Serum-free cultivation of anchorage-dependent cells on microcarrier: effective production of human macrophage colony-stimulating factor.
For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalizing the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10(7) cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.
Satoh
S
S
Research Laboratories, Toyo Jozo Co., Ltd., Shizuoka-ken, Japan.
Kobayashi
J
J
Mizoguchi
J
J
Nogawa
M
M
Otani
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D002448:Cell Adhesion; D003470:Culture Media; D004247:DNA; D005784:Gene Amplification; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
D003470:Culture Media; D016173:Macrophage Colony-Stimulating Factor; D004247:DNA