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1.
搅拌式生物反应器悬浮培养水母雪莲细胞的研究 总被引:7,自引:0,他引:7
应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r min、70 0~1000L min和 4.0~ 5.0gDCW L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW L ,黄酮产量 416mg L ,黄酮含量占细胞干重的 30%。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现 ,流变压力使细胞聚集体分裂 ,使反应器中细胞生长受到影响 ,黄酮产量较摇瓶中降低 相似文献
2.
Trejo-Tapia G Cerda-García-Rojas CM Rodríguez-Monroy M Ramos-Valdivia AC 《Biotechnology progress》2005,21(3):786-792
Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor. 相似文献
3.
Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells 总被引:2,自引:0,他引:2
This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies. 相似文献
4.
J. Meghrous A. Kamen S. R. Palli S. S. Sohi G. F. Caputo S. C. Bedard 《Biocontrol Science and Technology》2000,10(3):301-313
The purpose of this study was to develop a cell culture process in a bioreactor for the production of a viral insecticide for the spruce budworm, Choristoneura fumiferana . Several cell lines were tested for their growth in serum-free medium suspension cultures. One cell line, CF-124T-2C1 (CF-2C1), was successfully adapted to grow in suspension cultures in SFM. Serum-free Ex-Cell 405 medium produced a much higher cell density (6.3 x 10 6 cells ml -1 ) than the Grace's medium supplemented with 10% fetal bovine serum (2.5 x 10 6 cells ml -1 ). Also, a higher yield of virus was obtained in the former medium. Ex-Cell 405, was used to study the growth of CF-2C1 cells and the production of C. fumiferana nucleopolyhedrovirus (CfMNPV) in a 3 l bioreactor. Under these conditions, a specific growth rate ( μ) of 0.027 h -1 was obtained during the exponential growth phase, and the specific carbon dioxide evolution rate, as determined by on-line measurement, was 0.9 x 10 -16 mol cell -1 s -1 and 1.78 x 10 -16 mol cell -1 s -1 during growth and infection phases, respectively. Virus production in bioreactor cultures infected at 1.3 x 10 6 cells ml -1 was consistently lower than that obtained in Erlenmeyer shake flasks. Only 26% of the cells were infected in the bioreactor compared to 44% in the shake flasks. However, a higher yield of occluded virus was obtained in the bioreactor cultures than in shake flasks. The production of occlusion bodies (OB) achieved in bioreactor cultures was 2 x 10 6 OB ml -1 . 相似文献
5.
The initiation of the suspension culture of Camptotheca acuminata M42 cell line was reported. Isocamptothecin A (ICPTA) and isocamptothecin B (ICPTB), two alkaloids that do not exist in the parent plant were identified in the suspension cell culture of Camptotheca acuminata. Suspension cell culture was carried out in both flasks and a 5-l airlift bioreactor and the time course for cell growth and accumulations of ICPTA and ICPTB were investigated. It was found that accumulations of ICPTA and ICPTB was partly associated with cell growth and the size of cell aggregates affected the contents of ICPTA and ICPTB in the cultures to some extent (cell aggregates with a size of 2--4 mm in diameter presented the highest ICPTA and ICPTB contents). Although the cell dry weight of 15.1 g l-1 achieved in a 5-l airlift bioreactor was lower than that in flasks, 18.4 g l-1, the contents of ICPTA and ICPTB were almost the same levels. 相似文献
6.
Archambault J Williams RD Lavoie L Pépin MF Chavarie C 《Biotechnology and bioengineering》1994,44(8):930-943
Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc. 相似文献
7.
Comparison of manufacturing techniques for adenovirus production 总被引:2,自引:0,他引:2
We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors
for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred
tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in
standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The
stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers.
The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and
7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell.
Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption
and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant.
Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293
cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
8.
