13C labeling dynamics of intra- and extracellular metabolites in CHO suspension cells

Materials and methods Experimental set-up CHO-K1 cells were cultivated in protein free TC-42 medium (TeutoCell, Bielefeld, Germany) in 250 ml baffled shake flasks. For the non-stationary experiment the cultures were inoculated at a start cell density of 2 × 10 cells/ml in a start volume of 120 ml. Four parallel cultivations were performed, two with 100% [U-C6] glucose and two with 100% [U-C5]glutamine, respectively. Extracellular samples were taken from all four cultivations every 6 h for cell counting and determination of extracellular metabolite concentrations and extracellular labeling dynamics. Intracellular samples were taken alternately from the two replicates. After 2 min, 10 min, 20 min, 30 min, 60 min, 2 h, 4 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 48 h, a sample of 5 ml cell suspension was quenched in 45 ml ice-cold 0.9% sodium chloride solution, centrifuged for 1 min at 2000 × g, washed once by rinsing the cell pellet with 50 ml ice-cold 0.9% sodium chloride solution, and frozen in liquid nitrogen. Intracellular metabolites were extracted in methanol and water by repeated freezethaw cycles, as described previously [2]. Extracts were dried in a centrifugal evaporator. Analytics Cell counting and viability determination was carried out using an automated cell counter (Invitrogen, Darmstadt, Germany). Quantification of extracellular glucose, organic acids and amino acids via HPLC was carried out as described recently [3]. For determination of extracellular labeling dynamics, lyophilized supernatants were resolved in dimethylformamid (0.1% pyridine) and derivatized with MBDSTFA (Macherey-Nagel, Düren, Deutschland). Dried cell extracts were resolved in pyridine (20 mg/ml methoxylamine) and derivatized with MSTFA (Macherey-Nagel, Düren, Deutschland). Samples were analyzed by GC-MS. Unique fragments containing the whole carbon backbone were chosen for excreted extracellular metabolites and selected intracellular metabolites of the central metabolism.