The protective effect of serum against hydrodynamic damage of hybridoma cells in agitated and surface-aerated bioreactors.

The effect of serum on the growth rate and metabolism of CRL-8018 hybridoma cells in an agitated, surface-aerated bioreactor was examined. In the employed well-controlled bioreactors at high agitation rates, hybridoma cells in medium containing 1% fetal bovine serum rapidly die in the presence of a vortex with accompanying gas-bubble entrainment, whereas non-agitated control cultures grow normally in medium containing 1% serum. Serum levels greater than 5% counteract the detrimental hydrodynamic effects due to agitation and bubble entrainment. The protective effect is present after short-term (less than 1 h) exposure to 10% serum concentration, suggesting a protection mechanism which is, at least in part, of a physical nature. The apparent cell yields on glucose, lactate, and glutamine decreased with decreasing growth rate due to low serum concentrations. The results are incorporated into a simple model in which the apparent growth rate is the sum of an invariable growth rate and a changing death rate.     

Biological responses of hybridoma cells to hydrodynamic shear in an agitated bioreactor.

Effects of long-term hydrodynamic shear on hybridoma cells were investigated in a 250-ml continuous stirred-tank reactor (CSTR). Cells grown at steady state were subjected to step changes in agitation rates. Cell viability, glucose consumption, and monoclonal antibody (MAb) production were determined at high agitation rates and compared with the control (100 rev min-1). Impeller tip speeds higher than 40 cm s-1 caused a significant drop in cell concentration and respiration activity, and increased lactate dehydrogenase (LDH) release to the culture medium. Also, high agitation speeds caused a decrease in MAb concentration and an increase in specific glucose consumption rate. The effects of dilution rate and serum concentration on the sensitivity of hybridoma cells to hydrodynamic shear were determined. Serum was found to protect the cells against shear damage and had a significant positive effect on hybridoma growth and MAb production. Shear damage on cells in CSTR was approximated to first-order kinetics. The death rate constant increased sharply at impeller tip speeds above 40 cm s-1.     

Influence of bovine serum albumin on the growth of hybridoma cells in airlift loop reactors using serum-free medium

Summary Hybridoma cells were grown in serum-free media using different culture systems: stationary culture, spinner flask and a laboratory-scale airlift loop reactor. Influence of bovine serum albumin (BSA) on growth and product formation was only found in the airlift loop reactor.     

Degradation of glycated bovine serum albumin in microglial cells

Glycated protein products are formed upon binding of sugars to lysine and arginine residues and have been shown to accumulate during aging and in pathologies such as Alzheimer disease and diabetes. Often these glycated proteins are transformed into advanced glycation end products (AGEs) by a series of intramolecular rearrangements. In the study presented here we tested the ability of microglial cells to degrade BSA-AGE formed by glycation reactions of bovine serum albumin (BSA) with glucose and fructose. Microglial cells are able to degrade BSA-AGEs to a certain degree by proteasomal and lysosomal pathways. However, the proteasome and lysosomal proteases are severely inhibited by cross-linked BSA-AGEs. BSA-AGEs are furthermore able to activate microglial cells. This activation is accompanied by an enhanced degradation of BSA-AGE. Therefore, we conclude that microglial cells are able to degrade glycated proteins, although cross-linked protein-AGEs have an inhibitory effect on proteolytic systems in microglial cells.     

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.     

Raman studies of bovine serum albumin.

The Raman Spectra of bovine serum albumin have been obtained in the solute state, in alkaline and acidic solutions, and in the gel. The reversible denaturations of bovine serum albumin solutions by heat, acid's, and alkali were studied and a new mechanism for heat denaturation has been proposed based on a continuous unfolding of the α-helices.     

ATP binding to bovine serum albumin.

Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.     

The interaction between bovine serum albumin and surfactants.

1. Potassium n-decyl phosphate binds exothermically to bovine serum albumin at pH 7.0 to form a specific complex containing approx. 60 phosphate anions. 2. The formation of the complex is accompanied by changes in the u.v. difference spectrum of the protein. 3. At higher phosphate concentrations (above 0.4mM) surfactant molecules continue to be bound, and the protein undergoes a gross change in conformation. 4. n-Dodecyltri-methylammonium bromide binds endothermically to bovine serum albumin at pH7.0 but the extent of binding for a given free surfactant concentration is less than for the phosphate surfactant. 5. Binding is accompanied by a small change in the specific viscosity and by changes in the u.v. difference spectrum of the protein. 6. It is suggested that over the surfactant concentration ranges studied n-decyl phosphate ions first bind to the C-terminal part of the protein and then to the more compact N-terminal part whereas n-dodecyltrimethylammonium ions bind only to the C-terminal part of bovine serum albumin.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements

Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers. (c) 1992 John Wiley & Sons, Inc.     

Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection

The effect of a well-defined mild shear stress on hybridoma cell viability (HB-8852) in a serum-free culture medium has been analysed, and the role as shear protector of different concentrations of fetal bovine serum have been studied. Samples harvested from cultures in their late exponential growth phase, were subjected in a rheometer to a constant shear stress of 0.41 ± 0.02 Pa, and the evolution of viable and total cell concentrations was determined and compared with static controls. A simple segregated kinetic model for the viable and dead cells was used to know the effect of serum concentration on the specific cell growth and death rate of the cells.     

Aggregation and fibrillation of bovine serum albumin

The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology and characteristic amyloid X-ray fiber diffraction peaks. Fibrillation occurs over minutes to hours without a lag phase, is independent of seeding and shows only moderate concentration dependence, suggesting intramolecular aggregation nuclei. Nevertheless, multi-exponential increases in dye-binding signal and changes in morphology suggest the existence of different aggregate species. Although beta-sheet content increases from 0 to ca. 40% upon aggregation, the aggregates retain significant amounts of alpha-helix structure, and lack a protease-resistant core. Thus BSA is able to form well-ordered beta-sheet rich aggregates which nevertheless do not possess the same structural rigidity as classical fibrils. The aggregates do not permeabilize synthetic membranes and are not cytotoxic. The ease with which a multidomain all-alpha helix protein can form higher-order beta-sheet structure, while retaining significant amounts of alpha-helix, highlights the universality of the fibrillation mechanism. However, the presence of non-beta-sheet structure may influence the final fibrillar structure and could be a key component in aggregated BSA's lack of cytotoxicity.     

Immunochemical studies of fragments of bovine serum albumin.

Antigenic properties of 14 fragments of bovine albumin were measured using antisera to albumin and to two of its fragments. All seven fragments larger than 21,000 daltons formed immune precipitates. Although immune precipitates were not formed with smaller fragments, inhibition tests indicated the presence of antigenic sites on several of these fragments as well. The results predict the occurrence of six or more antigenic determinants and allow assignment of their positions in the parent molecule. These sites are distributed along the entire protein chain, with the sites of greatest antibody affinity situated in the COOH-terminal region. Evidence is presented that some sites are homologous, reacting with the same populations of antibodies, and that other sites are unique, binding to an exclusive population of antibodies.     

The specific hydraulic conductivity of bovine serum albumin.

Previous studies of extracellular matrix hydraulic conductivity have characterized the flow resistance of glycosaminoglycans, proteoglycans and collagen. This work focuses on serum albumin, present in significant quantities in many connective tissues, but not previously considered for its role in determining connective tissue flow resistance. The specific hydraulic conductivity of bovine serum albumin solutions, as a function of concentration, was calculated from sedimentation and ultrafiltration data available in the literature. A rigid particle hydrodynamic model compared favorably with these results. Experimental measurements on an albumin ultrafiltration cell were in agreement with this model (within experimental error); furthermore, the experimental data confirmed the theoretical prediction that there is no (or negligible) pressure drop through the concentration polarization layer. Use of the hydrodynamic model for albumin specific hydraulic conductivity with literature values for the hindrance of albumin when passing through a glycosaminoglycan (GAG) matrix allows an estimate of the relative importance of the albumin on tissue hydraulic conductivity: in non-cartilaginous tissues with moderate GAG concentrations, tissue levels of albumin can generate flow resistance effects comparable to those of the GAGs, although well less than the flow resistance of these tissues.     

The acylation of bovine serum albumin with diacetylcycloserine.

The reaction of the amino groups of bovine serum albumin (BSA) with diacetylcycloserine (I) at pH 7.2-9.0 proceeded with both acylation by the diacetyl-beta-aminooxy-D-alanyl (DAA) group and acetylation. The number of DAA groups was determined by their conversion to cycloserine (III) which can be accurately measured in micromolar amounts. The method was developed using the model compound diacetyl-beta-aminooxy-D-alanine butyl amide (II) which was converted to beta-aminooxy-D-alanine butyl amide (IV) by methanolysis and then to cycloserine by basic ring-closure of IV. Calculations using results obtained by this method combined with the experimentally determined number of free amino groups in the modified BSA indicated that the reaction of excess I with BSA effected the acetylation of about 35 and the acylation with DAA groups of about 22 of the 59 amino groups. These findings were supported by experiments demonstrating that the amount of acetic acid formed by hydrolysis of the modified BSA was approximately that predicted from the results of the cycloserine analyses.