Hybridoma Growth Research Articles

Cytokines in normal and abnormal parturition: Elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection

The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pg/ml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AF from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p less than 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM.(ABSTRACT TRUNCATED AT 250 WORDS)

Iteration of hybridoma growth and productivity in hollow fiber bioreactors using 31P NMR

Applications of nuclear magnetic resonance (NMR) spectroscopy to isolated or cultured mammalian cells have been limited because of technical difficulties in maintaining cultures at the extremely high densities required by NMR. Among the well-engineered systems available for such analyses, hollow fiber bioreactors (HFBRs) can maintain the greatest cell density. This attribute of HFBRs makes them ideal for application to NMR-based studies. These systems are currently being applied in biotechnology, where they are used for the production of mammalian cell-derived products, such as monoclonal antibodies. In this paper, the application of a HFBR system designed especially for NMR-based investigations is described. Performance of this system is monitored by NMR and the resulting stability and density of hybridoma cultures are reported. The resulting signal-to-noise per unit time is the highest seen to date for a mammalian cell system.

The influence of temperature on a mouse–mouse hybridoma growth and monoclonal antibody production

The aim of that study was to determine conditions that potentially could prolong the time that the cells remained viable. Thus, high inoculum concentrations (1.5 × 10 6 cell/mL) were chosen which obviated cell growth characterization. In contrast, we examine the entire growth phase and MAb production at various temperatures in this study, in order to investigate the effect of temperature on MAb production.

Influence of inoculum age on hybridoma culture kinetics.

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.

Effect of the double-stranded polynucleotide complex polyadenylate-polyuridylate (Poly A-U) on interleukin-6 production by mouse fibroblasts

Cultures of mouse embryonic fibroblasts (L 929) have been shown to produce a factor which promotes the growth of B cell hybridoma (hybridoma growth factor, HGF) i.e, interleukin 6 (IL-6). The aim of the present study was to investigate the effect of Poly A-U on IL-6 production by this cell type. After incubation for 48 h at 37°C of confluent (1 week old) L 929 fibroblasts in the presence or in the absence of Poly A-U, IL-6-like activity in supernatants was measured by the proliferation assay of the IL-6-dependent B cell hybridoma cell line, 7TD1. Poly A-U increased IL-6 activity in supernatants in a dose-dependent manner at doses higher than 50 μg/ml, the maximum activity being observed at the highest concentration of Poly A-U used, i.e. 500 μg/ml. β Interleukin-1 (β IL-1) and poly-cytidylic-polyinosinic (Poly I-C) have been shown to be inducers of IL-6 in fibroblast culture and thus their effect was compared to that of Poly A-U. The IL-6 activity in supernatants induced by 500 μg/ml Poly I-C (58.4 ± 16.4 U/ml; n = 4) was higher than that evoked by 100 U/ml β IL-1 (5.7 ± 0.4 U/ml) or 500 μg/ml Poly A-U (39.6 ± 7.8 U/ml). The increased production of IL-6 by Poly A-U may explain part of its previously re[orted immunomodulatory effects.

Activation of Monocytes during Inflammatory Bowel Disease

Inflammatory bowel diseases lead to a systemic acute-phase response. Monocyte activation plays a central role during systemic acute-phase response via secretion of inflammatory cytokines. We determined the activation of peripheral-blood monocytes in patients with inflammatory bowel diseases by measuring their interleukin-6 (IL-6) secretion. Blood was obtained from patients with active Crohn's disease before treatment [mean Crohn's disease activity index (CDAI) = 332 +/- 34] and from patients after treatment with prednisolone (mean CDAI index = 139 +/- 20). The mean serum IL-6 levels measured by a hybridoma growth assay (B9) were 23 +/- 4 U/ml before therapy and fell to 16 +/- 3 U/ml after treatment with prednisolone. Healthy persons and patients with inactive Crohn's disease usually had serum IL-6 levels below the detection limit of 4 U/ml. An ex vivo whole-blood system was used to measure IL-6 secretion by peripheral-blood monocytes with and without stimulation. Spontaneous IL-6 secretion in this system was about 9 U/ml in patients with Crohn's disease and below the detection limit of 4 U/ml in healthy controls. Moderate stimulation of blood cells [100 pg/ml lipopolysaccharide (LPS)] from patients with active Crohn's disease before and after treatment led to mean IL-6 concentrations of 1,160 +/- 514 and 131 +/- 54 U/ml, respectively. Maximal stimulation of peripheral blood before and after therapy by LPS (100 ng/ml) led to mean IL-6 concentrations of 5,570 +/- 1,660 and 6,220 +/- 1,630 U/ml, respectively. Thus, administration of glucocorticoids led to a rapid down-regulation of IL-6 synthesis by peripheral-blood monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of programmed death and G1 arrest in B‐cell hybridomas by interleukin‐6 is not accompanied by altered expression of immediate early response genes

