¹H NMR spectroscopy profiling of metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture

Abstract

We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.

Figure 1. Growth profiles and cell viability of CHOK1 (A + B) and CHOS (C + D).

Cells were grown at 37°C (•) or shifted to 27°C (■) or 10°C (▲). The timepoint of culture temperature shifting is indicated by a long dashed grey line. The cells were cultured for a further 24 hours before one flask at 27°C (□) and one at 10°C (△) were recovered to 37°C, the remaining flasks were maintained at the previous culture temperatures. The timepoint of culture temperature recovered is indicated by a short grey dashed line.

Figure 2. ¹H NMR spectra of cell culture media.

The extracellular metabolite profile of (A) CHOK1 and (B) CHOS cultures 1D ¹H NMR spectra. Samples taken at 0 h are shown in black, and 216 h in red. All spectra are shown on the same intensity scale.

Figure 3. The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) glucose concentration.

The concentration of glucose in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.

Figure 4. The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) lactate concentration.

The concentration of lactate in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.

Figure 5. The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) Alanine concentration.

The concentration of alanine in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.

Figure 6. The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) Isoleucine concentration.

The concentration of isoleucine in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.

Figure 7. The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) glutamic acid concentration.

The concentration of glutamic acid in CHOK1 (A+B) and CHOS (C+D) media and lysed cells is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.

Figure 8. Principal component analysis of extracellular metabolites of CHOK1 (A+B) and CHOS (C+D) cultures.

The analysis of the identified extracellular metabolites by PCA is plotted by either culturing conditions (A+C) or timepoint (B+D).