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1.
Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 106 cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 106 cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production.
Offprint requests to: Y. Shoham 相似文献
2.
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
3.
Obligatory anchorage dependant Vero cells were successfully grown in gelatinous-like macrocarriers made of calcium alginate. Entrapped single cells were immobilized within the polymerized alginate matrix divide to form large spherical clumps of cells. A cell density of 17×106 cells/ml of alginate with over 95% viability was obtained after 14 days in spinner flasks. When subjected to poliovirus type I infection, spherical masses of Vero cells progressively showed extensive cytopathic effect but remained entrapped in the alginate matrix of the macrocarriers. Virus was released into a cellular debris free-like supernatant and reached a peak titer of 108.0 TCID50/ml after 72 hours. 相似文献
4.
Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity 总被引:1,自引:0,他引:1
Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×106 to 7.9×105 cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 106 viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins. 相似文献
5.
Polystyrene microcarriers were prepared in four size ranges (53–63 m, 90–125 m, 150–180 m and 300–355 m) and examined for ability to support attachment and growth of human diploid fibroblasts. Cells attached rapidly to the microcarriers and there was a direct relationship between cell attachment and microcarrier aggregation. Phasecontrast and scanning electron microscopic studies revealed that while aggregation was extensive, most of the aggregate consisted of void volume. Cell growth studies demonstrated that human diploid fibroblasts proliferated well in microcarrier aggregates, reaching densities of 2.5–3×106 cells per 2 ml dish after 6 days from an inoculum of 0.5×106 cells per dish. When cells were added to the microcarriers at higher density (up to 5×106 cells per 2-ml culture), there was little net growth but the cells remained viable over a 7-day period. In contrast, cells died when plated under the same conditions in monolayer culture. When the microcarriers were used in suspension culture, rapid cell attachment and rapid microcarrier aggregation also occurred. In 100-ml suspension culture, a cell density of 0.7×106 cells per ml was reached after 7 days from an inoculum of 0.1×106 cells. Based on these data, we conclude that microcarrier aggregation is not detrimental to fibroblast growth. These data also indicate that small microcarriers (53–63 m) (previously thought to be too small to support the growth of diploid fibroblasts) can support fibroblast growth and this occurs primarily because microcarriers in this size range efficiently form aggregates with the cells. 相似文献
6.
N.S. Parker A.P. Negri D.M.F. Frampton L. Rodolfi M.R. Tredici S.I. Blackburn 《Journal of applied phycology》2002,14(5):313-324
Toxic dinoflagellates are important in natural ecosystems and are ofglobal economic significance because of the impact of toxic blooms onaquaculture and human health. Both the organisms and the toxins they producehave potential for biotechnology applications. We investigated autotrophicgrowth of a toxic dinoflagellate, Alexandrium minutum, inthree different high biomass culture systems, assessing growth, productivityandtoxin production. The systems used were: aerated and non-aerated2-L Erlenmeyer flasks; 0.5-L glass aerated tubes; anda 4-L laboratory scale alveolar panel photobioreactor. A range ofindicators was used to assess growth in these systems. Alexandriumminutum grew well in all culture conditions investigated, with amarked increase in both biomass and productivity in response to aeration. Thehighest cell concentration (4.9 × 105 cellsmL–1) and productivity (2.6 ×104cells mL–1d–1) was achieved inthe aerated glass culture tubes. Stable growth of A.minutum in the laboratory scale alveolar panel photobioreactor wasmaintained over a period of five months, with a maximum cell concentration of3.3 × 105 cells mL–1, a meanproductivity of 1.4 × 104 cells mL–1d–1, and toxin production of approximately 20g L–1 d–1 with weeklyharvesting. 相似文献
7.
The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×106 to 6–12×106 cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed. 相似文献
8.
