Importance and uses of citrate in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures
Citrate is an important water soluble tricarboxylic acid. It is not found in classical basal media formulations, except our Serum-free/Protein Free Hybridoma Media (S2897). 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.
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.
The following citrate products have been shown to enhance the growth of hybridoma, chinese hamster ovary (CHO) and other mammalian eukaryotic cells in serum-free cultures.