Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors.

A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase. Intracellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.     

The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures

Flow cytometric (FC) analysis was applied to determine changes at cellular level during the cultivation of hybridoma cell line MN12 in a suspension batch culture. The relative cell size, cytoplasmic and membrane IgG content and the viability were monitored. Besides, the specificity of the cytoplasmic and membrane IgG was ascertained by means of a synthetic peptide containing the antigenic epitope recognized by the antibody. Cell size was found to increase during the exponential growth phase. The viability as determined by FC follows a similar pattern with the viability data obtained by the conventional trypan blue exclusion test. The relative cytoplasmic and membrane IgG contents were high during the exponential growth and low during stationary phase. Measurement of cell cycle distribution and the antibody content in the culture fluid, indicated that the major part of the cytoplasmic IgG is secreted by cells in the G1-phase. It is concluded that flow cytometry is a useful tool to characterize hybridoma cell lines in a suspension batch culture.     

Cell size, macromolecular composition, and O2 consumption during agitated cultivation of Naegleria gruberi.

Cell size, macromolecular composition, carbohydrate utilization patterns, and O2 concentrations were measured throughout the growth stages of Naegleria gruberi in agitated culture in a complex medium. Biphasic logarithmic growth occurred during the intial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 X 10(6) amebae/ml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increase 31% to 35% per ameba at the end of each phase of log growth. DNA increased approximately 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O2 tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[¹⁴C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.     

Growth-related fluctuation in messenger RNA utilization in animal cells

Monkey fibroblasts maintained in culture regulate their levels of intracellular protein throughout the growth cycle by means of variations in the rate of protein biosynthesis. Cytoplasmic mRNA in stationary phase cells was compared to that in exponential phase cells. In stationary phase cells 56% of the cytoplasmic polyadenylated RNA was found in the 40--90S postpolysomal region of sucrose sedimentation gradients, while only 23% was found in this region in exponential phase cells. Analysis of electron micrographs of sectioned exponential and stationary phase cells revealed that this shift in polyadenylated RNA location is accompanied by a loss of polysome-like aggregates of ribosomes. Most if not all of this species of postpolysomal polyadenylated RNA is not being translated by single ribosomes since no detectable amounts of nascent peptide were present in this region. This nonpolysomal polyadenylated RNA is comparable in size to polysomal polyadenylated RNA. The length of the 3'-poly(A) tract was also comparable for these two species. The extent of capping of poly(A)- containing molecules was also comparable for these two species. The template activity of nonpolysomal RNA in a wheat germ extract was comparable to that of polysomal RNA. The peptides produced by these two preparations were of a similar large size. Furthermore, most of the nonpolysomal polyadenylated RNA of stationary phase cells was driven into polysomes in the presence of a low dose of cycloheximide. Therefore, we conclude that the untranslated mRNA that accumulates in stationary phase cells is structurally intact, is fully capable of being translated, and is not being translated due to the operation of a translational initiation block.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Cell Size,Macromolecular Composition,and O2 Consumption During Agitated Cultivation of Naegleria gruberi*

SYNOPSIS. Cell size, macromolecular composition, carbohydrate utilization patterns, and O₂ concentrations were measured throughout the growth stages of Naegleria gruberi in agitated cultures in a complex medium. Biphasic logarithmic growth occurred during the initial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 × 10* amebaeJml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increased 31% to 35% per ameba at the end of each phase of log growth. DNA increased ～ 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O₂ tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

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.     

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.     

Growth kinetics under adenine-limiting conditions of Bacillus subtilis KYA 741, an adenine-requiring strain

The growth kinetics of Bacillus subtilis KYA 741, an adenine-requiring strain, was investigated under adenine-limiting conditions. The concentration of adenine (the limiting substrate for cell growth) in the culture filtrate remained constant during the stationary phase. In this phase, DNA turnover was active and the DNA content per cell was constant throughout the cultivation period. When cells were transferred to medium without adenine, the cell concentration began to decrease immediately and then reached a constant level due to the supply of adenine from lysing to growing cells. The rates of degradation of cells and DNA were both found to be 0.2 hr^?1. An equation for cell growth in this pseudostationary phase was obtained by combining Contois' equation, in which the apparent saturation constant was a function of the cell concentration, with a term for cell degradation. This equation satisfactorily expressed the feature of cell growth and adenine consumption by B. subtilis KYA 741 under adenine-limiting conditions.     

