Investigation of Growth Properties of BHK-21 Cells with Different Characteristics to Comprise a Cell Bank in Low Serum-Supplement Medium in The Monolayer Culture System

BHK-21 cell lines with distinct growth characteristics are widely used in the production of Foot-and-Mouth Disease vaccines, and seed cell stocks are traditionally cultured in a monolayer system in GMEM medium containing 10% FBS. This study investigated the adaptation of BHK-21 An30 and BHK-21 An73 cell lines to Cellvento™ BHK-200 medium containing 2% FBS and examined the effects on their growth characteristics. Both cell lines underwent serial passages in serum-free medium supplemented with low serum, followed by analyses of viability, apoptosis, cell-cycle distribution, and colony-formation efficiency. Population doubling times were determined by generating growth curves after adaptation. BHK-21 An30 (t(33) = 2.410, p = .022*) and BHK-21 An73 (t(33) = 5.116, p < .001**) cultured in Cellvento™ BHK-200 medium showed higher growth than those grown in GMEM medium containing 10% FBS. Similarly, generation-dependent growth analyses demonstrated higher cell numbers for An30 (t(6) = 5.945, p = .012*) and An73 (t(6) = 2.882, p = .028*) in Cellvento™ BHK-200 compared with the control group. Cells grown in GMEM with 10% FBS exhibited higher colony-forming efficiency. Although viability and apoptosis rates did not differ significantly between the two media, the proportion of apoptotic cells decreased, and overall viability increased with additional passages in the low-serum-supplemented medium for both cell lines. The findings demonstrate that cell banks can be established in low-serum-supplemented, serum-free media, offering a feasible solution for large-scale cell culture production of Foot-and-Mouth Disease vaccines.