De Dobbeleer C Cloutier M Fouilland M Legros R Jolicoeur M 《Biotechnology and bioengineering》2006,95(6):1126-1137
A perfusion bioreactor allowing continuous extraction of secondary metabolites was designed and challenged for Eschscholtzia californica plant cell suspensions. Four sedimentation columns mounted inside a 2.5-L bioreactor separated single cells and cell aggregates from the culture medium. Cells were elicited with chitin at day 4 and the liquid medium free of cells and debris was then continuously pumped to the extraction columns containing fluidized XAD-7 resins, and then recirculated back to the cell suspension. A medium upward velocity corresponding to cell sedimentation velocity maintained a stable cell/medium separation front in the columns for sedimented cell volume (SCV) of 90% (70% packed cell volume, PCV). Two perfusion bioreactor cultures of 10 and 14 days were performed. A maximum dilution rate of 20.4/day was reached from day 4 to day 6, and was then reduced to 5/day at day 9 for 55% SCV. Control cultures were performed without and with free extraction resins into the cell suspension. Perfusion cultures showed similar specific growth rates of 0.24 +/- 0.04/day before and after elicitation. However, production level in the perfusion cultures was similar to that from the culture without resins with a maximum of 2.06 micromole/gDW total alkaloids, with 1.54 micromole/gDW in the resins. Cultures with free resins resulted in 30.94 micromole/gDW with 28.4 +/- 8.8 micromole/gDW in the resins. Difference in the cells nutritional state from elicitation was identified as a major cause in the production reduction. However, pathway to chelilutine was favored in the continuous extraction culture. 相似文献
9.
Airlift bioreactor operations have been studied for the growth-associated production of secondary metabolites from plant cell suspension cultures. The model system used in this work was Thalictrum rugosum producing berberine, an isoquinoline alkaloid. The airlift system was well suited for growth of Thalictrum cell suspension cultures unless the cell density was high. At high cell density, the airlift system with a draught tube was not adequate due to large aggregates clogging the recirculation paths. This was overcome by use of a cell scraper in the reactor. For berberine production, gas-stripping also played a significant role and it was discovered that CO(2) and ethylene were important for product formation. By supplying a mixture of CO(2) and ethylene into the airlift system, the specific berberine content was increased twofold. It is evident that continuous gas sparging was harmful for the production of berberine without supplementation with other gases. 相似文献
10.
Laminin 5 is a multifunctional extracellular matrix protein, which supports epithelial cell adhesion through multiple cell binding sites. For elaborate studies, a 35 kDa fragment localized at the C-terminal extremity of the molecule, the LG4/5 fragment was recombinantly expressed in mammalian HEK293-EBNA cells. As the production of the LG4/5 fragment by adherent cell monolayers was very low (<1 microg/ml), we used the commercially available small scale bioreactor system miniPERM. The HEK293-EBNA transfectants were adapted to grow in suspension in defined medium containing low level of fetal calf serum and produced the recombinant LG4/5 protein with quality consistent with that produced in conventional static cell culture conditions. Cells grew without forming aggregates in the bioreactor and the resulting HEK293-EBNA-LG4/5 cell line was suitable for unlimited passages in the bioreactor. 2.5x10(5) cells/ml were cultured for 25 days to reach the maximal cell density of 1.6x10(7) cells/ml. The quantification of protein synthesis revealed that the highest level of 2.4 mg of recombinant LG4/5 protein was harvested when 10(7) transfected cells/ml were injected in the bioreactor and allowed to grow for 2 days. The mean daily recombinant LG4/5 fragment product yield of 1.2 mg of protein per minifermenter shows that cultivation of HEK293-EBNA transfectants in suspension is highly convenient for the production of recombinant laminin fragments. 相似文献
11.
Three-dimensional growth of endothelial cells in the microgravity-based rotating wall vessel bioreactor 总被引:10,自引:0,他引:10
Sanford GL Ellerson D Melhado-Gardner C Sroufe AE Harris-Hooker S 《In vitro cellular & developmental biology. Animal》2002,38(9):493-504
We characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d. Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d. These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology. The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope. Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material. These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB). The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures. The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME [N(G)-nitro-L-arginine-methylester]. Cells in the SFB responded similarly. The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression. We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function. 相似文献
12.
