Kinetic analysis of hybridoma growth and monoclonal antibody production in semicontinuous culture

Hybridoma I.13.17 was grown in semicontinuous culture in an attempt to investigate the steady-state concentrations of key components of monoclonal antibody (MAb) synthesis (e.g., intracellular MAb, IgG messenger RNAs) at different dilution rates between 0.008 and 0.055 h(-1). There was a general trend of increasing steady-state levels of total cytoplasmic RNA, total cell-associated MAb or cytoplasmic MAb, DNA synthesis rate, cellular metabolic activity, heavy (H-) and light (L-) chain IgG mRNAs with the increase in dilution rates. Increase in the half-lives of H- and L-chain mRNAs with increase in dilution rates may be sufficient to account for their increasing levels found under the same conditions. The specific growth rate was profoundly affected by the dilution rate, particularly near the lower end of the dilution rate range. Linear relationships were observed between the steady-state amounts of total cell-associated MAb and the relative levels of H- and L-chain mRNAs. Material balances on intracellular MAb demonstrated an increasing percentage of antibody not released into the growth medium (e.g., stored within the cell or anchored to the cell membrane) with increasing dilution rate. The MAb production rate per cell decreased significantly with the increase in dilution rates. No correlation was found between the relative levels of H- or L-chain mRNAs and the specific MAb production rate. Possible implications of rate-limiting steps in MAb synthesis and secretion are discussed.     

A semicontinuous culture model that links cell growth to extracellular nutrient concentration

During semicontinuous culture, a sample of fixed volume is removed at regular time intervals to make measurements and/or harvest culture components, and an equal volume of fresh medium is immediately added to the culture, thereby instantaneously enhancing nutrient concentrations and diluting cell concentration. The resulting cell concentration versus time curve (i.e., the actual cell growth curve) has a saw-toothed appearance because of the periodic dilution of cell concentration. The observed cell concentrations correspond to the peaks of the saw-toothed curve. Cell growth rates are estimated from the locus of observed cell concentrations (i.e., from the apparent growth curve obtained by connecting the peaks of the saw-toothed curve). The sole preexisting model (Fencl's mode) for estimating cell growth rate is valid only when the cells are growing exponentially at a constant rate between samplings. This model has limited validity: despite the periodic enhancement of nutrient concentration, cell growth between samplings eventually causes nutrient depletion, and the cells cease to grow exponentially. Failure to recognize the limits of validity for Fencl' model has resulted in many erroneous applications of the model and, consequently, many incorrect estimates of cell growth rates. To provide a means for correctly estimating cell growth rates, Fencl's exponential model was extended, and a new model that describes the effects of nutrient depletion on cell growth in semi-continuous culture was obtained. The new model shows that exhaustion of a single growth-limiting nutrient in semicontinuous culture causes the locus of cell concentrations observed at time intervals of Deltat to follow a logistic growth curve. The actual cell growth rate was shown to equal the apparent logistic growth rate plus the effective dilution rate -Deltat(-1) In (1 - f), where f is the ratio of sample volume to total culture volume. Moreover, the model predicts that both the apparent logistic growth rate and the apparent steady-state cell concentration should rise linearly with the concentration of growth-limiting nutrient in the input medium, but fall linearly with increases in the effective dilution rate. The new logistic model for nutrient-limited cell growth in semicontinuous culture was successfully tested using published data for Asterionella formosa, Cyclotella meneghiniana, Daucus carota, and strain L mouse cells.     

Vertical tubular photobioreactor for semicontinuous culture of Cyanobium sp

We evaluated the kinetic culture characteristics of the microalgae Cyanobium sp. grown in vertical tubular photobioreactor in semicontinuous mode. Cultivation was carried out in vertical tubular photobioreactor for 2 L, in 57 d, at 30 °C, 3200 Lux, and 12 h light/dark photoperiod. The maximum specific growth rate was found as 0.127 d⁻¹, when the culture had blend concentration of 1.0 g L⁻¹, renewal rate of 50%, and sodium bicarbonate concentration of 1.0 g L⁻¹. The maximum values of productivity (0.071 g L⁻¹ d⁻¹) and number of cycles (10) were observed in blend concentration of 1.0 g L⁻¹, renewal rate of 30%, and bicarbonate concentration of 1.0 g L⁻¹. The results showed the potential of semicontinuous cultivation of Cyanobium sp. in closed tubular bioreactor, combining factors such as blend concentration, renewal rate, and sodium bicarbonate concentration.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Fermentation by the human large intestine microbial community in an in vitro semicontinuous culture system.

