Bhk 21 c 13

BHK-21 suspension cells were developed from the original adherent cell line, BHK-21 (C-13) (ATCC, Manassas, VA, USA) by the Animal and Plant Quarantine Agency and the Korea Research Institute of Bioscience & Biotechnology for use in suspension culture with serum-free media [15 ]. ProVero™ 1 cell culture medium (Lonza, Basel, Switzerland), a serum-free cell culture medium, was used to culture the BHK-21 suspension cells by incubation at 110 rpm in a shaking incubator at 37 °C with 5% CO₂. In this study, three different strains of serotype O FMDV were used. Two strains were domestic isolates called FMDV O/Boeun/SKR/2017 and FMDV O Jincheon/SKR/2014. The other strain was a genetically engineered virus called the FMDV Om-O-PanAsia2 recombinant strain that was made from a recombination of the backbone region of the O1 Manisa/Turkey/69 strain with the P1 region only being replaced by the O PAK/44/2008 strain [16 (link)].

The cell line “KCTC 12945BP” that was developed from the original adherent cell line, BHK-21 [C-13] (ATCC, Manassas, VA, USA), by the Animal and Plant Quarantine Agency (APQA) and the Korea Research Institute of Bioscience & Biotechnology for use in suspension culture with serum-free media, was adapted for growth in Cellvento^TM BHK-200 cell culture medium (Merck, Darmstadt, Germany) by incubation at 110 rpm in a shaking incubator at 37 °C with 5% CO₂. Cell numbers and viability were analyzed via the trypan blue exclusion method using an automated cell counter (Vi-Cell XR; Beckman Coulter Inc., Brea, CA, USA). A 70% volume of the total cells with 3 × 10⁵ cells/mL was grown for 3.5 days up to approximately 3 × 10⁶ cells/mL, and 30% volume of fresh Cellvento media was added without removing the spent media, followed by inoculation of FMDV into a flask or bioreactor.

Baby hamster kidney-21 (BHK-21) suspension cells were developed from the original adherent cell line, BHK-21 (C-13) (ATCC, Manassas, VA, USA) by the Animal and Plant Quarantine Agency and the Korea Research Institute of Bioscience & Biotechnology for use in suspension culture with serum-free media [10 ]. The cells reached at least 1.5 × 10⁶ cells/mL from a seeding density of 3 × 10⁵ cells/mL within 72 h. The type O FMDV strains (O/Boeun/SKR/2017 and O/Andong/SKR/2010), isolated in Korea, were used to inoculate BHK-21 suspension cells (3 × 10⁶ cells/mL) at a multiplicity of infection (MOI) of 0.001 on a shaking platform in an incubator held at 37 °C. Viruses were harvested at 16 h post-infection and clarified by centrifugation at approximately 3000× g for 20 min at 4 °C to remove cell debris. Binary ethylenimine (BEI, Sigma-Aldrich, St. Louis, MO, USA) was added at 3 mM to the virus culture supernatant to inactivate FMDV, followed by incubation at 100 rpm for 28 h at 26 °C. Subsequently, the BEI was neutralized by adding 2% sodium thiosulfate (Daejung Chemicals & Metals, Siheung, Korea).