A Computer Model for Intracellular pH Regulation in Chinese Hamster Ovary Cells

Wu, P.; Ray, N. G.; Shuler, Michael L.

doi:10.1021/bp00022a004

Cited by 23 publications

(30 citation statements)

References 28 publications

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“…The recovery of pH in could be a result of metabolic consumption of intracellular fatty acids and/or a proton leak caused by various mechanisms (48). However, because the decrease in unesterified oleate (Fig.…”

Section: Discussionmentioning

confidence: 99%

Fatty acid transport and metabolism in HepG2 cells

Guo

Huang

Cai

et al. 2006

American Journal of Physiology-Gastrointestinal and Liver Physi

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The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.

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Section: Discussionmentioning

confidence: 99%

Fatty acid transport and metabolism in HepG2 cells

Guo

Huang

Cai

et al. 2006

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Therefore, a complete medium exchange was performed on days 5.8 and 7.7 for both the AHFBR and the slabs. The intracellular pH of CHO cells should not be affected at pH 6.8 31 but their growth rate may have been reduced during the last day of culture at pH \ 7.0. 32 CHO cells in alginate slabs grew exponentially at least until Day 7.8, just before harvest on Day 8.3 ( Figure 6A).…”

Section: Ahfbr Immobilized Culture Of Cho Cellsmentioning

confidence: 99%

A novel alginate hollow fiber bioreactor process for cellular therapy applications

Hoesli

Luu

Piret

2009

Biotechnology Progress

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Gel-matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet-like clusters to treat Type 1 diabetes, the need for large-scale production can be anticipated. The throughput of the commonly used nozzle-based devices for cell encapsulation is limited by the rate of droplet formation to approximately 0.5 L/h. This work describes a novel process for larger-scale batch immobilization of mammalian cells in alginate-filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intra-capillary space medium flow was negligible, (2) extra-capillary alginate gelling was complete and (3) 83 +/- 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate-forming cell line that grew from mostly single cells to pancreatic islet-sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small-scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate-immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel-matrix assisted cultures for cellular therapy.

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“…This might induce a cytoplasmic alkalanization (Frelin et al, 1988). Ammonium ions might acidify the cell cytoplasm, by a mechanism called “proton shuttle” (McQueen and Bailey, 1990b; Wu et al, 1993): the ammonium ions, having an extracellular origin when one is studying the effect of this component and having an intracellular origin under normal culture conditions, can penetrate into the cells. In the last case, the ammonium ions issued from the cell metabolism are evacuated from the cells under the form NH 3 by passive diffusion.…”

Section: Introductionmentioning

confidence: 99%

“…If this charge is too important compared to the evacuation of protons from the cell, pH i will decrease. Some studies reported already results where one or more of the above‐mentioned culture variables were studied in relation to pH i and where their effect on cell and culture behavior could indeed be associated with an effect on pH i (McQueen and Bailey, 1991; Ozturk et al, 1992; Wu et al, 1993). They dealt however with very short‐term experiments on cells resuspended in buffers, while our work aims at studying cells in normal real culture conditions for longer exposure times, since we have evidence to believe that such measurements reflect better the real cell behavior.…”

Section: Introductionmentioning

confidence: 99%

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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A Computer Model for Intracellular pH Regulation in Chinese Hamster Ovary Cells

Cited by 23 publications

References 28 publications

Fatty acid transport and metabolism in HepG2 cells

Fatty acid transport and metabolism in HepG2 cells

A novel alginate hollow fiber bioreactor process for cellular therapy applications

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

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