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

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.