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

Abstract

To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10 serum medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 x 10(5) cells/ml, a maximum viable cell concentration of 6.4 x 10(3)) cells/ ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1 x 10(5) cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

“…However, serum-free media implies a case to case design based on the particular supplements required for each strain. Chemically defined serum-free media have been extensively studied and several reviews have been published on the subject (Keen and Rapson, 1995;Lao and Shalla, 1996;Kim et al 1999). Hormones and growth factors requirements are specific for each type of cell (Freshney, 2000;Xie et al 2003).…”

5.3 Nutrient Media for Cell Culture Technology

“…The identification of defined supplements that improve growth, productivity, and consistency is highly attractive for cell culture optimisation strategies. These types of media and specific defined supplements have been extensively studied and several reviews have been published on the subject [30][31][32].…”

Cell culture medium improvement by rigorous shuffling of components using media blending

“…With factorial designs, concentrations of several components are varied simultaneously and thus represent the method of choice to identify interactions and to find optimal combinations of components. Factorial designs have been used as medium development tools for bacterial systems (Wejse et al 2003;Kiviharju et al 2004;Zhao et al 2005) and for mammalian cells such as CHO cells (Castro et al 1992;Kim et al 1998Kim et al , 1999Lee et al 1999;Liu et al 2001;Chun et al 2003).…”