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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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Related in: MedlinePlus

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.
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pone-0077195-g002: 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.

Mentions: The 0 h 1D proton spectra shown in black in Figure 2 immediately highlight the differences in composition of the two media types considering all data in Figure 2 is reported on the same intensity scale and after identical scan number. Visual observations of the CHOK1 medium (with the serum protein components of the media removed by chloroform:methanol extraction) revealed a more sparsely populated spectrum in comparison to the CHO-S medium. Each sample was spiked with the same concentration of DSS and as the volume of each peak is proportional to the total number of protons that contribute to it, it is clear that the CHO-S medium is formulated with a much higher metabolite concentration than that found in the CHOK1 medium. The protein extraction process also contributed to differences between the two media type’s spectra as after extraction the metabolites were dried to remove the large amount of methanol then re-suspended in deuterium oxide (2H2O), which cannot be seen in the 1H NMR spectra. As a result there is no large solvent peak at 4.65 ppm in the CHOK1 spectra. In contrast, the CHO-S medium spectra displays a residual 1H2O peak because these samples contain only 10% deuterium oxide for NMR spectrometer locking. However, the uniform presence of the DSS spike peak confirms 1H2O solvent suppression for CHO-S data does not have an adverse effect on quantitation as the scan number was identical and signal to noise comparable between CHOK1 and CHO-S data. Also, a residual chloroform peak was evident in the CHOK1 spectra at 7.68 ppm as highlighted in Figure 2A.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0077195-g002: 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.
Mentions: The 0 h 1D proton spectra shown in black in Figure 2 immediately highlight the differences in composition of the two media types considering all data in Figure 2 is reported on the same intensity scale and after identical scan number. Visual observations of the CHOK1 medium (with the serum protein components of the media removed by chloroform:methanol extraction) revealed a more sparsely populated spectrum in comparison to the CHO-S medium. Each sample was spiked with the same concentration of DSS and as the volume of each peak is proportional to the total number of protons that contribute to it, it is clear that the CHO-S medium is formulated with a much higher metabolite concentration than that found in the CHOK1 medium. The protein extraction process also contributed to differences between the two media type’s spectra as after extraction the metabolites were dried to remove the large amount of methanol then re-suspended in deuterium oxide (2H2O), which cannot be seen in the 1H NMR spectra. As a result there is no large solvent peak at 4.65 ppm in the CHOK1 spectra. In contrast, the CHO-S medium spectra displays a residual 1H2O peak because these samples contain only 10% deuterium oxide for NMR spectrometer locking. However, the uniform presence of the DSS spike peak confirms 1H2O solvent suppression for CHO-S data does not have an adverse effect on quantitation as the scan number was identical and signal to noise comparable between CHOK1 and CHO-S data. Also, a residual chloroform peak was evident in the CHOK1 spectra at 7.68 ppm as highlighted in Figure 2A.

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