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1.
Radiation survival curves of EMT6/Ed spheroids have been obtained under conditions which eliminate changes in oxygen concentration between growth and irradiation. These curves show a high-dose, resistant component which is nearly parallel to the curves obtained when spheroids were irradiated under nitrogen. Thus EMT6 spheroids appear to model accurately the radiation responses of EMT6 tumors. In contrast, when spheroids were grown to relatively high density (300-400 spheroids per 250-ml spinner flask), then separated into several flasks for irradiation, an increase in oxygen concentration in the medium occurred which fully oxygenated the previously hypoxic cells. The two causes for the oxygen depletion in sealed growth flasks were quantitated. Depletion of total oxygen in the flask occurred, and, more importantly, oxygen consumption kept the growth medium well below equilibrium with the oxygen in the gas phase. Smaller but similar effects on oxygen concentration were found in flasks containing V79 spheroids. 相似文献
2.
Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. (c) 1996 John Wiley & Sons, Inc. 相似文献
3.
In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO3-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO2 to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO2 is required by cells as an essential nutrient for growth, and that the desired level of CO2 in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO2 tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO2. 相似文献
4.
A 24-microwell plate with improved mixing and scalable performance for high throughput cell cultures
Multi-well plates are widely used in high throughput drug screening, cell clone development, media design and cell culture optimization in the biotechnology industry. The reproducibility and data quality of cell cultures in multi-well plates are greatly affected by mixing, aeration, and evaporation. A novel 24-microwell plate (MWP) with static mixers for improved mixing and aeration, and gas permeable lids for reduced evaporation was developed for cell cultures. Mixing, oxygen transfer, evaporation, and cell proliferation as affected by the static mixer, shape of the well and agitation rate were studied. The static mixer improved mixing pattern and reduced cell aggregation under orbital shaking conditions. Consequently, the static mixer also improved cell proliferation with a significantly higher specific growth rate in round wells. In general, consistent growth kinetics was observed for cells cultured on the plate. Overall, the MWP improved the data quality with smaller standard deviations and better reproducibility. Furthermore, CHO cells cultured in the MWP gave similar kinetics in glucose consumption, lactate production, cell growth and viability, and antibody production in a serum-free medium to those cultured in spinner flasks, demonstrating its scalable performance and potential application in high throughput screening for cell culture process development. 相似文献
5.
Fabrication of viable and functional pre‐vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks
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Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre‐vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two‐stage (proliferative‐osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. Conclusion: Collectively, this work established an effective method to fabricate pre‐vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration. 相似文献
6.
Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. 相似文献
7.
In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation. 相似文献
8.
C K?sehagen F Linz G Kretzmer T Scheper K Schügerl 《Enzyme and microbial technology》1991,13(11):873-881
The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens 总被引:7,自引:5,他引:2
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The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium. 相似文献
10.
Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002 相似文献
11.
The production of hybridoma cell lines secreting antibody against foot-and-mouth disease virus (FMDV) was more difficult than the production of similar cell lines secreting antibody against vesicular stomatitis virus or measles virus. A rapid and efficient protocol for the selection and culturing of 'anti-FMDV' hybridoma cultures was therefore developed and is described. This required the determination of the optimal culture medium (commercially available), source of serum supplement, line of myeloma cells, type of culture and routine for the subculturing of the hybridoma cells. The protocol consisted of fusion between immune splenocytes and NS-1 mouse myeloma cells, seeding into the wells of 24-well (24W) plates, culturing in RMPI 1640 medium supplemented with either foetal or donor calf serum, and passaging through 24W plates, 6W plates and 100 ml flasks (20 ml medium), respectively. The time at which aminopterin was added to kill unfused myeloma cells was also critical, with the optimum time being 24 h after fusion. In contrast, the B lymphocyte growth stimulant (2-mercaptoethanol) had no beneficial effects on the growth of the hybridomas. 相似文献
12.
Christoph Ries Gernot John Corinne John Regine Eibl Dieter Eibl 《Engineering in Life Science》2010,10(1):75-79
While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (kLa‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×107 cells/mL) and for the efficient expression of an intracellular model protein. 相似文献
13.
B. Mered P. Albrecht Hope E. Hopps 《In vitro cellular & developmental biology. Plant》1980,16(10):859-865
Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was
DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute
of Technology.
The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the
beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue
culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary
and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles
and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0
mg/ml, respectively.
In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and
stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production
units. 相似文献
14.
