Euploid segregation through multipolar mitosis in mammalian cell cultures

Cultures were made of kidney cells of male Rhesus monkey and a karyological analysis of these cells was carried out at various times after plating using the chromosome banding method of Seabringht (1972), in order to study the segregation phenomena which take place in cell cultures in vitro through multipolar mitoses and to identify segregating cells. — Several segregating cells were found: haploid cells, diploid cells with two X chromosomes and triploid cells which were strictly euploid. Polyploidization-segregation cycles in the cell cultures and their mechanism and chronological sequence were analyzed. — On the basis of these results it was suggested that the genome is organized in haploid sets capable of segregating as units and of producing strictly euploid segregating daughter cells.     

Regulating apoptosis in mammalian cell cultures

Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay apoptosis and increase the longevity of cell cultures can lead to more economical processes. Researchers have shown that both genetic and chemical strategies to block apoptotic signals can increase cell culture productivity. Here, we discuss various strategies which have been implemented to improve cellular viabilities and productivities in batch cultures.     

Defining viability in mammalian cell cultures

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

Further studies on a mutant mammalian cell line defective in mitosis

Experiments have been performed on a temperature-sensitive hamster cell line, ts-546. After the cells are switched to the non-permissive temperature, interphase cells continue through the cell cycle until the cells enter metaphase. Normal mitotic events then fail to occur. Metaphase chromosomes in the cells condense and coalesce into chromatin aggregates. Nuclear membrane re-forms around the aggregates resulting in the formation of mono-, bi- or multi-nucleate interphase-like cells. The conversion of mitotic cells to interphase-like states is completed within a few hours. The initial characterization of the mutant cell line was based on the observation that rounded-up cells accumulate in culture at the non-permissive temperature. The mitotic roundingup process may be utilized as a useful marker for selective isolation of mutant cell lines defective in mitosis.     

No elevated cell surface charge at mitosis in X-ray-irradiated mammalian cells

Rounded mitotic cells showed 30% enhanced electrophoretic mobility (EPM) when compared to spindle-formed interphase cells. This increase in EPM that was not present in interphase cells that had been rounded chemically by EDTA is considered to reflect a structural change in the cell membrane during mitosis. X-ray irradiation induced a dose-dependent EPM decrease in both interphase and mitotic cells during a 4-hour period. During the next 20 h of incubation, EPM recovery took place in cells irradiated with 250R, but not in cells exposed to 1000R. EPM was enhanced during mitosis in cells irradiated with low doses, but was absent in cells irradiated with 1000R. The ratio of colony-forming cells and of electrophoretically recovered mitotic cells after 24 h of exposure showed a good statistical correlation. These results indicate that unrepaired membrane damage contributes to mitotic cell death after irradiation.     

Temperature compensation in the mammalian cell cycle

Random and synchronous V79 cells were shifted from 37.5 °C to temperatures between 29 ° and 41 °C. Intermitotic time determinations of random cultures showed an increase in generation time and a broadening in the distribution of generation times in cells whose cycle spanned the temperature shift, but only a slight increase in generation time after one generation at temperatures between 34 °–40 °C. At 33.5 °C and below there was a stepwise increase in generation time. When cells grown at non-standard temperatures were allowed to habituate for 48 h at the altered temperature prior to analysis, the increase in median intermitotic time was slightly less in comparison to analyses done after only one generation following the temperature step. The Q₁₀ for cell division of cells growing at temperatures from 34 ° to 40 °C was between 1.15 and 1.26, suggesting that the mammalian cell cycle is temperature compensated over a limited (6–7 °C) temperature span. Mammalian cells in culture appear to have the same capacity for temperature compensation in their cell cycle as do unicellular eukaryotes. The fact that cycle time at lower temperatures increases in a discrete manner is taken as evidence for a quantal clock.     

Metabolic screening of mammalian cell cultures using well-plates

For use in a broad spectrum of cell culture applications, we have devised a novel method, termed High-Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells. This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering. First, HTMS makes use of a simplified metabolic network for metabolic flux analysis. Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network. Second, HTMS makes use of microtiter well-plate technology and adaptations of well-known enzymatic assays to increase precision and throughput for cell culture experiments. Multireplicate, multiparallel cultures in the sub-milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T-flasks, spinner flasks, or bioreactor systems. The simplicity of the network and the well-plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses. The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.     

NMR-based metabolomics of mammalian cell and tissue cultures

NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94?h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.     

On-line monitoring of cell mass in mammalian cell cultures by acoustic densitometry

Acoustic resonance densitometry (ARD) provides a highly reproducible and stable method for on-line measurement of culture biomass density. The technique provides a direct determination of changes in relative density of culture medium and cell mass. At cell concentrations higher than 10(6) cells mL(-1)this method can replace cell counts and provide a continuous measure of total cell mass. In cultures of hybridomas or U937 human lymphoma cells, the ARD value correlates well with cell number except when the average cell size changes during culture. It is argued that cell mass determined by ARD rather than cell number should be used as the basis for measurements of specific biological activity.     

Ammonia removal using hepatoma cells in mammalian cell cultures

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.     

Quantitative analyses of circadian gene expression in mammalian cell cultures

The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform–nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by the cAMP and glucocorticoid pathways are different, implying that actions of different genes stimulated by these pathways lead to distinctive programs of circadian synchronization.