The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Higareda, Ana E.; Possani, Lourival D.; Ramı́rez, Octavio T.

doi:10.1002/(sici)1097-0290(19971205)56:5<555::aid-bit9>3.0.co;2-h

Cited by 31 publications

(8 citation statements)

References 37 publications

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“…A detailed description of the system can be found elsewhere (33,34). A similar DOT control system has proven its reliability for other cultures with substantially different dynamics, including bacteria, hybridomas, insect cells, and human hematopoietic cell cultures (33)(34)(35)(36)(37).…”

Section: Microorganismmentioning

confidence: 99%

Effect of Oscillating Dissolved Oxygen Tension on the Production of Alginate by Azotobacter vinelandii

et al. 2001

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The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.

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Section: Microorganismmentioning

confidence: 99%

Effect of Oscillating Dissolved Oxygen Tension on the Production of Alginate by Azotobacter vinelandii

et al. 2001

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“…Description of the culture systems, conditions used, and other media components are detailed elsewhere (Higareda et al 1997;Mendonça et al 1998). …”

Section: Cell Lines Culture Media and Cell Culturesmentioning

confidence: 99%

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Meneses‐Acosta

Mendonça

Merchant³

et al. 2001

Biotechnol. Bioeng.

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Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub‐G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub‐G1 population and onset of death. The fraction of particles in the smaller peak (6–11 μm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 441–457, 2001.

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“…Batch cultures of noninfected insect cells (Spodoptera frugiperda (Sf9), ATCC1711) and of a murine hybridoma (Higareda et al, 1997) were studied. TNM-FH and DMEM media were used for insect and hybridoma cell cultures, respectively.…”

Section: Cell Lines Culture Media and Cell Culturesmentioning

confidence: 99%

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Meneses‐Acosta

Mendonça

Merchant³

et al. 2001

Biotechnol. Bioeng.

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Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.

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The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Cited by 31 publications

References 37 publications

Effect of Oscillating Dissolved Oxygen Tension on the Production of Alginate by Azotobacter vinelandii

Effect of Oscillating Dissolved Oxygen Tension on the Production of Alginate by Azotobacter vinelandii

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

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