Effects of Calcium Ions on Cell Growth and Aggregation of a Suspension-adapted Recombinant CHO Cell Line

초록

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical industries for the production of recombinant therapeutic proteins. CHO cells can grow in suspension without serum and many biopharmaceutical companies have taken advantage of this cell line for their approved therapeutic drugs. Some CHO-derived cells adapted to suspension culture form aggregates. Cell aggregation hinders mass transfer of nutrients and dissolved oxygen and lowers viable cell density. A low-calcium medium generally suggested for suspension cultures has therefore been employed for large-scale cultivation as well as for adaptation procedures. Despite the importance of its addition to suspended CHO cells, there have been few detailed studies that investigate the influence of calcium ions on adapted CHO cell aggregates grown in serum-free medium. In this work, we studied the effect of calcium ions on cell growth, cell size distribution, and cell aggregation of a serum-free suspension-adapted CHO-DG44 cell lineage. Suspension cultures were grown in 125 mL Erlenmeyer flasks at an initial density of 2.5 X105 cells/mL in modified DMEM/F12 media. Different media containing various amounts of calcium chloride from 0 to 200 mg/L were prepared. In the batch suspension cultures with 10mg/L or 30mg/L CaCl2, a cell density of more than 10X105 cells/mL was obtained after 48hr, while increasing the calcium chloride concentration above 50mg/L decreased cell growth. In cultivations using 50, 100, or 200 mg/L calcium chloride, the viable cell numbers did not exceed 8 X105 cells/mL. Cell aggregation in a modified DMEM/F12 medium containing 50 mg/L and 100 mg/L of CaCl2 was confirmed with microscopic observation. Using a Coulter counter, it was also observed that 100 mg/L CaCl2 increased the average mean diameter of the cells from 14μm up to 18μm after 72hr cultivation. When dextran sulfate (25mg/L, Mw > 500,000) was added single-cell formation was induced in media with calcium chloride corresponding to up to 100 mg/L. The cells in the relatively high-calcium media exhibited improved cell growth compared with those in same medium without dextran sulfate. In the presence of 100 mg/L CaCl2, for example, growth was 58% higher than that without dextran sulfate. To clarify the observed influence of calcium ions, a long passage culture experiment was carried out. During nine consecutive passages (one time per 2days), the adapted CHO DG-44 cells showed a similar growth rate within serum-free media having 10 ~ 30 mg/L calcium chloride, whereas in the case of calcium-free or high-calcium medium (100mg/L) the growth ceased at 7 and 14 days, respectively. Moreover, CHO cells in 10mg/L CaCl2 showed the best result with a maximal cell density of 12X105 cells/mL at 96hrs following subcultivation of the final passage. These results indicate that reducing the calcium concentration in the growth medium is required for suspension cultivation. Low-calcium medium, with a CaCl2 concentration much lower than that of commercial serum-free media, may be preferential for large scale suspension culture.