Abstract
An efficient mammalian cell system for producing bioproducts should retain high cell viability and efficient use of energy sources rendering the need to understand the effects of various variables on the cell system. In this study, global metabolite (metabolomics) analysis approach was used to try and understand the relationships between types of media used, culture growth behavior and productivity. CHO-KI cells producing IGF-1 were obtained from ATCC and grown in T-flask (37 °C, 5 % CO2) until 70–80 % confluent in RPMI 1640 and Ham’s F12, respectively. Samples were taken at 8-hourly intervals for routine cell counting, biochemical responses, insulin like growth factor—1 (IGF-1) protein concentration and global metabolite analysis (gas chromatography mass spectrometry, GCMS). Conditioned media from each time point were spun down before injection into GCMS. Data from GCMS were then transferred to SIMCA-P + Version 12 for chemometric evaluation using principal component analysis. The results showed that while routine analysis gave only subtle differences between the media, global metabolite analysis was able to clearly separate the culture based on growth media with growth phases as confounding factor. Different types of media also appeared to affect IGF-1 production. Asparagine was found to be indicative of healthiness of cells and production of high IGF-1. Meanwhile identification of ornithine and lysine in death phase was found to be associated with apoptosis and oversupplied nutrient respectively. Using the biomarkers revealed in the study, several bioprocessing strategies including medium improvement and in-time downstream processing can be potentially implemented to achieve efficient CHO culture system.
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Allen J, Davey HM, Broadhurst D, Heald JK, Rowland JJ, Oliver SG, Kell DB (2003) High- throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nat Biotech 21:692–696
Altamirano C, Paredes C, Cairo JJ, Godia FF (2000) Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine. Biotechnol Prog 16:69–75
Altamirano C, Cairo JJ, Godia F (2001) Decoupling growth and product formation in Chinese hamster ovary cells through metabolic control. Biotechnol Bioeng 76:351–360
Altamirano C, Paredes C, Illanes A, Cairo JJ, Godia F (2004) Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. J Biotechnol 110:171–179
Bradley SA, Ouyang A, Purdie J, Smitka TA, Wang T, Kaerner A (2010) Fermentanomics: monitoring mammalian cell cultures with NMR spectroscopy. J Am Chem Soc 132:9531–9533
Cartwright T (1994) Animal Cells as Bioreactors. Cambridge University Press, Cambridge
Chong WPK, Goh LT, Reddy SG, Yusufi FNK, Lee DY, Wong NSC, Chew KH, Yap MGS, Ying SH (2009) Metabolomics profiling of extracellular metabolites in recombinant Chinese Hamster Ovary fed-batch culture. Rapid Commun Mass Spectrometry 23:3763–3771
Chong WPK, Reddy ST, Yusufi FNK, Lee D-Y, Wong NSC, Chew KH, Yap MGS, Ying SH (2010) Metabolomics-driven approach for the improvement of Chinese hamster ovary cell growth: overexpression of malate dehydrogenase II. J Biotechnology 147:116–121
Dippel B (2011) http://www.raman.de/htmlEN/home/advantageEn.html. Accessed 25 September 2012
Fernandez C, Fransson U, Hallgard E, Spegel P, Holm C, Krogh M, Warell K, James P, Mulder H (2008) Metabolomic and Proteomic Analysis of a Clonal Insulin-Producing β-Cell Line (INS-1 832/13) J Proteome Res 7:400–411
Filipiak W, Sponring A, Filipiak A, Clemens A, Schubert J, Miekisch W, Amann A, Troppmair J (2010) TD-GC MS analysis of volatile metabolites in human lung cancer and normal cells in vitro. Cancer Epidemiol Biomarkers 19:182–195
Gibney M (2005) Metabolomics in human nutrition: opportunities and challenges. J Clin Nutr 82:497–503
Goodacre R, Seetharaman V, Dunn WB, Harrigan GG, Kell DB (2004) Metabolomics by numbers: acquiring and understanding global metabolite data. Review. Trends Biotechnol 22:245–252
Griffin JL (2003) Metabonomics: NMR spectroscopy and pattern recognition analysis of body fluids and tissues for characterisation of xenobiotic toxicity and disease diagnosis. Curr Opin Chem Biol 7:648–654
Hinger S, Reiter M, Gaida T, Kral G, Gruber G, Weigang F, Katinger H (1989) Cultivation of hybridomas producing IgG at low serum concentration. Cytotechnology 2(Suppl):78–79
Khoo SHG, Al-Rubeai M (2007) Metabolomics as a complementary tool in cell culture. Biotechnol App Biochem 47:71–84
Kumar N, Gammell P, Clynes M (2007) Proliferation control strategies to improve productivity and survival during CHO based production culture. Cytotechnology 53:33–46
Kuystermans D, Krampe B, Swiderek H, Al-Rubeai M (2007) Using cell engineering and omic tools for the improvement of cell culture processes. Cytotechnology 53:3–22
Lindon JC, Nicholson JK, Holmes E (2006) The handbook of metabonomics and metabolomics. Elsevier, Amsterdam
Pope GA, Mackenzie DA, Defernez M, Aroso MAAM, Fuller LJ, Mellon FA, Dunn WB, Brown M, Goodacre R, Kell DB, Marvin ME, Louis EJ, Roberts IN (2007) Metabolic footprinting as a tool for discriminating between brewing yeasts. Yeast 24:667–679
Quek LE, Dietmair S, Kromer JO, Nielsen LK (2010) Metabolic flux analysis in mammalian cell culture. Metab Eng 12:161–171
Silas GVB, Sandrine M, Mats A, Jorn S, Jens N (2005) Mass spectrometry in metabolome analysis. Mass Spectrometry Rev 24:613–646
Takeda S, Sim PG, Horrobin DF, Sanford T, Chisholm KA, Simmons V (1993) Mechanism of lipid peroxidation in cancer cells in response to gamma-linolenic acid (GLA) analyzed by GC-MS(I): conjugated dienes with peroxyl (or hydroperoxyl) groups and cell-killing effects. Anticancer Res 13:193–199
Tobias KE, Kahana C (1995) Exposure to ornithine results in excessive accumulation putrescine and apoptotic cell death in ornithine decarboxylase overproducing mouse myeloma cells. Cell Growth Differ 6:1279–1285
Wishart DS, Knox C, Guo AC et al (2009) HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 37:D603–D610
Zhang F, Sun X, Yi X, Zhang Y (2006) Metabolic characteristics of recombinant Chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine. Cytotechnology 51:21–28
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This research was funded by IIUM Research Matching Grant Scheme (RMGS). We would like to thank everyone who contributed to the research on effects of different types of growth media on metabolites profile of CHO-K1 cells.
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Mohmad-Saberi, S.E., Hashim, Y.Z.HY., Mel, M. et al. Metabolomics profiling of extracellular metabolites in CHO-K1 cells cultured in different types of growth media. Cytotechnology 65, 577–586 (2013). https://doi.org/10.1007/s10616-012-9508-4
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DOI: https://doi.org/10.1007/s10616-012-9508-4