title: Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.
abstract: Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.
authors.affiliation: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bedoya-López
authors.affiliation: Unidad Universitaria de Apoyo Bioinformático, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México.
authors.forename: Karel
authors.initials: K
authors.lastname: Estrada
authors.affiliation: Unidad Universitaria de Apoyo Bioinformático, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sanchez-Flores
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México.
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.affiliation: Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile.
authors.forename: Claudia
authors.initials: C
authors.lastname: Altamirano
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Segovia
authors.affiliation: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
authors.forename: Juan
authors.initials: J
authors.lastname: Miranda-Ríos
authors.affiliation: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
authors.forename: Mauricio A
authors.initials: MA
authors.lastname: Trujillo-Roldán
authors.affiliation: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
authors.forename: Norma A
authors.initials: NA
authors.lastname: Valdez-Cruz
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D005947:Glucose