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1.
Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved.Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead.The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.  相似文献   

2.
Cell harvesting technique is of considerable importance in the scale-up of microcarrier culture of anchorage-dependent cells. The traditional methods are often time- and labor-consuming and cause physiological damage to the cells. Bead-to-bead cell transfer provides an attractive solution to the scale up process. By intermittent agitation, successful cell transfer was achieved. Significant cell growth was observed where bare beads contacted with confluent ones. Most of the fresh microcarriers reached near confluence four days after addition into the culture medium.  相似文献   

3.
Perfusion culture with microcarriers was conducted to produce cell-associated and cell-free Varicella-Zoster virus (VZV) with human embryonic lung cells. After the cells were infected with VZV infected cells, glucose in the medium decreased rapidly, suggesting that VZV propagation was related closely to the use of glucose. While the yield of cell-associated VZV in microcarriers was 9,350 PFU/cm2, almost two-thirds of that in T-80 flask and cell factory, the yield of cell-free VZV in microcarriers was only about 10% of that in T-80 flask and cell factory.  相似文献   

4.
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   

5.
Pilot production of u-PA with porous microcarrier cell culture   总被引:8,自引:0,他引:8  
A recombinant DNA CHO cell line secretingurokinase-type plasminogen activator (u-PA) wascultivated with Cytopore cellulose porousmicrocarriers in a 30l Biostat UC stirred tankreactor. After 26 days of culture, using a spinfilter toretain cells in bioreactor, the cell density couldreach 1.33 × 107 ml-1. The maximal u-PAactivity in supernatant was 7335 IU·ml-1, and204l supernatant containing 7.1 g u-PA was harvested.After 100 days of culture with 0.1% fetal bovineserum medium, a modified cell retention system whichcan be washed-out backward, substituted thespinfilter to prevent filter clogging. The maximalcell density was over 107 ml-1, the maximalu-PA activity in supernatant reached 6250IU·ml-1, and 1604l supernatant containing about51 g u-PA was harvested. Compared to perfusionculture, batch medium-replaced culture could raiseutilizing efficiency of the medium, increase cell specificproductivity and improve the quality of the product which wasnot steady in a 37 °C environment. Cells can movefrom seed porous microcarriers occupied by cells tovacant microcarriers spontaneously, withouttrypsinization, and continue to grow until all microcarriers contained cells. It shows that Cytoporeporous microcarriers are very useful and convenient toscale up cultivation step by step.  相似文献   

6.
A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 107 cells per ml were obtained. The described system is scaleable.  相似文献   

7.
Chitosan/gelatin composite microcarrier for hepatocyte culture   总被引:2,自引:0,他引:2  
Li K  Wang Y  Miao Z  Xu D  Tang Y  Feng M 《Biotechnology letters》2004,26(11):879-883
Solid and porous chitosan/gelatin (CG) composite microcarriers were prepared by a water-in-oil emulsion process with additional freezing and lyophilization. Adult rat hepatocytes (10(6) cells ml(-1)) attached on CG microcarriers maintained at least 15 d of viability and differentiated functions. Over 15 d, unimmobilized hepatocytes released 1.34-fold less lactate dehydrogenase (LDH), and retained 1.63-, 1.51- and 1.28-fold higher albumin secretion, urea synthesis and 7-ethoxycoumarin deethylation activities, respectively, than those on collagen-coated microcarriers. The CG matrix is therefore a promising microcarrier for hepatocyte culture.  相似文献   

8.
Summary Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis. For reprint requests address: Dr. Keith Jensen, Pfizer, Inc., Groton, CT 06340. This work was supported in part under contract N01-CP-33234 within the Virus Cancer Program of the National Cancer Institute.  相似文献   

9.
目的应用生物反应器培养Vero细胞制备EV71病毒。方法以3 L生物反应器采用4 g/L、8 g/L Cytodex-1微载体培养比较Vero细胞比生长率,并以4 g/L微载体培养EV71病毒。结果 4 g/L微载体培养Vero细胞3~4 d微载体细胞密度达2.3×106/mL,按0.001的感染复数(MOI)接种EV71病毒,病毒收获液的滴度最高达7.90 lgPFU/mL,较静置培养平均高出0.92 lgPFU/mL。结论初步建立了3 L生物反应器微载体培养Vero细胞制备EV71病毒的工艺,为进一步放大生产规模奠定了基础。  相似文献   

10.
This article describes a three-dimensional culture system for the expansion of anchorage-dependent cells using fiber-shaped microcarrier (MC; Cytodex3) aggregates, termed “MC fibers.” The fiber encapsulates the cells, the MC aggregates, and collagen and is covered with a poly-l -lysine membrane. The thin structure of the fiber enables sufficient supply of O2 and nutrients to the cell. Using the MC fiber, we demonstrated the efficient expansion of C2C12 cells with high viability through serial passaging. Therefore, our culture system is useful for various applications where large-scale cell expansion is required, such as in pharmaceutical technologies, regenerative medicine, and cultured meat production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2755, 2019.  相似文献   