The murine B-cell hybridoma B9 requires interleukin-6 (IL-6) for its survival and proliferation in vitro. We show here that withdrawal of IL-6 from B9 cultures results in programmed death, concomitant with arrest of the cells in the G1 phase of the cell cycle. Unlike several other systems that undergo programmed cell death, no induction of transcripts corresponding to the testosterone-repressed message-2 or transglutaminase genes is observed during this process. Upon readdition of IL-6 to G1-arrested B9 cells, viability is maintained and entry into S phase occurs after a lag period of 10 to 12 hr. Northern blot analysis showed that the immediate-early mRNAs normally induced shortly after growth factor stimulation in quiescent fibroblasts (c-fos, c-jun, Egr-1, c-myc, JE, and KC), and other growth-related genes (2F1, c-Ha-ras, and p53), are either not induced or remain unchanged during G1 to S phase progression. A correlation was found, however, between the temporal pattern of expression of several G1/S phase genes (dihydrofolate reductase, thymidine kinase, transferrin receptor, and histone H3) and DNA synthesis. These results demonstrate that IL-6-induced viability and growth of hybridoma (and, presumably, plasmacytoma) cells is mediated via novel signal transduction pathways.

Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production

A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 10 2 to 10 5 cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 10 6 cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 10 3 cells per ml grew 30% slower than those at 10 4 or 10 5. This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.

C5a-mediated release of interleukin 6 by human monocytes

Recombinant human C5a (rC5a) was assessed for its ability to induce interleukin 6 (IL-6) production in human peripheral blood-derived mononuclear cell (PBMC) cultures. rC5a was observed to induce IL-6 production as measured by hybridoma growth promotion (B9.9 assay) and human B cell differentiation (SKW6.4 assay). Optimal IL-6 production was obtained after 24 hr stimulation with 0.1–0.5 μg/ml rC5a. In addition, natural human C5a des Arg and natural porcine C5a were able to induce a similar level of IL-6. The observed IL-6 activity appeared not to be due to endotoxin contamination since heat treatment ( 100°C 15 min ) inhibited rC5a induction of IL-6. The rC5a stimulation also induced an increase in steady-state IL-6 mRNA as determined by Northern blot analysis. Pretreatment of PBMC with leucine-methyl ester to deplete monocytes reduced the rC5a-induced IL-6 production to background levels. In addition, stimulation of purified T cell preparations with rC5a produced little IL-6 activity, suggesting that monocytes are the major source of IL-6 in this system. These results suggest that the inflammatory and immunoregulatory activities of C5a may in part be due to the stimulation of IL-6 release, a cytokine which possesses potent pleiotropic functions.

Interleukin-6 release by rat liver macrophages

Tissue macrophages of the liver (Kupffer cells) release interleukin-6 (IL-6) in vitro. Since Kupffer cells reside in close proximity to hepatocytes, which are major target cells of IL-6, the regulation of IL-6 release by hepatic macrophages has been investigated in this study. Using the hybridoma growth test to detect IL-6, we found that Kupffer cells already maximally release IL-6 at endotoxin concentrations as low as 1.0 ng/ml. The stimulated secretion of IL-6 was increased 4-8-fold by endotoxin when compared to the control macrophages incubated in serum-containing medium alone. The preincubation of macrophages with interferon-gamma enhanced the capacity of Kupffer cells to respond to endotoxin. The secretion of IL-6 could also be induced by interleukin (IL)-1 beta and tumor necrosis factor (TNF-alpha). The most potent inducers, however, were the paramyxoviruses Newcastle Disease Virus and Sendai Virus. The release of IL-6 by macrophages upon stimulation with endotoxin was almost completely inhibited by 1 microM dexamethasone. Whereas 100 nM of prostaglandin E2 (PGE2) inhibited the release of TNF-alpha in rat Kupffer cells, it did not affect the secretion of IL-6.

Fluorescein-specific hybridomas derived from primary mice exhibit more stringent growth requirements than do hybrids from pre-immune animals

Although the generation of antigen-specific hybridoma cell lines from animals which have been multiply immunized is now a routine procedure, the derivation of hybridomas following a single in vivo antigen injection has proven to be much more difficult to accomplish. We show that the addition of an interleukin-containing supernatant derived from rat spleen cells which have been stimulated with concanavalin A (ConA SN) to hybrids after the initial cloning step results in the consistently successful isolation of IgM anti-fluorescein specific hybridomas. However, addition of the same supernatant to fused cultures simultaneously with the addition of the HAT selection medium results in the loss of growing cells In contrast to the situation with primary hybridomas, the growth of secondary hybridomas is inhibited by the addition of ConA SN at the cloning step. Following successful cloning, there is a time-dependent variation in the sensitivity of all cell lines to ConA SN.

Generation of Monoclonal Anti-Digoxin Antibodies

Anti-digoxin monoclonal antibodies are a useful model for basic immunochemical studies, for investigation of endogenous digoxin-like substances and in immunoassay for cardiac glycosides. The complete phenotypic characterization is a requisite for the selection of antibodies with desired binding parameters for different purposes. Twenty-two high-affinity monoclonal antibodies specific for digoxin were obtained in two fusion experiments. Treatment of antigen-antibody complex with potassium thiocyanate (KSCN) and absorption ELISA were used for the selection of high-affinity antibodies at the earliest stages of hybridoma growth. The true affinity constants of selected antibodies were determined in ELISA. They had proven to vary between 10(-7) and 10(-10) M. The fine specificity of 21 anti-digoxin monoclonal antibodies was determined by cross-reactivity experiments with 25 structurally related compounds. Cardiac glycosides, digoxin metabolites, endogenous steroids and spironolactone were used in the elucidation of the antigenic recognition pattern of antibodies. The elucidation of the binding characteristics of anti-digoxin monoclonal antibodies makes possible the selection of antibodies possessing binding characteristics appropriate for a wide range of designations.

Hybridoma growth, metabolism, and product formation in HEPES-buffered medium: II. Effect of pH

The effects of pH on cell metabolism during the batch growth of hybridomas in flasks were evaluated. Maintaining the pH at 7.2 resulted in a reduction of the maximum antibody and viable cell concentrations by ca. 40% and increased glucose and amino acid metabolic quotients. When the pH was allowed to fall to 6.7 there was further growth and antibody production after glutamine was exhausted using branched-chain amino acids as substrates. Y'[lac/gluc] increased from 1.32 at pH 6.7 to 1.50 at pH 7.2.

Hybridoma growth, metabolism, and product formation in HEPES-buffered medium: I. Effect of passage number

Shake flask studies with a murine hybridoma cell line show drastically increased cell yields per mL of culture medium as function of passage number (“cell age”), whereas the maximum antibody concentrations at the end of batch growth decreased from 180 μg/mL to 75 μg/mL after 10 months of serial propagation in flasks. The results are presented in the light of differences in cell metabolism with glucose, lactate, ammonia, glutamine, and selected amino acids being monitored.

Growth and product inhibition kinetics of T-cell hybridomas producing lymphokines in batch and continuous culture

The kinetics of growth and lymphokine formation by T-cell hybridomas were investigated in batch and continuous culture. Product inhibitions by ammonia and lactic acid were considered in the proposed model and experimental data were used to determine the kinetic and inhibition constants. The effect of dilution rate on specific substrate consumption and product formation rates was investigated.