Transient gene expression in CHO cells monitored with automated flow cytometry

Transient gene expression is frequently used in industry to rapidly generate usable quantities of a protein from cultured cells. In gene therapy applications it is used to express a therapeutic protein in vivo. A quantitative assessment of the expression kinetics is important because it enables optimization and control of culture conditions for higher productivity. Previous experimental studies show a characteristic peak in average protein expression per cell after transfection followed by an exponential decrease of the expressed protein. Here, we show that the exponential decrease in single cell expression of enhanced Green Fluorescent Protein (eGfp) occurs in discrete steps. We attribute this to the absence of plasmid replication and to symmetric partitioning of plasmid and eGfp between dividing cells. This is reflected in the total eGfp in the bioreactor, which increased at a constant rate throughout the experiment. Additionally, the data provide a detailed time course of cell physiology during recovery from electroporation. The time course of cell physiology precisely indicates when the culture shifts growth phases. Furthermore, the data indicate two unique stationary phases. One type of stationary phase occurs when proliferation ceases while cells decrease their cell size, maintain granularity, and mean eGfp content decreases. The second type occurs when proliferation ceases while cells increase their cell size, increase granularity, and surprisingly maintain eGfp content. The collected data demonstrate the utility of automated flow cytometry for unique bioreactor monitoring and control capabilities in accordance with the US Food and Drug Administration’s Process Analytical Technology initiative.     

Cell Size,Macromolecule Composition,Nuclear Number,Oxygen Consumption and Cyst Formation During Two Growth Phases in Unagitated Cultures of Acanthamoeba castellanii1

SYNOPSIS. The size, composition and physiology of average cells have been studied in cultures of Acanthamoeba castellanii during the phases of logarithmic growth and population growth deceleration (PGD). Most of the features examined were relatively constant during log phase, but had significant changes during PGD. Average cell volume increased about 60% and total dry mass about 15–20% during the latter period. Total protein content remained constant thruout both growth phases, but cytochrome oxidase doubled during PGD. DNA, RNA and glycogen levels began to change during late log phase. DNA decreased about 50% and RNA increased about 75%. Glycogen decreased 50% during the RNA build-up and then increased to a plateau above the log phase level. A final decrease in glycogen followed an increase in the relative numbers of cysts in late PGD. It was found that PGD begins when O₂ becomes limiting and evidence that the subsequent changes in macromolecule composition are related to encystation is discussed.     

Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells

Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.     

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Regulation of synthesis of a brain-specific protein in monolayer cultures of clonal rat glial cells

C6 cells were grown in monolayer culture under conditions permitting continued exponential cell division after attainment of a density at which extensive intercellular contacts were formed. An increase in the relative synthesis of S100 protein coincided with the time of formation of extensive intercellular contacts and preceded the onset of the stationary phase of growth by three generations. These observations suggested that the induction of S100 protein synthesis was mediated by cell contact and not by an arrest of cellular growth. The mechanism of this induction was first studied in a homologous non-initiating cell-free protein-synthesizing system from C6 cells, using fixed amounts of free amino acids or fully charged rat liver aminoacyl-tRNA as a source of precursors for protein synthesis. Real synthesis of total soluble proteins decreased as the cells progressed from logarithmic to stationary growth while synthesis of S100 protein increased during this period. The capacity of poly(A)+ RNA from logarithmic and stationary cultures to direct the synthesis of S100 protein was estimated in a cell-free protein-synthesizing system derived from wheat embryos. Increased synthesis of S100 protein in stationary cultures was directly correlated with an increase in translatable S100 protein mRNA.     

Studies on macronuclear DNA from Paramecium aurelia

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.     

Effect of light quality on the growth and proximal composition of <Emphasis Type="Italic">Amphora</Emphasis> sp.

We determined the effects of various light spectra (white, blue, green, yellow, and Grolux) on the growth rate, proximate composition, pigment content, and cell size of the marine benthic diatom Amphora sp. during two growth phases of batch cultures. The growth rate was higher under green light and lowest with Grolux and yellow light. Cell size differed significantly between growth phases but remained unaffected by light spectra; the smallest cells were observed on the initial day of culture, whereas larger cells developed in the stationary phase under Grolux treatment. The proximate composition was modified by the light spectra and growth phase. In the exponential growth phase, the protein content was higher with yellow and white light, and lipid content peaked with Grolux. The pigment content (chlorophyll a, carotenoids) was also higher under yellow light. In the stationary growth phase, we noted a higher carbohydrate content under Grolux and yellow light. Our results show that light spectra and growth phase influence the metabolism of Amphora sp., changing its proximate composition, pigment content, growth rate, and cell size.