A semicontinuous culture of the microbial community of the human large intestine that was maintained over 81 days is described. The initial inoculum was feces, and about 200 ml of nutrient suspension was fed to 500 ml of fermentor contents once or twice daily. The nutrient suspension contained comminuted fibrous food, sodium deoxycholate, urea, acid-hydrolyzed casein, vitamins, and salts. The fermentation was monitored, and the major products were acetate, propionate, butyrate, methane, hydrogen, and carbon dioxide. The concentration of anaerobic bacteria was 2 X 10(9) per ml of culture contents and was 100 times that of fecal coliforms. When the nutrient suspension contained lettuce, celery, carrots, and unsweetened applesauce, the predominant nonsporeforming anaerobes isolated were Bacteroides species. When carrots and applesauce were omitted, the predominant nonsporeforming isolates were Fusobacterium species. On both diets, clostridia were isolated that resembled Clostridium clostridiiforme. The fermentation and bacteriological analyses indicated that the in vitro ecosystem appears to be a reasonable facsimile of the large intestine ecosystem.     

The cell composition of Nannochloropsis sp. changes under different irradiances in semicontinuous culture

Nannochloropsis sp. was grown semicontinuously with a rate of daily renewal of the culture media of 40% of the volume of the culture under different irradiances (40, 60, 80, 220 and 480 mol quanta m^–2 s^–1). Under the conditions tested, light saturation was achieved at 220 mol quanta m^–2 s^–1 with no significant increase in steady-state cell density or of dry weight productivity with higher irradiance, reaching values of 115 × 10⁶ cells ml^–1 and 375 mg l^–1 day^–1 respectively. C/N ratios clearly indicated the point of light saturation, decreasing with increasing irradiance for light-limited conditions and increasing for light-saturated conditions. Under light-limited conditions, an increase in the irradiance produced an increase in the protein percentage of the organic fraction to the detriment of lipids and carbohydrates, while small changes were recorded under light-saturated conditions. The degree of unsaturation of fatty acids was lower with increasing irradiance, with a three-fold decrease of the percentage of total n–3 fatty acids, from 29 to 8% of total fatty acids, caused mainly by a decrease of eicosapentaenoic acid (EPA) (20:5n–3). The microalga reached its maximal value of dry weight productivity (375 mg l^–1 day^–1), EPA productivity (3.2 mg l^–1 day^–1) and maximal protein content (36% of the organic content) at the point at which light saturation was achieved. Results demonstrate the efficiency of the use of the irradiance for the modification of the biochemical composition of Nannochloropsis sp.     

In vivo metabolism of 5'-methylthioadenosine in lemna

Evidence is presented that Lemna converts 5′-methylthioadenosine (MTA) to methionine. The methylthio moiety and four of the ribose carbons of the nucleoside contribute the methylthio and the four-carbon moieties of methionine. Plants grown in the presence of inhibitors which block methionine biosynthesis convert MTA to methionine at a rate sufficient to sustain normal growth (at least 4.4 nanomoles per colony per doubling with a molar yield of at least 65%). The pathway for conversion is shown to be constitutive in plants grown in standard medium and to function at a rate sufficient to dispose of MTA arising as a result of polyamine synthesis, and to explain the observed rate (1.4 nanomoles per colony per doubling) of preferential recycling of methionine sulfur (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839). Rapid entry of methionine methyl into S-adenosylmethionine and phosphorylcholine was observed for plants grown in standard medium. Adenine generated during this cycle is efficiently salvaged into ADP and ATP.

