Importance and uses of citrate in serum-free eucaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures

Citrate, a Serum-Free Medium Supplement, Useful In Biomanufacturing; Tissue Engineering and Specialty Media:

Citrate is an important water soluble tricarboxylic acid. It is not found in classical basal media formulations, except Sigma's Serum-free/Protein Free Hybridoma Media (S2772, S2897 and S8284). However, it is frequently added to serum containing media as a serum, and to serum-free media as an albumin contaminant. A significant number of new serum-free or protein-free media used for bio-manufacturing and tissue engineering are supplemented with citrate as a chelator of iron. Whatever it source, citrate has important roles in the cell culture. In addition to its function as an iron chelator, which may or may not be beneficial depending on specific applications, citrate can support cholesterol/sterol, ubiquinone and isoprenoid biosynthesis. Citrate can also be linked to protein glycosylation via the dolichol pathway. Consequently, citrate may be a valuable supplement for use in the biomanufacture of glycoproteins.

For a more complete discussion of citrate as a cell culture component go to Sigma's Media Expert.

Primary Functions of Citrate in Cell Culture Systems:

  • Citric acid is produced in the mitochondria by the donation of a 2-carbon residue from acetyl-CoA to oxaloacetic acid. Through a series of reactions, known as the tri-carboxylic acid (TCA) cycle, citric acid is metabolized to oxaloacetic acid. During this process, electron equivalents are transferred to NAD and FAD and carbon dioxide is produced. These electron equivalents subsequently flow down the cytochrome facilitated respiratory chain resulting in the reduction of oxygen to water, and storage of energy in the form of ATP.
  • Excess citric acid formed in the mitochondria during the TCA cycle can also be transported to the cytoplasm where it is converted into oxaloacetate and acetyl-CoA. The resulting acetyl-CoA is used in fatty acid and isoprenoid synthesis. These process store energy derived from ATP’s high-energy phosphate bond and NAD’s electron equivalents and support cell signaling, ubiquinone and sterol biosynthesis and glycosylation.
  • In serum, citrate is an anti-coagulant. It functions by chelating calcium required by enzymes involved in the coagulation cascade.
  • In vitro, in serum-free cell culture systems, citric acid is sometimes used as a chelator for iron. It binds iron with moderately high efficiency. Unfortunately, citrate bound iron can participate in a number of oxidative stress reactions and may contribute to toxic occurrences in media.
  • In vitro, citrate can displace bicarbonate as the coordinating anion in transferrin. Citrate may be transferred into the cell as a coordinating anion of transferrin. It may also negatively affect the efficiency with which transferrin binds and delivers iron.
  • Citrate is a feedback inhibitor of phosphofructokinase (EC and it may block or slow down glycolysis if it builds up in the cytoplasm.

Chemical Attributes of Citrate that make it a Useful Serum-Free Medium Supplement:

Citrate is a 6 carbon tri-carboxylic acid (PK1 =3.13, pK2 = 4.76 and pK3 = 6.4). At physiological pH, citrate exists as a polyvalent anion that can effectively bind divalent and trivalent cations, especially iron. Citrate has an affinity for ferric and ferrous iron and it has been used as a chelator for iron in serum-free cell culture systems.

Citrate is also a chelator of calcium. Both calcium and iron are important metals for cell growth and survival. Calcium plays many important roles in cell function, communication and differentiation and it is often used in cell culture at rate limiting concentrations. Hence, the presence of citrate may substantially affect those processes controlled by calcium.

Citrate is present in serum and albumin containing cell culture systems primarily as a contaminant or an unintended supplement. Historically, citrate found its way into cell culture as a component of serum. Serum contains citrate as a nutrient and as a contaminant. The contamination of human serum with citrate results from the use of sodium citrate as an anti-coagulant. Citrate binds fairly tightly to albumin and is often added to cell culture in this form. It has been shown to co-purify with serum albumin and it is frequently a contaminant of human Cohn fraction V derived material.

Citrate Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eucaryotic Cells in Serum-free Cultures


Sigma's Cell Culture Media Expert provides in depth discussion of this and other serum-free and protein-free media supplements. The Media Expert contains additional sections on raw materials, component use recommendations, formulation strategies and references. Whenever you have a questions about or problems with your eucaryotic mammalian cell culturing system visit the Media Expert for helpful guidance.