Monitoring of Biosynthetic and Metabolic Activity in Animal Cell Culture Using Flow Cytometric Methods

Al‐Rubeai, Mohamed; Klöppinger, Martina; Fertig, Georg; Emery, A. N.; Miltenburger, H.G.

doi:10.1016/b978-0-7506-0421-5.50076-3

Cited by 8 publications

(2 citation statements)

References 4 publications

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“…Before the maximal viable cell concentration was reached, the pH i of the cells began to decrease and a value of about 7.4 was measured at the end of the culture for the remaining viable cells. A similar pH i evolution profile has been reported by Al-Rubeai et al (1992) for a batch culture without medium pH control of another hybridoma cell line but within a wider range of pH values varying from 6.7 to 7.3.…”

Section: Resultssupporting

confidence: 82%

Intracellular pH Monitoring as a Tool for the Study of Hybridoma Cell Behavior in Batch and Continuous Bioreactor Cultures

Cherlet¹,

Marc²

1998

Biotechnology Progress

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We show in this paper the results obtained when studying the behavior of hybridoma cells by monitoring of the pHi during batch and continuous bioreactor cultures. A first set of experiments, consisting of a batch culture and a continuous culture at variable dilution rate, was set up under normal physiological operating conditions. Significant pHi variations were measured during these cultures. For the batch culture, maximal pHi values around 7.60 were associated with the middle of the growth phase, while lower values were found in the culture beginning and during the decay phase, respectively 7.47 and 7.40. For the continuous culture, pHi increased with increasing dilution rate, ranging between 7.40 and 7. 60 for dilution rates between 0.010 and 0.040 h-1. These pHi variations were found for both cultures to be linked to variations of the specific growth rate of the cells. The observed link between pHi and cell growth provided us a general framework for the study of the effect of suboptimal operating conditions on cell behavior, all having a particular interest in animal cell culture technology. First, our results indicate that a decrease of the medium pH of its normal value of 7.00 to 6.70 did not necessarily result in cytoplasmic acidification, at least not after prolonged exposition times in continuous culture, and this despite a pronounced growth inhibitory effect. This effect can therefore rather be explained by the combination of the increased demand for maintenance energy associated with the higher DeltapH gradient maintained across the cell membrane and the decreased supply of energy, in particular via the glucose metabolism. Second, our results indicate that also increased ammonium ion concentrations do not lower pHi. This observation, together with the results of batch cultures carried out at a more alkaline pH, indicates that the form NH3, and not the form NH4+, has a negative effect on cell growth of our hybridoma cell line. Third, in the case of an osmolality increase, a significant pHi increase was observed, with mean values of 7.35 at the lower osmolalities 335 and 370 mOsm/kg and 7.45 at the higher osmolalities of 400, 425, and 450 mOsm/kg. This higher pHi might account at least partially for the increased monoclonal antibody production observed at hyperosmolality and coincided furthermore with a faster glutamine consumption rate.

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Section: Resultssupporting

confidence: 82%

Intracellular pH Monitoring as a Tool for the Study of Hybridoma Cell Behavior in Batch and Continuous Bioreactor Cultures

Cherlet¹,

Marc²

1998

Biotechnology Progress

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“…Flow cytometric analysis was performed using Coulter EPICS Elite Analyser (Coulter Electronics) equipped with an argon laser emitting 15 mW at 488 nm, as described by Al-Rubeai et al (1992). Passing the cells individually through the argon laser beam activated the relative cellular DNA content of 10 6 stained cells.…”

Section: Preparation and Analysis Of Cells Using Flow Cytometrymentioning

confidence: 99%

Characterisation of Tetraploid and Diploid Clones of Spodoptera frugiperda Cell Line

Jarman-Smith¹,

Mannix²,

Al‐Rubeai

2004

Cytotechnology

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We have isolated and characterised diploid and tetraploid clones from the normally heterologous Spodoptera frugiperda (Sf-9) cell line by dilution cloning technique. Tetraploid clones were found to have cell sizes in excess of 35% larger than that of the diploid clones. In contrast, the maximum cell numbers achieved in batch cultures of diploid clones were on average 185% higher than the tetraploid cell numbers. Growth rates and metabolic quotients during the exponential phase were similar for both clones. Tetraploid cells infected with wild-type and recombinant green fluorescent protein (GFP) baculovirus, resulted in more polyhedra or GFP product per cell. Importantly, the difference between the clones either completely diminished or reduced to 50% when the yield was assessed in terms of the amount of polyhedra or GFP per mL of medium, respectively. These results indicate that the existing heterogeneity in insect cell populations with respect to ploidy level, are correlated to cell growth and product yield.

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

et al. 1991

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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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Monitoring of Biosynthetic and Metabolic Activity in Animal Cell Culture Using Flow Cytometric Methods

Cited by 8 publications

References 4 publications

Intracellular pH Monitoring as a Tool for the Study of Hybridoma Cell Behavior in Batch and Continuous Bioreactor Cultures

Intracellular pH Monitoring as a Tool for the Study of Hybridoma Cell Behavior in Batch and Continuous Bioreactor Cultures

Characterisation of Tetraploid and Diploid Clones of Spodoptera frugiperda Cell Line

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

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