Summary Ethanol-induced death rate was higher for cells ofSaccharomyces bayanus orKluyveromyces marxianus in spanse suspensions (2×104 cells/ml) compared with dense suspensions (2×105–2×107 cells/ml). Specific death rates of sparse suspensions decreased to values similar to dense suspensions if ethanol-induced death experiments were undertaken in the media obtained after harvesting the cells previously incubated with the same lethal concentration of ethanol. 相似文献
9.
Marina Radrizzani Carlo Gambacorti-Passerini Giorgio Parmiani Giuseppe Fossati 《Cancer immunology, immunotherapy : CII》1989,29(1):67-73
Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19+ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19+ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 109 normal MNC, obtained by leukapheresis, yielded 5 × 108 A-LAK cells with a total of 5.7 × 105 lytic units of cytotoxicity against K562 and a total of 3.3 × 105 lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer. 相似文献
10.
Isabelle Chevalot Athanase Visvikis Pierre Nabet Jean-Marc Engasser Annie Marc 《Cytotechnology》1994,16(2):121-129
Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h–1, the highest cell density (near 1.3×106 cells ml–1), and the highest enzyme activity around 300 mU ml–1, which corresponded to a specific cellular level of 20 mU 10–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems. 相似文献
11.
Scale-up of biopesticide production processes using wastewater sludge as a raw material 总被引:1,自引:0,他引:1
Yezza A Tyagi RD Valèro JR Surampalli RY Smith J 《Journal of industrial microbiology & biotechnology》2004,31(12):545-552
Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e+08 cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity. 相似文献
12.
J. L. Moreira P. M. Alves J. G. Aunins M. J. T. Corrondo 《Applied microbiology and biotechnology》1994,41(2):203-209
Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical aissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1. 2 · 106 cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 · 108 cells/ml, with a porosity of 55%. Viability was consistently in the range 85–90%, both for aggregate and suspended cells. 相似文献
13.
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
14.
The addition of 2.8 g/ml algal extracts enhanced both scu-PA production and cell growth in a serum-free medium, compared to a conventional serum-free medium for the cultivation of recombinant CHO cells. The growth rate and scu-PA production were relatively lower in the serum-free medium than 5% serum containing medium: however, specific scu-PA production rate was higher in the serum-free medium due to the long-term period of cultivation (3.66×10–4 vs. 2.48×10–4 IU/cell/day). Overall scu-PA production rate was also greater in an enforced serum-free medium as 25,000 IU/day over 50 d of perfusion cultivation. The conversion ratio of scu-PA to tcu-PA was greatly reduced in the serum-free medium during perfusion cultivation (10% compared to 20% conversion in a serum containing medium). 相似文献
15.
Tsuyoshi Akiyoshi Shinya Arinaga Hideo Tsuji 《Cancer immunology, immunotherapy : CII》1987,24(3):259-262
Summary The effects of mitomycin C (MMC) on the generation of cell-mediated cytotoxicity in primary stimulation culture of human peripheral blood mononuclear cells (PBM) with the B lymphoblastoid Raji cell line were assessed. The cell-mediated cytotoxicity induced in culture was significantly augmented when MMC was added to cultures on day –1 to day 3 for 24 h at concentrations of 2.5×10–2 g/ml and 2.5×10–3 g/ml. To identify the cell populations affected by MMC, PBM were separated by adherence to plastic after treatment with MMC for 24 h (day –1). The two populations were recombined with untreated separated cells and stimulated with antigen. The ability to develop an augmented cell-mediated cytotoxicity was associated with the adherent cell fraction of MMC-treated PBM. Therefore, the ability of MMC-treated adherent cells to produce interleukin 1 (IL 1) was examined. Significantly higher levels of IL 1 were produced by treated cells as compared to untreated adherent cells. The results appear to indicate that the selective effects of MMC on the adherent cell fraction, especially the modification of IL 1 production, may be involved in the mechanisms of MMC-induced augmented cell-mediated cytotoxicity. 相似文献
16.
Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l–1 2,4-D and 0.2 mg l–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml–1, in the presence of the non-pathogenic Ehg154 aggregates ml–1 and in the control 115 aggregates ml–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell. 相似文献
17.
Mahalakshmi Atchison Michael L. Atchison Benjamin L. Van Duuren 《Cell biology and toxicology》1985,1(4):323-331
The mouse skin cocarcinogens fluoranthene, pyrene, and undecane were used with the indirect-acting carcinogen, benzo(a)pyrene (BP), and the direct-acting alkylating carcinogen, ß-propiolactone (BPL), in an in vitro transformation assay. Dose response, cytotoxicity, and transformation studies with these compounds were performed with a subclone (A31-1-1) of the Balb/3T3 cell line. Transformation frequencies were found to increase with increasing concentrations of BP used up to 1.0 µg/ml or when BPL was used up to 4.0 µg/ml. A significant increase (P<0.05) in the transformation frequency over that seen with carcinogen alone was observed when cells were exposed to a combination of fluoranthene (4.0 µg/ml) and BP (0.063 µg/ml) or pyrene (5.0 µg/ml) and BP (0.063 µg/ml). Thus, the transformation frequency obtained with BP + fluoranthene was 3.8 × 10–4 compared to 1.2 × 10–4 when BP was tested alone. Similarly, the transformation frequency using BP + pyrene was 2.8 × 10–4 vs 1.2 × 10–4 when BP was tested alone. Undecane did not exert any cocarcinogenic effect with BP in the dose range tested. In this in vitro assay, no cocarcinogenic effect was observed when BPL was used with any of the above mouse skin cocarcinogens. All cells isolated from transformed foci showed characteristics of transformed cells including anchorage-independent growth.Abbreviations BP
benzo(a)pyrene
- BPL
ß-propiolactone
- CE
cloning efficiency
- CE50
median CE
- RCE
relative CE
Department of Cell Biology, New York University Medical CenterInstitute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA.Contribution No. L217 from the Laboratory of Organic Chemistry and Carcinogenesis. 相似文献
18.
The growth rates and attainable cell densities were compared in Grace and TC-100 media in spinner flasks of various sizes as well as in a 10-l bioreactor. In all mentioned reactors, a higher cell growth rate and final cell concentration could be obtained in the TC-100 medium than in the Grace medium. In bigger flasks, the cell concentration was higher. It was possible to maintain a constant viable cell concentration (2.45 · 106 ml–1) in continuous cultures (without perfusion) at a dilution rate of 0.01 h–1. 相似文献
19.
BelloCell is a novel packed bed bioreactor that allows alternating nutrient and gas transfer to a culture. Spodoptera frugiperda Sf-9 grown in the BelloCell (300 ml culture) reached 1.3–1.5×107 cells ml−1 in 7–8 days and the total baculovirus-expressed protein yield was 2.3-times that in a stirred tank bioreactor (600 ml culture). The superior cell and protein yields underline the potential of BelloCell for cell culture and recombinant protein production. 相似文献
20.
Hideki Yamaji Hideki Fukuda Yasuhiro Nojima Colin Webb 《Applied microbiology and biotechnology》1989,30(6):609-613
Summary Immobilisation of anchorage-independent animal cells using Biomas Support Particles (BSPs) was investigated. Mouse myeloma MPC-11 cells were physically entrapped in three-dimensional reticulated polyvinyl formal (PVF) resin PSPs (3×3×3 mm) with matrices of relatively small pores (30–100 m) by filtering medium containing cells or incubating in a shake flask for inoculation. Physically entrapped cells became immobilised in the BSPs by forming aggregates within the matrices of the reticulated PVF resin, and cell density in the BSPs reached at least 107 cells/cm3 BSP. Immobilised cells in the BSPs were successfully cultivated in static and/or shake-flask cultures with regular replacement of medium for a long period. 相似文献