Cell culture
Suspension-adapted CHO-DG44 (A1100001, Thermo Fisher, Darmstadt, Germany) and CHO-K1 (CCL-61TM, ATCC) cells were cultured in chemically defined HyClone™ SFM4CHO™ medium (Cytiva, Marlborough, MA, USA) supplemented with 10 g/L glucose (Roth, Karlsruhe, Germany) and 4 mM l-glutamine (Lonza, Basel, Switzerland) at 37 °C, 5% CO2, 85% humidity, and 140 rpm (25 mm orbit) in shake flasks (Corning Inc, New York, USA) using a Kuhner incubate shaker (Adolf Kühner AG, Basel, Swiss). Subculturing was performed every 3–4 days to a viable cell density (VCD) of 0.5 × 106 cells/mL. VCD and viability were determined by trypan blue exclusion using CEDEX XS Cell Analyzer (Roche Diagnostics, Mannheim, Germany). CHO cells were transfected with 15 µg vector using the NEON® transfection system (Thermo Fisher Scientific, Waltham, MA, USA). Selection of polyclonal cell lines stably expressing derivatives of the pEF-myc-cyto-mCMV-d2GFP vector (Addgene, Watertown, MA, USA) was performed using 500 µg/mL G418 sulfate (Genaxxon Bioscience, Ulm, Germany) and/or 400 µg/mL Zeocin (InvivoGen, Toulouse, France). All utilized vectors are listed in Table 1. To induce fluorescent protein expression, CHO cells were seeded with a VCD of 0.5 × 106 cells/mL and grown statically for several days at undefined hypoxic conditions (37 °C, 5% CO2, and 85% humidity) in a Forma™ Steri-Cycle™ incubator (Thermo Fisher Scientific, Waltham, MA, USA) where a static cultivation and sedimentation of the cells was sufficient to generate hypoxic conditions. In addition, a shaken control batch was inoculated with a VCD of 0.5 × 106 cells/mL to meme normoxic conditions. To induce fluorescent protein expression by OREs, hyperosmotic conditions (up to 0.45 osm/kg) were created by addition of sodium chloride (NaCl) or glucose and compared to an isotonic (0.3 osm/kg) cultured control. To analyze the reversibility of the fluorescent protein expression after hyperosmotic induction of 0.4 osm/kg, the cells were centrifuged and the media was changed to isotonic media with 0.3 osm/kg. The osmolality of culture supernatants was measured using OSMOMAT 030 (Gonotec, Berlin, Germany).
Batch fermentation
For cultivation at defined O2 concentrations and hyperosmotic conditions, CHO cells were inoculated at a VCD of 0.7 × 106 cells/mL in a volume of 1 L using 2 L stirred tank benchtop bioreactors (Sartorius, Göttingen, Germany). Process conditions were controlled at a stirring speed of 100 rpm, pH of 7.15, and temperature of 37 °C. The O2 set point was adjusted at different time points by gassing with N2 to reach a pO2 of 1%. To restore normoxia (pO2 = 40%) the gas mix was adjusted by the fermentation head-unit and air was used to enhance the desired pO2. If air was not sufficient to provide the desired O2 concentration, the fermentation head-unit mixed pure O2 into the gas mix. Osmolality was increased by the addition of a 3 M NaCl solution with a peristaltic pump and the volume of added NaCl was observed by a scale. We aimed to provide an increased osmolality of around 0.4 osm/kg and confirmed a final osmolality after NaCl addition of 0.41 osm/kg by external measurement of the cell culture suspension.
