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Mammalian cell culture

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https://www.readbyqxmd.com/read/28720219/monitoring-leachables-from-single-use-bioreactor-bags-for-mammalian-cell-culture-by-dispersive-liquid-liquid-microextraction-followed-by-ultra-high-performance-liquid-chromatography-quadrupole-time-of-flight-mass-spectrometry
#1
N Dorival-García, J Bones
A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media...
July 3, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28719670/suppressive-effect-of-amp-activated-protein-kinase-on-the-epithelial-mesenchymal-transition-in-retinal-pigment-epithelial-cells
#2
Ryo Matoba, Yuki Morizane, Yusuke Shiode, Masayuki Hirano, Shinichiro Doi, Shinji Toshima, Ryoichi Araki, Mika Hosogi, Tomoko Yonezawa, Fumio Shiraga
The epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a central role in the development of proliferative vitreoretinopathy (PVR). The purpose of this study was to investigate the effect of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis, on the EMT in RPE cells. In this study, EMT-associated formation of cellular aggregates was induced by co-stimulation of cultured ARPE-19 cells with tumor necrosis factor (TNF)-α (10 ng/ml) and transforming growth factor (TGF)-β2 (5 ng/ml)...
2017: PloS One
https://www.readbyqxmd.com/read/28716374/high-throughput-quantitation-of-fc-containing-recombinant-proteins-in-cell-culture-supernatant-by-fluorescence-polarization-spectroscopy
#3
Ben Thompson, Jerry Clifford, Mike Jenns, Andrew Smith, Ray Field, Kalpana Nayyar, David C James
Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (<2 min per 96 samples) 96-well microplate-based assay that enables high-throughput quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5-80 mg/L, using microplate fluorescence polarization (FP) spectroscopy...
July 14, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28715496/transition-of-phase-response-properties-and-singularity-in-the-circadian-limit-cycle-of-cultured-cells
#4
Satoshi Koinuma, Hiroshi Kori, Isao T Tokuda, Kazuhiro Yagita, Yasufumi Shigeyoshi
The circadian system has been regarded as a limit cycle oscillator constructed by the integrated interaction of clock genes and proteins. Here, we investigated a mammalian circadian oscillation geometrically before and after a perturbation. We detected the singular point and transition from a type 1 to type 0 phase response curve (PRC) and determined the embedding dimension to show how many variables are needed to describe the limit cycle oscillation and relaxation process after a perturbation. As a perturbation, forskolin (FK) was administered to Rat-1 cells expressing the Per2::luc gene...
2017: PloS One
https://www.readbyqxmd.com/read/28715390/mammalian-cell-encapsulation-in-alginate-beads-using-a-simple-stirred-vessel
#5
Corinne A Hoesli, Roger L J Kiang, Kamini Raghuram, René G Pedroza, Karen E Markwick, Antonio M R Colantuoni, James M Piret
Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets...
June 29, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28714852/the-small-molecule-mimetic-agonist-trimebutine-of-adhesion-molecule-l1-contributes-to-functional-recovery-after-spinal-cord-injury-in-mice
#6
Junping Xu, Chengliang Hu, Qiong Jiang, Hongchao Pan, Huifan Shen, Melitta Schachner
Curing spinal cord injury (SCI) in mammals is a daunting task because of the lack of permissive mechanisms and strong inhibitory responses at and around the lesion. The neural cell adhesion molecule L1CAM (L1) has been shown to favor axonal regrowth and enhance neuronal survival and synaptic plasticity, and thus constitutes a viable target to promote regeneration after SCI. Since delivery of full-length L1 or its extracellular domain could encounter difficulties in translation to therapy in humans, we have identified several small organic compounds that bind to L1 and stimulate neuronal survival, neuronal migration, and neurite outgrowth in an L1-dependent manner...
July 14, 2017: Disease Models & Mechanisms
https://www.readbyqxmd.com/read/28711596/the-relevance-of-contact-independent-cell-to-cell-transfer-of-tdp-43-and-sod1-in-amyotrophic-lateral-sclerosis
#7
REVIEW
Maya A Hanspal, Christopher M Dobson, Justin J Yerbury, Janet R Kumita
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving the formation of cytoplasmic aggregates by proteins including TDP-43 and SOD1, in affected cells in the central nervous system (CNS). Pathology spreads from an initial site of onset to contiguous anatomical regions. There is evidence that for disease-associated proteins, including TDP-43 and SOD1, non-native protein conformers can promote misfolding of the natively folded counterparts, and cell-to-cell transfer of pathological aggregates may underlie the spread of the disease throughout the CNS...
