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1.
Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in
suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 106 cells/ml, maximal cell densities of 16 × 106 and 6 × 106 cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures
remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities
reduced the duration of the lag phase. After inoculation at 2.5 × 106 cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 106 cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent
protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale
cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production. 相似文献
2.
Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc. 相似文献
3.
J. L. Moreira A. S. Feliciano P. C. Santana P. E. Cruz J. G. Aunins M. J. T. Carrondo 《Cytotechnology》1994,15(1-3):337-349
Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate chacteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this selection procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures. 相似文献
4.
Ana I. Calejo Eleazar Rodriguez Virgília S. Silva Jernej Jorgačevski Matjaž Stenovec Marko Kreft Conceição Santos Robert Zorec Paula P. Gonçalves 《Cell biology and toxicology》2010,26(4):341-353
Prolonged exposure to aluminium may impact health. Aluminium’s deleterious effects are mostly attributed to its selective
accumulation in particular organs and cell types. Occupational exposure to aluminium is allied with a reduced level of serum
prolactin, a stress peptide hormone mainly synthesised and secreted by the anterior pituitary lactotrophs. Our aim was to
study the effect of aluminium on the viability of rat lactotrophs in primary suspension cultures where multicellular aggregates
tend to form, comprising approximately two thirds of the total cell population as confirmed by confocal microscopy. Flow cytometric
light scattering of calcein acetoxymethyl ester and ethidium homodimer-1 labelled cells was used to define subpopulations
of live and dead cells in heterogeneous suspensions comprised of single cells and multicellular aggregates of distinct size.
Concentration-dependent effects of AlCl3 were observed on aggregate size and cell survival. After 24-h exposure to 3 mM AlCl3, viability of single cells declined from 5% to 3%, while in multicellular aggregates, viability declined from 23% to 20%.
The proportion of single cells increased from 30% to 42% within the same concentration range, while in large aggregates, the
proportion remained approximately constant representing 35% of the cell suspension. In large aggregates, cell viability (75%)
remained unaltered after exposure to AlCl3 concentrations up to 300 μM, while in single cells, viability was halved at 30 μM. In conclusion, our finding indicates that
prolonged exposure to aluminium may lead to significant loss of pituitary cells. 相似文献
5.
H. Herbst H. -U. Peters I. -S. Suh A. Schumpe W. -D. Deckwer 《Biotechnology Techniques》1988,2(2):101-104
Summary Xanthan concentration and molecular weight distribution are determined by size exclusion chromatography in the fermentation medium after dilution and cell removal by centrifugation. The analysis is rapid enough for process control. During a batch fermentation, the average molecular weight is found to be in the range of 7.2–9.3·106 g/mole and to run through a maximum. 相似文献
6.
Summary Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1×106 cells/ml. Concentrations of monoclonal antibody (MAb) reached 140 mg/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during active exponential growth and not during the stationary phase. 相似文献
7.
Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH4Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH4Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH4Cl even under the ammonia serial change.List of symbols DFeed
d-1 Dilution rate of fresh feed medium (=Fo/V)
- DOut
d-1 Dilution rate of cell suspension (=F1/V)
- F1
ml·d-1 Volumetric discharge rate of cell suspension
- F0
ml·d-1 Volumetric flow rate of fresh feed medium
- kD
h-1 Specific death rate
- P
mmol·l-1 Product concentration
- S
mmol·l-1 Substrate concentration in culture broth
- S0
mmol·l-1 Substrate concentration in feed medium
- t
d Cultivation time
- V
ml Working volume of reactor
- X0
cells·ml-1 Total cell density
- XV
cells·ml-1 Viable cell density
- YP/S
mmol·mmol-1 Yield of product from substrate
- YX/S
cells·mmol-1 Yield of cells from substrate
-
mmol·cell-1·h-1 Specific production rate
-
h-1 Specific growth rate
-
mmol·cell-1·h-1 Specific consumption rate of substrate 相似文献
8.
G. Amin R. De Mot K. Van Dijck H. Verachtert 《Applied microbiology and biotechnology》1985,22(4):237-245
Summary Direct alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l–1 of dextrin S. diastaticus produced 77 g·l–1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l–1 was obtained with a medium containing 400 g·l–1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l–1 of ethanol was produced from 400 g·l–1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l–1 of ethanol from 200 g·l–1 of dextrin, corresponding to an ethanol productivity of 7.6g·l–1·h–1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l–1 and 80 g·l–1 of ethanol using initial dextrin concentrations of 200 and 250 g·l–1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l–1·h–1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation. 相似文献
9.