Molecular biology
The starting plasmid 5HRE/GFP was a gift from Martin Brown & Thomas Foster (Addgene plasmid # 46926)40. Constructs were cloned using HRE sequences of VEGF and ORE sequences of AR published by Javan et al. 201716 and Ferraris et al. 199941, respectively. The oligos used are listed in Table 2. Prior to cloning, oligonucleotides were annealed by heating to 95 °C followed by slow cooling to 25 °C, phosphorylation using T4 PNK (New England Biolabs, Ipswich, MA, USA), and ligation by T4 ligase (New England Biolabs, Ipswich, MA, USA). After removing the 5HREs by digestion with XhoI and BglII (both New England Biolabs, Ipswich, MA, USA), the randomly ligated double-stranded oligonucleotides were cloned into the pEF-myc-cyto-mCMV-d2GFP vector. The origin of the destabilized blue fluorescent protein variant FKBP-mTagBFP2 and zeocin resistance for the exchange of fluorescent protein and selectable markers were the vectors pAW63.YY1.FKBP.knock-in. BFP (Addgene plasmid #104371)29 and pcDNA3.1/Zeo(+) (Thermo Fisher Scientific, Waltham, MA, USA), respectively. FKBP-mTagBFP2 and zeocin resistance were amplified via PCR using the following primers: FKBP-BFP Fwd (5-ATATCCATGGGTGCCCCTTCGACGGTTGTA-3); FKBP-BFP Rev (5-ATATTCTAGACTTCCCGGGTCGAGAAGGTC-3); Zeo Fwd (5′-ATATTCTAGACCCGTTTAAACCCGCTGA-3′); and Zeo Rev (5′-ATATTTCGAACTTTCATAGAAGGCGGCGGT-3′). Cloning of fluorescent protein and selectable markers into the pEF-myc-cyto-mCMV backbone was performed after digestion with NcoI and XbaI as well as XbaI and BstBI (New England Biolabs, Ipswich, MA, USA).
RNA isolation
Total RNA was isolated from 3 × 106 cells using the miRNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration and purity were determined using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) by absorbance at 260 nm.
RT-PCR
After RNA isolation, reverse transcription to cDNA was performed with the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Quantitative real-time PCR on NFAT5 and GAPDH as a loading control was performed using GreenMasterMix No ROX (Genaxxon Bioscience, Ulm, Germany) and the following primers: NFAT5 Fw (5′-TACCACGGACAACAAAGGCA-3′); NFAT5 Rev (5′-AAGTCGATGCCCTTCAGCTC-3′); GAPDH Fw (5′- GACTCTACCCATGGCAAGTTCA-3′); and GAPDH Rev (5′-TCGCTCCTGGAAGATGGTGATG-3′). A LightCycler® 480 Instrument II (Roche Diganostics, Mannheim, Germany) was used for gene expression analysis.
Flow cytometry analysis
The mean fluorescence intensity of d2EGFP and FKBP-mTagBFP2 expressed under hypoxic and hyperosmotic conditions, respectively, was measured using MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch-Gladbach, Germany). D2EGFP was measured using a 488 nm laser and the 525/50 nm filter setting (Channel B1), and FKBP-mTagBFP2 was detected by a 405 nm laser and a 450/50 nm filter setting (Channel V1). Subsequent data analysis was performed with MACSQuantify™ Software.
Live fluorescence microscopy
For live fluorescence microscopy, CHO cells were cultivated in a 2 L bioreactor, and deprivation conditions were induced as described in the methods section Batch fermentation. Automated and continuous sampling was performed by utilizing a peristaltic pump controlled by a socket timer to sample the cell culture suspension every 1 h 30 min. Fluorescence microscopy was performed by integrating a coated µ-slide with Luer adapters and a 0.2 mm channel height (Ibidi GmbH, Gräfelfing, Germany) into a BZ X 800 fluorescence microscope (Keyence, Neu-Isenburg, Germany). Images were automatically recorded by fluorescence microscopy at 5 fixed points in the µ-slide every 1 h 30 min, 3 min after the peristaltic pump stopped sampling to ensure clear good-quality pictures. Fluorescence intensity was qualitatively analyzed by imaging cytometry using Keyence BZ X 800 Analyzing software.
Statistical analysis
Two-tailed unpaired t test with Welsh’s correction was applied for statistical analysis using GraphPad Prism 6. Data are presented as the mean ± standard deviation.