The biomass of sage (Salvia officinalis L.) cell suspension culture was composed of single cells and cell aggregates. The development of aggregated cell culture from a single-cell suspension was monitored by particle size distribution for four particle size classes. Particle size distribution was compared between the biomass grown in bioreactor and shake flasks. The size of the particles had a strong influence on content of secondary metabolite, ursolic acid (UA). The single cell biomass fraction accumulated up to 7.7 mg UA g–1 DW which was up to 50 times higher compared to aggregated biomass fractions. 相似文献
13.
Bleckwenn NA Bentley WE Shiloach J 《Bioscience, biotechnology, and biochemistry》2005,69(6):1065-1072
Adaptation of the vaccinia virus expression system to HeLa S3 suspension bioreactor culture for the production of recombinant protein was conducted. Evaluation of hollow fiber perfusion of suspension culture demonstrated its potential for increased cell density prior to infection. The hollow fiber was also used for medium manipulations prior to infection. Two process parameters, multiplicity of infection (MOI) and temperature during the protein production phase, were evaluated to determine their effect on expression of the reporter protein, enhanced green fluorescent protein (EGFP). An MOI of 1.0 was sufficient for infection and led to the highest level of intracellular EGFP expression. Reducing the temperature to 34 degrees C during the protein production phase increased production of the protein two-fold compared to 37 degrees C in spinner flask culture. Scaling up the process to a 1.5-liter bioreactor with hollow fiber perfusion led to an overall production level of 9.9 microg EGFP/10(6) infected cells, or 27 mg EGFP per liter. 相似文献
14.
Effect of aggregate size in cell cultures of<Emphasis Type="Italic"> Saussurea medusa</Emphasis> on cell growth and jaceosidin production 总被引:3,自引:0,他引:3
Cell suspension cultures of Saussurea medusa were grown in shake flasks and a 5-l stirred tank bioreactor. Biomass and jaceosidin distribution in cell aggregates of different sizes were investigated during the cultivation period. The results showed that on day 10, jaceosidin accumulation showed an increase with increasing size of the cell aggregate to 4 mm in diameter, with the highest jaceosidin accumulation being 12.2 mg/g. An inverse tendency was observed with cell aggregates larger than 4 mm in diameter, with the lowest accumulation being 3.1 mg/g. However, all of the cell aggregates, despite their size, synthesized almost the same amount of jaceosidin at day 12. Oxygen diffusion limitation and cell-cell contact may explain this behavior. In comparison with cells cultivated in shake flasks, decreased biomass and decreased jaceosidin concentration were observed when the cells were cultivated in a stirred tank bioreactor. The sublytic effects caused by the hydrodynamic stress in combination with insufficient nutrients in the bioreactor may cause cell damage. 相似文献
15.
The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. (c) 1997 John Wiley & Sons, Inc. 相似文献
16.
Trejo-Tapia G Sepúlveda-Jiménez G Trejo-Espino JL Cerda-García-Rojas CM de la Torre M Rodríguez-Monroy M Ramos-Valdivia AC 《Biotechnology and bioengineering》2007,98(1):230-238
Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress. 相似文献
17.
Linardos TI Kalogerakis N Behie LA Lamontagne LR 《Biotechnology and bioengineering》1992,39(5):504-510
Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d(-1). Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations. 相似文献
18.
Ghani K Garnier A Coelho H Transfiguracion J Trudel P Kamen A 《Biotechnology and bioengineering》2006,95(4):653-660
Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture. 相似文献
19.
G.W. Bae C.S. Chung K.I. Kim C.H. Park H.J. Lee Y.A. Chae I.S. Chung 《Biotechnology Techniques》1998,12(11):843-845
Dimethylpolysiloxane (DMPS), used as a second phase in a suspension culture of Polygonum tinctorium, increased indirubin production by up to 259% and 16% in shake flask and air-lift bioreactors, respectively. In the shake flask culture, indirubin production increased by up to 248 mg/l using 90% v/v DMPS (volume of DMPS/volume of medium). In an air-lift bioreactor culture grown at a perfusion rate of 0.05 day–1 the indirubin concentration reached a maximum of 130 mg/l using 50% (v/v) DMPS. For the same DMPS content, the lower indirubin production in an air-lift bioreactor was probably due to the mass transfer limitation of indirubin between the two phases. © Rapid Science Ltd. 1998 相似文献
20.
The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures. 相似文献