On line estimation of oxygen uptake rate for insect cells growing in a modified spinner flask

The simple design of traditional spinner flasks makes the on-line estimation of cellular metabolism impossible. An on-line estimation system has been developed and used for the monitoring of oxygen uptake rate (OUR) for insect cells growing in a modified spinner flask. Neglect of oxygen desorption from culture media is a common source of error in OUR measurements for Sf21 cells. Therefore, an algorithm was developed to compensate for the affect of such desorption process on the determination of OUR. A modified spinner flask was successfully used as a low-volume bioreactor for insect cell cultivation and the OUR measurement developed here is both convenient and reliable.     

A gas-chromatographic method for the determination of oxygen uptake in aerated cultures

The oxygen uptake in aerated cultures (fermentor, shaken culture) is determined gas-chromatographically using Chromosorb 102(80/100 mesh) at -78 C. Nitrogen is used as internal standard making sampling simple and measurements of oxygen accurate.     

A new method for on-line measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures

Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liquid phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and constant k(L)a value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured on-line with a very good accuracy of +/-5%, and the specific OUR for a CHO cell line was determined during batch (growth phase) and continuous culture as, respectively, equal to 2. 85x10(-13) and 2.54x10(-13) mol O(2) cell(-1) h(-1). It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the cell concentration itself, especially during transition phases like the end of the growth phase in a batch culture.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

A non-radioactive method for measuring Cu uptake in HepG2 cells

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.     

Validation of manometric microrespirometers for measuring oxygen consumption in small arthropods

Scientists have used numerous techniques to measure organismal metabolic rate, including assays of oxygen (O(2)) consumption and carbon dioxide (CO(2)) production. Relatively few studies have directly compared estimates of metabolic rate on the same groups of animals as determined by different assay methods. This study directly compared measures of the metabolic rate of three lines of Drosophila simulans as determined either from direct measures of CO(2) production using infrared gas analysis (IRGA), or from estimates of O(2) consumption based on manometeric techniques. Determinations of metabolic rate of the same cohorts of flies using these two methods produced results that often differed widely. Typically metabolic rate as determined by the manometric method was significantly greater than that determined by CO(2) output. These differences are difficult to explain by simple biotic or abiotic factor(s). Because of the idiosyncratic nature of these differences it is not possible to use a simple factor to convert from metabolic rate measurements done using manometric techniques to those expected from direct measures of CO(2) output or O(2) consumption. Although manometric devices are simple to construct and use, measurements of metabolic rate made with this method can vary significantly from measurements made by directly assaying CO(2) production or O(2) consumption.     

A simple method for measuring the kinetics of the formation of oxygen radicals

Sodium 2, 6-dichloroindophenol (DCIP Na ) was used to measure the kinetics of the formation of oxygen radicals. The oxygen radicals react with DCIP - , resulting in a decrease in DCIP concentration which is monitored by the decrease in A . A method based on this principle was demonstrated with xanthine/xanthine oxidase giving a K = 4.9x10 M. Cu significantly inhibited the enzyme reaction.     

Suitability of the shaking flask for oxygen supply to microbiological cultures

Due of its simplicity the shaking flask is used in serial studies, e.g. in the screening for secondary metabolites or in the optimization of fermentation processes. Experimental investigations in these small bioreactors are often the first step in developing a large-scale fermentation process.Movement of the flask should produce sufficient mixing, supply of oxygen, and removal of carbon dioxide. In the case of fluids with low or moderate viscosity, gas transport is the most important aspect. This publication summarizes data necessary to calculate the gas transport. These data are derived from the consideration of the gas diffusions through the cotton plug as well as from the substance transport between the gas and liquid phases. As a result suitable fermentation conditions can be selected. Finally, the performance limits of the shaking flask are illustrated using the example of the oxygen supply in a Streptomyces tendae fermentation.List of Symbols A _s Cross section of plug - A Surface area of liquid in flask - a A/V _F specific phase interface area - c Concentration - c ^* Saturation concentration - c Plug diffusion term - D Widest diameter of flask - Diffusion coefficients in multicomponent gas mix tures - Diffusion coefficients in binary gas mixtures - Diffusion coefficient of oxygen in the liquid - d Diameter of neck of flask - e Eccentricity - G Volume-based mass flow - G _m Maximum volume-based mass flow - g Acceleration due to gravity - h Height coordinate - ¯H Mean height of plug - Hy p _i/c ^*, Henry constant - K Consistency index - k D _xy/D _xz, Ratio of diffusion coefficients in binary gas mixtures - k _M Monod constant - k _L a Mass transport coefficient: gas/liquid - M Molecular weight - m Flow exponent - n Speed of shaking - p Pressure - p _i Partial pressure of gas component i - q Area-based flow of volume - R , respiration ratio - Sc , Schmidt number - T Absolute temperature - V Flask volume - V _F Volume of liquid in flask - w Velocity of the Stefan flow - x, y, z Ratios of the partial pressures of the gases O₂, CO₂, N₂ - Rate of shear - Dynamic viscosity of the liquid - Kinematic viscosity of the liquid - Density of the liquid - _x, Density of O₂ gas - Surface tension Indices 0 State in gas volume of shaking flask - 1 State in outside air - G Gas volume - x, y, z O₂, CO₂, N₂     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

