title: Transcriptional profiling of batch and fed-batch protein-free 293-HEK cultures.
abstract: Dynamic nutrient feeding to control glutamine at low levels in protein-free fed-batch cultures of 293-human embryonic kidney (HEK) cells achieved cell concentrations of 6 x 10(6) cells/ml. This represented a 4-fold improvement in cell concentration compared to batch cultures. Reduction in glutamine and glucose consumption, as well as lactate and ammonia production, were also observed in these fed-batch cultures. High virus production titers of 3 x 10(11) pfu/ml were achieved in fed-batch cultures which were 10,000-fold higher than batch cultures. An investigation of the transcriptional regulation of the metabolic changes associated with the batch and the low-glutamine fed-batch cultures using DNA microarray was conducted. This analysis provides better understanding of the transcriptional regulatory mechanism resulting in the observed physiological changes. Transcriptional profiling of cells from the mid-exponential, late exponential and stationary phases of both the batch and fed-batch were undertaken using an 18,000 element human chip. Transcriptional profiles were ontologically classified to provide a global view of the genetic changes. Furthermore, a pathway-oriented analysis focusing on cellular metabolism was conducted to reveal the dynamic regulation of genes related to amino acid metabolism, tRNA synthetases, TCA cycle, electron transport chain and glycolysis.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Yih Yean
authors.initials: YY
authors.lastname: Lee
authors.forename: Kathy T K
authors.initials: KT
authors.lastname: Wong
authors.forename: Peter Morin
authors.initials: PM
authors.lastname: Nissom
authors.forename: Danny C F
authors.initials: DC
authors.lastname: Wong
authors.forename: Miranda G S
authors.initials: MG
authors.lastname: Yap
mesh_terms: D000260:Adenoviruses, Human / Q000254:growth & development; D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D002952:Citric Acid Cycle; D004579:Electron Transport; D020869:Gene Expression Profiling; D006019:Glycolysis; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction
chemical_list: D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases