Concept: Cell lines
Mammalian cell lines are characterized by a complex and flexible metabolism. A single model that could describe the variations in metabolic behavior triggered by variations in the culture conditions would be a precious tool in bioprocess development. In this paper, we introduce an approach to generate a poly-pathway model and use it to simulate diverse metabolic states triggered in response to removal, reduction or doubling of amino acids in the culture medium of an antibody-producing CHO cell line. Macro-reactions were obtained from a metabolic network via elementary flux mode enumeration and the fluxes were modeled by kinetic equations with saturation and inhibition effects from external medium components. Importantly, one set of kinetic parameters was estimated using experimental data of the multiple metabolic states. A good fit between the model and the data was obtained for the majority of the metabolites and the experimentally observed flux variations. We find that the poly-pathway modeling approach is promising for the simulation of multiple metabolic states.
In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10-15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na(+), osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.
Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.
A chemical investigation of the EtOAc-soluble fraction from the ethanol extract of the medullae of Juncus effusus led to the isolation of three new 9,10-dihydrophenanthrenes, juncuenins E-G (1-3); two new phenanthrenes, dehydrojuncuenins D-E (4-5); one new feruloylated glycoside (6); and one known 9,10-dihydrophenanthrene (7). The structures of these compounds were determined by analyzing their spectroscopic data. Metabolites 1-4 and 7 were further evaluated for their in vitro cytotoxic activities against seven human cancer lines (A549, MCF-7, BEL-7402, HeLa, COLO205, BGC-823, and SK-OV-3). Among them, compound 1 exhibited weak cytotoxicity against MCF-7 and HeLa cell lines. Compound 7 showed moderate cytotoxicity against MCF-7 and HeLa cell lines, with IC50 values of 9.17 and 19.6 µM, respectively.
Liquid marbles (LM) are non-sticky droplets covered by micro- or nanometrically scaled particles and obtained by simply rolling small amounts of a liquid in a very hydrophobic powder. Since pioneer work by Aussillous and Quéré, a wide palette of hydrophobic materials for the preparation of LM, as well as potential applications, has been reported. Due to the bioinspired origin of this concept, the applicability of LM in biomedicine are gaining increasing attention, with remarkable advances in their use as micro-bioreactors for blood typing, drug screening and tumor growth, among others. Herein, we explore the novel use of LM as a biotechnological tool for the cryopreservation of mammalian cells as an alternative to conventional methods, which typically require the use of cryopreservant agents that commonly associate with some degree of cell toxicity. Murine L929 fibroblasts, a reference cell line for cytotoxicity studies, and poly(tetrafluoroethylene), a hydrophobic polymer widely used in cardiovascular surgery, were selected for the preparation of the cell-containing LM. Our results reveal that there is a safe range of droplet volumes and cell densities that can be successfully used to cryopreserve mammalian cell lines and recover them after thawing without significantly affecting major cellular parameters such as adhesion, morphology, viability, proliferation, and cell cycle. We envision that progress in the exploration of cell-containing LM could also open their impact as micro-reactors for the miniaturization of cytotoxicity procedures of drugs and materials in which powerful tools for cell evaluation such as flow cytometry could be used due to the elevated amount of cells handled.
Nanoparticles are widely employed for many applications and the number of consumer products, incorporating nanotechnology, is constantly increasing. A novel area of nanotechnology is the application in medical implants. The widespread use of nanoparticles leads to their higher prevalence in our environment. This, in turn, raises concerns regarding potential risks to humans. Previous studies have shown possible hazardous effects of some nanoparticles on mammalian cells grown in two-dimensional (2D) cultures. However, 2D in vitro cell cultures display several disadvantages such as changes in cell shape, cell function, cell responses and lack of cell-cell contacts. For this reason, the development of better models for mimicking in vivo conditions is essential. In the present work, we cultivated A549 cells and NIH-3T3 cells in three-dimensional (3D) spheroids and investigated the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO2-NP).The results were compared to cultivation in 2D monolayer culture. A549 cells in 3D cell culture formed loose aggregates which were more sensitive to the toxicity of ZnO-NP in comparison to cells grown in 2D monolayers. In contrast, NIH-3T3 cells showed a compact 3D spheroid structure and no differences in the sensitivity of the NIH-3T3 cells to ZnO-NP were observed between 2D and 3D cultures. TiO2-NP were non-toxic in 2D cultures but affected cell-cell interaction during 3D spheroid formation of A549 and NIH-3T3 cells. When TiO2-NP were directly added during spheroid formation in the cultures of the two cell lines tested, several smaller spheroids were formed instead of a single spheroid. This effect was not observed if the nanoparticles were added after spheroid formation. In this case, a slight decrease in cell viability was determined only for A549 3D spheroids. The obtained results demonstrate the importance of 3D cell culture studies for nanoparticle safety testing, since some effects cannot be revealed in 2D cell culture.
This study was carried out to determine the cytotoxic effect of seven plant extracts and the isolated compounds - syringin and 4-methoxycinnamyl alcohol - on cancerous and non-cancerous cells. The ethanol extract of Foeniculum vulgare was found to exhibit the most significant toxicity with an IC50 value of 19.97 μg/mL on HeLa cells. Bioassay-guided fractionation led to the isolation of two compounds, syringin (1) and 4-methoxycinnamyl alcohol (2). Both compounds showed toxicity against MCF-7, HeLa and DU145 cancer cell line. The results showed that compound 2 showed high toxicity against all the cancer cell lines with IC50 values of 14.24, 7.82 and 22.10 μg/mL, respectively. 4-Methoxycinnamyl alcohol also showed no apoptotic effect in cell cycle analysis after 48 h at a concentration of 10 μg/mL. However, DNA fragmentation study revealed that necrosis took place at a concentration of 10 μg/mL after 48 h exposure.
Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity. The cell viability was instantly assessed through simultaneous perfusion with fluorescein diacetate (FDA) and propidium iodide (PI), both being dispensed through the same superfusion device used to porate and deliver the enzyme substrate. In this fluorescence assay, viable and non-viable cells were distinguished by their green and red emission, respectively, within 10 s. In addition, the enzyme activity was monitored over time as a secondary test for cellular activity. Our findings demonstrate that this microfluidic technology-assisted approach is a facile, rapid, and reliable means to determine the viability in single-cell experiments and that viability studies can be performed routinely alongside typical substrate delivery protocols. This approach would remove the need for global cell viability testing and would enable viability studies of only the cells under experimental analysis.
- Vector borne and zoonotic diseases (Larchmont, N.Y.)
- Published over 1 year ago
Rickettsia slovaca is transmitted by Dermacentor marginatus ticks, and is the causative agent of tick-borne lymphadenopathy and Dermacentor-borne necrosis erythema lymphadenopathy throughout Europe. It has not been found in New World ticks, nor have tick-borne lymphadenopathy or Dermacentor-borne necrosis erythema lymphadenopathy been reported in humans in the Americas. Here we describe the isolation of a R. slovaca-like agent from D. variabilis nymphs from a colony of ticks derived from field collected adults.
Aneuploidy and structural variations (SVs) generate cancer genomes containing a mixture of rearranged genomic segments with extensive somatic copy number alterations. However, existing methods can identify either SVs or allele-specific copy number alterations but not both simultaneously, which provides a limited view of cancer genome structure. Here, we introduce Weaver, an algorithm for the quantification and analysis of allele-specific copy numbers of SVs. Weaver uses a Markov random field to estimate joint probabilities of allele-specific copy numbers of SVs and their inter-connectivity based on paired-end whole-genome sequencing data. Weaver also predicts the timing of SVs relative to chromosome amplifications. We demonstrate the accuracy of Weaver using simulations and findings from whole-genome optical mapping. We apply Weaver to generate allele-specific copy numbers of SVs for MCF-7 and HeLa cell lines and identify recurrent SV patterns in 44 TCGA ovarian cancer whole-genome sequencing datasets. Our approach provides a more complete assessment of the complex genomic architectures inherent to many cancer genomes.