scholarly journals Fluid Menisci and In Vitro Particle Dosimetry of Submerged Cells

2021 ◽  
Author(s):  
Sandor Balog ◽  
Barbara Rothen-Rutishauser ◽  
Alke Fink
Keyword(s):  
Culture Media ◽  
In Vivo Studies ◽  
Cell Culture Media ◽  
Air Interface ◽  
Inner Surface ◽  
Particle Dosimetry ◽  
Meta Analyses ◽  
Cells Cultured

Understanding the mechanisms of interaction between cells and particulate nanomaterials lies in the heart of assessing the hazard associated with nanoparticles. The paradigm of toxicology requires quantifying and interpreting dose-response relationships, and cells cultured in vitro and exposed to particle dispersions rely on mathematical models that estimate the received nanoparticle dose. Yet, none of these models acknowledges the fact that aqueous cell-culture media wet the inner surface of hydrophilic open wells, which results in curved fluid-air interface called meniscus. We show that omitting this phenomenon leads to a nontrivial but systematic error and twists the fundamental concept of nanotoxicology. Given that reproducibility and harmonization between meta analyses, in vitro, in silico, and in vivo studies must be improved, we present an adequate mathematical model that greatly advances such efforts.

Evaluation of Annona muricata (Graviola) leaves activity against experimental trichinellosis: in vitro and in vivo studies

2021 ◽  
Vol 95 ◽  
Author(s):  
E.S. El-Wakil ◽  
H.F. Abdelmaksoud ◽  
T.S. AbouShousha ◽  
M.M.I. Ghallab
Keyword(s):  
Culture Media ◽  
Trichinella Spiralis ◽  
Drug Effects ◽  
In Vivo Studies ◽  
Control Group ◽  
Annona Muricata ◽  
Group I ◽  
Small Intestinal

Abstract Our work aimed to evaluate the possible effect of Annona muricata (Graviola) leaf extract on Trichinella spiralis in in vitro and in vivo studies. Trichinella spiralis worms were isolated from infected mice and transferred to three culture media – group I (with no drugs), group II (contained Graviola) and group III (contained albendazole) – then they were examined using the electron microscope. In the in vivo study, mice were divided into five groups: GI (infected untreated), GII (prophylactically treated with Graviola for seven days before infection), GIII (infected and treated with Graviola), GIV (infected and treated with albendazole) and GV (infected and treated with a combination of Graviola plus albendazole in half doses). Drug effects were assessed by adults and larvae load beside the histopathological small intestinal and muscular changes. A significant reduction of adult and larval counts occurred in treated groups in comparison to the control group. Histopathologically, marked improvement in the small intestinal and muscular changes was observed in treated groups. Also, massive destruction of the cultured adults’ cuticle was detected in both drugs. This study revealed that Graviola leaves have potential activity against trichinellosis, especially in combination with albendazole, and could serve as an adjuvant to anti-trichinellosis drug therapy.

scholarly journals Inflammatory Mediator Profiling Reveals Immune Properties of Chemotactic Gradients and Macrophage Mediator Production Inhibition during Thioglycollate Elicited Peritoneal Inflammation

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Derek Lam ◽  
Devon Harris ◽  
Zhenyu Qin
Keyword(s):  
Inflammatory Mediators ◽  
Inflammatory Mediator ◽  
Culture Media ◽  
Cell Culture Media ◽  
Inflammatory Mediator Production ◽  
Inflammation Resolution ◽  
Lps Treatment ◽  
Macrophage Accumulation

Understanding of spatiotemporal profiling of inflammatory mediators and their associations with MΦ accumulation is crucial to elucidate the complex immune properties. Here, we used murine thioglycollate elicited peritonitis to determine concentrations of 23 inflammatory mediators in peritoneal exudates and plasma before (day 0) and after (days 1 and 3) thioglycollate administration to peritoneal cavities; these mediators included TNF-α, FGF-9, IFN-γ, IP-10, RANTES, IL-1α, IL-6, IL-7, IL-10, IL-11, IL-12p70, IL-17A, lymphotactin, OSM, KC/GRO, SCF, MIP-1β, MIP-2, TIMP-1, VEGF-A, MCP-1, MCP-3, and MCP-5. Our results showed that concentrations of most mediators in exudates and plasma reached peak levels on day 1 and were significantly reduced on day 3. Conversely, MΦnumbers started to increase on day 1 and reached peak levels on day 3. Moreover, LPS treatmentin vitrosignificantly induced mediator productions in cell culture media and lysates from MΦisolated on day 3. Our results also showed that on day 0, concentrations of many mediators in plasma were higher than those in exudates, whereas on day 1, the trend was reversed. Overall, the findings from thioglycollate elicited peritonitis reveal that reversible chemotactic gradients between peritoneal exudates and blood exist in basal and inflamed conditions and the inflammatory mediator productionin vivois disassociated with macrophage accumulation during inflammation resolution.

