s):  
Gabriela de Oliveira Fernandes ◽  
Marcella Pecora Milazzotto ◽  
Andrei Antonioni Guedes Fidelis ◽  
Taynan Stonoga Kawamoto ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Markers ◽  
Culture Medium ◽  
Culture Media ◽  
Ionization Mass ◽  
Metabolic Profiles ◽  
Bovine Embryos ◽  
The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

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

2018 ◽  
Vol 19 (11) ◽  
pp. 3538 ◽  
Author(s):  
Brandon Lehrich ◽  
Yaxuan Liang ◽  
Pooya Khosravi ◽  
Howard Federoff ◽  
Massimo Fiandaca
Keyword(s):  
Cell Growth ◽  
Extracellular Vesicles ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Cellular Growth ◽  
Primary Astrocyte ◽  
Fetal Bovine ◽  
Cell Growth And Viability

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.

scholarly journals Impact of serum in cell culture media on in vitro lactate dehydrogenase (LDH) release determination

2017 ◽  
Vol 3 (1) ◽  
pp. 9-13 ◽  
Author(s):  
Bernhard Hiebl ◽  
Sinem Peters ◽  
Ole Gemeinhardt ◽  
Stefan M. Niehues ◽  
Friedrich Jung
Keyword(s):  
Cell Culture ◽  
Lactate Dehydrogenase ◽  
Culture Media ◽  
Cell Culture Media ◽  
Ldh Release

scholarly journals Oxygen and Glucose Levels in Cell Culture Media Determine Resveratrol’s Effects on Growth, Hydrogen Peroxide Production, and Mitochondrial Dynamics

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 157 ◽  
Author(s):  
Joao Fonseca ◽  
Fereshteh Moradi ◽  
Andrew Valente ◽  
Jeffrey Stuart
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Culture ◽  
Cell Growth ◽  
Cultured Cells ◽  
Culture Media ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Network ◽  
Hydrogen Peroxide Production ◽  
Network Characteristics ◽  
Glucose Levels

Resveratrol is a plant-derived polyphenol that has been widely studied for its putative health promoting effects. Many of those studies have been conducted in cell culture, in supra-physiological levels of oxygen and glucose. Resveratrol interacts with reactive oxygen species (ROS) as an antioxidant or pro-oxidant. Resveratrol affects the expression and activities of ROS-producing enzymes and organelles. It is therefore important to consider how cell culture conditions might determine the effects of resveratrol on cultured cells. We determined the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics in C2C12 mouse myoblasts and PC3 human prostate cancer cells under conditions of physiological (5%) and supra-physiological (18%) oxygen, and normo- (5 mM) and hyper-glycemia (25 mM). Interestingly, most effects of resveratrol on the parameters measured here were dependent upon prevailing oxygen and glucose levels during the experiment. Many of the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics that were seen in 25 mM glucose and/or 18% oxygen were absent under the physiologically relevant conditions of 5 mM glucose with 5% oxygen. These findings emphasize the importance of using physiologically meaningful starting conditions for cell-culture experiments with resveratrol and indeed any manipulation affecting ROS metabolism and mitochondria.

scholarly journals A High Throughput Approach Based on Dynamic High Pressure for the Encapsulation of Active Compounds in Exosomes for Precision Medicine

2021 ◽  
Vol 22 (18) ◽  
pp. 9896
Author(s):  
Eugenia Romano ◽  
Paolo Antonio Netti ◽  
Enza Torino
Keyword(s):  
Cell Culture ◽  
High Pressure ◽  
Culture Media ◽  
Preliminary Test ◽  
Bilayer Membrane ◽  
Microfluidic System ◽  
Culture Model ◽  
Before And After ◽  
Dynamic High Pressure

In recent decades, endogenous nanocarrier-exosomes have received considerable scientific interest as drug delivery systems. The unique proteo-lipid architecture allows the crossing of various natural barriers and protects exosomes cargo from degradation in the bloodstream. However, the presence of this bilayer membrane as well as their endogenous content make loading of exogenous molecules challenging. In the present work, we will investigate how to promote the manipulation of vesicles curvature by a high-pressure microfluidic system as a ground-breaking method for exosomes encapsulation. Exosomes isolated from Uppsala 87 Malignant Glioma (U87-MG) cell culture media were characterized before and after the treatment with high-pressure homogenization. Once their structural and biological stability were validated, we applied this novel method for the encapsulation in the lipidic exosomal bilayer of the chemotherapeutic Irinotecan HCl Trihydrate-CPT 11. Finally, we performed in vitro preliminary test to validate the nanobiointeraction of exosomes, uptake mechanisms, and cytotoxic effect in cell culture model.

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