Step-fortifications of nutrients in mammalian cell culture

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated. (c) 1993 John Wiley & Sons, Inc.     

Descriptive parameter evaluation in mammalian cell culture

Several methods exist for assessing population growth and protein productivity in mammalian cell culture. These methods were critically examined here, based on experiments with two hybridoma cell lines. It is shown that mammalian cell culture parameters must be evaluated on the same basis. In batch culture mode most data is obtained on a cumulative basis (protein product titre, substrate concentration, metabolic byproduct concentration). A simple numerical integration technique can be employed to convert cell concentration data to a cumulative basis (cell-hours). The hybridoma lines used in this study included a nutritionally non-fastidious line producing low levels of MAb and a nutritionally fastidious hybridoma with high productivity. In both cases the cell-hour approach was the most appropriate means of expressing the relationship between protein productivity and cell population dynamics. The cell-hour approach could be used as the basis for all metabolic population parameter evaluations. This method has the potential to be used successfully for both prediction and optimization purposes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Metabolic flux analysis in mammalian cell culture

Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while ¹³C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale ¹³C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.     

Design of a large-scale mammalian cell,suspension culture facility

The design of a suspension culture facility capable of producing approximately 10¹² cells per week has been developed on a small-scale system which has evolved from various architectural, engineering, biological, and biohazard considerations. The smaller system is composed of spinner flasks (50 ml to 8 liters) modified for semicontinuous culture conditions, metal reservoirs, a continuous flow centrifuge, and supportive equipment. The large system which is under construction is composed of metallic vessels of up to 500 liter working volume with hard plumbing, monitors, controllers, recorders, continuous flow centrifuge and other ancillary equipment. This system begins with medium preparation and ends with harvesting of cells and disposition of supernatant. The design of this turn-key operation was developed over a two and one-half year period through the cooperation of private industry, the federal government, and the academic community.     

The effect of alkaline pH on the cell growth of six different mammalian cells in tissue culture

The effect of high alkaline pH on the reinitiation of cell growth was studied in six different mammalian cells. We failed to confirm the observation of Zetterberg & Engström, Proc natl acad sci US 78 (1981) 4334 [17] and Exp cell res 144 (1983) 199 [18]. Treatment of quiescent cells at pH 9.5 did not stimulate cell growth when measured by total protein/flask or increase in cell number.     

Fate of juvenile hormone in mammalian cell culture.

The metabolism of juvenile hormone (JH) I has been examined in fetal mouse liver cells maintained in culture. Diffusion of the hormone into the cells appears to be passive. The hormone is metabolized essentially to organic-soluble metabolites (diol ester, diol acid and acid) by the action of epoxide hydrase and carboxylesterases. Conjugative reactions play a minor role, less than 3% of the hormone being excreted as conjugates (glucuronides, sulfates and mercapturic acid). About 0.8% of the cellular radioactivity is bound to macromolecules, mainly those of nuclear and mitochondrial origin. Metyrapone and SKF 525-A inhibit covalent binding of the hormone to cytoplasmic macromolecules, which suggests participation of the cytochrome P-450 system in covalent binding of the hormone.     

Population-based modeling of the progression of apoptosis in mammalian cell culture

The production of biopharmaceuticals from mammalian cell culture is hindered by apoptosis, which is the primary cause of cell death in these cultures. As a tool for optimization of culture yield, this study presents a population-based model describing the progression of apoptosis in a monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell culture. Because mAb production does not cease when apoptosis begins, the model was designed to incorporate subpopulations at various stages in the progression of apoptosis. The model was validated against intracellular measurements of caspase activity as well as cell density, nutrient levels, and toxic metabolites. Since the specific details of apoptotic mechanisms have not been elucidated in this cell line, we employed a model comparison analysis that suggests the most plausible pathways of activation.     

A mammalian cell culture collection for biotechnology

Conclusion Culture collections are key to the success of biotechnology companies. Protection of patent strains and manufacturing inoculum; standardization of biological materials for research, development and manufacturing; and documentation of organism transfers are essential functions provided by a culture collection in a biotechnology company. Certified, stable mammalian cell cultures will continue to be key in research advances and in manufacturing of biotechnology products in the future.     

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.     

