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dc.rights.licenseReconocimiento 4.0 Internacional. (CC BY)es
dc.contributor.authorPerelmuter, Karenes
dc.contributor.authorTiscornia, Inéses
dc.contributor.authorComini, Marcelo A.es
dc.contributor.authorBollati-Fogolín, Marielaes
dc.date.accessioned2023-06-05T16:28:31Z-
dc.date.available2023-06-05T16:28:31Z-
dc.date.issued2022-02-09-
dc.identifier.urihttps://hdl.handle.net/20.500.12381/3244-
dc.description.abstractCellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.es
dc.description.sponsorshipAgencia Nacional de Investigación e Innovaciónes
dc.description.sponsorshipFOCEM (MERCOSUR Structural Convergence Fund)es
dc.language.isoenges
dc.publisherMDPI, Basel, Switzerland.es
dc.rightsAcceso abiertoes
dc.sourceSensorses
dc.subjectGM-CSFes
dc.subjectBioprocess development-
dc.subjectBiosensor-
dc.subjectRedox metabolism-
dc.subjectrxYFP-
dc.titleGeneration and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevancees
dc.typeArtículoes
dc.subject.aniiCiencias Médicas y de la Salud-
dc.subject.aniiBiotecnología de la Salud-
dc.subject.aniiTecnologías que involucran la manipulación de células, tejidos, órganos o todo el org-
dc.identifier.aniiPR_FMV_2009_1_261es
dc.identifier.aniiMERCOSUR Structural Convergence Fund, COF 03/11es
dc.type.versionPublicadoes
dc.identifier.doihttps://doi.org/10.3390/s22041324-
dc.anii.institucionresponsableInstitut Pasteur de Montevideoes
dc.anii.subjectcompleto//Ciencias Médicas y de la Salud/Biotecnología de la Salud/Tecnologías que involucran la manipulación de células, tejidos, órganos o todo el orges
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