Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

• Published in: Cytotechnology (Dordrecht) 1991, Vol.5 (S2), p.35-51
• Main Authors: MINAMOTO, Y, OGAWA, K, ABE, H, IOCHI, Y, MITSUGI, K
• Format: Article
• Language: English
• Subjects: Animal cells; Animals; B-Lymphocytes - cytology; Biological and medical sciences; Biotechnology; Blood; Cells, Cultured; Culture Media; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Fibroblasts - cytology; Fundamental and applied biological sciences. Psychology; Glutamine; Humans; Methods. Procedures. Technologies; Sterilization
• ISSN: 0920-9069; 1573-0778
• DOI: 10.1007/BF00573879

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium. As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37 degrees C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640 + 10% FBS. It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable. Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast. Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2 x 10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day. The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.