Skip to main content
SpringerLink
Log in

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

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 equalising 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 107 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

CSF:

Colony-Stimulating Factor

M-CSF:

Macrophage Colony-Stimulating Factor

EGF:

Epidermal Growth Factor

FCS:

Fetal Calf Serum

dhfr:

dihydrofolate reductase

MTX:

Methotrexate

HEPES:

N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)

KLa:

oxygen mass transfer coefficient

cDNA:

complementary DNA

CAT:

Chloramphenicol Acetyltransferase

pfu:

plaque forming unit

KDa:

Kilo-daltons

References

  1. BarnesD and SatoG (1980a) Methods for growth of cultured cells in serum free medium. Anal. Biochem. 102: 255–270.

    Google Scholar 

  2. BarnesD and SatoG (1980b) Serum-free cell culture: A unifying approach. Cell 22: 649–655.

    Google Scholar 

  3. BoosmanA, StricklerJ and WilsonK (1987) Partial primary structures of human and murine macrophage colony stimulating factor (CSF-1). Biochem. Biophys. Res. Commun. 144: 74–80.

    Google Scholar 

  4. BoshartM, WeberF, JahnG, Dorsch-HaslerK, FleckensteinB and SchaffnerW (1985) A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41: 521–530.

    Google Scholar 

  5. BottensteinJ, HayashiI, HutchingsS, MasuiH, MatherJ, McClureDB, OhasaS, RizzinoA, SatoG, SerreroG, WolfeR and WuR (1979) The growth of cells in serum-free hormone-supplemented media. Methods Enzymol. 58: 94–109.

    Google Scholar 

  6. BurrowsMT (1924) Relation of oxygen to the growth of tissue cells. Am. J. Physiol. 68: 110 (abstract).

    Google Scholar 

  7. ButlerM, ImamuraT, ThomasJ and ThillyWG (1983) High yields from microcarrier cultures by medium perfusion. J. Cell Sci. 61: 351–363.

    Google Scholar 

  8. GassonCG, WeibartRH, KaufmanSE, ClarkSC, HewichRM, WongGG and GolbeDW (1984) Purified human granulocyte-macrophage colony-stimulating factor: Direct action on neutrophils. Science 226: 1339–1342.

    Google Scholar 

  9. GentryLE, WebbNR, LimGJ, BrunnerAM, RanchalisJE, TwardzikDR, LioubinMN, MarquardtH and PurchioAF (1987) Type I transforming growth factor beta: Amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells. Mol. Cell Biol. 7: 3418–3427.

    Google Scholar 

  10. GluzmanY (1981) SV40-trasformed simian cells support the replication of early SV40 mutants. Cell 23: 175–182.

    Google Scholar 

  11. GormanCM, MoffatLF and HowardBH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2: 1044–1051.

    Google Scholar 

  12. GraffS and McCartyKS (1957) Sustained cell culture. Exp. Cell Res. 13: 348–457.

    Google Scholar 

  13. GrifinBE (1981) In: ToozeJ (eds.) DNA tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 61–123.

    Google Scholar 

  14. GublerU and HoffmanBJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269.

    Google Scholar 

  15. HayashiI and SatoGH (1976) Replacement of serum by hormones permits growth of cells in a defined medium. Nature 259: 132–134.

    Google Scholar 

  16. HimmelfarbP, ThayerPS and MartinHE (1969) Spin filter culture: The propagation of mammalian cells in suspension. Science 164: 555–557.

    Google Scholar 

  17. JayeM, HowkR, BurgessW, RiccaGA, ChiuIM, RaveraMW, O'BrienSJ, ModiWS, MaciagT and DrohanWN (1986) Human endotherial cell growth factor: Cloning, nucleotide sequence, and chromosome localization. Science 233: 541–548.

    Google Scholar 

  18. JensenMD, WallachDFH and LinPS (1974) Comparative growth characteristics of Vero cells on gas-permeable and conventional supports. Expl. Cell Res. 84: 271–281.

    Google Scholar 

  19. JonesM and BontingSL (1956) Some relations between growth and carbohydrate metabolism in tissue cultures. Exp. Cell Res. 10: 631–639.

