Development and validation of a methodology for intracellular pH measurements of hybridoma cells under bioreactor culture conditions

The intracellular pH (pH(i)) is an important factor in the regulation of di fferent cellular processes. It might therefore be used as a marker of the p hysiological state of cells cultivated in a bioreactor environment. We deve loped and validated therefore a methodology that permits a reproducible and reliable pHi measurement under such bioreactor culture conditions, contrar y to earlier reported measurements, carried out on cells resuspended in buf fers under nongrowth conditions. The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM BCECF = 2',7'-bis-carbo xyethyl-5,6-carboxyfluorescein and analyzed by flow cytometry. Such a measu rement is perfectible to changes of the cells between the moment of samplin g and of final analysis on the flow cytometer. All intermittent steps were for this reason studied in detail, either to determine the optimal conditio ns to be used or to characterize their influence on the final pH(i) value m easured. Additional experiments were carried out, showing the representativ eness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.