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 requ
ired to obtain and maintain a constant concentration of ascorbate in the cu
lture medium using ascorbate and ascorbate-phosphate. The study was carried
out with human fibroblasts and the amounts of ascorbate in the culture med
ium were determined by high performance liquid chromatography. A mixture of
0.25 mmol/L ascorbate and 0.45 mmol/L ascorbate-phosphate provided a const
ant concentration of ascorbate in the culture medium. This constant ascorba
te concentration proved to be nontoxic for cells and stimulated cell growth
in the short term and long term.