scholarly journals Dynamic interactions and intracellular fate of label-free GO within mammalian cells: role of lateral sheet size

2019 ◽  
Author(s):  
Yingxian Chen ◽  
Livia Elena Crică ◽  
Vinicio Rosano ◽  
Adrián Esteban Arranz ◽  
David Spiller ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Analytical Techniques ◽  
Transport Systems ◽  
Dependent Manner ◽  
Label Free ◽  
Fluorescent Properties ◽  
Uptake Mechanism ◽  
Mammalian Cell Lines ◽  
Intracellular Fate

AbstractGraphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is a prerequisite for successful clinical translation. In this study, we exploited intrinsic fluorescent properties of GO to establish the relationship between lateral dimensions of the material, its cellular uptake mechanism and intracellular fate. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO), was synthesized and thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was interrogated using a range of pharmaceutical inhibitors for main endocytic pathways (ethyl-isopropyl amiloride, monodansylcadaverine, chlorpromazine, genistein, cytochalasin D, latrunculin A, dynasore and sodium azide), and validated using negatively charged polystyrene beads with different diameters (0.1 and 1 μm). Regardless of lateral dimension, both types of GO were found to interact with the plasma membrane and to be efficiently taken up by a panel of cell lines in a time- and dose-dependent manner. s-GO was internalised mainly via macropinocytosis while us-GO used mainly clathrin- and caveolae-mediated endocytosis. Lastly, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results aim to offer significant insight into the mechanism of interaction of GO with non-phagocytic cell lines that can be exploited for the design of biomedically applicable 2D transport systems.

Cytotoxicity Study of Textile Fabrics Impregnated With CuO Nanoparticles in Mammalian Cells

2017 ◽  
Vol 36 (6) ◽  
pp. 478-484 ◽  
Author(s):  
Gagandeep Singh ◽  
James Beddow ◽  
Christopher Mee ◽  
Lidia Maryniak ◽  
Eadaoin M. Joyce ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Dermal Fibroblast ◽  
Copper Oxide Nanoparticles ◽  
Growth Media ◽  
Indirect Contact ◽  
Cuo Nps ◽  
Mammalian Cell Lines ◽  
Textile Fabrics ◽  
Hdf Cells

Copper and copper compounds have multifunctional properties (antibacterial, antiviral, and antifungal) with promising applications. Copper in its nanoparticle (Cu NPs) forms has been widely used in various industrial and commercial applications. In the current research, the cytotoxic effects of textile fabrics impregnated with copper oxide nanoparticles (CuO NPs) were studied in mammalian cell lines. CuO NPs were impregnated onto textile substrates using 2 different techniques: the sonochemical generation and impregnation of NPs from metal complexes ( insitu) and a “throwing the stones” technology using commercially prepared CuO NPs. The cytotoxicity of these 2 textile fabric types was assayed on human dermal fibroblast (HDF) cells and human hepatocellular carcinoma cells (HepG2) and was evaluated by indirect contact using an MTT assay. The impregnated fabrics were not exposed to the cells, rather their leachates were used to test cytotoxicity. The fabrics were soaked into the growth media for up to 7 days, and the leachates from day 1 and day 7 were incubated with the cell lines for 24 hours prior to the testing. The discharge or leaching from antimicrobial nanomaterials into the surroundings and surface waters is posing a serious environmental threat, which needs to be addressed. Hence, with regard to product safety, it is a good approach to study the fabric leachates rather than the intact material. The results showed that CuO NPs are not toxic to HDF cells. However, cytotoxicity was seen in HepG2 cells with cell viability decreasing by 20% to 25% for all the fabrics after 24 hours.

scholarly journals ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

2008 ◽  
Vol 76 (10) ◽  
pp. 4600-4608 ◽  
Author(s):  
Karin Heine ◽  
Sascha Pust ◽  
Stefanie Enzenmüller ◽  
Holger Barth
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Clostridium Botulinum ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Adp Ribosylation ◽  
Mammalian Cell Lines ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

scholarly journals Cell Line Classification Using Electric Cell-Substrate Impedance Sensing (ECIS)

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Megan L. Gelsinger ◽  
Laura L. Tupper ◽  
David S. Matteson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Statistical Tests ◽  
Classification Methods ◽  
Impedance Sensing ◽  
Mammalian Cell Lines ◽  
Cell Substrate ◽  
Multiple Cell ◽  
Line Classification