Biochemical and biological analysis of human interleukin 6 expressed in rodent and primate cells

The cDNA for human interleukin 6 (IL 6) was stably expressed at high levels in the three mammalian cell lines COS-7, PA317, and GH3 to yield IL 6 proteins of 25 to 27, 26, 22 to 24, and 23 kDa molecular mass. Both size and relative amounts of the recombinant IL 6 (rIL 6) species produced correspond to those of natural IL 6 secreted by LPS-stimulated monocytes. Oligosaccharide analysis of recombinant IL 6 utilizing tunicamycin and endoglycosidases revealed O- and N-linked glycosylation that is comparable to that of natural IL 6 derived from human monocytes and fibroblasts. IL 6 expressed in each of the three cell lines was phosphorylated similarly to the IL 6 produced in human monocytes and fibroblasts. IL 6 secreted by the three different cell lines have marked differences in specific biological activities. COS-7 IL 6 appeared to be 12-fold more active in its hybridoma growth factor activity than that made in PA317 or GH3 cells. In contrast, PA317 and GH3 IL 6 were 230 and 6.7 times more effective than COS-7 IL 6 in inducing Ig production in CESS cells. Also, PA317 and GH3 IL 6 were more effective than COS-7 IL 6 in inducing the acute-phase protein fibrinogen in human hepatocytes. The rIL 6 species exhibited no antiviral activity.

A protein-free medium for the growth of hybridomas and other cells of the immune system

A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.

Effects of cytokines on human thymic epithelial cells in culture: II. Recombinant IL1 stimulates thymic epithelial cells to produce IL6 and GM-CSF

Our earlier study reported the ability of interleukin 1 (IL1) to promote proliferation and to induce morphological changes of human thymic epithelial cells (TEC) in culture. The present study was undertaken to examine the effects of IL1 on the secretory function of TEC. Both human recombinant IL1 α and IL1 β induced TEC to produce molecules in the culture supernatant fluids (TES) which displayed marked thymocyte proliferative capacities. This activity was specifically induced by IL1 since other TEC growth factors such as epidermal growth factor and a bovine pituitary extract had no effect on promoting secretion of T cell-activating molecules by TEC. Using specific radioimmunoassays for both forms of IL1, we found that unstimulated TEC produced negligible amounts of IL1 α and IL1 β in TES, which were not increased by IL1 stimulation, and we concluded that the IL1-induced TES molecules were not IL1. IL1 induced TEC to produce IL6, as detected by the hybridoma growth factor biological activity. Neutralizing anti-IL6 antibodies completely blocked the thymocyte activating capacities of the IL1-induced TES thus implying a major role for IL6 in TEC-derived T cell activation. IL1 also induced TEC to produce GM-CSF as measured by bioassay and confirmed by an immunoenzymetric assay. Our results confirm that TEC are a source of cytokines and show that TEC respond to IL1 by producing cytokines with consequences on the thymic lymphoid population. This further emphasizes the importance and complexity of paracrine molecular interactions involved in intrathymic development.

Glutamine‐limited batch hybridoma growth and antibody production: Experiment and model

The influence of glutamine (a major energy source) on both hybridoma growth and monoclonal antibody production was examined. A series of batch experiments were performed in T-flasks containing initial glutamine levels ranging from 0.5 to 4.0 mM in RPMI 1640 with 20% v/v fetal calf serum. The maximum final cell concentration increased with initial glutamine levels in the range of 0.5-2 mM; further glutamine increases had little or no effect. Earlier studies in our laboratories demonstrated that serum component(s) strongly influence the maximum specific growth rate. Here, the present studies reveal also the stoichiometric limitation by glutamine in the later stages of growth when its concentration is drastically reduced. For 0.5 to 1.5 mM initial glutamine, complete substrate utilization coincided with the cessation of cell growth and the onset of the death phase. For initial glutamine concentrations higher than 2.0 mM, growth halted prior to glutamine exhaustion, presumably because serum or RPMI component(s) were exhausted. The specific antibody secretion rate was essentially non-growth-associated above a critical low glutamine concentration in both the growth and death phases. At or below this critical value, an apparent emergence of stoichiometnc or energy limitation resulted in a dramatic drop in the secretion rate to zero. A simple unstructured model was developed that simulates these trends well. All parameters were determined using only subsets of the data. Nevertheless, these parameter values provided simulations in good agreement with all the glutamine-limited cultures.

A flow‐cytometric analysis of hybridoma growth and monoclonal antibody production

A flow cytometric kinetic study of hybridoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody-producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourage consideration of structured kinetic studies which recognize the quality of the biophase.