Glutamine decomposition in DMEM: Effect of pH and serum concentration

Summary The pH and serum dependence of the glutamine decomposition rate constant, K_gln, in Dulbecco's Modified Eagle's Medium, DMEM, was determined. The findings indicate that K_gln increases with increase in pH and fetal calf serum (FCS) concentration in DMEM. At a constant pH of 7.25, K_gln increases from 5.7×10^–4 to 13.2×10^–4 h^–1 as FCS content of the medium increases from 0.0 to 10.0 (% v/v). Moreover, at a constant FCS of 10 (% v/v), K_gln increases from 11.5×10^–4 to 33.6×10^–4 h^–1 as pH of the medium increases from 7.2 to 7.6.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Colorimetric pH measurement of animal cell culture media

Most animal cell culture media can be buffered using bicarbonate and high pressure CO₂ in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO₂ pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.     

Secretion of beta-hexosaminidase by cultured human skin fibroblasts. Kinetics, effect of temperature, cell density, serum concentration and pH.

The temporal relation between the fusion of mononucleated myoblasts into multinucleated fibres and the quantitative changes in the activity of creatine kinase isoenzymes was determined in rat skeletal muscle cell cultures. The effect of actinomycin D on the isoenzyme transition was investigated. The activity of creatine kinase in cultures prior to the onset of cell fusion is predominantly of the BB type. During the phase of cell fusion, there is a manyfold increase in creatine kinase activity. This is due to the appearance or great increase in the activity of the MM isoenzyme. During this period, increase in the BB isoenzyme activity is very small. Inhibition of RNA synthesis by actinomycin D shortly before the onset of cell fusion did not prevent cell fusion and isoenzyme transition during the first 6 h following application of the drug.     

Measurement of ammonia and glutamine in cell culture media by gas sensing electrodes

Summary This paper describes the use of a commercially available off-line gas sensing electrode for determination of ammonia and glutamine in cell culture media. The measurement technique was tested in different media preparations with different serum concentrations. The glutamine decomposition was studied as a function of pH for cell culture medium and the results were compared to those obtained by conventional methods,i.e., HPLC. Finally, glutamine and ammonia metabolism were studied during the cultivation of a hybridoma cell line.     

Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media

We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.     

The effect of salt concentration on auxin stability in culture media

The concentrations of indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were followed for 35 days in cell-free liquid medium containing 100, 50, or 0% Murashige-Skoog (MS) salt base. Although the concentrations of NAA or 2,4-D remained constant the level of IAA decreased to only 11% of the original concentration after 35 days in the presence of 100% MS salt base. The observed rate of IAA degradation was accelerated by the presence of MS salts.     

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

Simultaneous measurement of glucose and glutamine in insect cell culture media by near infrared spectroscopy

The purpose of this study was to develop non-invasive techniques to monitor the composition of cell culture media in insect cell bioreactors. Such a monitor could be used in conjunction with a fed-batch feeding scheme to ensure that cells are maintained in an optimal environment for growth and protein production. Glucose and glutamine concentrations in an insect cell culture bioreactor were determined off-line with near-infrared (NIR) absorption spectroscopy. Spectra were collected from 5000 to 4000 cm(-1) with a 1.5-mm optical path length. Partial least squares (PLS) regression was applied to correlate the collected spectra with the concentration of the desired analytes. Under the culture conditions evaluated here, glucose and glutamine concentrations ranged from 38 to 55 mM and from 3 to 13 mM, respectively. Accurate measurements of glucose and glutamine in insect cell culture samples were possible over these entire ranges. The standard error of prediction (SEP) and mean percent error (MPE) for glutamine were 0.52 mM and 5.3%, respectively. Glucose could be measured with an SEP of 1.30 mM and an MPE of 2.3%. These levels of error are quite low considering the changing complexity of the growth media due to the shifting levels of amino acids, carbohydrates, yeastolate, proteins, and cell debris. This study represents an important step in the development of noninvasive on-line monitoring devices for cell culture bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 11-15, 1997.     

Limb bud chondrogenesis in cell culture, with particular reference to serum concentration in the culture medium

When limb bud mesodermal cells of stages 23–24 chick embryos were plated at low cell density (2 × 10⁵ cells/cm²) and cultured in medium containing 10% fetal calf serum (FCS) (serum-rich medium), all cells became fibroblastic and no chondrocyte differentiation occurred in the culture. However, when cells of the same origin were cultured in a medium containing only 0.1% FCS (serum-poor medium), almost all the cells formed aggregates which developed further to form cartilage nodules. The loss of chondrogenic activity in serum-rich medium culture was irreversible: cultivation of the limb bud cells in serum-rich medium for 12 h abolished chondrogenic activity completely and these cells could not resume activity on re-cultivation in serum-poor medium. Calf, horse and chick serum at a concentration of 10% also induced the loss of chondrogenic activity in low cell density culture. Failure of chondrogenesis in serum-rich medium culture seemed to be due to the commitment of bipotential limb bud mesodermal cells to fibroblastic cells rather than to selective detachment of pre-committed chondroblasts.     

alpha-Oligodeoxynucleotide stability in serum, subcellular extracts and culture media

Degradation of a synthetic alpha-oligodeoxynucleotide was studied in order to compare its survival with naturally occurring beta-oligodeoxynucleotides in five systems used for antisense hybridization arrest experiments. In contrast to beta-oligodeoxynucleotides, alpha-oligodeoxynucleotides were not detectably degraded over 24 h at 37 degrees C in HeLa cell postmitochondrial cytoplasmic extract or RPMI 1640 with 10% fetal bovine serum, and showed significant survival after 24 h at 37 degrees C in rabbit reticulocyte lysate, fetal bovine serum and human serum.     