AbstractWe present new methods for cell line classification using multivariate time series bioimpedance data obtained from electric cell-substrate impedance sensing (ECIS) technology. The ECIS technology, which monitors the attachment and spreading of mammalian cells in real time through the collection of electrical impedance data, has historically been used to study one cell line at a time. However, we show that if applied to data from multiple cell lines, ECIS can be used to classify unknown or potentially mislabeled cells, factors which have previously been associated with the reproducibility crisis in the biological literature. We assess a range of approaches to this new problem, testing different classification methods and deriving a dictionary of 29 features to characterize ECIS data. Most notably, our analysis enriches the current field by making use of simultaneous multi-frequency ECIS data, where previous studies have focused on only one frequency; using classification methods to distinguish multiple cell lines, rather than simple statistical tests that compare only two cell lines; and assessing a range of features derived from ECIS data based on their classification performance. In classification tests on fifteen mammalian cell lines, we obtain very high out-of-sample predictive accuracy. These preliminary findings provide a baseline for future large-scale studies in this field.

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity

1977 ◽  
Vol 23 (2) ◽  
pp. 183-189 ◽  
Author(s):  
John L. Middlebrook ◽  
Rebecca B. Dorland
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Corynebacterium Diphtheriae ◽  
Pseudomonas Exotoxin ◽  
Intact Cells ◽  
Mammalian Cell Lines ◽  
First Order Kinetics ◽  
Wide Range ◽  
Species Specific

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1–4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat-and mouse-derived cell lines were very in sensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line–specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.

scholarly journals Stable Expression of a Hepatitis E Virus (HEV) RNA Replicon in Two Mammalian Cell Lines to Assess Mechanism of Innate Immunity and Antiviral Response

2020 ◽  
Vol 11 ◽  
Author(s):  
Ling-Dong Xu ◽  
Fei Zhang ◽  
Lei Peng ◽  
Wen-Ting Luo ◽  
Chu Chen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Innate Immune ◽  
Type I ◽  
Dependent Manner ◽  
Genotype 3 ◽  
Mammalian Cell Lines ◽  
Rna Replicon

Hepatitis E virus (HEV) is one of the major etiological agents responsible for acute hepatitis. Hepatitis E virus does not replicate efficiently in mammalian cell cultures, thus a useful model that mimics persistent HEV replication is needed to dissect the molecular mechanism of pathogenesis. Here we report a genotype-3 HEV RNA replicon expressing an EGFP-Zeocin (EZ) resistant gene (p6-EZ) that persistently self-replicated in cell lines of human (Huh-7-S10-3) or hamster (BHK-21) origin after transfection with in vitro RNA transcripts and subsequent drug screening. Two cell lines, S10-3-EZ and BHK-21-EZ, stably expressed EGFP in the presence of Zeocin during continuous passages. Both genomic and subgenomic HEV RNAs and viral replicase proteins were stably expressed in persistent HEV replicon cells. The values of the cell models in antiviral testing, innate immune RNA sensing and type I IFN in host defense were further demonstrated. We revealed a role of RIG-I like receptor-interferon regulatory factor 3 in host antiviral innate immune sensing during HEV replication. We further demonstrated that treatment with interferon (IFN-α) or ribavirin significantly reduced expression of replicon RNA in a dose-dependent manner. The availability of the models will greatly facilitate HEV-specific antiviral development, and delineate mechanisms of HEV replication.

scholarly journals Targeted Comparative RNA Interference Analysis Reveals Differential Requirement of Genes Essential for Cell Proliferation

2006 ◽  
Vol 17 (11) ◽  
pp. 4837-4845 ◽  
Author(s):  
Yuichi J. Machida ◽  
Yuefeng Chen ◽  
Yuka Machida ◽  
Ankit Malhotra ◽  
Sukumar Sarkar ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Cancer Cell Line ◽  
Prostate Cancer Cell Line ◽  
Breast Epithelial Cell ◽  
Epithelial Cell Line ◽  
Dependent Manner

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53−) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type–specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.

NanoLiterBioReactor: Monitoring of Long-Term Mammalian Cell Physiology at Nanofabricated Scale

2004 ◽  
Vol 820 ◽  
Author(s):  
Ales Prokop ◽  
Zdenka Prokop ◽  
David Schaffer ◽  
Eugene Kozlov ◽  
John Wikswo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Cell Counting ◽  
Cell Physiology ◽  
Mammalian Cell Lines ◽  
Cell Counts ◽  
Multiple Cell

AbstractThere is a need for microminiaturized cell-culture environments, i.e., NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment--e.g., to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

scholarly journals Growth and eGFP Production of CHO-K1 Suspension Cells Cultivated From Single Cell to Laboratory Scale