July 12, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28710856/quantitative-intracellular-flux-modeling-and-applications-in-biotherapeutic-development-and-production-using-cho-cell-cultures
#8
REVIEW
Zhuangrong Huang, Dong-Yup Lee, Seongkyu Yoon
Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse...
July 15, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28710759/rapid-detection-of-%C3%AE-h2ax-by-flow-cytometry-in-cultured-mammalian-cells
#9
Denis Firsanov, Liudmila Solovjeva, Olga Lublinskaya, Valeriy Zenin, Igor Kudryavtsev, Maria Serebryakova, Maria Svetlova
Methods commonly used for detection of DNA double-strand breaks (DSBs) and analysis of cell death are generally time-consuming, and, therefore, any improvements in these techniques are important for researchers and clinicians. At present, flow cytometry is the most rapid method for detection of DSBs and cell viability. In this chapter, we provide our experience and methodological modification of flow cytometry protocol for the detection of γ-H2AX, a well-known marker of DSBs, in fixed mammalian fibroblasts...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28710756/rapid-detection-of-apoptosis-in-cultured-mammalian-cells
#10
Igor Kudryavtsev, Maria Serebryakova, Liudmila Solovjeva, Maria Svetlova, Denis Firsanov
Flow cytometry is a powerful tool for the analysis of apoptosis, the process that directly determines cell fate after the action of different stresses. Here, we describe a flow cytometry method for the assessment of early and late stages of apoptosis in non-fixed cultured cells using SYTO16, DRAQ7, and PO-PRO1 dyes simultaneously. This multicolor flow cytometry procedure requires 45 min for completion and provides a quantitative assessment of cell viability. It can be useful in evaluating the cytotoxic properties of new drugs, and antitumor interventions...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28708530/bambi-promotes-porcine-granulosa-cell-steroidogenesis-involving-tgf-%C3%AE-signaling
#11
Long Bai, Guiyan Chu, Wusu Wang, Aoqi Xiang, Gongshe Yang
Transforming growth factor β (TGF-β), acting as the auto/para endocrine factors, has multi-function in mammalian follicle development. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is considered as a pseudoreceptor in the TGF-β signal pathway which has the similar extracellular structure of TGF-β receptor but lack of intracellular serine/threonine kinase domains. However, the biological function of BAMBI involved in porcine granulosa cell steroidogenesis remains unknown. This study was thus carried out to explore the effect of BAMBI on the steroidogenesis process in porcine primary granulosa cells...
May 28, 2017: Theriogenology
https://www.readbyqxmd.com/read/28707715/myelinating-cocultures-of-rodent-stem-cell-line-derived-neurons-and-immortalized-schwann-cells
#12
Tomohiro Ishii, Emiko Kawakami, Kentaro Endo, Hidemi Misawa, Kazuhiko Watabe
Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines...
July 14, 2017: Neuropathology: Official Journal of the Japanese Society of Neuropathology
https://www.readbyqxmd.com/read/28701781/identification-for-differential-localization-of-putative-corneal-epithelial-stem-cells-in-mouse-and-human
#13
Jin Li, Yangyan Xiao, Terry G Coursey, Xin Chen, Ruzhi Deng, Fan Lu, Stephen C Pflugfelder, De-Quan Li
Human Corneal epithelial stem cells (CESCs) have been identified to reside in limbus for more than 2 decades. However, the precise location of CESCs in other mammalian remains elusive. This study was to identify differential localization of putative CESCs in mice. Through a series of murine corneal cross-sections from different directions, we identified that anatomically and morphologically the murine limbus is composed of the thinnest epithelium and the thinnest stroma without any palisades of Vogt-like niche structure...
July 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28701514/talin-the-master-of-integrin-adhesions
#14
REVIEW
Benjamin Klapholz, Nicholas H Brown
Talin has emerged as the key cytoplasmic protein that mediates integrin adhesion to the extracellular matrix. In this Review, we draw on experiments performed in mammalian cells in culture and Drosophila to present evidence that talin is the most important component of integrin adhesion complexes. We describe how the properties of this adaptor protein enable it to orchestrate integrin adhesions. Talin forms the core of integrin adhesion complexes by linking integrins directly to actin, increasing the affinity of integrin for ligands (integrin activation) and recruiting numerous proteins...
