2.1.3. Proper Use of Ascorbate in Cell Culture

Cell culture study designs may have a large impact on the results obtained with ascorbic acid. To minimize artifacts, cell culture experiments should replicate *in vivo* conditions as closely as possible. Ascorbate levels in media should be maintained within the physiological range of human plasma (about 5–100 μM), and the use of supraphysiological concentrations should be avoided, unless conditions of intravenous vitamin C infusion are being mimicked [24]. When ascorbic acid is added to cell culture, the loss of ascorbate in the media competes with the intracellular accumulation of ascorbate. Although cells may accumulate ascorbate, once the media is depleted of ascorbic acid, intracellular ascorbate levels decline slowly through oxidation or efflux [33–35,38], once again returning cells to a depleted state. Meanwhile, degradation products may accumulate in the media or cells, which would normally be removed under physiological conditions. In addition, cells without

ascorbic acid are not a proper control for ascorbate treatment, as some level of ascorbate is always present in all cells of the human body.

Ascorbate should be added to culture media in a way that limits the rate of ascorbate oxidation and the effects of ROS that may be formed. The use of serum-free media that has been supplemented with transferrin to control iron or copper redox chemistry shows great promise in stem cell studies [46]. The stability of ascorbate can be enhanced by low oxygen growth conditions and the use of stabilized derivatives of ascorbate such as ascorbate-2-phosphate (AAP) that cannot participate in redox chemistry outside the cell yet can maintain physiological intracellular ascorbate levels [9]. Furthermore, pyruvate or α-ketoglutarate in cell culture media can be used to blunt the effects of any hydrogen peroxide formed [21,22], although they will not prevent the loss of ascorbic acid.

Due to the inherent instability of the molecule, there is an absolute necessity for monitoring ascorbic acid levels in media and cells during cell culture experiments. As with animal and human studies described below, this is the only method currently available to assess the vitamin C status of cells, and is a valuable tool for understanding the mechanisms of ascorbate's biological actions. Unfortunately, ascorbate levels are rarely measured in cell culture, animal, or human studies, which severely limits their validity and any conclusions that can be drawn.

Despite precautions, conventional cell culture conditions will promote an environment in which ascorbate artifacts are commonplace. Culturing cells with vitamin C requires control over many aspects of the media and culture conditions that has heretofore been lacking. Monitoring ascorbate levels and limiting oxidation may not be sufficient to fully recapitulate the physiological roles of vitamin C. While redesigning cell culture systems to support biologically relevant reactions of ascorbic acid and eliminate artifacts may limit the practicality of experimental designs, these changes are necessary for cell culture models to have continued use in vitamin C research.