2021 ◽  
Vol 9 ◽  
Author(s):  
Julian Schmitz ◽  
Oliver Hertel ◽  
Boris Yermakov ◽  
Thomas Noll ◽  
Alexander Grünberger
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Mammalian Cell Culture ◽  
Laboratory Scale ◽  
Cellular Heterogeneity ◽  
Suspension Cells ◽  
Stirred Tank Bioreactor ◽  
Mammalian Cell Lines

Scaling down bioproduction processes has become a major driving force for more accelerated and efficient process development over the last decades. Especially expensive and time-consuming processes like the production of biopharmaceuticals with mammalian cell lines benefit clearly from miniaturization, due to higher parallelization and increased insights while at the same time decreasing experimental time and costs. Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices have been proved to be valuable to miniaturize the cultivation of mammalian cells. So far, growth characteristics of microfluidic cultivated cell lines were not systematically compared to larger cultivation scales; however, validation of a miniaturization tool against initial cultivation scales is mandatory to prove its applicability for bioprocess development. Here, we systematically investigate growth, morphology, and eGFP production of CHO-K1 cells in different cultivation scales ranging from a microfluidic chip (230 nl) to a shake flask (125 ml) and laboratory-scale stirred tank bioreactor (2.0 L). Our study shows a high comparability regarding specific growth rates, cellular diameters, and eGFP production, which proves the feasibility of MSCC as a miniaturized cultivation tool for mammalian cell culture. In addition, we demonstrate that MSCC provides insights into cellular heterogeneity and single-cell dynamics concerning growth and production behavior which, when occurring in bioproduction processes, might severely affect process robustness.

scholarly journals Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park

2002 ◽  
Vol 68 (9) ◽  
pp. 4559-4566 ◽  
Author(s):  
Jason A. Simser ◽  
Ann T. Palmer ◽  
Volker Fingerle ◽  
Bettina Wilske ◽  
Timothy J. Kurtti ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ixodes Ricinus ◽  
Mammalian Cells ◽  
Citrate Synthase ◽  
Spotted Fever ◽  
Spotted Fever Group ◽  
Mammalian Cell Lines ◽  
Isolation And Characterization ◽  
Rickettsia Monacensis ◽  
City Park

ABSTRACT We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/MunichT) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

scholarly journals Interaction of Recombinant Norwalk Virus Particles with the 105-Kilodalton Cellular Binding Protein, a Candidate Receptor Molecule for Virus Attachment

2000 ◽  
Vol 74 (24) ◽  
pp. 11589-11597 ◽  
Author(s):  
Masaru Tamura ◽  
Katsuro Natori ◽  
Masahiko Kobayashi ◽  
Tatsuo Miyamura ◽  
Naokazu Takeda
Keyword(s):  
Mammalian Cells ◽  
Protein A ◽  
Binding Activity ◽  
Conformational Structure ◽  
Dependent Manner ◽  
Norwalk Virus ◽  
Capsid Protein Gene ◽  
Virus Attachment ◽  
Protein Protein Interaction ◽  
Mammalian Cell Lines

ABSTRACT Norwalk virus (NV), responsible for outbreaks of acute gastroenteritis, comprises the species of the genus Norwalk-like viruses in the family Caliciviridae. Although the study of the molecular biology of NV has been hampered by a lack of culture systems or small experimental animal models, virus-like particles (VLPs) generated with recombinant baculoviruses harboring the capsid protein gene of NV provide a useful tool for investigating NV-cell interactions. In this study, the attachment of the recombinant VLPs derived from the Ueno virus (UEV), a strain belonging to the genogroup II NVs, to mammalian and insect cells was examined. Kinetic analyses of the binding of the recombinant VLPs of the UEV (rUEVs) to Caco-2 cells demonstrated that the binding was specific and occurred in a dose-dependent manner. Approximately 7.5% of the prebound rUEVs were internalized into the Caco-2 cells. Enzymatic and chemical modification of Caco-2 cell surface molecules suggested that the binding was directly mediated by a protein-protein interaction. A virus overlay protein-binding assay (VOPBA) indicated that rUEVs appeared to bind to a 105-kDa molecule, designated as the NV attachment (NORVA) protein. Furthermore, the assay indicated that its native conformational structure was indispensable for the binding activity. In Caco-2 cells, the NORVA protein was detected when VOPBA was carried out with the VLPs from Seto and Funabashi viruses, which are serologically different NVs from UEV, used as probes. The binding of rUEVs to NORVA protein was also observed in six mammalian cell lines other than Caco-2. These data suggest that the attachment of NV to mammalian cells is mediated by NORVA protein, which is ubiquitously expressed in the mammalian cells. The present study is the first report on the role of the cellular molecule in the binding of recombinant VLPs of NV.

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