Conversion of MTA to methionine completes the steps in methionine thiomethyl recycling (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839) in which the sulfur of methionine is retained while the four-carbon moiety is not. The findings further show that the four-carbon moiety of methionine can be derived via the ribose moiety of MTA in addition to the established route from O-phosphohomoserine via transsulfuration. Previous observations (Giovanelli, Mudd, Datko 1980 Biochemistry of Plants pp 453-505) can now be interpreted as establishing that exogenous methionine down-regulates its own net synthesis via the transsulfuration pathway.

     

Use of a semicontinuous culture system as a model for determining the role of human intestinal microflora in the metabolism of xenobiotics

The use of semicontinuous culture system for determining the role of the human intestinal microflora int he biotransformation of xenobiotics is discussed. This model system, which stimulates the lumen of the large intestine, has been used to investigate human intestinal microflora metabolism of compounds representative of three different classes of chemicals; a benzidine-based azo dye, Direct Black 38, a nitropolycyclic aromatic hydrocarbon, 1-nitropyrene and a substituted pyrimidine, 5-fluorocytosine. Metabolites of each of the test compounds were identified, and the kinetics of production and biological activity of the metabolites determined. Metabolic adaptation was observed with 1-nitropyrene and 5-fluorocytosine incubations. This microbial culture system could be quite useful, especially in concert with other in vitro models and animal studies, for determining the pharmacological and toxicological role of the human intestinal microflora in the transformation of xenobiotics. Particular emphasis on the application of this method for toxicological studies in elucidating the role of the intestinal microflora in the etiology of cancer is described.     

Growth kinetics of microorganisms with various ecological strategies in a dialysis culture at low specific growth rates

A dialysis culture was found to be most suitable for studying the metabolism of microorganisms at a low specific growth rate. The biomass of all microorganisms studied in the dialysis culture increased with time in a linear fashion; hence, the energy spent for growth decreased in proportion to a decrease in the specific growth rate. Microorganisms growing in oligotrophous environment (Arthrobacter globiformis and Lipomyces tetrasporus ) spent much less energy comparing to microorganisms from eutrophic habitats (Pseudomonas fluorescens and Debaryomyces formicarius ).     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture,clonal growth in low density culture,and stimulation to enter S phase in resting culture

Summary A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin transferrin epidermal growth factor, poly-d-lysine, bovine albumin, oleic acid, and bovine α-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetat bovine serum (FBS), and colonies, albeit of smaller sizes, diddform. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking α-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding α-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Altered synthesis of heparan sulfate proteoglycans at low sulfate concentration

The structural alterations in heparan sulfate produced by sulfate deprivation were studied in cell cultures of the Engelbreth-Holm-Swarm tumor. Tumor cells were labeled in vitro with [3H]glucosamine and/or [35S]sulfate in media containing either 300 microM MgSO4 or no added carrier sulfate, and the newly synthesized proteoglycans isolated by chromatography on DEAE-Sephacel. The proteoglycans isolated from low sulfate cultures showed a reduced affinity for the column eluting at lower salt concentrations compared with the proteoglycans isolated from cultures containing sulfate, suggesting that the former were undersulfated. Analysis of the isolated heparan sulfate side chains indicated that two pools of heparan sulfate were present which differed in their degree of sulfation. Both pools were synthesized by both high sulfate and low sulfate cultures, but the highly sulfated pool was the predominant form produced in sulfate containing cultures, while the undersulfated pool was the predominant form synthesized in low sulfate cultures. The more sulfated pool contained more N-sulfate than the less sulfated pool. Few if any free amino groups were detected in either pool, suggesting that the initial deacetylation step in the biosynthesis of heparan sulfate is tightly coupled to the N-sulfation step in the cells.     

