Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.     

Influence of amino acids on hybridoma cell viability and antibody secretion

It is generally accepted that the phase of cell decline observed in batch culture of mammalian cells is related to exhaustion of medium nutrients (principally glucose and glutamine) and/or to waste products accumulation. In the present paper, we have studied the influence of glutamine on the proliferation of mouse hybridoma cells. We showed that repeated addition of glutamine prolonged the life span of the culture and significantly increased the secretion of monoclonal antibody. Flow cytometry analysis suggests that this effect of glutamine is related to a delay in cell death rather than to a stimulation of proliferation.Addition of glutamine and glucose failed however to prevent the death of the culture. Determinations of amino acid consumption in glutamine-supplemented samples and experiments carried out with complementary sources of amino acids (e.g. tryptose phosphate) strongly suggest that amino acid supply is a critical factor governing cell growth and productivity.     

Avoiding rapid growth at high cell densities: A potentially important optimisation criterion for hybridoma cultures

Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002–0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4×10⁹ cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.     

A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells

A three compartment model (ER --> Golgi --> extracellular medium) is used here to describe the interorganelle transport and final secretion of an IgG(2a) monoclonal antibody (MAb) in 9.2.27 murine hybridoma cells. Model simulations of pulse-chase and continuous labeling experiments are used to gain a better understanding of the kinetics of MAb interorganelle traffic. Simulation results for the continuous labeling case compare well with experimental data obtained during continuous labeling of 9.2.27 hybridoma cells. Incorporation of this compartmental transport model into our previously developed model of MAb synthesis and assembly can provide a useful tool for analyzing the dynamics and regulation of the complete antibody secretory pathway under different growth and/or nutritional conditions.     

Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures.

The kinetics of monoclonal antibody synthesis and secretion have been studied in synchronous and asynchronous mouse hybridoma cell cultures. Pulse-labelling of IgG followed by immunoprecipitation and quantitation of synthesized and secreted IgG in synchronous cultures show maximum production during G1/S phases. Secretion takes place through exocytotic release of vesicle contents. Pulse-chase experiments show that 71% of the synthesized IgG is secreted within 8 h of the pulsing period and only a further 4% is secreted by 22 h. Higher specific antibody production (QA) is obtained if (a) cells are arrested and then maintained in G1/S phases, (b) viability is decreased during the death phase of batch culture, (c) the dilution rate is decreased in continuous culture or (d) cells are subjected to hydrodynamically induced stress. The increase in QA in all these cases is mainly due to the passive release of the accumulated intracellular antibody. DNA and protein synthetic activity peak during the early exponential phase and decline rapidly during mid and late exponential and death phases. Metabolic activity however peaks up to 20 h after the peak in DNA synthesis, and declines similarly during the death phase. The data are consistent with the idea that slow growth and higher death rates increase QA and that Ig secretion is probably subject to complex intracellular control.     

Metabolic shift analysis at high cell densities

Abstract: In high cell density cultures it is virtually inevitable that the environment to which the cells are exposed is heterogeneous. Thus, with suspended cultures, individual cells are subject to temporal changes in their environment whereas with aggregated or immobilized cells, the culture can be considered as being formed by a number of subpopulations, each with its own environmental characteristics. In addition, in a high cell density environment, high concentrations of end products may negatively influence the growth rate. This may result in the selection of organisms with an altered metabolic behaviour or with a decreased sensitivity to the adverse effects of the product. We discuss the consequences of this heterogeneity with regard to carbon source metabolism in view of the ability of many bacterial species to adapt to environmental conditions. Selection of variant organisms was found to occur with Clostridium butyricum when grown for a prolonged time in a medium containing approx. I-50 mM glucose. In contrast to the original strain, these variants could sustain a high maximal growth rate in the presence of butyric acid. In addition, they had acquired the capacity to spontaneously form aggregates and were able to carry out a completely solventogenic fermentation. Heterogeneous metabolic activity in aggregated cells is demonstrated with cultures of Lactobacillus laevolacticus , an aggregateforming lactic acid bacterium that converts glucose completely to o-lactate. By using microelectrodes, we show that the fraction of metabolically active cells decreases with increasing aggregate size: in larger aggregates steep pH gradients occur with the effect that only the outer layer of the aggregate is metabolically active, i.e. contributes to lactic acid formation, whereas with smaller aggregates all cells remain active. As a result, the net specific lactic acid production rate of the population as a whole is not invariably increased with increased aggregate size.     

Thymidine secretion by hybridoma and myeloma cells

Secretion of thymidine appeared to be a common property of hybridoma and myeloma cells, but not of other cell types, which were tested. Of three hybridoma cell lines tested, all secreted thymidine in amounts resulting in the accumulation of thymidine to concentrations of 10-20microM in the culture medium. Also three of five myeloma cell lines that were analyzed secrete thymidine, but none of the other cell types that were studied. Thymidine was purified to homogeneity (4mg purified from 3l of culture medium) and identified as such by nuclear magnetic resonance spectroscopy. The cells that secreted thymidine showed high resistance to the growth inhibitory effect of thymidine.     

