Abstract
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°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 2×108/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.
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Abbreviations
- CD:
-
Cyclodextrin
- DEX:
-
Dexamethasone
- EDTA:
-
Ethylene Diamine Tetra Acetate
- EGF:
-
Epidermal Growth Factor
- GG:
-
Glysyl-Glysine
- GP:
-
beta-Glycerophosphate
- FBS:
-
Fetal Bovine Serum
- FGF:
-
Fibroblast Growth Factor
- FN:
-
Fibronectin
- IFN:
-
Interferon
- PBS:
-
Phosphate Buffer Saline
References
BarnesD and SatoGH, (1980) Serum-free cell culture: a unifying approach. Cells 22: 649–655.
FinterNB, FantesKH, AllenG, JohnstonMD, BallGD and LockyerMJ (1983) Mass human cell culture as a source of interferons. In: Proc. Biotech. '83 pp. 389–396. Academic Press N.Y.
HamRG and McKeehanWL (1978) Nutritional requirements for clonal growth of non-transformed cells. In: Nutrition Requirements of Cultured Cells pp. 63–115. Japan Scientific Societies Press Tokyo.
HoshiH, KanM, MinamotoY and YamaneI (1982) Hydrocortisone potentiates cell proliferation and promotes cell spreading on tissue culture substrata of human diploid fibroblasts in a serum-free hormone-supplemented medium. Biomed. Res. 3: 546–552.
KanM, MinamotoY, SunamiS, YamaneI and UmedaM. (1982) The effects on cell adhesion of fibroconectin and gelatin in a serum-free, bovine serum albumin medium. Cell Struct. Funct. 7: 245–252.
KawamotoT, SatoJD, McCleureDB and SatoGH (1983) Development of a serum-free medium for growth of NS-1 mouse myeloma cells and its application to the isolation of NS-1 hybridomas. Anal. Biochem. 130: 445–453.
KobayashiS, IizukaM, HaraM, OzawaH, NagashimaT and SuzukiJ (1980) Preparation of human fibroblast interferon for clinical trials. In: The Clinical Potential of Interferons, pp. 57–68. Univ. Tokyo Press, Tokyo.
LimF and SumA (1980) Microencapsulated islet as bioartificial endocrine pancreas. Science 210: 908–910.
LyersenBK, PutnamJ, BognarE, PattersonM, PughGG and NollLA (1985) Large-scale mammalian cell culture, pp. 39, Academic Press, NY.
MiyoshiI, HirakiS, TsubotaT, KubonishiI and MasujiH (1977) Human B cell, T cell and null cell leukaemic cell lines derived from acute lymphoblastic leukaemias. Nature 267: 843–844.
HurakamiH, MasuiH, SatoGH, SueokaN, ChowTP and Seoka (1982) Growth of hybridoma cells in serum-free medium: Ethanolamine is an essential component. Proc. Natl. Acad. Sci. U.S.A. 79: 1158–1162.
NilausenK (1978) Role of fatty acid in growth-promoting effect of serum albumin on hamster cells in vitro. J. Cell. Physiol. 96: 1–13.
RosenbergSA, LotzeMT, MuulL, MleitmanS, ChangAE, EttingghausenSE, MantoryYL, SkibberJT ShiloniET, VetteoJ, SeippCA, SimpsonC and RiechertCM (1985) Observation on the systematic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. New Engl. J. Med. 313: 1485–1492.
SatoT, MinamotoY, YamaneI, KudoT and TachibanaT (1982) Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium. Exp. Cell. Res. 138: 127–134.
StehleP, KuhneB, KubinW, FurstP and FaenderP (1982) Synthesis and characterization of tryosine- and glutamine-containing peptides. J. Appl. Biochem. 4: 280–286.
SzejtliJ and Banky-ElodE (1975) Inclusion complexes of unsaturated fatty acids with amylose and cyclodextrin. Die Starke 27: 368–376.
TolbertWR, FederJ and KimesRC (1981) In Vitro 17: 885.
YamaneI, MurakamiO and JinboK (1968) An autoclavable powdered culture medium for mammalian cells. Proc. Soc. Exp. Biol. Med. 127: 335–336.
YamaneI, MurakamiO and KatoM (1976) Serum-free culture of various mammalian cells and the role of bovine albumin. Cell. Struct. Funct. 1: 279–284.
YamaneI, KanM, MinamotoY and AmatsujiY (1981a) Alpha-cyclodextrin, a novel substitute for bovine albumin in serum-free culture of mammalian cells. Proc. Jpn. Acad., Ser. B 57: 385–389.
YamaneI, KanM, Hoshi and MinamotoY (1981b) Primary culture of human diploid cell and its long-term transfer in a serum-free medium. Exp. Cell. Res. 134: 470–474.
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Minamoto, Y., Ogawa, K., Abe, H. et al. Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture. Cytotechnology 5 (Suppl 2), 35–51 (1991). https://doi.org/10.1007/BF00573879
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DOI: https://doi.org/10.1007/BF00573879