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

Wagstaff JL, Masterton RJ, Povey JF, Smales CM, Howard MJ - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.

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.

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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.
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pone-0077195-g003: 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.

Mentions: The DSS peak area within each spectra represents the equivalent of 90 μM of protons and so by knowing the number of protons within each media component that contribute to the associated peaks within the 1D spectra the molar concentration of each component can be established. The concentrations of each quantifiable metabolite identified within the extracellular media across all time points and growth conditions are listed in Tables S1, S2, S3 and S4 in File S1 with selected metabolite data shown in figures 3, 4, 5, 6, and 7 for glucose, lactate, alanine, isoleucine and glutamic acid respectively. The metabolites identified were selected based upon those that have previously been implicated as changing during mammalian cell culture and where we did observe changes. Figure 3A and 3C shows the effect of incubation temperature on the consumption of glucose over 216 h for the CHOK1 and CHO-S culture media respectively. The 37°C time course for both cell types show that all available glucose had been consumed by 216 h. The normalised data reveals a steady consumption of glucose in both cultures up to the point of the first temperature shift at 72 h. It is of note that at this first temperature shift time point more than 60% of the total glucose has been depleted from the CHOK1 media where as less that 40% of the original glucose has been used in the CHO-S medium. This reflects the large differences in the initial concentrations of 1.3 mM and 73 mM in the CHOK1 and CHO-S media respectively. 24 h after the temperature shifts similar trends of a reduction in glucose usage was observed in both media types and for both shifted temperatures, with the 10°C cultures using less glucose in comparison to the 27°C shifted cells. Cultures of both cell types show very little glucose consumption when shifted and kept at 10°C until the end of the culture and the final concentration of glucose for the 27°C shifted cultures is approximately 50% of that seen at 10°C. Data from temperature recovered cultures again follow a similar trend in both cell types. The recovery of the cells to 37°C highlights an increase in glucose demand relative to the shifted cultures with the 10°C shift and recovered cells having used less glucose than the 27°C shift and recovered cells at 120 h. By the end of the culture glucose concentrations of these cultures matches that of the 37°C control culture (i.e. all glucose depleted).


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

Wagstaff JL, Masterton RJ, Povey JF, Smales CM, Howard MJ - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3795012&req=5

pone-0077195-g003: 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.
Mentions: The DSS peak area within each spectra represents the equivalent of 90 μM of protons and so by knowing the number of protons within each media component that contribute to the associated peaks within the 1D spectra the molar concentration of each component can be established. The concentrations of each quantifiable metabolite identified within the extracellular media across all time points and growth conditions are listed in Tables S1, S2, S3 and S4 in File S1 with selected metabolite data shown in figures 3, 4, 5, 6, and 7 for glucose, lactate, alanine, isoleucine and glutamic acid respectively. The metabolites identified were selected based upon those that have previously been implicated as changing during mammalian cell culture and where we did observe changes. Figure 3A and 3C shows the effect of incubation temperature on the consumption of glucose over 216 h for the CHOK1 and CHO-S culture media respectively. The 37°C time course for both cell types show that all available glucose had been consumed by 216 h. The normalised data reveals a steady consumption of glucose in both cultures up to the point of the first temperature shift at 72 h. It is of note that at this first temperature shift time point more than 60% of the total glucose has been depleted from the CHOK1 media where as less that 40% of the original glucose has been used in the CHO-S medium. This reflects the large differences in the initial concentrations of 1.3 mM and 73 mM in the CHOK1 and CHO-S media respectively. 24 h after the temperature shifts similar trends of a reduction in glucose usage was observed in both media types and for both shifted temperatures, with the 10°C cultures using less glucose in comparison to the 27°C shifted cells. Cultures of both cell types show very little glucose consumption when shifted and kept at 10°C until the end of the culture and the final concentration of glucose for the 27°C shifted cultures is approximately 50% of that seen at 10°C. Data from temperature recovered cultures again follow a similar trend in both cell types. The recovery of the cells to 37°C highlights an increase in glucose demand relative to the shifted cultures with the 10°C shift and recovered cells having used less glucose than the 27°C shift and recovered cells at 120 h. By the end of the culture glucose concentrations of these cultures matches that of the 37°C control culture (i.e. all glucose depleted).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus