Development and Validation of a Methodology for Intracellular pH Measurements of Hybridoma Cells under Bioreactor Culture Conditions

• Published in: Biotechnology progress Vol. 15; no. 4; pp. 630 - 639
• Main Authors: Cherlet, Marc, Franck, Patricia, Nabet, Pierre, Marc, Annie
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 01.07.1999; American Institute of Chemical Engineers
• Subjects: Animal cells; Animals; Biological and medical sciences; Bioreactors; Biotechnology; Buffers; Calibration; Cell culture; Cell Survival; Cells, Cultured; Cold Temperature; Culture Media; Cytology; Enzyme inhibition; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Growth kinetics; Hybridomas - cytology; Hybridomas - metabolism; Hydrogen-Ion Concentration; Kinetics; Metabolism; Methods. Procedures. Technologies; Mice; Reproducibility of Results; Staining and Labeling - methods; Time Factors; Performance evaluation; Cell culture; Flow cytometry; Fluorescent tracer; Rodentia; Hybridoma; Vertebrata; Bioreactor; Mammalia; Mouse; Animal; pH; Intracellular; Measurement method
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp990047s

The intracellular pH (pHi) is an important factor in the regulation of different cellular processes. It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment. We developed and validated therefore a methodology that permits a reproducible and reliable pHi measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions. The hybridoma cells were sampled from the culture, stained with the pH‐sensitive dye BCECF‐AM (BCECF = 2′,7′‐bis‐carboxyethyl‐5,6‐carboxyfluorescein), and analyzed by flow cytometry. Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer. All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pHi value measured. Additional experiments were carried out, showing the representativeness of the measured pHi value for the pHi the cells possess really in the culture at the moment of sampling.