    Google Scholar 

  20. KashiwaguraT, WilsonDF and ErecinskaM (1984) Oxygen dependence of cellular metabolism: The effect of O2-tension on gluconeogenesis. J. Cell. Physiol. 120: 13–18.

    Google Scholar 

  21. KawasakiES, LandnerMB, WangAM, ArsdellJV, WarrenMK, CoyneMY, SchweickartVL, LeeMT, WilsonKJ, BoosmanA, StanleyER and MarkDF (1985) Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1). Science 230: 293–296.

    Google Scholar 

  22. KnazekRA, GullinoPM, KohlerPO and DedrickRL (1972) Cell culture on artficial capillaries: An approach to tissue growthin vitro. Science 178: 65–67.

    Google Scholar 

  23. KnopRH, ChenCW, MitchellJB, RussoA, McPhersonS and CohenJS (1984) Metabolic studies of mammalian cells by31P-NMR using a continuous perfusion technique. Biochim. Biophys. Acta 804: 275–284.

    Google Scholar 

  24. KrebsHA (1950) Body size and tissue respiration. Biochim. Biophys. Acta 4: 249–269.

    Google Scholar 

  25. KumazakiT, TerasawaK and IshiiS (1987) Affinity chromatography on immobilized anhydrotrypsin: General utility for selective isolation of C-terminal peptides from protease digests of proteins. J. Biochem. 102: 1539–1546.

    Google Scholar 

  26. LederLD (1987) Der Blutmonocyt. Springer Berlin-Heidelberg-New York, p. 223.

    Google Scholar 

  27. ManosMM (1988) Expression and processing of a recombinant human macrophage colony-stimulating factor in mouse cells. Mol. Cell. Biol. 8: 5035–5039.

    Google Scholar 

  28. McCormickF, TraheyM, InnisM, DiekmannB and RingoldG (1984) Inducible expresslon of amplified human beta interferon genes in CHO cells. Mol. Cell. Biol. 4: 166–172.

    Google Scholar 

  29. McLimansWF, BlumensonLE and TunnahKV (1968) Kinetics of gas diffusion in mammalian cell culture systems. II. Theory. Biotechnol. Bioeng. 10: 741–763.

    Google Scholar 

  30. MolonyWB, McPhersonK and FliegelmanL (1980) Esterase activity in leukocytes demonstrated by the use of naphtol AS-D chloroacetate substrate. J. Histochem. Cytochem. 8: 200.

    Google Scholar 

  31. OnomichiK, EtoY and ShibaiH (1987) Expression of amplified cDNA sequences encoding human interleukin 2 in human cells. Agric. Biol. Chem. 51: 1385–1392.

    Google Scholar 

  32. PikeBL and RobinsonWA (1970) Human bone marrow colony growth in agar-gel. J. Cell Physiol. 78: 77–84.

    Google Scholar 

  33. RamabhadranTV, ReitzBA and ShahDM (1985) Highlevel expression of the bovine growth hormone gene in heterologous mammalian cells. Gene 38: 111–118.

    Google Scholar 

  34. RaskL, AnudiH and PetersonPA (1979) The primary structure of human retinol-binding protein. FEBS. Lett. 104: 55–58.

    Google Scholar 

  35. RettenmierCW, RousselMF, AshmunRA, RalphP, PriceK and SherrCJ (1987) Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes. Mol. Cell. Biol. 7: 2378–2387.

    Google Scholar 

  36. RichterA, SanfordKK and EvansVJ (1972) Influence of oxygen and culture media on plating efficiency of some mammalian tissue cells. J. Natn. Cancer Inst. 49: 1705–1712.

    Google Scholar 

  37. SakaiN, UmedaT, SuzukiH, IshimatsuY and ShikitaM (1987) Macrophage colony-stimulating factor purified from normal human urine. 222: 341–344.

    Google Scholar 

  38. SangerF and CoulsonAR (1978) The use of thin acrylamide gel for DNA sequencing. FEBS Lett. 87: 107–110.

    Google Scholar 

  39. Sato GH (1975) The role of serum in cell culture. In: Litwack G (ed.) Biochemical actions of hormones. Vol. 3 pp. 391-396.

