In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture

被引:32
作者
Behjousiar, Alireza [1 ,2 ,3 ]
Kontoravdi, Cleo [3 ]
Polizzi, Karen M. [1 ,2 ]
机构
[1] Univ London Imperial Coll Sci Technol & Med, Div Mol Biosci, Dept Life Sci, London, England
[2] Univ London Imperial Coll Sci Technol & Med, Ctr Synthet Biol & Innovat, London, England
[3] Univ London Imperial Coll Sci Technol & Med, Dept Chem Engn, Ctr Proc Syst Engn, London SW7 2AZ, England
基金
英国生物技术与生命科学研究理事会; 英国工程与自然科学研究理事会;
关键词
TRANSPORT; LACTATE;
D O I
10.1371/journal.pone.0034512
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The development of processes to produce biopharmaceuticals industrially is still largely empirical and relies on optimizing both medium formulation and cell line in a product-specific manner. Current small-scale (well plate-based) process development methods cannot provide sufficient sample volume for analysis, to obtain information on nutrient utilization which can be problematic when processes are scaled to industrial fermenters. We envision a platform where essential metabolites can be monitored non-invasively and in real time in an ultra-low volume assay in order to provide additional information on cellular metabolism in high throughput screens. Towards this end, we have developed a model system of Chinese Hamster Ovary cells stably expressing protein-based biosensors for glucose and glutamine. Herein, we demonstrate that these can accurately reflect changing intracellular metabolite concentrations in vivo during batch and fed-batch culture of CHO cells. The ability to monitor intracellular depletion of essential nutrients in high throughput will allow rapid development of improved bioprocesses.
引用
收藏
页数:9
相关论文
共 18 条
[1]   Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors [J].
Ai, Hui-wang ;
Hazelwood, Kristin L. ;
Davidson, Michael W. ;
Campbell, Robert E. .
NATURE METHODS, 2008, 5 (05) :401-403
[2]   Considerations on the lactate consumption by CHO cells in the presence of galactose [J].
Altamirano, Claudia ;
Illanes, Andres ;
Becerra, Silvana ;
Cairo, Jordi Joan ;
Godia, Francesc .
JOURNAL OF BIOTECHNOLOGY, 2006, 125 (04) :547-556
[3]  
Aurilia V, 2009, CELLULAR BIOMOLECULA
[4]   Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors [J].
Bermejo, Clara ;
Haerizadeh, Farzad ;
Takanaga, Hitomi ;
Chermak, Diane ;
Frommer, Wolf B. .
BIOCHEMICAL JOURNAL, 2010, 432 :399-406
[5]   Fluorescent Nano-Optodes for Glucose Detection [J].
Billingsley, Kelvin ;
Balaconis, Mary K. ;
Dubach, J. Matthew ;
Zhang, Ning ;
Lim, Ed ;
Francis, Kevin P. ;
Clark, Heather A. .
ANALYTICAL CHEMISTRY, 2010, 82 (09) :3707-3713
[6]   Optical determination of glutamine using a genetically engineered protein [J].
Dattelbaum, JD ;
Lakowicz, JR .
ANALYTICAL BIOCHEMISTRY, 2001, 291 (01) :89-95
[7]   Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering [J].
Deuschle, K ;
Okumoto, S ;
Fehr, M ;
Looger, LL ;
Kozhukh, L ;
Frommer, WB .
PROTEIN SCIENCE, 2005, 14 (09) :2304-2314
[8]   Expression systems for therapeutic glycoprotein production [J].
Durocher, Yves ;
Butler, Michael .
CURRENT OPINION IN BIOTECHNOLOGY, 2009, 20 (06) :700-707
[9]   Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors [J].
Fehr, M ;
Takanaga, H ;
Ehrhardt, DW ;
Frommer, WB .
MOLECULAR AND CELLULAR BIOLOGY, 2005, 25 (24) :11102-11112
[10]   Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels [J].
Han, Yi ;
Liu, Xing-Mao ;
Liu, Hong ;
Li, Shi-Chong ;
Wu, Ben-Chuan ;
Ye, Ling-Ling ;
Wang, Qu-Wei ;
Chen, Zhao-Lie .
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2006, 102 (05) :430-435