Inhibition of enterotoxigenic Escherichia coli by the microflora of the porcine ileum, in an in vitro semicontinuous culture system

An in vitro fermentation system capable of maintaining at least part of the microbial population of the contents of the porcine ileum has been developed. The system was tested over the pH range 6.0-8.5, anaerobically and at dissolved oxygen concentrations within the ranges detected in the ileum of piglets at weaning (50 and 100 μmol 1^-1). The results demonstrated that changes in pH and dissolved oxygen within these ranges had relatively little effect on the total numbers of aerobic and anaerobic bacteria. Lactic acid bacteria, enumerated anaerobically, showed changes in viable counts in response to pH changes but were apparently unaffected by changes in dissolved oxygen, although the proportion of aerotolerant species within this group was increased at high concentrations of dissolved oxygen. The relative proportions of lactobacilli and coliforms were similar to those reported in vivo when dissolved oxygen was present at a concentration of 50 μmol 1^-1. Under these conditions the simulated population was consistently found to resist colonization by an enterotoxigenic Escherichia coli , of a serotype known to cause weaning scours in the piglet.     

Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor

The microalga incorporated photobioreactor is a highly efficient biological system for converting CO2 into biomass. Using microalgal photobioreactor as CO2 mitigation system is a practical approach for elimination of waste gas from the CO2 emission. In this study, the marine microalga Chlorella sp. was cultured in a photobioreactor to assess biomass, lipid productivity and CO2 reduction. We also determined the effects of cell density and CO2 concentration on the growth of Chlorella sp. During an 8-day interval cultures in the semicontinuous cultivation, the specific growth rate and biomass of Chlorella sp. cultures in the conditions aerated 2-15% CO2 were 0.58-0.66 d(-1) and 0.76-0.87 gL(-1), respectively. At CO2 concentrations of 2%, 5%, 10% and 15%, the rate of CO2 reduction was 0.261, 0.316, 0.466 and 0.573 gh(-1), and efficiency of CO2 removal was 58%, 27%, 20% and 16%, respectively. The efficiency of CO2 removal was similar in the single photobioreactor and in the six-parallel photobioreactor. However, CO2 reduction, production of biomass, and production of lipid were six times greater in the six-parallel photobioreactor than those in the single photobioreactor. In conclusion, inhibition of microalgal growth cultured in the system with high CO2 (10-15%) aeration could be overcome via a high-density culture of microalgal inoculum that was adapted to 2% CO2. Moreover, biological reduction of CO2 in the established system could be parallely increased using the photobioreactor consisting of multiple units.     

Isolation and characterization of low sulfated heparan sulfate sequences with affinity for lipoprotein lipase

Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains.     

Enzymes of phosphatidylcholine synthesis in lemna, soybean, and carrot

Cell-free extracts from Lemna and suspension cultured carrot (Daucus Carota L.) catalyze S-adenosylmethionine-dependent N-methylations of phosphoethanolamine, phosphomethylethanolamine, and phosphodimethylethanolamine; extracts of suspension cultured soybean (Glycine max), of phosphoethanolamine only. Material pelleted from each tissue between 15,000 and 100,000g catalyzes S-adenosylmethionine-dependent N-methylations of phosphatidylmethylethanolamine and phosphatidyl-dimethylethanolamine, but not phosphatidylethanolamine. Extracts from each tissue catalyze CTP-dependent cytidylyltransfers to each of the three methylated phosphoethanolamine derivatives, forming the corresponding CDP derivatives. Some of the properties of the activities investigated are reported. On the basis of in vivo labeling experiments, we have proposed (AH Datko, SH Mudd 1988 Plant Physiol 88: 854-861) differing pathways for phosphatidylcholine synthesis in which, after a common committing step, N-methylation of phosphoethanolamine, subsequent methylations occur in Lemna almost exclusively at the phospho-base level; in soybean, at the phosphatidyl-base level; and in carrot, at both levels. Thus, among the activities investigated, at least those required for the operation of the proposed pathways have been positively demonstrated. The extent to which the present results explain the differences between these pathways is discussed, and a speculation offered as to how these differences may have arisen phylogenetically.     

Synthesis of methylated ethanolamine moieties: regulation by choline in lemna

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[(3)H(3)C]methionine, l-[(14)CH(3)]methionine, or [1,2-(14)C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.