scholarly journals Engineering the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

2021 ◽  
Author(s):  
◽  
Alistair Brown
Keyword(s):  
Substrate Specificity ◽  
Culture Media ◽  
Protein Domain ◽  
In Vivo Studies ◽  
Blue Pigment ◽  
Carrier Protein ◽  
Type I ◽  
Phosphopantetheinyl Transferase

<p>Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that synthesise bioactive peptides using an assembly line architecture, wherein each module is responsible for the incorporation of a monomer into the growing chain. Present in both fungi and bacteria, NRPSs are responsible for a wide variety of secondary metabolites and bioactive compounds including siderophores, antibiotics, anti-cancer compounds and immunosuppressants. For functionality, NRPSs require the attachment of a phosphopantetheine moiety to their peptidyl carrier protein domains. This reaction is catalysed by a phosphopantetheinyl transferase (PPTase).  The NRPS blue pigment synthetase A (BpsA) is unusual in that it is comprised of only a single module. BpsA contains an adenylation domain that recognises and sequentially binds two molecules of L-glutamine, an oxidation domain that is believed to oxidise each glutamine monomer, a peptidyl carrier protein domain that binds the phosphopantetheine moiety, and a thioesterase domain that cyclises each glutamine and releases the final bicyclic product from the enzyme. This final product is a blue pigment called indigoidine, and its synthesis from two molecules of L-glutamine is powered by ATP. Comparatively to other NRPSs BpsA is easy to manipulate and work with both in vitro and in vivo. Here, the ability to easily detect synthesis of indigoidine was utilised to provide a versatile reporter to detect a variety of biochemical activities.  PPTases are essential enzymes that are promising drug targets in the clinically important bacteria Pseudomonas aeruginosa and Mycobacterium tuberculosis. BpsA can be purified in the inactive apo form, which then requires a PPTase to activate it to enable indigoidine synthesis. Here it was shown that mixing BpsA, a PPTase, the enzymatic substrates, and a potential inhibitor enables screening for PPTase inhibition by monitoring the rate or extent of indigoidine synthesis. This method was optimised and used to screen commercial drug libraries against two PPTases, PcpS from P. aeruginosa and PptT from M. tuberculosis. Several novel inhibitors were identified and pilot in vivo studies were performed. M. tuberculosis also possesses a second essential PPTase called TB-AcpS, which has very narrow substrate specificity and cannot post-translationally modify BpsA. In an attempt to widen the substrate specificity a combination of rational engineering and directed evolution was employed. These attempts did not yield significant improvements in the ability of TB-AcpS to activate modified BpsA, however they did yield mutants that were more effective substrates for other type I PPTases.  The easily detectable nature of indigoidine also enabled application of BpsA as a reporter for a range of different substrates. Particularly effective was development of a commercially applicable method using BpsA to quantify L-glutamine in a range of conditions, including cell culture media and blood. The assay developed offers several advantages over currently available kits. BpsA was also used to detect and quantify ATP, and this was applied to monitor adenylation reactions. Finally, the ability of BpsA to synthesise indigoidine-like compounds from glutamine analogues was explored.</p>

scholarly journals Morphological and Physicochemical Characterization of Agglomerates of Titanium Dioxide Nanoparticles in Cell Culture Media

2016 ◽  
Vol 2016 ◽  
pp. 1-19 ◽  
Author(s):  
Verónica Freyre-Fonseca ◽  
Darío I. Téllez-Medina ◽  
Estefany I. Medina-Reyes ◽  
Maribel Cornejo-Mazón ◽  
Edgar O. López-Villegas ◽  
...  
Keyword(s):  
Titanium Dioxide ◽  
Culture Media ◽  
Titanium Dioxide Nanoparticles ◽  
Physicochemical Characterization ◽  
Cell Culture Media ◽  
Size Stability ◽  
Maximum Stability

Titanium dioxide nanoparticles (TiO2NP) are possible carcinogenic materials (2B-IARC) and their toxicity depends on shape, size, and electrical charge of primary NP and on the system formed by NP media. The aim of this work was to characterize agglomerates of three TiO2NP by evaluating their morphometry, stability, and zeta potential (ζ) in liquid media and their changes with time. Sizes of agglomerates by dynamic light scattering (DLS) resulted to be 10–50 times larger than those obtained by digital image analysis (DIA) given the charged zone around particles. Fractal dimension (FD) was highest for agglomerates of spheres and belts in F12K, and in E171 in FBS media. E171 and belts increased FD with time. At time zero, using water as dispersant FD was larger for agglomerates of spheres than for of E171. Belts suspended in water had the smallest values of circularity (Ci) which was approximately unchanged with time. All dispersions hadζvalues around −30 mV at physiological pH (7.4) and dispersions of NP in water and FBS showed maximum stability (Turbiscan Lab analysis). Results help in understanding the complex NP geometry-size-stability relationships when performingin vivoandin vitroenvironmental-toxicity works and help in supporting decisions on the usage of TiO2NP.

