The conditions found in a typical cell culture environment promote the oxidation and subsequent degradation of ascorbic acid. Therefore, ascorbate is not usually added to cell culture media, as it often leads to the production of deleterious free radicals and reactive oxygen species (ROS). However, such conditions are non-physiological with respect to vitamin C, which is found in all extra- and intracellular, aqueous solutions in vivo. The consequences of such “a-scorbic”––or scorbutic––cell culture environment are not fully understood, but it is obvious that any “ascorbate-dependent” enzymatic reactions, the cells’ redox “milieu”, and antioxidant network must be severely impaired. Furthermore, reintroducing vitamin C to such a cell culture system may give rise to additional artifacts. In this section, we examine the factors that lead to ascorbate oxidation in cell culture media, the artifacts related to the absence of ascorbate in cultured cells or its addition under normal cell culture conditions, and methods that are currently being developed to allow safe addition of ascorbate to cells for physiologically relevant research. Overall, studying vitamin C in cell culture is fraught with many pitfalls and results need to be approached and interpreted with care.
2.1.1. Ascorbate Stability in Cell Culture
The primary concern with the use of ascorbic acid in cell culture is the stability of the molecule under typical incubation conditions. Cell culture incubators use approximately 90%–95% air and 5%–10% CO
2, resulting in oxygen levels approximately 10–100 times greater than those found in the circulation and in tissues. Increased oxygen tension can promote a pro-oxidant environment in cell culture media [
6], possibly generating a wide variety of ROS that can react with, and hence deplete, ascorbate [
7]. However, high oxygen levels alone are not sufficient to cause substantial ascorbic acid oxidation. Although the reduction potential of the reduced form of vitamin C, the ascorbate mono-anion (AscH
−), is sufficient to reduce molecular oxygen to superoxide radicals (Equation 1), the reaction kinetics make this process very slow (estimated second-order rate constant, k
2 ≈ 10
−4 M
−1 s
−1) [
8].
The stability of the ascorbate mono-anion is apparent in deionized water or simple salt solutions: ascorbate added to phosphate-buffered saline (PBS) shows minimal oxidation over a six-h time period in a cell culture incubator (
Figure 1, PBS) or when exposed to ambient air at room temperature (data not shown). In contrast, ascorbate incubated in cell culture media under standard conditions is rapidly oxidized [
9,
10] (
Figure 1, RPMI). The composition of the cell culture media plays an important role in the rate of ascorbate oxidation: in serum-free RPMI medium the half-life of ascorbate is about 1.5 h (
Figure 1); however, a more rapid loss of ascorbate has been noted in other cell culture media formulations such as MEM or Williams E media (data not shown), including more complex solutions containing serum [
6,
11,
12].
Figure 1.
Ascorbate oxidation in buffer or cell culture medium. Ascorbate (100 μM) was added to RPMI 1640 or phosphate-buffered saline (PBS) and monitored over time. Chelated RPMI was used after overnight treatment with Chelex 100 resin and the addition of diethylenetriaminepentaacetic acid (DTPA, 1 mM) as described in Methods. RPMI RT represents media not incubated under 5% CO2 but under ambient air at room temperature.
Figure 1.
Ascorbate oxidation in buffer or cell culture medium. Ascorbate (100 μM) was added to RPMI 1640 or phosphate-buffered saline (PBS) and monitored over time. Chelated RPMI was used after overnight treatment with Chelex 100 resin and the addition of diethylenetriaminepentaacetic acid (DTPA, 1 mM) as described in Methods. RPMI RT represents media not incubated under 5% CO2 but under ambient air at room temperature.
Iron and copper are present in cell culture media, either added as part of the media formulation or appearing fortuitously, as they are required for normal cell growth and function [
13,
14]. However, the reduced forms of iron (ferrous iron, Fe
2+) and copper (cuprous copper, Cu
+) are able to reduce molecular oxygen to superoxide and, hence, participate in the production of ROS in cell culture systems. The Haber-Weiss reaction (also known as the superoxide-driven Fenton reaction) involves the reduction of the oxidized forms of iron (ferric iron, Fe
3+) or copper (cupric copper, Cu
2+) by superoxide (Equation 2) and the subsequent conversion of hydrogen peroxide (formed, e.g., by dismutation of superoxide radicals) to hydroxyl radicals and hydroxide by the reduced metal ions (Fenton reaction, Equation 3) (
Scheme 1). Hence, the metal ions act as catalysts and are required only in trace amounts for the Haber-Weiss reaction (Equation 4).
