Skip to main content
Log in

Monitoring of ascorbate at a constant rate in cell culture: Effect on cell growth

  • Cell Growth/Differentiation/Apoptosis
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

Ascorbic acid (vitamin C) is a primary antioxidant for cells. But, ascorbic acid added to culture medium is not readily available to cells in culture, because it is unstable in aqueous media. We determined the conditions required to obtain and maintain a constant concentration of ascorbate in the culture medium using ascorbate and ascorbate-phosphate. The study was carried out with human fibroblasts and the amounts of ascorbate in the culture medium were determined by high performance liquid chromatography. A mixture of 0.25 mmol/L ascorbate and 0.45 mmol/L ascorbate-phosphate provided a constant concentration of ascorbate in the culture medium. This constant ascorbate concentration proved to be nontoxic for cells and stimulated cell growth in the short term and long term.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Austria, R.; Semenzato, A.; Bettero, A. Stability of vitamin C derivatives in solution and topical formulations. J. Pharm. Biomed. Anal. 15:795–801; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Brigelius-Flohe, R.; Flohe, L. Ascorbic acid, cell proliferation, and cell differentiation in culture. Subcell. Biochem. 25:83–107; 1996.

    PubMed  CAS  Google Scholar 

  • Buettner, G. R. Ascorbate autoxidation in the presence of iron and copper chelates, Free Radic. Res. Commun. 1:349–353; 1986.

    PubMed  CAS  Google Scholar 

  • Buettner, G. R. In the absence, of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. J. Biochem. Biophys. Methods 16:27–40; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Buettner, G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300:535–543; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Buettner, G. R.; Jurkiewicz, B. A. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145:532–541; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Diliberto, E. J., Jr.; Daniels, A. J.; Viveros, O. H. Multicompartmental secretio of ascorbate and its dual role of dopamine beta-hydroxylation. Am. J. Clin. Nutr. 54(Suppl. 6):1163S-1172S; 1991.

    PubMed  CAS  Google Scholar 

  • Ernster, L.; Forsmark-Andree, P. Ubiquinol: an endogenous antioxidant in aerobic organisms. Clin. Investig. 71:60–65; 1993.

    Article  Google Scholar 

  • Geesin, J. C.; Gordon, J. S.; Berg, R. A. Regulation of collagen synthesis in human dermal fibroblasts by the sodium and magnesium salts of ascorbyl-2-phosphate. Skin Pharmacol. 6:65–71; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Hata, R.; Senoo, H. l-Ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation, of a three-dimensional tissue like substance by skin fibroblasts. J. Cell. Physiol. 138:8–16; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Hata, R.; Sunada, H.; Arai, K.; Sato, T.; Ninomiya, Y.; Nagai, Y.; Senoo, H. Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts. Eur. J. Biochem. 173:261–267; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Herbert, V.; Shaw, S.; Jayatilleke, E. Vitamin C-driven free radical generation from iron. J. Nutr. 126:1213–1220; 1996.

    Google Scholar 

  • Ishikawa, O.; Kondo, A.; Okada, K.; Miyachi, Y.; Furumura, M. Morphological and biochemical analyses on fibroblasts and self-produced collagens in a novel three-dimensional culture. Br. J. Dermatol. 136:6–11; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Ivanov, V. O.; Ivanova, S. V.; Niedzwiecki, A. Ascorbate affects proliferation of guinea pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects. J. Mol. Cell. Cardiol. 29:3293–3303; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Kitts, D. D. An evaluation of the multiple effects of the antioxidant vitamins. Trends Food Sci. Technol. 8:198–203; 1997.

    Article  CAS  Google Scholar 

  • Kurata, S.; Senoo, H.; Hata, R. Transcriptional activation of type I collage genes by ascorbic acid 2-phosphate in human skin fibroblasts and its failure in cells from a patient with alpha 2 (I)-chain-defective Ehlers-Danlos syndrome. Exp. Cell Res. 206;63–71; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Legrand, C.; Bour, J. M.; Jacob, C., et al. Lactate dehydrogenase (LDH) activity of the number of dead cells in the medium of cultured eukaryotic cells as marker. J. Biotechnol. 25:231–243; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Liebler, D. C. The role of metabolism in the antioxidant function of vitamin E. Crit. Rev. Toxicol. 23:147–169; 1993.

    PubMed  CAS  Google Scholar 

  • Michiels, C.; Toussaint, O.; Remacle, J. Comparative study of oxygen toxicity in human fibroblasts and endothelial cells. J. Cell. Physiol. 144:295–302; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Navas, P.; Villalba, J. M.; Cordoba, F. Ascorbate function at the plasma membrane. Biochim. Biophys. Acta 1197:1–13; 1994.

    PubMed  CAS  Google Scholar 

  • Ono, M.; Aratani, Y.; Kitagawa, I.; Kitagawa, Y. Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates, adipose conversion of 3T3-L1 cells. Exp. Cell Res. 187:309–314; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Padh, H. Cellular functions of ascorbic acid. Biochem. Cell. Biol. 68:1166–1173; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Petrrkofsky, B. The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch. Biochem. Biophys. 152:318–328; 1972.

    Article  Google Scholar 

  • Phillips, C. L.; Combs, S. B.; Pinnell, S. R. Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts. J. Investig. Dermatol. 103:228–232; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Phillips, C. I.; Tajima, S.; Pinnell, J. R. Ascorbic acid and transforming growth factor beta 1 increase collagen biosynthesis via different mechanisms: coordinate regulation of pro-alpha 1 (I) and pro-alpha 1 (III) collagens. Arch. Biochem. Biophys. 295:397–403; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Sakagami, H.; Satoh, K.; Ohata, H., et al. Relationship between ascorbyl radical intensity and apoptosis-inducing activity. Anticancer Res. 16:2635–2644; 1996.

    PubMed  CAS  Google Scholar 

  • Speek, A. J.; Schrijver, R.; Schreurs, W. H. Fluorimetric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization. J. Chromatogr. 305:53–60; 1984.

    Article  PubMed  CAS  Google Scholar 

  • Washko, P. W.; Wang, Y.; Levine, M. Ascorbic acid recycling in human neutrophils. J. Biol. Chem. 268:15,531–15,535; 1993.

    CAS  Google Scholar 

  • Yamamoto, I.; Muto, N.; Murakami, K.; Akiyama, J. Collagen synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-o-alpha-d-gluco-pyranosyl-l-ascorbic acid. J. Nutr. 122:871–877; 1992.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to A. Chamson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chepda, T., Cadau, M., Girin, P. et al. Monitoring of ascorbate at a constant rate in cell culture: Effect on cell growth. In Vitro Cell.Dev.Biol.-Animal 37, 26–30 (2001). https://doi.org/10.1290/1071-2690(2001)037<0026:MOAAAC>2.0.CO;2

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1290/1071-2690(2001)037<0026:MOAAAC>2.0.CO;2

Key words

Navigation