Biomedical subjects

M al-Rubeai

Publications and source records attributed to M al-Rubeai.

23 records · Page 2Linked to original sources

PubMed1992

Further studies of the culture of mouse hybridomas in an agitated bioreactor with and without continuous sparging.

TB/C3 mouse hybridoma cells have been grown at 2 controlled dO2 conditions by headspace and sparged oxygenation. Also a variety of sparging rates and sparger sizes and positions have been employed. Headspace oxygenation at dO2 levels from 5% to 100% of saturation give essentially the same performance as controls. Sparging is generally damaging to cells, the extent of damage decreasing with reduced sparging rate until at below about 0.02 vvm results equivalent to the unsparged conditions are obtained. Damage is clearly linked with bubble-cell interactions at the air-medium interface where bubbles bursting in clusters and of a size less than 5 mm appear to be the most lethal. When the interaction of air sparging with the agitator flow leads to an increase in the number of smaller bubbles and cluster bursts, cell damage is further increased. Pluronic F-68 reduces damage very significantly. Biological aspects are briefly discussed in the light of various biological tests. The practical implications of this work for large scale, free suspension cell culture are outlined.

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PubMed1991

Cell cycle, cell size and mitochondrial activity of hybridoma cells during batch cultivation.

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G2 fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.

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PubMed1991

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.

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PubMed1990

Electron microscopy of hybridoma cells with special regard to monoclonal antibody production.

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.

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PubMed1990

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.

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