All cultivations were carried out in 250-ml flasks with 50 ml filling volume, being shaken on an orbital shaker with a 50 mm shaking diameter. The shaking frequency was set to 140 rpm and the temperature to 36.5°C. Flasks for passaging and manual sampling with subsequent offline analysis were cultured in an incubator (ISF1-X, Kühner AG, Switzerland) at a controlled relative humidity of 70% and a controlled CO2 concentration of 5%, respectively. Flasks for time-resolved monitoring of the OTR were operated in parallel in an incubator (ISF1-X, Kühner AG, Switzerland) under the same conditions but without CO2 and humidity control. Instead, these flasks were directly gassed with a gas mixture of 5% CO2 in synthetic air (19.5% O2) from a gas bottle (Figure 1A ). Cultivations were either carried out in PowerCHOTM two serum-free chemically defined culture medium, containing HEPES buffer and Pluronic® F68 (Lonza AG, Switzerland) (“medium 1”) or in EX-CELL® Advanced™ CHO Fed-batch Medium (Sigma-Aldrich, United States) (“medium 2”). Both culture media were supplemented with 6 mM L-glutamine from a 200 mM stock solution (Gibco Life Sciences, Thermo Fisher Scientific, United States) before cultivation. Directly before use, culture media and supplements were pre-heated to 36.5°C for about 30 min in a water bath (VWB2 12, VWR, USA).
Pluronic f68
Manufactured by Lonza
Sourced in Switzerland
Pluronic® F68 is a non-ionic surfactant manufactured by Lonza. It is a block copolymer of polyethylene glycol and polypropylene glycol that can be used as a stabilizing agent in various applications.
Automatically generated - may contain errors
2 protocols using pluronic f68
1
Optimized Cultivation of CHO Cells
2
Transient Transfection of CHO-S Cells
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Twenty-four hours prior to transfection, CHO-S cells (Thermo Fisher, R800-07) were cultured (1 × 106 cells per mL) in ProCHO4 medium (without l -glutamine, nucleosides (HT), with HEPES, Pluronic F-68, Lonza) supplemented with 1% Pro-HT and 4% l -glutamine. For transient transfection of 1 × 108 cells, 500 μg polyethylenimine (PEI) transfection solution (1 μg/μL) was diluted in sterile 2.5 mL 150 mM NaCl and mixed with 125 μg plasmid DNA. The mixture was incubated for 10 min at room temperature. Cells were centrifuged for 10 min at 1,000 RPM and then resuspended in 50 mL of ProCHO4 medium. The PEI-DNA mixture was added carefully to the cells with subsequent incubation at 37 °C with shaking. After 4 h, 50 mL PowerCHO-2CD medium (without l -glutamine, nucleosides (HT), with HEPES, Pluronic F-68) supplemented with 1% Pro-HT and 4% l -glutamine was added to the cells, and cells were incubated at 32 °C with shaking. After 5–6 days, supernatant was harvested, and a low yield of protein was obtained. Supernatant was purified using immobilized metal-affinity chromatography, and purity was validated by western blot and Coomassie staining under reducing and non-reducing conditions. Recombinant protein concentration was determined using a NanoDrop (ND-1000 spectrophotometer), bicinchoninic acid assay (ThermoScientific), and Western blot.
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