11.
Summary A technique has been devised to attach adult rat hepatocytes to collagen-coated dextran microcarriers. Cells were cultured serum-free for 2 d and their viability, enzyme activities, glucose metabolism, and hormone responsiveness were compared to data obtained from conventional dish cell culture. The two diffeent culture methods showed no difference in cell viability and morphology. Microcarrier-cultured cells exhibited hormone responsiveness comparable to dish cultures; glycolysis could be activated three-fold by the sole addition of insulin, and gluconeogenesis was increased by 40 to 50% by glucagon. During the 48-h culture glucokinase and phosphoenolpyruvate carboxykinase activities declined at a similar rate in both culture systems. Long-tem culture wih 0.1 μM insulin prevented the decrease of glucokinase activity. Insulin responsiveness (activation of glycolysis) was still pronounced after 48 h in culture. The microcarrier technique establishes a new in vitro liver system in which acute and long-term hormonal actions can be investigated using the technical advantages of a suspensions cultures. This study had been supported by the Deutsche Forschungsgemeinschaft.  相似文献   

12.
By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage.  相似文献   

13.
14.
A cellular automaton model for microcarrier cultures   总被引:2,自引:0,他引:2  
In order to achieve high cell densities anchoragedependent cells are commonly cultured on microcarriers, where spatial restrictions to cell growth complicates the determination of the growth kinetics. To design and operate large-scale bioreactors for microcarrier cultures, the effect of this spatial restriction to growth, referred to as contact inhibition, must be decoupled from the growth kinetics. In this article, a cellular automaton approach is recommended to model the growth of anchorage-dependent cells on microcarriers. The proposed model is simple to apply yet provides an accurate representation of contact-inhibited cell growth on microcarriers. The distribution of the number of neighboring cells per cell, microcarrier surface areas, and inoculation densities are taken into account with this model. When compared with experimental data for Vero and MRC-5 microcarrier cultures, the cellular automaton predictions were very good. Furthermore, the model can be used to generate contact-inhibition growth curves to decouple the effect of contact-inhibition from growth kinetics. With this information, the accurate determination of kinetic parameters, such as nutrient uptake rates, and the effects of other environmental factors, such as toxin levels, may be determined. (c) 1994 John Wiley & Sons, Inc.  相似文献   

15.
By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesio  相似文献   

16.
Scale-up of suspension and anchorage-dependent animal cells   总被引:12,自引:0,他引:12  
Alternative culture processes for laboratory scale-up (to 20 L) are described for both suspension and anchorage-dependent cells. Systems range from simple multiple culture units such as the roller bottle, through stirred suspension and microcarrier unit bioreactors, to highly sophisticated perfusion culture capable of maintaining cells at densities of about 108/mL. Critical parameters in scale-up are discussed, and the advantages and disadvantages of each culture system are critically evaluated.  相似文献   

17.
High-density microcarrier cell cultures for influenza virus production   总被引:4,自引:0,他引:4  
Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.  相似文献   

18.
Summary The growth of Madin Darby canine kidney cells on microcarriers was studied at different cell seeding densities. Maximum growth was attained when a cell-to-bead ratio of 7∶1 was used. Under these conditions an initial concentration of above 3×105 cells/ml resulted in a yield of over 2×106 cells/ml in 2 d. The amino acid utilization of cells from Dulbecco's modified Eagle medium was studied under the above conditions. Eight amino acids (arg, cys, gln, ile, leu, met, ser, and val) showed rapid and near complete depletion from the medium. Five amino acids (his, lys, phe, thr, and tyr) showed limited depletion. Two amino acids (ala and gly) were released into the medium by the cells.  相似文献   

19.
The effect of medium viscosity on the specific death rate of bovine embryonic kidney (BEK) cells cultured in spinner flask microcarrier cultures has been examined for various impeller speeds. Two types of media were used, a serum-containing growth medium and a serum-free maintenance medium. The latter does not support cell growth. We found that increasing medium viscosity suppresses cell death rates in both growth and maintenance medium cultures in an agitation-intensity-dependent fashion; the beneficial effect of medium viscosity in reducing the specific death rate is amplified as the agitation rate is increased. Furthermore, increasing medium viscosity has no effect on the specific death rate of the cells when the agitation rate is below a critical level. A model based on the turbulent energy content of eddies in the dissipation spectrum of turbulence of length scales on the order of magnitude of the microcarrier diameter and lower has been developed to account for cell death due to both bead-to-bead and bead-to-eddy interactions. The model constitutes a significant departure from previous efforts first because both types of interactions are accounted for simultaneously and second because the properties of a spectrum of eddies instead of the Kolmogorov-scale eddy size alone are used in the model. The model explains the functional dependence of the specific death rates on the medium viscosity at varying agitation intensities.  相似文献   

20.
Summary Dispersed adult rat pancreatic islet cells were incubated with Cytodex-3 microcarrier beads for 72 h, during which time single cells adhered firmly to bead surfaces. Electron microscopy revealed well-preserved ultrastructure of attached A, B, and D cells. Perifusion of these cultures showed stable basal insulin release, brisk, biphasic insulin responses to 30-min glucose stimulation, and consistent, monophasic spikes of insulin release in response to repeated, brief pulses of glucose. These results indicate that adult rat islet cells attach to microcarriers and remain viable in culture. This preparation offers advantages for studies of hormone secretory dynamics of differentiated single islet cells, free from cell-to-cell interactions. This study was supported by grants from the Medical Research Council of New Zealand. D.W.H. was the recipient of a Novo Diabetes Research Scholarship.  相似文献   

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