A pilot plant for mammalian cell culture

Studies of the possible viral etiology of human leukemia have required large quantities of cultured cells derived from human hematopoietic tissues. Since cultures sufficiently large and free from contamination could not readily be produced according to existing methods, a pilot, cell culture plant has been constructed for the production of mammalian cells in mass quantity. 500-ml to 20-liter trophocell units have already proved to be scientifically and economically practical, as they provide good reliability, excellent growth rates, and sustained yield of human cells. 200-liter stainless steel culture units have now been added to the trophocell system. Five complete 200 liter units are now in operation. The design of the original stainless steel unit was based on that of a stainless steel, jacketed soup kettle. There are no openings in the vessel other than those in the lid, which provide convenient access points for sampling, sensor probes, etc. Environmental parameters, e.g., liquid level, temperature, and pH, are monitored and controlled with commercially available apparatus. Many initial problems connected with the new 200 liter units have been resolved, but operational and design problems remain in the areas of stable instrumentation, cell harvesting, salvaging and reuse of unspent media components, establishment of physiologic steady stale, recovery of virus-containing cells with reculture of the remaining unaffected cells, and the recovery and separation of cell components and special products such as immunoglobulins, interferons, and hormones. A definitive cell plant with culture units of 20, 50, 250, and 1250 liters is now being constructed.     

A cartridge oxygenator for mammalian cell culture in a stirred tank bioreactor

Summary Tubing made of membrane with high oxygen permeability is often used in supplying oxygen to animal cell culture bioreactors. We have fabricated the tubing into a cartridge configuration. Such an arrangement allows damaged tubing to be replaced conveniently and eases the maintenance of such an oxygenator in bioreactors.     

Depression of the radioprotective effect of isoproterenol on mammalian cells in vitro after desensitization of the cAMP system

A short (5 min) incubation of cultured Chinese hamster fibroblasts with the specific beta-agonist isoproterenol (1 microM) leads to an increase in the intracellular content of cAMP and a decrease in radiosensitivity of the cells. Prolonged (up to 1 h) incubation induces a desensitization of the cAMP system to isoproterenol and causes a decrease both in the cAMP-stimulating and radioprotective effect of isoproterenol. There were no detectable changes in the beta-adrenoreceptor number or binding affinity of beta-receptors to the radiolabelled beta-antagonist dihydroalprenolol in desensitized cells; cAMP-phosphodiesterase activity was also the same as in intact cells. It is proposed that a 1 h incubation of the cells with isoproterenol induces the first step of desensitization, i.e. the functional "uncoupling' of beta-receptors from adenylate cyclase. Thus, the presence of beta-receptors in cells is not enough for the realization of the radioprotective potency of isoproterenol; an intact, non-desensitized, state of the cAMP system is obligatory.     

大规模动物细胞培养的问题及对策

大规模动物细胞培养在生物技术产业化进程中显示出强大的潜力。本文综述了大规模动物细胞培养过程中出现的问题及其解决办法 ,包括细胞培养环境、基因工程途径改建细胞系及过程监控等。对于这些进展的充分了解对优化细胞培养工艺、提高产品质量具有重要意义     

Beta-adrenergic modulation of the polymorphonuclear leukocyte respiratory burst is dependent upon the mechanism of cell activation

Although inhibition of polymorphonuclear leukocyte activation by beta-adrenoceptor agonists has been recognized for over a decade, effects have only been observed at high drug concentrations and in the presence of theophylline. In this study, catecholamine and prostaglandin modulation of the respiratory burst was evaluated with respect to the mechanism of polymorphonuclear leukocyte activation. Very low concentrations of isoproterenol and prostaglandin E2 inhibited the respiratory burst when induced by chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) or calcium ionophore (A23187, ionomycin), but not when initiated by synthetic diacylglycerol. Because formyl-methionyl-leucyl-phenylalanine and ionophore mobilize calcium and arachidonic acid generation follows an increase in intracellular calcium, the arachidonic acid metabolite leukotriene B4 was studied. Isoproterenol at a very low (0.1 nM) concentration also rapidly inhibited leukotriene B4 generation. Since cyclic AMP was increased by isoproterenol regardless of the means of cell activation, modulation of intracellular calcium was evaluated with the fluorescent probe indo-1. A transient increase in calcium after formyl-methionyl-leucyl-phenylalanine or ionophore (but not oleoyl acetylglycerol) cell activation was inhibited by isoproterenol or prostaglandin E2. These results suggest that adrenergic agonists specifically modulate calcium-dependent polymorphonuclear leukocyte function. Because marked inhibition was observed at very low drug concentrations, cyclic AMP-dependent effects may be important in both homeostatic and therapeutic modulation of inflammatory response.