July 12, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28698531/microfluidic-platform-for-the-long-term-on-chip-cultivation-of-mammalian-cells-for-lab-on-a-chip-applications
#15
Frank Bunge, Sander van den Driesche, Michael J Vellekoop
Lab-on-a-Chip (LoC) applications for the long-term analysis of mammalian cells are still very rare due to the lack of convenient cell cultivation devices. The difficulties are the integration of suitable supply structures, the need of expensive equipment like an incubator and sophisticated pumps as well as the choice of material. The presented device is made out of hard, but non-cytotoxic materials (silicon and glass) and contains two vertical arranged membranes out of hydrogel. The porous membranes are used to separate the culture chamber from two supply channels for gases and nutrients...
July 10, 2017: Sensors
https://www.readbyqxmd.com/read/28697965/beyond-organoids-in-vitro-vasculogenesis-and-angiogenesis-using-cells-from-mammals-and-zebrafish
#16
REVIEW
Muhammad Ibrahim, Michael K Richardson
The ability to culture complex organs is currently an important goal in biomedical research. It is possible to grow organoids (3D organ-like structures) in vitro; however, a major limitation of organoids, and other 3D culture systems, is the lack of a vascular network. Protocols developed for establishing in vitro vascular networks typically use human or rodent cells. A major technical challenge is the culture of functional (perfused) networks. In this rapidly advancing field, some microfluidic devices are now getting close to the goal of an artificially perfused vascular network...
July 8, 2017: Reproductive Toxicology
https://www.readbyqxmd.com/read/28696558/mammalian-cell-tissue-culture
#17
Katy Phelan, Kristin M May
Cultured mammalian cells are used extensively in the field of human genetics. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2017 by John Wiley & Sons, Inc.
July 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28694505/presence-of-diabetes-autoantigens-in-extracellular-vesicles-derived-from-human-islets
#18
Craig P Hasilo, Sarita Negi, Isabelle Allaeys, Nathalie Cloutier, Alissa K Rutman, Marco Gasparrini, Éric Bonneil, Pierre Thibault, Éric Boilard, Steven Paraskevas
Beta-cell (β-cell) injury is the hallmark of autoimmune diabetes. However, the mechanisms by which autoreactive responses are generated in susceptible individuals are not well understood. Extracellular vesicles (EV) are produced by mammalian cells under normal and stressed physiological states. They are an important part of cellular communication, and may serve a role in antigen processing and presentation. We hypothesized that isolated human islets in culture produce EV that contain diabetes autoantigens (DAA) from these otherwise normal, non-diabetic donors...
July 10, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28693723/determination-of-formylated-dna-and-rna-by-chemical-labeling-combined-with-mass-spectrometry-analysis
#19
Han-Peng Jiang, Ting Liu, Ning Guo, Lei Yu, Bi-Feng Yuan, Yu-Qi Feng
Nucleic acids carry diverse chemical modifications that exert critical influences in a variety of cellular processes in living organisms. In addition to methylation, the emerging DNA and RNA formylation has been reported to play functional roles in various physiological processes. However, the amounts of formylated DNA and RNA are extremely low and detection of DNA and RNA formylation is therefore a challenging task. To address this issue, we developed a strategy by chemical labeling combined with in-tube solid-phase microextraction - ultra high performance liquid chromatography - electrospray ionization - tandem mass spectrometry (in-tube SPME-UPLC-ESI-MS/MS) analysis for the sensitive determination of DNA and RNA formylation...
August 15, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28693562/activation-of-the-alpha-globin-gene-expression-correlates-with-dramatic-upregulation-of-nearby-non-globin-genes-and-changes-in-local-and-large-scale-chromatin-spatial-structure
#20
Sergey V Ulianov, Aleksandra A Galitsyna, Ilya M Flyamer, Arkadiy K Golov, Ekaterina E Khrameeva, Maxim V Imakaev, Nezar A Abdennur, Mikhail S Gelfand, Alexey A Gavrilov, Sergey V Razin
BACKGROUND: In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2...
July 11, 2017: Epigenetics & Chromatin
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