František Franěk

Iron compounds at high concentrations enable hybridoma growth in A protein-free medium

SummarySoluble iron salts at very high concentrations (50 μM to 5 mM), substituting for the only indispensable protein transferrin, enable hybridoma growth and monoclonal antibody production in a chemically defined protein-free medium.

Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical “ladder” of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.

Serum-free medium for hybridoma and parental myeloma cell cultivation: A novel composition of growth-supporting substances

Serum-free chemically defined medium for hybridoma and parental myeloma cultivation was developed on the basis of testing of individual substances supporting hybridoma growth under serum-free conditions. Optimized concentrations of transferrin, insulin, ethanolamine, linoleic acid, serum albumin, ascorbic acid, hydrocortisone, and trace elements could substitute serum. Developed serum-free hybridoma (SFH) medium differs from analogous previously described media mainly by a more complete combination of growth-supporting supplements and by the presence of ascorbic acid and hydrocortisone. Growth comparable with that in the medium supplemented with 10% bovine serum was achieved with four hybridomas and two myelomas. SFH medium was also suitable for long-term cultivation of hybridomas without cessation of monoclonal antibody production. Growth potency and the specific growth requirements of hybridomas in serum-free medium are, to a large degree, determined by parental myeloma.

Kinetics of development of spontaneous apoptosis in B cell hybridoma cultures

The kinetics of the development of apoptosis was studied in mouse B cell hybridoma batch cultures carried out in the iron-rich protein-free medium. One of the markers of apoptosis, the apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride, was found to rise significantly at 144 h, i.e., in the late stationary phase. At the decline of the culture (216 h) the value of the apoptotic index reached 29.1%. Analysis of another marker, the degree of DNA fragmentation determined on the basis of chromatographic resolution of isolated cellular DNA, revealed a significant increase as early as 96 h, i.e., at the end of the exponential phase. At 216 h the net value of the fragmented DNA fraction was about 30% of cellular DNA. The values of both markers were found to be very similar when the iron-rich protein-free supplement was replaced with conventional 10% foetal calf serum. This finding suggested that the growth factors present in the serum were not able to abolish the tendency of the hybridoma culture to undergo spontaneous apoptosis. The timing of the spontaneous onset of apoptosis in the exponential phase indicated that B cell hybridoma apoptosis was a process associated with cell proliferation and full metabolic activity rather than with the decline of cell vitality.

Growth-stimulating effect of transferrin on a hybridoma cell line: Relation to transferrin iron-transporting function

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.

Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity.

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG1 subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.

Neutron small-angle scattering study on two different precipitin types of pig Anti-Dnp antibodies

Anti-Dnp antibodies elicited in pigs at an early phase of the immune response were previously shown to react with polyvalent high molecular weight Dnpantigens in a different way than antibodies elicited at a later phase [l] . While the early antibodies form mostly insoluble complexes (i.e., precipitates) when interacting with heavily Dnp-substituted proteins, the late antibodies mostly form soluble complexes with the same antigens. It has been suggested that early and late antibodies differ in their shapes and/or the flexibilities of their F, arms [l] . Neutron smallangle scattering experiments were carried out to test this assumption. The present paper reports that the early and late antibody molecules do differ by their radii of gyration. Moreover, it is shown that the radii of gyration change upon complexing the antibodies with a low molecular weight hapten, the extent of change being approximately the same for both the early and late antibodies.

Slow conformational change in anti-dansyl antibody as a consequence of hapten binding: demonstration by ESR spectra.

Attempts to detect conformational changes in antibody molecules following antigen binding led to various results depending on the particular antibodyantigen model system (reviewed in [ 1,2]). One of the most sensitive tools for the study of changes of protein conformation is the spin-label method. Changes of ESR spectra of spin&belled antibodies induced by binding of protein antigens were determined in (31. Here we demonstrate that occupation of combining sites of pig anti-am ~tibody by e-Dns-lysine results in a change of ESR spectrum of spin&belled antibody. Whereas the binding of the hapten is instantaneous, as evidenced by the measurement of fluorescence enhancement, the changes of the ESR spectrum proceed for several minutes after addition of the hapten. Presumably, the change of the ESR spectrum refIects a relatively slow reversible conformational change in the antibody molecule foliowing the interaction with the hapten.

Distance between two binding sites of the same antibody molecule: A neutron small-angle scattering study of pig anti-Dnp antibody complexed with mono-Dnp-dextran

Although the general features of the antibody molecules are known, considerable uncertainty still exists as to their shape when present in immune complexes. Small-angle X-ray scattering analysis employed [ 1,2] showed that the radii of gyration of rabbit antibodies decrease upon binding low-molecular weight haptens. With the use of neutron small-angle scattering, we have shown [3]. that pig antibodies which occur early and late during an immune response to Dnp-antigens and differ by their ability to precipitate antigen [4], differ by their radii of gyration as well; when reacting with a low-molecular weight hapten, the radii of gyration of the early and late antibodies decrease by 4.6% and 7.5%, respectively. This paper reports on neutron small-angle scattering experiments aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. The contrast variation method offers a possibility to measure the distance between two ligands bound to the same molecule when the condition is fulfilled that the ligands have a sufficiently large volume and that their neutron scattering density differs from that of the antibody. To this end, a high molecular weightAlthough the general features of the antibody mole- cules are known, considerable uncertainty still exists as to their shape when present in immune complexes. Small-angle X-ray scattering analysis employed [ 1,2] showed that the radii of gyration of rabbit antibodies decrease upon binding low-molecular weight haptens. With the use of neutron small-angle scattering, we have shown [3]. that pig antibodies which occur early and late during an immune response to Dnp-antigens and differ by their ability to precipitate antigen [4], differ by their radii of gyration as well; when reacting with a low-molecular weight hapten, the radii of gyra- tion of the early and late antibodies decrease by 4.6% and 7.5%, respectively. This paper reports on neutron small-angle scattering experiments aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. The contrast variation method offers a possibility to measure the distance between two ligands bound to the same molecule when the condition is fulfilled that the ligands have a sufficiently large volume and that their neutron scattering density differs from that of the antibody. To this end, a high molecular weight