Development of an intracellular pH measuring method and subsequent application in a CHO batch fermentation process

Abstract

Intracellular pH (pHi) has a major impact on various cellular functions and parameters as growth, viability and productivity of the culture. Its close relationship to a wide array of biological processes make it a potential marker for the physiological state of the cells. pHi measurements done so far were performed on cells resuspended in buffers and therefor withdrawn from their culture systems. Incubation times with fluorescent dyes of 30 min and longer are reported, carrying the risk of high rates of dye leakage. Hence it was concluded that these data do not necessarily reflect what happens inside cells in a culture systems. In this work following issues are studied: (1) the development of a rapid pHi measuring method to determine pHi under culture conditions. Therefor cell culture samples were instantly loaded after sample drawing with the fluorescent dye 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF-AM) and the initial rate of rise in fluorescence was measured.; (2) revealing the effect of medium pH alterations on intracellular pH. Batch cultured chinese hamster ovary (CHO) cells were exposed to a pH shift on day 3 of the fermentation process. pHi was measured twice a day over the entire fermentation process and additionally high frequent during the transition phase to a new medium pH; (3) finding a possible

link between cytosolic pH and specific rates. The procedure described made it possible to obtain pHi values within 3 min after sample drawing. The rapid pHi measuring method provided reliable data of pHi under culture conditions without consuming sample preparations or long-term incubation with the fluorescent dye. pHi was instantly affected by medium pH perturbations. Short-term pHi regulation systems as intracellular buffering capacity were sufficient to counteract an acute alkali or acid load, but these mechanisms were finite. The long-term response of the pHi to an ongoing stress induced by a pH shift can be summed up to: a shift in medium pH led to a shift in the steady state pHi. pHi in cells undergoing a pH shift significantly differed from pHi values in their control reactors. ¿ pH (pHe-pHi) increased at pH 6.8 and decreased at a more alkaline medium pH. Differences of specific rates between the shifted reactors and their control vessels could not be clearly attributed to pHi. It must be considered that depletion of nutrients and accumulation of metabolites in a batch system have a major impact on cell metabolism. The fact, that extracellular pH changes instantly affected pHi, pH could be of fundamental interest for large scale animal culture systems where cells are repeatedly exposed to non-physiological pH values in the base feeding zone.