Adaptation of dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time-consuming and labor-intensive process because nonadapted DHFR-deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension-adapted DHFR-deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2-359 mu M enhanced cell growth in in-house CDM. Addition of 3.6 mu M THF to in-house CDM resulted in a more than 2.5-fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 mu M THF yielded up to 2.9-fold enhancement of maximum viable cell density. An anchorage-and serum-dependent DHFR-deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in-house CDM supplemented with 3.6 mu M THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 mu M THF can help achieve a high density of suspension-adapted DHFR-deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR-deficient CHO DG44 cells to suspension culture. (C) 2016 American Institute of Chemical Engineers