Asbestos-Mediated transfection of mammalian cell cultures

Summary The capacity of asbestos to mediate transfection was tested in a rapid and relatively simple system: picornavirus RNAs and mammalian cells in vitro. Thirteen asbestos samples, including amosite, anthophyllite, chrysotile, and crocidolite, 4 picornaviruses (poliovirus 1 and 2, echovirus 7, and encephalomyocarditis virus), and 4 cell lines (CLI, chimpanzee liver; KB, human carcinoma eta, monkey kidney; NIH 3T3, mouse embryo) were tested. The results showed that all asbestos samples mediated transfection and that all cell lines were transfectible by viral RNA with asbestos. Transfection was much greater with asbestos added to the viral RNA inoculum than to the cells before or after the RNA. Transfection was directly proportional to asbestos concentration. Initiation of transfection events was rapid, with half becoming irreversible by washing 2 min postinoculation. DNA in the inoculum strongly interfered with asbestos-mediated transfection by the RNA but was ineffective when added, with or without asbestos, to the cells before or after the inoculum. Asbestos compared with six classical “insoluble” facilitators (bentonite, calcium phosphate, chromic oxide, ferric oxide, kaolin, talc) was of intermediate rank in transfection mediation. It is hypothesized that the prominence of asbestos in carcinogenesis is due to a combination of properties, including transfection mediation as well as chromosome mutagenicity, fiber dimensions, biological durability, hydrocarbon transport, and prevalence.     

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.     

A direct nitric oxide gas delivery system for bacterial and mammalian cell cultures.

Nitric oxide (NO) is the smallest known gaseous signaling molecule released by mammalian and plant cells. To investigate the pathophysiologic role of exogenous NO gas (gNO) in bacterial and mammalian cell cultures, a validated in vitro delivery method is required. The system should be able to deliver gNO directly to bacterial and/or cell cultures in a continuous, predictable, and reproducible manner over a long period of time (days). To accomplish this, a gas delivery system was designed to provide optimal growth conditions for bacteria and/or mammalian cells. Parameters for cell exposure, such as concentration of gNO, nitrogen dioxide (NO(2)), oxygen (O(2)), temperature, and relative humidity (RH) were continuously monitored and evaluated. Uptake of gNO into various media was monitored by measuring the nitrite concentration using the Griess reagent technique. A selection of standard growth media [saline, tryptic soy broth (TSB), Middlebrook 7H9 (MB 7H9), and Dulbecco's modified Eagle's medium (DMEM)] exposed to various concentrations of gNO revealed a steady and consistent transfer of gNO into the aqueous phase over a 48-h period. Validation of optimal growth conditions within the device, as compared to a conventional incubator, were accomplished by growing and observing viability of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and human fibroblast cultures in the absence of gNO. These results indicate that an optimal growth environment for the above tested cells was accomplished inside the proposed delivery system. Dose-dependent toxicological data revealed a significant bacteriostatic effect on P. aeruginosa and S. aureus with continuous exposure to 80 ppm gNO. No toxic effects were observed on dermal fibroblast proliferation at concentrations up to 400 ppm gNO for 48 h. In conclusion, the designed gNO exposure system is capable of supporting cellular viability for a representative range of prokaryote and eukaryotic cells. The exposure system is also capable of obtaining toxicological data. Therefore, the proposed device can be utilized to continuously expose cells to various levels of gNO for up to 72 h to study the in vitro effects of gNO therapy.     

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.     

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.     

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.     

A simple device for planting replicate mammalian cell cultures

Summary The design and use of a unit for planting uniform inocula for replicate cultures are described. Its design permits continuous gassing of suspensions of mammalian cells with humidified CO₂, thus stabilizing the pH (±<0.05 pH unit) of culture media buffered with sodium bicarbonate. The unit can be readily modified to deliver different volumes; identical samples can be dispensed simply and rapidly, with minimal cell damage and chance of microbial contamination. Quantitative data regarding sample uniformity and growth subsequent to planting with this unit are presented.     

A simple device for planting replicate mammalian cell cultures

Summary The design and use of a unit for planting uniform inocula for replicate cultures are described. Its design permits continuous gassing of suspensions of mammalian cells with humidified CO₂, thus stabilizing the pH (±<0.05 pH unit) of culture media buffered with sodium bicarbonate. The unit can be readily modified to deliver different volumes; identical samples can be dispensed simply and rapidly, with minimal cell damage and chance of microbial contamination. Quantitative data regarding sample uniformity and growth subsequent to planting with this unit are presented.     

A high-throughput media design approach for high performance mammalian fed-batch cultures

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.     

Metabolic flux rewiring in mammalian cell cultures

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Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.     

Mechanisms involved in activity-dependent synapse formation in mammalian central nervous system cell cultures

Differences in neuronal activity produced by electrical stimulation lead to competition between synapses from sensory afferents converging on a common spinal cord neuron. Studies were performed on neurons dissociated from the mouse spinal cord and grown in culture dishes with three compartments. Synaptic efficacy from stimulated afferents was increased compared with unstimulated convergents, and the number of functional connections was increased by stimulation compared with control cultures. Blocking NMDA channel activation with 100 microM APV in medium containing 1.8 mM calcium inhibited this synaptic plasticity, but plasticity was not blocked by APV in medium in which the calcium concentration was elevated to 3 mM. These experiments support the view that electrical activity differentially influences processes that cause a persistent decrease in synaptic efficacy or lead to synapse elimination and those that increase synaptic strength or lead to synapse augmentation. We interpret our results in terms of a model in which these antagonistic mechanisms are both regulated via changes in calcium levels and second messengers that are modulated by electrical activity. A significant portion of the activity-related calcium influx relevant to synaptic plasticity passes through the NMDA channel, but other sources of calcium are involved. In particular, competitive elimination of synapses appears to occur during blockade of NMDA channels if the extracellular concentration of calcium is elevated moderately. The outcome of competition between the two calcium-dependent but antagonistic processes may depend either on their differential sensitivity to intracellular calcium concentration or separate specificities to NMDA and non-NMDA receptor-linked mechanisms.     

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.     

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.