  40. Satoh S, Kobayashi J and Otani M (1990) Isolation and characterization of a growth-promoting factor from calf serum, and identification to retinol-binding protein.In Vitro in press.

  41. Satoh S and Mizoguchi J (1989) Improved extracellular secretion of M-CSF by deleting transmembrane region of the molecule. Cell Culture Engineering II Abst., Santa Barbara, CA, p. 7.

  42. SchahillSJ, DevosR, Van derheydenJ and FiersW (1983) Expression and characterization of the product of a human immune interferon cDNA gene in chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 80: 4654–4658.

    Google Scholar 

  43. SchwalzelF and BrannsteinH (1968) Cytochemishe Darstellung von Esteraseactivaten in Blutund Knochenmarkszellen. Klin. Wschr. 48: 642.

    Google Scholar 

  44. SimonsenCC and LevinsonAD (1983) Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc. Natl. Acad. Sci. USA 80: 2495–2499.

    Google Scholar 

  45. SourthernPJ and BergP (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1: 327–341.

    Google Scholar 

  46. SouzaLM, BooneTC, GagriloveJ, LaiPH, ZseboKM, MurdockDC, ChazinVR, BruszewskiJ, LuH, ChenKK, BarendtJ, PlatzerE, MooreMAS, MetelsmannR and WelteK (1986) Recombinant human granulocyte colonystimulating factor: Effects on normal and leukemic myeloid cells. Science 232: 61–65.

    Google Scholar 

  47. SpierRE and GriffithsB (1983) An examination of the data and concepts germane to the oxygenation of cultured animal cells. Dev. Biol. Stand. 55: 81–92.

    Google Scholar 

  48. StevensKM (1965) Oxygen requirements for liver cellsin vitro. Nature 206: 199.

    Google Scholar 

  49. SundelinJ, LaurentBC, AnudiH, TragardhL, LarhammarD, BjorckL, ErikssonU, AkerstromB, JonesA, NewcomerM, PetersonPA and RaskL (1985) Amino acid sequence homologies between rabbit, rat, and human serum retinolbinding proteins. J. Biol. Chem. 260: 6472–6480.

    Google Scholar 

  50. TempletonD and EckhartW (1984) N-terminal amino acid sequence of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation. Mol. Cell. Biol. 4: 817–821.

    Google Scholar 

  51. TsuneokaK and ShikitaM (1985) A colony-stimulating factor for neutrophil granulocytes: A marked increase of its production by addition of sodium butyrate and lipopolysaccharide in serum-free culture of RSP-2·P3 cells. J. Cell. Physiol. 125: 436–442.

    Google Scholar 

  52. UrlaubG and ChasinLA (1980) Isolation of chinese hamster cell mutants deficient in dehydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77: 4216–4220.

    Google Scholar 

  53. WerrleinRJ and GlinosAD (1974) Oxygen microenvironment and respiratory oscillations in cultured mammalian cells. Nature 251: 371–379.

    Google Scholar 

  54. WongG, TemplePA, LearyAC, Witek-GiannottiJS, YangYC, CiarlettaAB, ChungM, MurthaP, KrizR, KaufmanRJ, FerenzCR, SibleyBS, TurnerKJ, HewickRM, ClarkSC, YanaiN, YokotaH, YamadaM, SaitoM, MotoyosiK and TakakuF (1987) Human CSF-1: Molecular cloning and expression of 4-kb cDNA encoding the human urinary protein. Science 235: 1504–1508.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research Laboratories, Toyo Jozo Co., Ltd., 632-1 Ohito-cho, Tagata-gun, 410-23, Shizuoka-ken, Japan

    Sakae Satoh, Junji Kobayashi, Junzo Mizoguchi, Makoto Nogawa & Masaru Otani

Authors
  1. Sakae Satoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Junji Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junzo Mizoguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Makoto Nogawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masaru Otani
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Satoh, S., Kobayashi, J., Mizoguchi, J. et al. Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor. Cytotechnology 5 (Suppl 2), 95–114 (1991). https://doi.org/10.1007/BF00573882

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00573882

Key words

Search

Navigation