Cell culture and media for maintenance of CHO K1 master cell line (MCL)

The CHO K1 master cell line (MCL) was generated by nucleofection of a landing pad vector into CHO K1 cells (ATCC), followed by screening clones for single copy integration by southern blotting. The landing pad vector expressed a hygromycin resistant gene (HYG) using a chimeric promoter (ChiP) which consisted of the murine CMV enhancer ({"type":"entrez-nucleotide","attrs":{"text":"M11788","term_id":"330622","term_text":"M11788"}}M11788), the hCMV core promoter and the hCMV intron A ({"type":"entrez-nucleotide","attrs":{"text":"M60321","term_id":"330624","term_text":"M60321"}}M60321). The HYG expression cassette was flanked by FRT3 and FRT. An impaired puromycin resistant gene lacking start codon ((ATG-)Puro) followed by the simian virus 40 (SV40) polyadenylation signal (pA) was placed downstream of FRT for selecting correct cassette exchange by RMCE (Fig. 1A). It was confirmed that the MCL contained only one copy of landing pad vector at a single integration site by southern blotting and targeted locus amplification (TLA) analyses (Cergentis). The MCL was grown in a protein-free medium (maintenance media) consisting of 50% HyQ PF (GE Healthcare Life Sciences) and 50% CD CHO (Thermo Fisher Scientific) supplemented with 1 g/L sodium carbonate (Sigma), 6 mM glutamine (Sigma) and 0.1% Pluronic F-68 (Thermo Fisher Scientific) in a humidified Kuhner shaker (Adolf Kühner AG) with 8% CO₂ at 37 °C. Routine subculture was conducted every 3 to 4 days by seeding cells at density of 3 × 10⁵ cells/mL in 15 mL of fresh medium in 125 mL shake flasks (Corning). Cell density and viability were determined by trypan blue exclusion method on Vi-Cell XR viability analysers (Beckman Coulter).