Joint effects of sodium chloride,glutamine, and glucose in mouse preimplantation embryo culture media

A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCl, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage. Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium. Even in the presence of 5 mM glucose, 70% of the embryos develop to at least four cells, and 60% reach the blastocyst stage. Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development. Moreover, the response to glutamine depends on the concentration of NaCl in the medium. When the NaCl concentration is low, glutamine inhibits development. In contrast, when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt. We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte.     

Rapid sodium cyanide depletion in cell culture media: outgassing of hydrogen cyanide at physiological pH

During the course of in vitro studies on cyanide exposure with SH-SY5Y human neuroblastoma cells, we found that sodium cyanide (NaCN) up to a concentration of 10 mM had no significant toxic effect under our culture conditions. Further investigation of this apparent cyanide resistance revealed that the sodium cyanide was being rapidly depleted from the cell culture medium. Cyanide was interacting with constituents of the cell culture medium and was somehow being detoxified or removed from solution. The reaction of cyanide with cell culture media in 96-well culture plates reduced cyanide concentrations rapidly (80-90% in 2 h at 37 degrees C). Running the same reaction in capped tubes significantly reduced cyanide loss from solution. Incubation of cyanide with individual constituents of the cell culture medium in solution showed that glucose, phenol red, and amino acids all acted to detoxify or remove cyanide from solution. When amino acids or buffers were incubated with sodium cyanide in aqueous solution at pH 7.4, hydrogen cyanide (HCN) was found to degas from the solutions. We compared HCN outgassing over a range of pH values. As expected, HCN remained very soluble at high pH, but as the pH was reduced to 7.0, the rate of HCN formation and outgassing increased dramatically. Acid-base reactions involving cyanide and proton donors, such as amino acids and other cell culture media constituents, at physiological pH result in rapid HCN outgassing from solution at 37 degrees C. These results indicate that previous in vitro cyanide toxicity studies done in standard culture media with prolonged incubation times using gas-exchanging culture containers might have to be reevaluated in light of the fact that the effective cyanide concentrations in the culture media were significantly lower than reported.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

The influence of serum proteins and pH on the effective amino acid concentration in tissue culture medium

Amino acid binding by calf serum proteins in Dulbecco's modified Eagle medium has been studied by an ultrafiltration method. For some of the amino acids studied, up to 20 % may be unavailable to the cell because of non-specific, reversible binding to serum proteins. The extent of binding varies over the pH range ordinarily encountered during cell growth. A method is presented by which the degree of binding may be determined. The greatest degree of binding is noted for tryptophan, arginine and lysine.     

Alveolar type II-like cell colonies: effect of alveolar macrophages and macrophage-conditioned media

Alveolar type II-like colonies were obtained after a low density plating (5 X 10(3)/60 mm tissue culture dish) of primary type II cells. These colonies were formed only when type II cells were either cocultured with alveolar macrophages or with conditioned media generated by alveolar macrophages. Cells in the colonies appeared homogeneous and kept their lamellar bodies over a period of 8 weeks and more, as observed by electron microscopy. These cells reacted immunocytochemically with antibodies directed against the 32-38 kDa protein fractions of rat surfactant.     

Flax anther culture: effect of genotype,cold treatment and media

We report on screening of wide range of flax cultivars for androgenic response and on testing of induction conditions for flax (Linum usitatissimum L.) anther culture and plant regeneration. Anthers were cultured on four different media: Mo, N6, MS and N&N supplemented with various combinations of growth regulators. The induction of callus formation from cultured anthers was the highest on N6 (with cultivar PR FGL 77 – 12 %) and N&N media (with cultivar Carolin – 2.8 %), preferentially after cold pretreatment (7days at 8 °C). Shoots were formed on calli derived from the microspores inside the cultured anthers on media N&N and N6 supplemented with 1mgl^–1 zeatin or 1mgl^–1BAP + 1mgl^–1NAA, respectively and elongated on MS medium supplemented with 2mgl^–1 zeatin. The highest number of shoots (120) was observed with cultivar Red Wing. Shoots were rooted on MS medium supplemented with 2mgl^–1IAA. Our experiments resulted in total in 62 % anther response and 155 plants regenerated and transferred into soil.     

Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha‐ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2‐methyl‐1,3‐thiazolidine‐2,4‐dicarboxylic acid and 2‐(2‐carboxyethyl)?1,3‐thiazolidine‐2,4‐dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha‐ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l ‐methionine (met) and l ‐tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed‐batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti‐oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant‐free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759–770, 2017