scholarly journals In vitro culture of Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): Influence of temperature and culture media

2018 ◽  
Vol 27 (4) ◽  
pp. 562-569 ◽  
Author(s):  
Carinne Moreira de Souza Costa ◽  
Talissa Beatriz Costa Lima ◽  
Matheus Gomes da Cruz ◽  
Daniela Volcan Almeida ◽  
Maurício Laterça Martins ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Treatment Strategies ◽  
Infected Fish ◽  
In Vivo Studies ◽  
Colossoma Macropomum ◽  
Tank Water

Abstract Infection by the acantocephalan Neoechinorhynchus buttnerae is considered one of most important concerns for tambaqui fish (Colossoma macropomum ) production. Treatment strategies have been the focus of several in vivo studies; however, few studies have been undertaken on in vitro protocols for parasite maintenance. The aim of the present study was to develop the best in vitro culture condition for N. buttnerae to ensure its survival and adaptation out of the host to allow for the testing of substances to be used to control the parasite. To achieve this, parasites were collected from naturally infected fish and distributed in 6-well culture plates under the following treatments in triplicate: 0.9% NaCl, sterile tank water, L-15 Leibovitz culture medium, L-15 Leibovitz + agar 2% culture medium, RPMI 1640 culture medium, and RPMI 1640 + agar 2% culture medium. The plates containing the parasites were maintained at 24 °C, 28 °C, and 32 °C. The RPMI 1640 + agar 2% culture medium showed the best survival of 24 days at 24 °C. No body alterations such as swollen parasites, body deformation, dehydration and hardening were observed in the RPMI 1640 + 2% culture medium.

scholarly journals Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes, induced by culture substrates

1984 ◽  
Vol 219 (3) ◽  
pp. 793-799 ◽  
Author(s):  
S O Kolset ◽  
R Seljelid ◽  
U Lindahl
Keyword(s):  
Foreign Body ◽  
Culture Media ◽  
Chondroitin Sulphate ◽  
Morphological Changes ◽  
Artificial Substrates ◽  
Plastic Substrate ◽  
Incubation Periods ◽  
Cells Cultured

Monocytes were isolated from human blood and cultured in vitro on plastic culture dishes or on fibronectin-coated dishes. After 5 days in vitro, the cells on plastic dishes displayed marked morphological changes compared with day 1, with an epithelioid appearance resembling that of foreign-body cells. This transition was inhibited in cells cultured on fibronectin-coated dishes. 35S-labelled polysaccharides were isolated from the culture media after 24h incubation periods with inorganic [35S]sulphate. The cells cultured for 5 days on a plastic substrate synthesized, and secreted into the medium, an oversulphated galactosaminoglycan previously shown to contain 4,6-di-O-sulphated N-acetylgalactosamine units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. In contrast, 35S-labelled polysaccharide produced by cells cultured on plastic for 1 day only, or on fibronectin for either 1 or 5 days, contained only minor amounts of such disulphated sugar units. These findings indicate that the formation of oversulphated chondroitin sulphate is coupled to the conversion of monocytes into epithelioid cells. Furthermore, they suggest that the overall process is induced by contact with artificial substrates, and that it may be regarded as the equivalent of a foreign-body reaction in vivo.

scholarly journals In Vitro Bioavailability of the Hydrocarbon Fractions of Dimethyl Sulfoxide Extracts of Petroleum Substances

2020 ◽  
Vol 174 (2) ◽  
pp. 168-177 ◽  
Author(s):  
Yu-Syuan Luo ◽  
Kyle C Ferguson ◽  
Ivan Rusyn ◽  
Weihsueh A Chiu
Keyword(s):  
Cell Culture ◽  
Protein Binding ◽  
Aromatic Compounds ◽  
Culture Media ◽  
In Vitro Testing ◽  
Cell Culture Media ◽  
Chemical Profiles ◽  
In Vivo Extrapolation