In vitro culture of hybridoma cells in agarose beads producing antibody secretion for two weeks

A new process for embedding cells in agarose is described. Beads were obtained by extruding an ultralow gelling temperature agarose solution in a capillary containing a hydrophobic medium flowthrough. The toxicity of the procedure has been evaluated by monitoring the energy status of agarose-embedded C(6) glioma cells with (31)P nuclear magnetic resonance (NMR). Suspension and microbead cultures of hybridoma cell line were compared. In suspension culture the number of cells and the antibody concentrations increased for 5 days before the stationary phase began, when the cultures were stopped. In agarose bead cultures, the gel provided an enormous support surface area (50 m(2)/ mL of gel). It was possible to seed 20-fold more cells. The gel pressure modified the proliferative process and antibody pattern secretion. In particular, the antibodies could be harvested for two weeks.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.     

Protein synthesis and antibody production in hybridoma cells under hyperthermia

The relation between rates of protein synthesis and antibody production was studied for hybridoma cells treated at 42 degrees and 44 degrees C. Both the biosynthetic parameters were shown to recover after a mild heat shock at 42 degrees C with approximately the same kinetics. The treatment at 44 degrees C led to a full inhibition of Ig production, and the protein electrophoretic pattern was not recovered to normal state within 4-6 hours. The synthesis of heat shock proteins (HSP) was found only after the treatment at 42 degrees C. It is suggested that the expression of HSP is necessary for the recovery of hybridoma cell activities.     

Drosophila cells can be grown to high cell densities in a bioreactor

Summary Cell cultures of the fruitfly, Drosophila melanogaster, were grown in a bioreactor to high cell densities: 5.7×10⁷ cells/ml, in batch, semi-batch and continuous flow modes. Using a semi-batch culture mode 500g wet weight of cell mass was produced in 16 days, using 5 l of cheap, commercially available, serum-free medium.     

Inhibition of influenza-immune T cell effector function by virus-specific hybridoma antibody.

The in vitro activity of influenza-specific cytotoxic T cells can be inhibited by incubation of the target cells with monoclonal anti-influenza antibodies. Hybridoma antibodies that bind to the virus HA inhibit the cytotoxic activity of TDL for the virus-infected target by as much as 80%, whereas these same antibodies never reduce splenic T cell function by more than 40%. This reflects the fact that TDL from anti-influenza strain A/WSN/33 (HON1) are highly subtype-specific, whereas splenic effector cells from the same mice are cross-reactive for target cells infected with heterologous influenza A viruses. These findings are discussed in the light of previous failures to block virus-immune T cell effector function with heterogeneous antisera produced in vivo, and are considered to favor the idea that at least some of the "virus-immune" T cells are indeed recognizing viral antigens.     

Factors controlling cell proliferation and antibody production in mouse hybridoma cells: I. Influence of the amino acid supply

To investigate the influence of medium amino acid resources on hybridoma cell proliferation and monoclonal antibody secretion, we first determined, in two different cell lines, the pattern of amino acid consumption. We then showed that complementation of each cell line by an amino acid solution prepared to cover its needs led in both cases to a marked increase in cell density and monoclonal antibody production. This observation suggests that the amino acid supply is one of the factors limiting cell growth and productivity in typical batch processes, and we demonstrated that a similar limitation occurs in a semicontinuous culture. Finally, we showed that the cell decline observed after a period of amino acid deficiency is not irreversible and that proliferation could be resumed if the required amino acids were added.     

Monocytes as accessory cells in the human mixed lymphocyte response: Lack of requirement at high cell densities

One-way mixed lymphocyte responses (MLR) were observed in microtiter plates with round-bottomed, but not flat-bottomed, wells between human peripheral blood mononuclear cell populations depleted of monocytes. The response in flat-bottomed wells was reconstituted when either responder or stimulator monocytes were added or when the cell density was increased. Possible implications of these results concerning the monocyte requirements for generating an MLR are discussed.     

Determination of specific monoclonal antibody secretion rate during very slow hybridoma growth

A total cell recycling suspension perfusion reactor has been constructed for investigation of specific monoclonal antibody secretion rate of the 9.2.27 murine hybridoma under conditions of a very low growth rate. By rapidly recycling hybridoma cells using a thermostated tangential flow filter, 3.6 mg cell dry weight/cm³ could be maintained at growth rate of <0.05 _max for over 250 h. Under these conditions, secretion of lactate, ammonia and l-alanine were directly related to the rate of l-glutamine supply. Monoclonal antibody accumulated in the reactor to levels in excess of 1400 g/ cm³. Surprisingly, as specific growth rate decreased, the specific immunoglobulin secretion rate remained constant, implying that monoclonal antibody synthesis could be uncoupled from growth.List of Symbols CMF cm³/(min · cm²) cross membrane flow rate - D h^–1 dilution rate - DOT % air saturation dissolved oxygen tension - F _R cm³/min perfusion rate - GlcPR mg/min glucose provision rate - GlnPR mg/min l-glutamine provision rate - N _A mmoles O₂/(dm³ · h) oxygen transfer rate - q _ala mmoles/h l-alanine secretion rate - q _MAB mg MAB · 10^–8 viable cells ^–1 · day^–1 specific MAB secretion rate by viable cells - ¯q _MAB (dimensionless) ¯q _MAB/¯q _{MAB MAX} - ¯q _{NH 3} mmoles/h ammonia secretion rate - S _R mg/cm³ limiting substrate concentration - h^–1 specific growth rate - _app h^–1 apparent growth rate - ¯ (dimensionless) / _MAX - VC cells/cm³ viable cell number     

High cell density perfusion culture of hybridoma cells recycling high molecular weight components

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.