介绍一种简便的红细胞无氧糖酵解率测定方法

目的为了简化现有的华伯氏呼吸仪检测红细胞无氧糖酵解的方法,进行此项研究.方法实验用葡萄糖试剂盒,高氯酸,可见光分光光度计,氮气瓶和摇床.实验分4步进行任氏液.Trls-HCI溶液等试液的配制,任氏血的制备,红细胞无氧糖酵解率的测定和红细胞无氧糖酵解率的计算.结果用SOD保养液.在4℃条件下保存入红细胞75d时糖酵解率为86.2%±5.0%,明显优于GMA保养液39.2%±8.9%.结论不用华伯氏仪的红细胞无氧糖酵解率的测定方法,操作简便,方法准确可靠,在普通实验室即可进行.     

Polarographic measurement of oxygen uptake by astrocytes in primary cultures using the tissue-culture flask as the respirometer chamber

Summary Oxygen uptake was measured in primary cultures of astrocytes from the brain hemispheres of newborn DBA mice by the aid of an oxygen electrode inserted directly into the culture flastk, i.e. using the flasks, completely filled with MEM medium, as the respirometer chamber. The respiration was inibitally intense (300 μmol per hr per 100 mg protein) but delined somewhat during the 6 hr of measurement, probably due to a depletion of intermediary metabolites released to the large surplus of medium. The respiratory rates were approximately identical in the presence of a CO₂/ bicarbonate and a HEPES buffer. Exposure to a high concentration of potassium led to a transient stimulation of the oxygen uptake of almost 100%, a response that was very easily observed using the present method. Since no mechanical damage was inflicted upon the cells, culturing could be continued, if so desired, after the measurement.     

A sensitive method for measuring oxygen consumption

A technique is presented whereby oxygen consumption rates of the order of 10⁻⁶ μl/hr can be measured, thus providing a means for studying the respiration rates of single cells, even slowly respiring ones. The technique is based on the principle of incubating cells in extremely small chambers containing highly concentrated hemoglobin solutions. The absorbance shift occurring when hemoglobin is transformed from its oxygenated to its deoxygenated form is recorded microspectrophotometrically. The results obtained by this technique seem to be well in accordance with those of the Warburg manometric method. The technique is convenient and easy to handle and the sensitivity can be varied within wide limits, permitting different types of materials to be studied. In experiments using yeast cells, respiration rates from 1.0 × 10⁻⁶ to 1.8 × 10⁻⁶ μl O₂/cell/hr were revealed.     

Polarographic measurement of oxygen uptake by astrocytes in primary cultures using the tissue-culture flask as the respirometer chamber.

Oxygen uptake was measured in primary cultures of astrocytes from the brain hemispheres of newborn DBA mice by the aid of an oxygen electrode inserted directly into the culture flask, i.e. using the flask, completely filled with MEM medium, as the respirometer chamber. The respiration was initially intense (300 mumol per hr per 100 mg protein) but declined somewhat during the 6 hr of measurement, probably due to a depletion of intermediary metabolites released to the large surplus of medium. The respiratory rates were approximately identical in the presence of a CO2/bicarbonate and a HEPES buffer. Exposure to a high concentration of potassium led to a transient stimulation of the oxygen uptake to almost 100%, a response that was very easily observed using the present method. Since no mechanical damage was inflicted upon the cells, culturing could be continued, if so desired, after the measurement.     

A simple method for freeze-fracture of monolayer cultures

A simple method is described for the freeze-fracture in situ of monolayer cultures grown on gold carriers coated with a thin layer of silicon monoxide. Preliminary observations on 3T3 mouse embryo fibroblasts indicate that this technique exposes large areas of cell membrane, making it possible to determine how areas of membrane specialization are related to the cell as a whole and to regions of cellular interaction. 3T3 cells cultured on silicon monoxide show no modification of growth properties compared to cells growing on Falcon plastic, and other cell lines also appear to grow well on this substrate.     

A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells

Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.     

小篮子法测定植物种子呼吸速率的方法改进

利用CO2红外气体分析仪对测定植物种子CO2呼吸速率的小篮子法进行了改进。结果表明:改良小篮子法操作简便,可实时监测数据变化,测定的实验数据准确度较高,变异系数较小,在一定程度上克服了传统小篮子法的缺陷,使实验测定更具科学性和严谨性,可用于本科生植物生理学实验教学。