Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Sep;78(9):5588–5592. doi: 10.1073/pnas.78.9.5588

Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone.

W J Bettger, S T Boyce, B J Walthall, R G Ham
PMCID: PMC348796  PMID: 7029539

Abstract

A serum-free, hormone-supplemented medium, enriched with a mixture of lipids, has been developed that supports rapid clonal growth of human diploid fetal lung fibroblasts (Flow 2000, WI-38, MRC-5, and IMR-90) and of low-passage human foreskin fibroblasts. The medium, which contains less than 1 microgram of total protein per ml, also supports serial passage of Flow 2000 cells under totally serum-free conditions. It provides lipid at a total of 10 micrograms/ml as a liposome prepared from a mixture of soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. The soybean lecithin, which contains a variety of naturally occurring phospholipids, can be replaced with a mixture of highly purified phospholipids. Except for possible contaminants in the substances used in its preparation, the serum-free medium is fully defined chemically. It consists of an optimized basal nutrient medium, MCDB 110, supplemented with insulin, epidermal growth factor, dexamethasone, prostaglandins E1 and F2 alpha, phosphoenolpyruvate, dithiothreitol, glutathione, and the lipids listed above.

Full text

PDF
5588

Images in this article

5590Image
on p.5590

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barnes D., Sato G. Methods for growth of cultured cells in serum-free medium. Anal Biochem. 1980 Mar 1;102(2):255–270. doi: 10.1016/0003-2697(80)90151-7. [DOI] [PubMed] [Google Scholar]
  2. Bottenstein J., Hayashi I., Hutchings S., Masui H., Mather J., McClure D. B., Ohasa S., Rizzino A., Sato G., Serrero G. The growth of cells in serum-free hormone-supplemented media. Methods Enzymol. 1979;58:94–109. doi: 10.1016/s0076-6879(79)58127-0. [DOI] [PubMed] [Google Scholar]
  3. EAGLE H. Nutrition needs of mammalian cells in tissue culture. Science. 1955 Sep 16;122(3168):501–514. doi: 10.1126/science.122.3168.501. [DOI] [PubMed] [Google Scholar]
  4. FISHER H. W., PUCK T. T., SATO G. Molecular growth requirements of single mammalian cells. III. Quantitative colonial growth of single S3 cells in a medium containing synthetic small molecular constituents and two purified protein fractions. J Exp Med. 1959 Jun 1;109(6):649–660. doi: 10.1084/jem.109.6.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ham R. G. Dermal fibroblasts. Methods Cell Biol. 1980;21A:255–276. [PubMed] [Google Scholar]
  6. Ham R. G., McKeehan W. L. Development of improved media and culture conditions for clonal growth of normal diploid cells. In Vitro. 1978 Jan;14(1):11–22. doi: 10.1007/BF02618170. [DOI] [PubMed] [Google Scholar]
  7. Ham R. G., McKeehan W. L. Media and growth requirements. Methods Enzymol. 1979;58:44–93. doi: 10.1016/s0076-6879(79)58126-9. [DOI] [PubMed] [Google Scholar]
  8. Hayashi I., Sato G. H. Replacement of serum by hormones permits growth of cells in a defined medium. Nature. 1976 Jan 15;259(5539):132–134. doi: 10.1038/259132a0. [DOI] [PubMed] [Google Scholar]
  9. Heldin C. H., Wasteson A., Westermark B. Growth of normal human glial cells in a defined medium containing platelet-derived growth factor. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6611–6615. doi: 10.1073/pnas.77.11.6611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Higuchi K. Cultivation of animal cells in chemically defined media, a review. Adv Appl Microbiol. 1973;16:111–136. doi: 10.1016/s0065-2164(08)70025-x. [DOI] [PubMed] [Google Scholar]
  11. Holmes R., Helms J., Mercer G. Cholesterol requirement of primary diploid human fibroblasts. J Cell Biol. 1969 Jul;42(1):262–271. doi: 10.1083/jcb.42.1.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Katsuta H., Takaoka T. Cultivation of cells in protein- and lipid-free synthetic media. Methods Cell Biol. 1973;6:1–42. doi: 10.1016/s0091-679x(08)60046-1. [DOI] [PubMed] [Google Scholar]
  13. Kritchevsky D., Howard B. V. The lipids of human diploid cell strain WI-38. Ann Med Exp Biol Fenn. 1966;44(2):343–347. [PubMed] [Google Scholar]
  14. Mather J. P., Sato G. H. The use of hormone-supplemented serum-free media in primary cultures. Exp Cell Res. 1979 Nov;124(1):215–221. doi: 10.1016/0014-4827(79)90271-4. [DOI] [PubMed] [Google Scholar]
  15. McKeehan W. L., Ham R. G. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J Cell Biol. 1976 Dec;71(3):727–734. doi: 10.1083/jcb.71.3.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McKeehan W. L., Hamilton W. G., Ham R. G. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2023–2027. doi: 10.1073/pnas.73.6.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McKeehan W. L., McKeehan K. A., Hammond S. L., Ham R. G. Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro. 1977 Jul;13(7):399–416. doi: 10.1007/BF02615100. [DOI] [PubMed] [Google Scholar]
  18. McKeehan W. L. The effect of temperature during trypsin treatment on viability and multiplication potential of single normal human and chicken fibroblasts. Cell Biol Int Rep. 1977 Jul;1(4):335–343. doi: 10.1016/0309-1651(77)90063-7. [DOI] [PubMed] [Google Scholar]
  19. Orly J., Sato G. Fibronectin mediates cytokinesis and growth of rat follicular cells in serum-free medium. Cell. 1979 Jun;17(2):295–305. doi: 10.1016/0092-8674(79)90155-7. [DOI] [PubMed] [Google Scholar]
  20. Pagano R. E., Weinstein J. N. Interactions of liposomes with mammalian cells. Annu Rev Biophys Bioeng. 1978;7:435–468. doi: 10.1146/annurev.bb.07.060178.002251. [DOI] [PubMed] [Google Scholar]
  21. Peehl D. M., Ham R. G. Clonal growth of human keratinocytes with small amounts of dialyzed serum. In Vitro. 1980 Jun;16(6):526–540. doi: 10.1007/BF02626466. [DOI] [PubMed] [Google Scholar]
  22. Savage C. R., Jr, Cohen S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J Biol Chem. 1972 Dec 10;247(23):7609–7611. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES