NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes

Abstract

A fiber-optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic-anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose-depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine-depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate-level phosphorylation, as shown by decreases of 35-52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6-38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

“…In addition, in group 3 we observed low production of ammonia (8 nmoles/(h 10 6 cells)) and low average ammonia levels (1.2 × 10 −2 g/L) implying low glutamine utilization via aerobic metabolism, which is consistent with previous studies of chick chondrocytes (Rajpurohit et al, 1996) and mouse hybridoma cells (Miller et al, 1987;Siano and Mutharasan, 1991). It was suggested that low oxygen concentrations inhibit the TCA cycle resulting in ␣-ketoglutarate accumulation and low NH 3 production (Miller et al, 1987;Rajpurohit et al, 1996;Siano and Mutharasan, 1991). The cells in constructs from group 3 thus behaved as if under hypoxic conditions even though the measured bulk phase oxygen tension was 43 mmHg.…”

“…Values of Y L/G higher than 2 (2.1-2.2) were also reported by Siano and Mutharasan (1991) in mouse hybridoma cells cultured under hypoxic conditions and attributed to the conversion of glutamine to lactate. In addition, in group 3 we observed low production of ammonia (8 nmoles/(h 10 6 cells)) and low average ammonia levels (1.2 × 10 −2 g/L) implying low glutamine utilization via aerobic metabolism, which is consistent with previous studies of chick chondrocytes (Rajpurohit et al, 1996) and mouse hybridoma cells (Miller et al, 1987;Siano and Mutharasan, 1991).…”

“…The ideally maximum value of Y L/G of 2 corresponds to the complete conversion of glucose into lactate, i.e., anaerobic metabolism of glucose. The amount of ammonia can be used as an indicator of glutamine utilization and TCA cycle activity (Miller et al, 1987;Siano and Mutharasan, 1991).…”

Improving an on-line feeding strategy for fed-batch cultures of hybridoma cells by dialysis and `Nutrient-Split'-feeding

“…Metabolic activity can not be measured directly. Respiration and energy conversion can be monitored by oxygen mass balance [13,27] or spectroscopic analysis of NAD/ NADH + H + respectively [41]. A strategy ®rst described by Zhou et al [44] determines the physiological state of the cells on-line during cultivation by the oxygen uptake rate OUR.…”

On-line Characterization of Metabolic State in Batch Cultivation of Clostridium diolis for 1,3-Propanediol Production Using NADH+H+ Fluorescence

“…Duysens and Amesz first reported the use of on-line fluorescence measurement technique in suspensions of baker's yeast and alga [15]. Thereafter it has been used by several researchers for in situ characterization of biomass and cellular metabolic state using different fermentation processes [13,[16][17][18][19]. The correlation between culture fluorescence and biomass concentration could be made on a reasonable assumption that during the balanced growth of the microorganism the intracellular concentration of NADH+H + remains constant.…”