Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.     

Characterization of the ssnA gene, which is involved in the decline of cell viability at the beginning of stationary phase in Escherichia coli

When grown in rich medium, Escherichia coli exhibits a drastic reduction of the number of viable cells at the beginning of stationary phase. The decline of cell viability was retarded by disruption of the ssnA gene, which was identified as a gene subject to RpoS-dependent negative regulation. Moreover, ssnA expression was induced at the time of decline of cell viability at early stationary phase. The viability decline was augmented in the rpoS background, and this augmentation was suppressed by ssnA mutation. Cloning of the ssnA gene in a multicopy plasmid, pBR322, caused small colony formation and slow growth in liquid medium. Cells harboring the ssnA clone showed aberrant morphology that included enlarged and filamentous shapes. The gene product was identified as a 44-kDa soluble protein, but its function could not be deduced by homology searching. From these results, we conclude that ssnA is expressed in response to a phase-specific signal(s) and that its expression level is controlled by RpoS, by a mechanism which may contribute to determination of cell number in the stationary phase.     

Hormonal interactions in human prostate tumor LNCaP cells

Melatonin, the hormone secreted by the pineal gland at night, has recently been found to attenuate growth and viability of benign human prostate epithelial cells. Estradiol suppressed these responses by efflecting a protein kinase C mediated inactivation of melatonin receptors. In the present study, the effects of melatonin on growth and viability of the human androgen-sensitive prostatic tumor cell line-LNCaP and the influence of estradiol on these responses were explored. Melatonin inhibited 3H-thymidine incorporation into LNCaP cells at physiological concentrations. This response decayed within 24 h. The inactivation of the response slowed down in the presence of the protein kinase C inhibitor GF-109203X. Estradiol also inhibited 3H-thymidine incorporation and its effects were additive to those of melatonin. Suppression of DNA content was observed in cells treated for 2 days with melatonin (0.1 nM); this suppression was maintained for longer periods in the presence than in the absence of estradiol. In addition, estradiol and melatonin slightly and additively decreased cell viability. These results demonstrate for the first time a direct interaction of melatonin with androgen-sensitive prostate tumor cells leading to attenuation of cell growth. They also show that unlike in benign prostate epithelial cells, estrogen attenuates LNCaP cell growth and supports rather than inactivates melatonin's action.     

Strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies

Summary Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1×10⁶ cells/ml. Concentrations of monoclonal antibody (MAb) reached 140 mg/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during active exponential growth and not during the stationary phase.     

Proteasome synthesis and assembly are required for survival during stationary phase

We examined the alterations in 20S proteasome homeostasis, protein oxidation, and cell viability that occur during the stationary phase or chronological model of yeast aging. Data in this report demonstrate that proteasome subunit expression is increased, proteasome composition is altered, and levels of individual proteasome proteolytic activities are elevated during stationary phase-induced aging in Saccharomyces cerevisiae. Despite such alterations, a progressive loss of proteasome-mediated protein degradation and a significant increase in protein oxidation were observed in cells maintained under stationary phase conditions. Deletion of UMP1, a gene necessary for 20S proteasome biogenesis, had no effect on cellular viability under normal growth conditions, but impaired the ability of cells to survive under stationary phase conditions. During stationary phase, the levels of oxidized protein increased more rapidly and to higher levels in the mutant lacking UMP1 than in the wild-type cells. Taken together, these data implicate a role for proteasome synthesis and altered 20S proteasome composition in maintaining viability during stationary phase, and demonstrate that even with these modifications a gradual loss of proteasome-mediated protein degradation occurs during stationary phase-induced aging. These data also suggest a role for impaired proteasome-mediated protein degradation in increased protein oxidation and cell death observed during the aging of eukaryotic cells.     

The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture.

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5- to 8-fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T-59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.     

The production of monoclonal antibody in growth-arrested hybridomas cultivated in suspension and immobilized modes.

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.     

Effect of process parameters and product-host-interaction on hVEGFA-production by recombinant Chinese hamster ovary cells

A potential producer clone was identified among recombinant, human vascular endothelial growth factor A (hVEGFA)-producing Chinese Hamster Ovary (CHO) K1 cells, using a recently established screening method. In batch spinner cultivations, the cells showed a maximum growth rate of 0.045 h(-1), a final total cell density of 5.3×10(6) mL(-1) (living cell density: 3.4×10(6) mL(-1)), and a final hVEGFA concentration of 207 μg L(-1). Living cell density and productivity in the spinner cultivations could be increased by glutamine feeding. Transfer of the process to the bioreactor (batch mode, control of pH, T, and O2) resulted in a reduction of the growth rate by roughly 50%, while overall living cell density and productivity increased, largely due to an extension of the production phase. When the bioreactor was run in the fed-batch mode, growth rates were further reduced, while productivity and living cell densities reached a maximum (hVEGFA: 358 μg L(-1), cells: 5.2×10(6) mL(-1)). In addition, the death rate of the hVEGFA-producing cells was considerably reduced compared with the parent cell line, most likely due to product-host-interaction. This hypothesis was corroborated when a second recombinant CHO cell line (antibody producer) was transfected with the hVEGFA gene and afterward consistently showed higher viable cell densities together with a significantly improved antibody titer.     

Colony dimorphism associated with stress resistance in Oenococcus oeni VP01 cells during stationary growth phase

The resistance to stresses as starvation, the presence of ethanol, sulfite and low pH, is a fundamental prerequisite for starter cultures used to induce malolactic fermentation in wine. In order to evaluate stress resistance of cells undergone starvation, cells viability in laboratory cultures of Oenococcus oeni VP01 strain was monitored during prolonged stationary growth phase. Once entered the stationary phase, strain VP01 showed 99% reduction of cell viability within 4 days. The remaining cells population maintained viability over 70 days and, when plated on agar medium, generated small colonies. The occurrence of this phenomenon was associated to stress resistance, since 10-day-old cells resulted more resistant than 3-day-old cells to ethanol and low pH conditions. No genomic mutations were revealed by pulse-field gel electrophoresis (PFGE) analysis in aged cultures. Total protein analysis by bidimensional electrophoresis highlighted differential protein expression in cultures differentially aged. It was demonstrated that O. oeni starving cultures at the stationary phase are constituted by dynamic cell populations. These results offer interesting perspective for a better understanding of cells behavior when inoculated in wine.     

Cultivation of fall armyworm ovary cells in simulated microgravity

Summary A methodology is presented to culture Fall Armyworm Ovary cells in simulated microgravity using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 × 10⁷ viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm² in shaker culture to under 0.5 dyne/cm² in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Effect of trisodium citrate treatment on hybridoma cell viability

Trisodium citrate has been widely used to dissolve calcium alginate beads in order to estimate cell concentration in the beads. To obtain an accurate measurement of viable cell concentration in calcium alginate beads, the effect of trisodium citrate solutions on hybridoma cell viability was studied with regard to stage of growth and trisodium citrate concentration. The cells in the decline phase of growth were more sensitive to 30 minutes of trisodium citrate treatment than the cells in the exponential phase of growth. The cell viability did not decrease rapidly during citrate treatment regardless of cell growth phase and trisodium citrate concentration in the range of 1–1.5%. By using the commercially available sodium alginate, Keltone LV, dissolution time can be kept short enough (below 5 minutes) to keep the effect of trisodium citrate negligible to cell viability.     

Maintaining high anaerobic succinic acid productivity by product removal

During dual-phase fermentations using Escherichia coli engineered for succinic acid production, the productivity and viable cell concentration decrease as the concentration of succinic acid increases. The effects of succinic acid on the fermentation kinetics, yield, and cell viability were investigated by resuspending cells in fresh media after selected fermentation times. The cellular succinic acid productivity could be restored, but cell viability continuously decreased throughout the fermentations by up to 80% and subsequently the volumetric productivity was reduced. Omitting complex nutrients in the resuspension media had no significant effect on cellular succinate productivity and yield, although the viable cell concentration and thus the volumetric productivity was reduced by approximately 20%. By resuspending the cells, the amount of succinate produced during a 100-h fermentation was increased by more than 60%. The results demonstrate that by product removal succinic acid productivity can be maintained at high levels for extended periods of time.     

Reaction of Chinese hamster cell culture to the cytotoxic action of imuran in the early and late stationary growth phases

It has been shown that when cells are incubated for a long time without medium change, imuran exerts a much greater effect on the viability of cells in the late stationary phase of growth than in the early stationary phase. When nutrient medium supplemented with 10% serum was changed for the medium with 0.5% serum, or when a partial change of medium (one third of the volume) was carried out daily from day 4, these differences were only slightly pronounced. Thus, the degree of cell response to imuran in the early and late stationary phases of growth depends upon the way of cell maintaining in the resting state.     

Decrease in cell viability in an RMF,sigma(38), and OmpC triple mutant of Escherichia coli

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.