ISSN:0006-3592    

DOI:10.1002/bit.260320802

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractHybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale‐up procedures. An SP2/0‐derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model1at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth‐associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1–7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional

ISSN:0006-3592    

DOI:10.1002/bit.260320803

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractNuclear magnetic resonance (NMR) spectroscopic analysis of whole cells is an important emerging technique for noninvasive and nondestructive monitoring of cell physiology. However, this technique requires extremely high cell densities. Attempts to maintain densities above the carrying capacity of a maintenance system result in the demise of the entire culture. To define conditions for maintaining mammalian cells at high densities for NMR studies, we have designed a bioreactor to operate under defined, oxygen‐limited conditions within an NMR spectrometer. The bioreactor utilizes hollow fibers to deliver nutrients and remove wastes from an agitated cell suspension. The mass transfer properties of the fibers with respect to oxygen were determined. Ehrlich Ascites Tumor (EAT) cells were supplied with glutamine as the respiratory carbon source. The maximum viable cell density supported by a given oxygen concentration in the fluid flowing through the fiber lumen was predicted and then confirmed experimentally on the bench and in the spectromete

ISSN:0006-3592    

DOI:10.1002/bit.260320804

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractResults are presented which show how the microcarrier concentration affects the hydrodynamic environment in animal cell bioreactors. At low levels of agitation, no physical effects of microcarrier concentration were found. However, cell growth was strongly influenced by cell concentration. At high levels of agitation, a strong detrimental effect of microcarrier concentration was found. A new mechanism of hydrodynamic damage was identified which is second order in microcarrier concentration. The identification of this mechanism adds to the fundamental understanding of hydrodynamic phenomena in microcarrier bioreactors.

ISSN:0006-3592    

DOI:10.1002/bit.260320805

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractA high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 × 108viable cells/mL in the extracapillary space—corresponding to an overall reactor density of 7 × 107cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later sta

ISSN:0006-3592    

DOI:10.1002/bit.260320806

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractMonoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300‐L scale. Suspension culture offers some advantages relative to high‐density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on‐line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 × 106cells/mL without causing apparent cell

ISSN:0006-3592    

DOI:10.1002/bit.260320807

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe negative effects of excessive agitation on tissue cells in microcarrier culture have often been ascribed to “shear.” Analysis of the fluid mechanics occurring suggests that there are actually three potential damage mechanisms: collisions of a cell‐covered microcarrier with other beads, collisions with parts of the reactor (primarily the impeller), and interaction with turbulent eddies the size of the microcarrier beads. Review of the available quantitative information on agitation effects in cell cultures does not establish which mechanism is predominant; the range of experimental variables reported emphasizes power input over the other reactor and impeller parameters. The bead–bead collision model is tentatively supported by the available data, but the other mechanisms may still be significant in some systems. The formation of bead aggregates by cellular bridging provides a parallel means of damaging cells. Breaking of these bridges by any of the three means identified earlier can cause cell destruction and/or the net transfer of cells to formerly bare beads. High concentrations of bridges are favored by lower agitation rates, presumably because the bridges are not as quickly destroyed after fo

ISSN:0006-3592    

DOI:10.1002/bit.260320808

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum‐free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum‐free formulations and selection rules for the serum‐free ingredients. In general, serum‐free media which are “lipid rich” can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum‐free media, irrespective of the

ISSN:0006-3592    

DOI:10.1002/bit.260320809

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractA stirred bath technique was developed for determining effective diffusivities in cell matrices. The technique involves cell immobilization in a dilute gel which has negligible effect on solute diffusion. Agar and collagen were tested as immobilizing gels. Agar gel was shown to have minor interactions with the diffusion of various biological molecules, and was used for immobilization of Ehrlich Ascites Tumor (EAT) cells. Diffusivities of glucose and lactic acid were measured in EAT matrices for cell loadings between 20 and 45 vol %. Treatment with glutaraldehyde was effective in quenching the metabolic activity of the cells while preserving their physical properties and diffusive resistance. The measured data agree favorably with predictions based on Maxwell's equation for effective diffusion in a periodic composite material. The stirred bath technique is useful for diffusivity determinations in immobilized matrices or free slurries, and is applicable to both microbial and mammalian cell systems.

ISSN:0006-3592    

DOI:10.1002/bit.260320810

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY

摘要:

AbstractThe ability to serially propagate mammalian cells in microcarrier cultures is essential for large‐scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE‐derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin‐coated microcarriers. By prewashing the cell‐laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS‐4, and continuous human kidney cells, TCL‐598 which produces prourokinase, were viably dissociated from commercially available gelatin‐coated microcarriers, Cytodex‐3. Cells dissociated from microcarriers reattached and grew on micro‐carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS‐4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS‐4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL‐598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inocula

ISSN:0006-3592    

DOI:10.1002/bit.260320811

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1988

数据来源: WILEY