Scheme 1.
Metal-dependent and metal-independent production of reactive oxygen species by ascorbate in cell culture media.
Scheme 1.
Metal-dependent and metal-independent production of reactive oxygen species by ascorbate in cell culture media.
One of the most important biological functions of ascorbate is the reduction of Fe
3+ or Cu
2+ in the active site of enzymes, providing electrons used either in the hydroxylation of the enzymes’ substrates or the maintenance of the active-site metal ion in the reduced state [
3]. In the case of enzyme-bound metals, the transfer of electrons occurs in a controlled manner, which minimizes deleterious side reactions. However, in a solution of non-protein bound iron or copper, added ascorbate will reduce these metal ions (Equation 5); replacing superoxide in Equation 2, leading to superoxide production and facilitating the flow of electrons into the Haber-Weiss reaction (Equation 4). Even if media formulations are carefully controlled, trace amounts of these transition metals may be found on cell culture glassware, plastic dishes, and reagents, fueling ascorbate oxidation and ROS production [
8,
15].
In biological fluids and inside cells
in vivo, a combination of low oxygen levels and protein-bound metal ions greatly reduce ascorbate-mediated pro-oxidant effects [
16,
17,
18]. Consequently, ascorbate in human plasma does not get readily oxidized [
19]. By contrast, ascorbate addition to cell culture media results in the production of ROS, including superoxide radicals, hydrogen peroxide (from the dismutation of superoxide radicals), and hydroxyl radicals (
Scheme 1) [
6,
15]. For this reason, ascorbic acid is often mistaken for inducing a pro-oxidant environment in cell culture systems [
17,
20], although the effects of hydrogen peroxide production specifically may be masked by other media components, such as serum proteins, pyruvate, or α-ketoglutarate [
6,
11,
21,
22]. A more accurate description may be that ascorbate unmasks the presence of catalytic transition metals in the cell culture environment [
15]. Treating cell culture media with metal chelating agents slows the rate of ascorbate oxidation (
Figure 1, Chelated RPMI), increasing the half-life from approximately 1.5 h in standard media (RPMI) to about 2.7 h. Interestingly, metal chelation does not completely prevent ascorbate oxidation (
Figure 1), suggesting that other media components also contribute to ascorbate loss [
6].
One such contributing factor may be ascorbate auto-oxidation: the direct reaction of ascorbate with molecular oxygen [
8]. As stated above, the direct interaction between the ascorbate mono-anion and oxygen is highly unfavorable; however, the ascorbate di-anion (Asc
2−) rapidly reacts with oxygen (Equation 6). At pH 7.0, the concentration of Asc
2− is low, but its contribution to the rate of ascorbate oxidation can become considerable under conditions when pH rises or with increasing concentrations of added ascorbate (
Scheme 1) [
23]. Cell culture media pH is often stabilized by the addition of sodium bicarbonate to offset the acidic effects of the high CO
2 environment. However, when this media is exposed to air outside the incubator, the pH value can rise rapidly to 8.0 or higher [
11]. RPMI media equilibrated to ambient air accelerates ascorbate oxidation when compared to media in a cell culture incubator (
Figure 1, RPMI RT
versus RPMI), decreasing the half-life from about 1.5 h to less than one hour. As the Asc
2− present in solution reacts with oxygen to form dehydroascorbic acid, additional Asc
2− ions are generated to re-establish the equilibrium driven by the pH (
Scheme 1 and Equation 7). This continuous production of Asc
2− would fuel auto-oxidation, which is prevalent when supraphysiological levels of ascorbate are added to cell culture media, since the Asc
2− concentration would increase proportionally and greatly enhance oxidation effects. Although concentrated ascorbic acid will lower the pH of cell culture media, investigators usually offset this by the addition of sodium hydroxide to limit the impact of the acidic pH on cells. However, any shift toward a neutral or slightly alkaline environment will increase the metal-independent, pH-driven pro-oxidant effects of ascorbate, which may explain the limited effect of metal chelators to reduce the cytotoxicity of millimolar concentrations of ascorbate towards cancer cells [
24].