Abstract Determining the in vitro bioavailable concentration is a critical, yet unmet need to refine in vitro-to-in vivo extrapolation for unknown or variable composition, complex reaction product or biological material (UVCB) substances. UVCBs such as petroleum substances are commonly subjected to dimethyl sulfoxide (DMSO) extraction in order to retrieve the bioactive polycyclic aromatic compound (PAC) portion for in vitro testing. In addition to DMSO extraction, protein binding in cell culture media and dilution can all influence in vitro bioavailable concentrations of aliphatic and aromatic compounds in petroleum substances. However, these in vitro factors have not been fully characterized. In this study, we aimed to fill in these data gaps by characterizing the effects of these processes using both a defined mixture of analytical standards containing aliphatic and aromatic hydrocarbons, as well as 4 refined petroleum products as prototypical examples of UVCBs. Each substance was extracted with DMSO, and the protein binding in cell culture media was measured by using solid-phase microextraction. Semiquantitative analysis for aliphatic and aromatic compounds was achieved via gas chromatography-mass spectrometry. Our results showed that DMSO selectively extracted PACs from test substances, and that chemical profiles of PACs across molecular classes remained consistent after extraction. With respect to protein binding, chemical profiles were retained at a lower dilution (higher concentration), but a greater dilution factor (ie, lower concentration) resulted in higher protein binding in cell medium, which in turn altered the ultimate chemical profile of bioavailable PACs. Overall, this case study demonstrates that extraction procedures, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the final bioavailable concentrations of bioactive constituents of UVCBs in vitro. Thus, in vitro-to-in vivo extrapolation for UVCBs may require greater attention to the concentration-dependent and compound-specific differences in recovery and bioavailability.

In vitro cytocompatibility testing of oxidative degradation products

2021 ◽  
pp. 088391152110031
Author(s):  
Scott M Herting ◽  
Mary Beth B Monroe ◽  
Andrew C Weems ◽  
Sam T Briggs ◽  
Grace K Fletcher ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Culture ◽  
Culture Media ◽  
Degradation Products ◽  
Oxidative Degradation ◽  
Cell Culture Media ◽  
Novel Approach ◽  
Gene And Protein Expression

Implantable medical devices must undergo thorough evaluation to ensure safety and efficacy before use in humans. If a device is designed to degrade, it is critical to understand the rate of degradation and the degradation products that will be released. Oxidative degradation is typically modeled in vitro by immersing materials or devices in hydrogen peroxide, which can limit further analysis of degradation products in many cases. Here we demonstrate a novel approach for testing the cytocompatibility of degradation products for oxidatively-degradable biomaterials where the materials are exposed to hydrogen peroxide, and then catalase enzyme is used to convert the hydrogen peroxide to water and oxygen so that the resulting aqueous solution can be added to cell culture media. To validate our results, expected degradation products are also synthesized then added to cell culture media. We used these methods to evaluate the cytocompatibility of degradation products from an oxidatively-degradable shape memory polyurethane designed in our lab and found that the degradation of these polymers is unlikely to cause a cytotoxic response in vivo based on the guidance provided by ISO 10993-5. These methods may also be applicable to other biocompatibility tests such as tests for mutagenicity or systemic toxicity, and evaluations of cell proliferation, migration, or gene and protein expression.

scholarly journals Development of novel MOG analogues with increased stability to explore MCT2 and α-ketoglutarate biology in vivo

2021 ◽  
Author(s):  
Louise Fets ◽  
Patrícia M. Nunes ◽  
Sebastien Campos ◽  
Mariana Silva dos Santos ◽  
Natalie Bevan ◽  
...  
Keyword(s):  
Culture Media ◽  
Pharmacokinetic Profile ◽  
Monocarboxylate Transporter ◽  
Metabolic Effects ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Culture Media ◽  
Pharmacokinetic Properties

ABSTRACTα-ketoglutarate (αKG) is a central metabolic node with far-reaching influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyloxalylglycine (DMOG) have been broadly used as tool compounds both in vitro and in vivo to study αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, a substrate of the monocarboxylate transporter MCT2. The expression level of MCT2 determines the intracellular concentration of NOG, and, as such, influences the molecular targets NOG engages with. Here we show that DMOG and MOG are highly unstable also in mouse blood. We therefore designed and characterised a series of MOG analogues with two aims: to improve pharmacokinetic properties, and to explore the pharmacophore of MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain MCT2-dependent uptake, including NOG-generating compounds that replicate the metabolic effects of MOG in a concentration-dependent manner. One such analogue, IPOG, shows significantly increased blood stability, and an improved overall pharmacokinetic profile, leading to increased NOG accumulation in MCT2-expressing tumours versus isogenic controls.

scholarly journals In vitro interaction network of a synthetic gut bacterial community

2021 ◽  
Author(s):  
Anna S. Weiss ◽  
Anna G. Burrichter ◽  
Abilash Chakravarthy Durai Raj ◽  
Alexandra von Strempel ◽  
Chen Meng ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Culture Media ◽  
Interference Competition ◽  
Interaction Network ◽  
In Vivo Studies ◽  
Metabolic Potential ◽  
Microbiome Research ◽  
Insight Into

AbstractA key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies.

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