Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10⁸ cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.     

The effects of temperature acclimation on the oxygen consumption and enzyme activity of red and white muscle fibres isolated from the tropical freshwater fish Oreochromis niloticus

The standard oxygen consumption rate and the activities of muscle citrate synthase, creatine phosphokinase and lactate dehydrogenase in the tropical fish Oreochromis niloticus acclimated to either 20.5 ± 0.3° C or 26.5 ± 0 ± 5 ± C for at least 3 months were investigated. The standard oxygen consumption rate of individual fish from the two acclimation temperatures was determined at 20, 25 and 30 ± C. At all experimental temperatures, the standard oxygen consumption rate of fish acclimated to 20.5 ± 0.3° C was significantly higher than that of fish kept at 26.5 ± 0.5 ± C. In both groups smaller individuals had a higher oxygen consumption rate than large ones.
Analyses of the activity levels of citrate synthase (CS), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) in both red and white muscles isolated from fish kept under the two temperature regimes were performed at 26 ± C. The activity of CS in both red and white muscles isolated from the 20.5 ± 0.3° C acclimated fish was significantly higher than that of muscles isolated from the 26.5 ± 0.5 ± C acclimation group. Similarly, the CPK activity in white muscles isolated from fish acclimated to 20.5 ± 0.3 ± C was higher than that of muscles obtained from the 26.5 ± 0.5 ± C acclimation group. However, the CPK activity in red muscles isolated from the two fish groups was not significantly different. The opposite results were obtained for LDH activity. For example, the LDH activity of white muscles isolated from fish acclimated to 26.5 ± 0.5 ± C was significantly higher than that of the same muscles but from the 20.5 ± 0.3 ± C acclimated fish. No differences were observed in the LDH activity of red muscles isolated from the two fish groups.     

温度、盐度对强壮箭虫耗氧率和窒息点的影响

研究了不同温度、盐度下强壮箭虫的耗氧率和窒息点.结果表明:温度和盐度均对强壮箭虫的耗氧率和比耗氧率有显著影响.试验温度在5℃-25℃,强壮箭虫个体耗氧率(IO)和比耗氧率(SO)开始随温度的升高而升高,之后呈明显下降趋势.其回归方程分别为y=0.0058x3 -0.2956x2+4.415x-8.7816(R2=0.99,P＜0.05)和y=0.0011x3 -0.0546x2+0.8161x-1.6232(R2=0.99,P＜0.05),数值分别为6.30～11.71μg·ind-1·h-1和1.22 ～2.16μg· mg-1·h-1,窒息点为4.18～6.87 mg·L-1.盐度10 ～40条件下,IO和SO随盐度的升高逐渐下降,回归方程分别为y=-0.0068x2 -0.1412x+21.702(R2=0.89,P＜0.05)和y=-0.0013x2-0.0261x+4.0114( R2 =0.89,P＜0.05),数值分别为4.98～17.73μg ·ind-1·h-1和0.92～3.56 μg·mg-1 ·h-1,窒息点为4.02～6.24 mg·L-1.对强壮箭虫与其他水生动物的耗氧率和窒息点进行比较得出,强壮箭虫是一种狭氧性浮游动物.     

温度、盐度和体长对西藏拟溞耗氧率的影响

在实验室内研究了盐度(S=5、10、15、20和25)、温度(T=3、8、14、20和22℃)和体长(L=0.83、1.25、1.49、1.87和2.42mm)对西藏拟耗氧率的影响。结果表明,在试验盐度内该的个体耗氧率(IO)和比耗氧率(SO)均随盐度(S)升高而升高,其回归方程分别为IO=0.0014S 0.0126和SO=0.115S0.5612,数值范围为0.02～0.045μg/(indh)和0.31～0.67μg/(mg·h)。个体耗氧率和比耗氧率在温度试验中有同样的趋势,回归方程分别为IO=0.0049e0.1574T和SO=0.0678e0.1605T,数值分别为0.0076～0.13μg/(ind·h)和0.10～1.71μg/(mg·h)。体长试验中个体耗氧率随体长增大而增大,而比耗氧率则降低,回归方程分别为:IO=0.0545L2.5962和SO=-0.2059L 0.7908,数值范围为0.036～0.68μg/(ind·h)和0.38～0.63μg/(mg·h)。讨论了盐度、温度和体长对西藏拟耗氧率的影响。     

Determination of oxygen profiles in agar-based gelled in vitro plant tissue culture media

Keeping account of the limited knowledge concerning the relevance of the oxygen status of the medium during in vitro culture, a technique was elaborated to systematically study the oxygen concentration in gelled media. In a first series of experiments, the Oxygen Diffusion Rate (ODR) technique was used to investigate the dissolved oxygen concentration as a function of time at different depths. The results obtained demonstrated that the oxygen concentration in agar media was reduced by 80% during the heating steps included in the preparation procedure. It took about one week to reach an oxygen concentration equal to 90% of the equilibrium value at a depth of 1 cm, irrespective of the brand of agar used. As the recovery of the oxygen concentration at various depths could be nicely modelled by Fick's law, it follows that this process is diffusion limited. In this respect, fluorescence recovery after photobleaching (FRAP) measurements revealed that the diffusion coefficient of oxygen in gelled media was only affected to a very small extent by the presence of up to 2% (w/v) agar. In a final experiment, explants of Ficus benjamina were cultured on a rooting medium. As the oxygen concentration in the gelled medium was not significantly affected by the presence of the biological material, it was concluded that the oxygen uptake by explants from gelled media is negligibly small and hence cannot be considered as being a growth-limiting factor during in vitro micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Routine metabolism of juvenile spot, Leiostomus xanthums (Lacépède), as a function of temperature, salinity and weight