Ascorbate levels can be maintained in cell culture media by frequent addition of vitamin C [
9], but the persistent oxidation will continuously generate dehydroascorbic acid [
25] and breakdown products, such as oxalate and threonate [
26]. Extracellular concentrations of dehydroascorbic acid in excess of 1–2 μM are considered non-physiological, given its short half-life [
25] and rapid uptake and by cells [
27,
28,
29]. Although cells can reduce the dehydroascorbic acid to ascorbic acid, it is currently unclear what effects constant exposure of cells to high levels of dehydroascorbic acid or its breakdown products may have. For example, dehydroascorbic acid exposure has induced stress signaling and cytotoxicity in some cell types, probably due to the loss of NADPH or glutathione needed for dehydroascorbic acid reduction [
30]. Oxalate has been shown to exert cytotoxic effects [
31], and threonate can impact cell signaling pathways [
32]. If anything, these exposures are more likely to generate artifacts as a consequence of the cell culture environment.
2.1.2. “Cellular Scurvy”
Even under conditions of severe vitamin C deficiency,
i.e., scurvy, some ascorbate is still present in cells and tissues of humans
in vivo. As discussed above, cell culture media are not usually supplemented with ascorbic acid, due to its inherent instability in these media. As a consequence, many researchers have reported that cells in culture are devoid of any detectible amounts of ascorbate, even with the use of extremely sensitive HPLC techniques [
24,
33,
34,
35,
36,
37,
38]. Similarly, many complete cell culture media containing fetal bovine serum (FBS) have no detectable amounts of ascorbate [
33,
34,
36,
38], and our own analysis of various commercial sources of media and FBS has shown identical results (unpublished observations). The effects of these ascorbate-free conditions are not well defined or understood. However, it should be recognized that many immortalized cell lines likely have been maintained under scorbutic conditions for generations. In this manner, a whole host of cell culture artifacts may be expected when ascorbate is reintroduced into the system.
Cells in culture can be maintained without ascorbic acid because it is not essential to cell growth and division. The biological functions of ascorbate as an electron donor in enzymatic synthesis pathways do not have an absolute requirement for ascorbate [
3,
39,
40]. These enzymes can use other reducing substrates as sources of electrons [
39,
41], and enzyme activity can still occur in the absence of ascorbate, albeit at a far decreased rate [
42]. In particular, the α-ketoglutarate-dependent dioxygenases, such as those involved in collagen synthesis and regulation of hypoxia-inducible factor 1α (HIF-1α), do not require ascorbate as part of the normal catalytic cycle; ascorbate is only needed to rescue the enzyme should an uncoupled enzymatic reaction occur [
3]. It has also been suggested that ascorbate may function to maintain intracellular iron in the ferrous state, making it available to replenish or replace ferric iron in the active site of these enzymes [
43]. It is possible that cells in culture adapt by increasing ferrous iron uptake and turn-over of iron-containing proteins, partially circumventing the need for ascorbic acid. Regardless of the mechanism, it is evident from cell culture studies that “ascorbate-requiring” enzymes, such as those involved in collagen synthesis [
44], degradation of HIF-1α [
43], norepinephrine and α-amidated peptide synthesis [
45], and histone and DNA demethylase activity [
46], still exhibit some residual activity in the absence of ascorbate. However, these enzymes have diverse effects in different tissues, and their activity in an ascorbate-free environment may not be reflective of their roles
in vivo.
On the other hand, normal physiological functioning of cells can be recapitulated when ascorbate is provided. Ascorbate appears to play an important role in the normal function of cultured endothelial cells, raising antioxidant protection, reducing oxidative stress and damage, and increasing eNOS activity when compared to cells devoid of vitamin C [
33,
38]. These effects on eNOS, at least, appear dependent on the ability of ascorbate to enhance the stability of tetrahydrobiopterin [
34] and influence AMP-activated kinase (AMPK) activity [
47]. In addition, ascorbate supplementation of cultured endothelial cells tightens cell-to-cell junctions that are critical for maintaining an endothelial barrier
in vivo [
48] and regulates NADPH oxidase activity [
49], a critical component of the inflammatory response.
It is important to note that the effect of ascorbate supplementation may also greatly vary by cell type. Many of the aforementioned effects of ascorbate are observed in primary cell lines. Although propagated in the absence of ascorbate, the response to ascorbate supplementation in these cells reflects responses seen
in vivo. Cancer cell lines and other immortalized cells, however, often show cytotoxic effects in response to ascorbate addition that are not observed in primary cell lines [
24]. This may be the result of adaptations that have accumulated in these cells due to the “culture shock” that alters the normal physiological responses to stimuli [
6], possibly involving iron dysregulation or aberrant cell signaling responses.
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,
34,
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.