VERO cells were cultivated in microcarriers (MCs) using a bioreactor with a working capacity of 3.7 l. We studied the effect of MCs concentration in the proportion of MC-bearing cells and on the kinetics of cell growth, as well as the cell growth with a batch, fed-batch and perfusion mode operation. The data show that, in a batch system, with an initial VERO cell inoculum of 3×104 cells/mg of MCs, 65%, 55% and 50% of the MCs were shown to be totally covered by cells after 7 days in cultures with respectively 2, 5 and 10 mg/ml of MCs. It was observed, that higher concentrations of MCs produced higher final yields of VERO cells. With 10 mg/ml of MCs, after 7 days of culture, a final yield of 1010 cells in the bioreactor, was obtained. The study of cell growth with a batch, fed-batch or perfusion system showed that the medium renewal allowed the continuous cell growth with the obtention of a final yield of 4×109 cells in a culture with 2 mg/ml of MCs. The number of cells that can be easily reached in these culture systems, which can be even further improved, indicates its usefulness for cell propagation and the preparation of cell products such as viral antigens.This work was supported in part by grants from the Fundação de Amparo à Pesquisa do Estado de S. Paulo (FAPESP), Fundação Instituto Butantan, and European Economic Community (EEC). C.A. Pereira is recipient of a Conselho Nacional de Pesquisa (CNPq) fellowship. We thank A.C. Barbosa for technical assistance. 相似文献
10.
Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc. 相似文献
11.
The present study is investigating the immobilization of Rubia tinctorum L. suspension cultures. The effects of three inoculation volumes and three immobilization materials (loofa, sisal and jute) on fresh and dry weights of biomass as well as on alizarin and purpurin production were determined in this study. Two grams of four-week old callus tissue were transferred to liquid medium to establish suspension cultures. After four weeks, suspension cultures of R. tinctorum at concentration of 8?×?105?living cells/ml were immobilized with lignocellulosic materials and the cells were attached to all immobilization materials at the end of the first week and started to form aggregates on them. At the fourth week of these batch systems, biomass was measured approximately three times higher than the starting suspension cultures. The highest fresh weight was obtained (339.40?g/l) from sisal with ? inoculation ratio. Immobilization materials and inoculation volumes had an effect on dry weights, and accordingly, the most effective combinations were jute with ? (J3) and ? (J1) inoculation volumes with 7.86 and 7.82?g/l dry weights, respectively. Alizarin and purpurin contents of immobilized cells, analyzed with U-HPLC method, were 6.05 and 22.91 times higher than inoculated cells. All immobilization materials used in this study had no negative effect on to cells and biomass accumulation was enhanced. Concomitantly with rapid biomass increase, alizarin and purpurin production was ascended. 相似文献
12.
Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7?l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10?mg/ml of MCs showed that a high concentration of MCs (10?mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6?×?106 cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2?mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH4 + which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed. 相似文献
13.
A. S. Afschar K. H. Bellgardt C. E. Vaz Rossell A. Czok K. Schaller 《Applied microbiology and biotechnology》1991,34(5):582-585
Summary
Klebsiella oxytoca fermented 199 g·l–1 high test or invert molasses using batch fermentation with substrate shift to produce 95.2–98.6 g 2,3-butanediol·l–1 and 2,4–4.3 g acetoin·l–1 with a diol yield of 96–100% of the theoretical value and a diol productivity of 1.0–1.1 g·l–1·h–1. Fermentation was performed numerous times with molasses in repeated batch culture with cell recovery. Such repeated batch fermentation, in addition to a high product yield, also showed a very high product concentration. For example, 118 g 2,3-butanediol·l–1 and 2.3 g acetoin·l–1 were produced from 280 g·l–1 of high test molasses. The diol productivity in this fermentation amounted to 2.4 g·l–1·h–1 and can undoubtedly be further increased by increasing the cell concentration. Because the Klebsiella cultures ferment 2,3-butanediol at an extremely high rate once the sugar has been consumed, the culture was inhibited completely by the addition of 15 g ethanol·l–1 and switching off aeration.
Offprint requests to: A. S. Afschar 相似文献
14.
Masahiko Ishida Ryoichi Haga Nobuko Nishimura Harumi Matuzaki Ryusei Nakano 《Cytotechnology》1990,4(3):215-225
Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×108 cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×107 cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×107 cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.Abbreviations Eagle's MEM
Eagle's minimal essential medium
- Dulbecco's modified Eagle MEM
Dulbecco's modified Eagle minimal essential medium 相似文献
15.