We developed procedures for large-scale cultivation of Helicobacter pylori in flasks and fermentors. Flasks incubated closed under a microaerophilic gas phase with a cotton plug covered by a plastic bag, followed by removal of the bag after 8 h, gave excellent growth. Growth in a 10-liter fermentor led to excessive foaming if the medium was sparged with gas; silicone- or polyglycol-based antifoaming agents were severely inhibitory. Use of fermentor surface gassing, first with a microaerophilic 6% oxygen gas mixture, then with air, and then with 95% oxygen, allowed the culture to grow to an A600 of 2.5 in < 24 h. This method was modified for scale-up to a 100-liter fermentor. 相似文献
15.
M L Meltz P Eagan C R Harris D N Erwin 《Physiological chemistry and physics and medical NMR》1988,20(1):23-30
A circulating water bath exposure system has been designed for in vitro radiofrequency radiation (RFR) exposure studies in the 915 to 2450 MHz range. A Styrofoam float, in which 10 T-25 plastic tissue culture flasks are embedded, is rotated at approximately 20 rpm in a Plexiglas water bath at a distance beneath a rectangular horn. The continuous circular rotation of the flasks is designed to "average out" the heterogeneity present in stationary flask exposures. The rotation also serves to prevent the establishment of chemical gradients in the medium within the flasks. Several factors have been demonstrated to affect the specific absorption rate (SAR) measured in the medium in the exposed flasks. These factors include: 1) the position of the exposure flasks relative to the long axis of the antenna horn; 2) whether the flasks are exposed while stationary or in rotation; 3) the volume of the medium contained in the flask; and 4) the depth in the medium in the flask at which temperatures for SAR calculation are measured. The presence of cells in the exposure flask (as attached monolayer or cell suspension) did not result in an SAR different from that measured in the same volume of medium without cells present. 相似文献
16.
Enhancement of in vitro growth of papaya multishoots by aeration 总被引:2,自引:0,他引:2
Lai Chuo-Chun Yu Tsong-Ann Yeh Shyi-Dong Yang Jiu-Sherng 《Plant Cell, Tissue and Organ Culture》1998,53(3):221-225
Efficient micropropagation of papaya (Carica papaya L.) has become crucial for multiplication of specific sex types of papaya
or transgenic lines resistant to virus infection. In this study, aeration at different intervals with a 0.02 μm filter disc
in the closure of culture flasks ensured exchange of gas components. The effect of aeration on development of multibuds to
multishoots was investigated. Multibuds grown in culture flasks after one-week without aeration followed by a two-week aeration
treatment caused a 41% increase in the number of shoots ≥0.5 cm, 42% increase in leaf expansion, and 17% increase in leaf
numbers in comparison with unaerated materials. Ethylene and oxygen concentrations in the culture flasks were measured by
gas chromatography and oxygen electrode at weekly intervals during the culture period. Oxygen concentrations were slightly
different between aerated and unaerated culture flasks. Ethylene in the unaerated flask reached the highest level (0.11 ppm)
2 weeks after the treatment, while accumulation of ethylene in the aerated flasks was not detected. The multishoots grown
for 3 weeks without aeration showed growth retardation on leaves and epinasty on petioles.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
17.
Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus-HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6-well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage-dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell-covered microcarriers suspended in culture medium in spinner flasks, suggested a re-examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 degrees C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 microg EGFP/10(6) infected cells. 相似文献
18.
To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages. 相似文献
19.
Doran PM 《Journal of biotechnology》2006,122(1):39-54
The role of surface adsorption in the disappearance of secreted foreign proteins from the medium of transgenic plant cell and organ cultures was investigated. When mouse monoclonal IgG1 was added to sterile plant culture media in glass shake flasks, the antibody concentration declined rapidly demonstrating that antibody was labile in the plant culture environment even in the absence of biomass and proteases. Elution of bound antibody from the surfaces of the flasks indicated that adsorption had contributed to the observed loss of antibody from solution. Antibody retention in sterile plant culture media was improved significantly when protein-resistant polymer coatings were applied to the glass vessels containing the antibody solutions. Pluronic F127 applied at a concentration of 1 mg mL(-1) to a primary dimethyldichlorosilane layer on glass yielded the best results in sterile Murashige and Skoog medium. When this coating was used in shake flasks for culture of transgenic tobacco hairy roots, there was a significant improvement in the accumulation of secreted recombinant antibody in the medium consistent with a reduction in antibody adsorption. Medium antibody levels eventually declined, however, as medium protease concentrations rose rapidly towards the end of the culture period. This work demonstrates that surface adsorption reduces the medium antibody titre observed in transgenic plant tissue cultures. 相似文献
20.
Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6-mm deep, stagnant, medium layer covering a cell culture. The measurement set-up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase. 相似文献