Routine oxygen consumption rates of juvenile spot, Leiostomus xanthums , were measured over a range of temperatures, salinities and fish weights. As predicted, Q O₂ increased with temperature and decreased with body weight. However, Q O₂ decreased with decreasing salinity and did not show the expected minimum at isosmotic concentrations. The data are best described by the relationship: log₁₀ Q O₂ (mg O₂ g⁻¹ h⁻¹) = 0.129 log_lo salinity (%0) + 1.604 log₁₀ temperature (°C)-0.1401og₁₀(g)-2.767.     

The limitations of heart rate as a predictor of metabolic rate in fish

Although telemetered heart rate (f_H) has been used as a physiological correlate to predict the metabolic rate (as oxygen consumption, V?O₂) of fish in the field, it is our contention that the method has not been validated adequately for fish. If f_H in fish is to be used to estimate V?O₂, a single linear (or log-linear) relationship must be established for each species between the two variables which allows V?O₂ to be predicted accurately under all environmentally relevant conditions. Our analyses of existing data indicate that while a good linear (or log-linear) relationship can be established between f_H and V?O₂, the conditions under which the relationship applies may be quite restricted. Physiological states and environmental factors affect the relationship between f_H and V?O₂ significantly such that several curves can exist for a single species. In addition, there are situations in which f_H and V?O₂ do not covary in a significant manner. In some situations f_H can vary over much of its physiological range while V?O₂ remains constant; in others V?O₂ may vary while f_H is invariate. The theoretical basis for this variability is examined to explain why the use of telemetered f_H in predicting V?O₂ of fish may be limited to certain specified applications.     

The effects of two levels of caffeine ingestion on excess postexercise oxygen consumption in untrained women

The effects of two levels of caffeine ingestion (5 mg.kg-1, CAF1, and 10 mg.kg-1, CAF2) on postexercise oxygen consumption was investigated in six untrained women aged 20.5 (SEM 0.5) years. After a test to determine maximal oxygen consumption (VO2max) each subject underwent three test sessions at 55% VO2max either in a control condition (CON) or with the CAF1 or CAF2 dose of caffeine. During exercise, oxygen consumption was found to be significantly higher in the CAF1 and CAF2 trials, compared to CON (P < 0.05). During the hour postexercise, oxygen consumption in CAF1 and CAF2 remained significantly higher than in CON (P < 0.05). At all times throughout the exercise, free fatty acid (FFA) concentrations were significantly higher in the caffeine trials than in CON. The FFA concentrations 1 h postexercise (+60 min) were further elevated above resting values for all three trials. Caffeine ingestion caused the greatest elevation above resting levels being 1.89 (SEM 0.19) mmol.l-1 and 1.96 (SEM 0.22) mmol.l-1 for the CAF1 and CAF2 trials, respectively. This was significantly higher (P < 0.0001) than the CON level which was 0.97 (SEM 0.19) mmol.l-1. Respiratory exchange ratio (R) values became significantly lower (P < 0.05) in CAF1 and CAF2 compared to CON at the onset of exercise and continued to decrease during the activity. Throughout the recovery period, R values were significantly lower for both caffeine trials compared to CON. The results of this study would suggest that caffeine is useful in significantly increasing metabolic rate above normal levels in untrained women during, as well as after, exercising at 55% VO2max.     

Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.     

低氧环境对细胞培养基内氧浓度的影响

目的:测定不同氧环境下细胞培养基内的实际氧浓度,并探讨在细胞培养过程中,影响培养基内氧含量的因素。方法:利用光纤氧传感探头,对不同氧环境下、不同细胞培养容器中,以及常氧或低氧环境下换液前后RPMI1640培养基内的氧含量进行测定。结果:培养基中的氧含量可以随着外界氧环境的变化而改变;低氧环境下24孔板和35 mm皿中的氧含量要比25 cm2培养瓶稳定;常氧环境下换液使得培养基内的氧含量明显升高,而在低氧环境下换液则对培养基内的氧含量无明显影响。结论:在不同氧浓度下的细胞培养模型研究中,严格控制外界环境中的氧浓度,选用合适的细胞培养容器,并且在换液过程中尽量避免或减少培养基与常氧环境的接触,是维持培养基内氧含量稳定的重要因素。     

Estimation of rates of oxygen uptake and carbon dioxide evolution of animal cell culture using material and energy balances

Material and degree of reductance balance equations are used to estimate the rates of oxygen uptake and carbon dioxide evolution of animal cell cultures. Lumped compositions, molecular weight and reductance degree of cellular protein, monoclonal antibody, biomass and amino acid consumption (excluding glutamine and alanine) are found to be relatively constant for different hybridoma cell lines and may be used as regularities. The calculated rates of oxygen uptake and carbon dioxide evolution agree well with experimental values of several different cultures reported in the literature. This simple method gives the same results as calculated on the basis of a detailed metabolic reaction network. This revised version was published online in July 2006 with corrections to the Cover Date.