A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10–3–1.45·10–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production. 相似文献
16.
Summary The effect of the intracellular concentration of Ca2+ on the cytoplasmic streaming of tonoplast-free cells ofChara australis was studied by intracellular perfusion. The perfusion media contained 1 mM Mg · ATP. Both cell ends were cut and left open. Media of different Ca2+ concentrations were perfused through the cell and the rate of the cytoplasmic streaming just after perfusion was measured. The critical concentration of Ca2+ for inhibiting the streaming was 5 × 10–4M, which was substantially higher than that found earlier byWilliamson (1975) andHayama
et al. (1979). Recovery from the inhibition occurred, though not completely, by removing Ca2+.In tonoplast-free cells the Ca2+ sensitivity differed according to the culture conditions. Cells cultured indoors exhibited a higher sensitivity than those cultured outdoors. Theformer cells contained granule-rich endoplasm aggregates after loss of the tonoplast, while the latter cells did no such aggregates. The aggregates were fixed to the cortical gel with a high dosage of Ca2+ and freed by removing it. 相似文献
17.
A diauxic fermentation was observed during batch fermentation of enzyme-hydrolyzed whey permeate to ethanol by Saccharomyces cerevisiae. Glucose was consumed before and much faster than galactose. In the continuous membrane recycle bioreactor (MRB), sugar utilization was a function of dilution rate and concentration of sugars. At a cell concentration of 160 kg/m3, optimum productivity was 31 kg/(m3 · h) at ethanol concentration of 65 kg/m3. Low levels of acetate (0.05–0.1 M) reduced cell growth during continuous fermentation, but also reduced galactose utilization. 相似文献
18.
Summary
Escherichia intermedia cells were immobilized by entrapment in a polyacrylamide gel and used for l-dopa synthesis from pyrocatechol, pyruvate and ammonia. An immobilized cell preparation containing 75 mg cells/g gel retained 45%–50% of the activity of free cells. The effect of temperature, pH and substrate concentration of the initial rate of l-dopa synthesis was very similar for free and immobilized cells. Substrate inhibition was observed for pyrocatechol, pyruvate and ammonia. In a batch reactor, 5.4 g·l-1
l-dopa was obtained, with 100% conversion yield of pyrocatechol and l-dopa productivity of 0.18 g·l-1·h-1. The use of a pyrocatechol-borate complex decreased by-product formation and catalyst inactivation. 相似文献
19.
Water conservation and protein metabolism in northern elephant seal pups during the postweaning fast
S. H. Adams D. P. Costa 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(5):367-373
Urine production and N output were monitored in northern elephant seal (Mirounga angustirostris) pups progressing through 10 weeks of a natural postweaning fast. Urine output declind by 84% (to 69±12 ml·day–1) at 10 weeks (P<0.05). Glomerular filtration rate at 10 weeks was 51% of the 67±3 ml serum·min–1 observed during week 1 (P<0.05). Urine N excretion fell by 69% to 1.2±0.17 g·day–1, while urinary concentration increased (P<0.05). Serum urea declined from an initial 11 mmol·1–1 to 5–7 mmol·1–1 by 5 weeks. The fall in urinary N loss (and thus amino acid oxidation) was concomitant with depressed metabolic rate. Therefore, protein contributed little toward meeting energy demands (i.e., <4% of average metabolic rate) throughout fasting. These data indicate that fasting pups improve water conservation and minimize protein catabolism during prolonged natural fasts without an exogenous source of water.Abbreviations AA
amino acid(s)
- AMR
average metabolic rate
- ANOVA
one-way analysis of variance
- BMR
basal metabolic rate
- BUN
blood urea nitrogen
- EP
end product
- EWL
evaporative water loss
- [Gr]s
serum creatinine concentration
- GFR
glomerular filtration rate
- LBM
lean body mass
- LML
Long Marine Laboratory
- MR
metabolic rate
- NEFA
non-esterified fatty acids
- RMR
resting metabolic rate
- TCA
tricarboxylic acid
- U:C
ulinary urea: creatinine concentration ratio 相似文献
20.
Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion
culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in
the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below
32°C resulted in growth suppression. However, specific productivity of FSH, q
FSH, increased as culture temperature decreased, and the maximum q
FSH of 43.4 ng/106 cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures,
we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 107 cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer
of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably
due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical
for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising. 相似文献