RESEARCH ARTICLE Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture Andrea Bedoya-López1, Karel Estrada2, Alejandro Sanchez-Flores2, Octavio T. Ramírez3, Claudia Altamirano4, Lorenzo Segovia5, Juan Miranda-Ríos1, Mauricio A. Trujillo-Roldán1, Norma A. Valdez-Cruz1* 1 Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México, 2 Unidad Universitaria de Apoyo Bioinformático, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México, 3 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México, 4 Escuela de Ingeniería Bioquímica, Pontificia OPEN ACCESS Universidad Católica de Valparaíso, Valparaíso, Chile, 5 Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor. México Citation: Bedoya-López A, Estrada K, Sanchez- Flores A, Ramírez OT, Altamirano C, Segovia L, et al. * [email protected] (2016) Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture. PLoS Abstract ONE 11(3): e0151529. doi:10.1371/journal. pone.0151529 Recombinant proteins are widely used as biopharmaceuticals, but their production by mam- Editor: Jonathan A Coles, Glasgow University, malian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly UNITED KINGDOM needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to Received: November 13, 2015 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 Accepted: February 28, 2016 during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant Published: March 18, 2016 human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-gen- Copyright: © 2016 Bedoya-López et al. This is an eration sequencing (NGS) was assessed to characterize the cellular behavior associated open access article distributed under the terms of the with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in medium, provided the original author and source are cultures with TDS compared to those at constant 37°C. In agreement with the extended cell credited. survival resulting from TDS, transcripts related to cell growth arrest that controlled cell prolif- Data Availability Statement: All relevant data are eration without the activation of the DNA damage response, were differentially expressed. within the paper and its Supporting Information files. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial Funding: This work was partially financed by biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for Consejo Nacional de Ciencia y Tecnología rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts (CONACyT, www.conacyt.mx/, projects 220795, encoding proteins involved in the secretory machinery, particularly in glycosylation. 104951-Z, 178528), and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Through NGS the dynamic processes caused by TDS were assessed in this biological Universidad Nacional Autónoma de México (PAPIIT- system. UNAM, www.dgapa.unam.mx, projects IN-210013 and IN-209113). ABL, thanks the “Programa de PLOS ONE | DOI:10.1371/journal.pone.0151529 March 18, 2016 1/26 Temperature Downshift Affects the CHO Transcriptome in rh-tPA Production Cultures Doctorado en Ciencias Biomédicas”, UNAM, and the Introduction scholarship from CONACYT-México. The funders had no role in study design, data collection and Recombinant proteins that require posttranslational modifications, similar to those found in analysis, decision to publish, or preparation of the humans, are preferentially produced in mammalian cells [1]. Chinese hamster ovary (CHO) manuscript. cells have been used as the preferred heterologous expression system to produce recombinant Competing Interests: The authors have declared glycoproteins, which are in increasingly high demand for the treatment of various diseases that no competing interests exist. [1,2]. Hence, there exists a critical need to improve these bioprocesses in order to increase recombinant protein yields while maintaining product stability, activity, and biosafety [1,3,4]. One strategy used to improve specific and volumetric productivity is to decrease the culture temperature [5–8]. Effects of temperature downshifts (TDS) from 37°C to temperatures rang- ing from 28°C to 34°C (a procedure known as “biphasic cultures under moderate hypother- mia”) have been reported, with the optimal conditions dependent on the product and cell line under investigation [9,10]. TDS usually reduces cell growth but increases cell viability, slows cell metabolism, delays cell death, and prolongs RNA half-life. Such effects have been related to increases in recombinant protein production [2,3,10–13]. Recent transcriptomic and proteomic studies have increased our understanding of the genetic and physiological processes in CHO cell cultures [14,15]. A transcriptomic analysis of a moderate hypothermic effect in CHO cells was performed using mouse and rat DNA microar- rays [16–18]. The expression of 301 genes (from 1,655 known genes in rat cDNA chips) and 162 genes (from 4,643 known genes in mouse cDNA chips) were modified in CHO cells that were cultured at 33°C, as compared to those cultured at 37°C [16]. Other recombinant CHO cells under TDS showed 237 genes that were differentially expressed from 4,509 well-annotated genes using CHO cDNA microarrays [18]. Next-generation sequencing (NGS) technologies are more effective for deep sequencing analyses of genomes and transcriptomes compared to Sanger-based sequencing techniques, and are not limited by DNA chips [19,20], allowing the identification of a large number of RNA transcripts. For the CHO-K1 cell line genome, Illumina´s sequencing platform showed an estimate size of 2.45-Gb and 24,383 predicted genes [21,22], allowing a better description of the transcriptome data set [19,23]. Recently, data were obtained using NGS on CHO cultures undergoing TDS from 36°C to 31°C, which caused a decrease in protein productivity [24]. Since high productivity is associated with particular cell responses and different transcrip- tomic states [18,25], we aimed to analyze and illustrate the effects of TDS on the transcriptomic profile of CHO TF70R cells. TDS performed in this study resulted in an increased production of recombinant rh-tPA and viability, as also observed by others using the same cell line [10,26,27]. The effects of TDS on cell metabolism and the secretion pathway through the endo- plasmic reticulum (ER) were also determined and described here. The NGS analysis shown is helpful for the development of clones with high productivity. In particular, it is proposed that specific changes in the ER and Golgi are the main responses associated to the increased recom- binant protein production observed in the biological system used here. Materials and Methods Cell lines and culture conditions The cell line, CHO TF70R, which produces rh-tPA, was obtained from Pharmacia & Upjohn S. A. Sweden and was a kind gift of Torsten Björlig. The cells were cultured in serum-free, chemi- 1 cally defined CD-OptiCHO medium (GIBCO ThermoFisher, Carlsbad, CA, USA), supple- mented with 6 mM glutamine (Biowest LLC, Kansas City, MO, USA), 4 mg/mL insulin HUMULIN (Eli Lilly, Indianapolis, IN, USA), 100 U/mL penicillin (Antibióticos de México, México D.F., México), 0.01 mg/mL streptomycin (Sigma-Aldrich, Hamburg, Germany), 0.001 PLOS ONE | DOI:10.1371/journal.pone.0151529 March 18, 2016 2/26 Temperature Downshift Affects the CHO Transcriptome in rh-tPA Production Cultures 1 g/L Pluronic F-68 (Sigma-Aldrich, Hamburg, Germany), 200 nM methotrexate (Pfizer, New York, NY, USA), and 4% yeast extract (Becton Dickinson, Franklin Lakes, NJ, USA) at pH 7.0. Inocula were prepared in 75-cm2 T-flasks (Nunc, Roskilde, Denmark) and incubated at 37°C 5 with humidified air containing 5% CO2. Six spinner flasks were inoculated with 3.5 × 10 cells/ mL at 90% viability in a working volume of 100 mL, and were cultured at 90 rpm with 5% CO2 atmosphere in a humidified incubator. After 48 h, three spinners were switched to 30°C (biphasic cultures) and three were kept at 37°C (control cultures). Samples were taken every 24 h until viability dropped to 40%. The cell concentration and cell viability were determined using a cell counter (TC10 BioRad, Hercules, CA, USA) and a Neubauer chamber following the trypan blue dye exclusion method. Determination of glucose, glutamine, lactate, and rh-tPA concentrations The concentration of glucose, glutamine, glutamate, and lactate was measured with a YSI 2900 (YSI Life Sciences, Yellow Springs, OH, USA). The concentration of rh-tPA in the culture supernatants was measured by ELISA using a monoclonal