Development of a serum-free medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44) using a statistical design: Beneficial effect of weaning of cells

To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chi nese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally mai ntained in 10% serum medium were gradually weaned to 0.5% serum medium to i ncrease the probability of successful growth in SF medium. A basal medium w as prepared by supplementing Dulbecco's modified Eagle's medium and Ham's n utrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Tw enty-eight different supplements; were selected as variables on the basis o f their growth-promoting abilities. From statistical analysis, leucine, try ptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phos phatidylcholine were identified as important components showing positive ef fects on cell growth. A new SF medium (SF-DG44) was formulated by supplemen ting the basal medium with these components. When the weaned cells were ino culated at 1.0 x 10(5) cells/ml, a maximum viable cell concentration of 6.4 x 10(5) cells/ml was achieved in SF-DG44 medium. In contrast, when the unw eaned cells were used, a concentration of only 4.1 x 10(5) cells/ml was rea ched under the same culture conditions, indicating that weaning of cells im proves cell growth in SI: medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman de sign technique and weaning of cells.