Copper in Cell Culture

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

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

Copper is an essential element in cell culture. However, classical media do not typically contain it in their basal formulations. This is likely because sera used at 5 to 10% provides sufficient copper for cell growth and survival, approx. 50 to 100 ng/mL. Efforts to develop serum-free media necessitate the addition of copper to the cell culture system. However, this requirement is not necessarily obvious. Certain media such as Iscove's Modified Dulbecco's Medium (IMDM) which is often used as a base for development of serum-free hybridoma media do not contain copper in their basal formulations. However, these media are often supplemented with albumin. Albumin is a physiological transport molecule for copper and purified natural albumins likely contain sufficient copper to, at least partially, support cells in culture.

William's E media is supplemented with 0.4 µM of copper. Most of the other classical media that contain copper were developed as modifications of Ham's F-10 or F-12 media for low serum and serum-free applications. The original Ham's F-10 and F-12 media were supplemented with 10 µM copper. These media were also often supplemented with serum, or sera derivatives. Other variations of Ham's F-12 also contain 10 µM copper: F-12 Coon's Modification; F-12 Kaighn's Modification (F12K) and MCDB-302 (developed for CHO cells). Serum-Free/Protein Free Hybridoma Medium is derived from F-12 and contains 10 µM of copper. DMEM/Ham's Nutrient Mixture F-12 (50:50) which is often used as a base for development of proprietary CHO media contains 0.0052 µM of copper contributed from the F-12 formulation.

Copper has an essential role in the growth and survival of cells in culture. However, unless it is properly chelated it is also toxic. Copper in sera is effectively chelated and delivered to cells by ceruloplasmin. Copper is also chelated by albumins and under the correct circumstances the copper requirement of cells in culture can at least partially be met by albumin bound copper. In the absence of serum and albumin the chelation of copper is dependent upon proprietary use of small molecules found in advanced formulations. Inappropriate management of copper delivery is a direct contributor to oxidative stress in cell culture. For a more comprehensive discussion on how copper functions and how it contributes to cell culture toxicity see below and for a more comprehensive discussion, visit our Media Expert.

Primary Functions of Copper, in Cell Culture Systems:

Copper is an essential element for the growth and maintenance of healthy cells in vivo and in vitro. Important Physiological Roles of Copper

• Copper plays a central role in the homeostasis of iron. As a component of ceruloplasmin, it assists with the transfer of iron from the storage molecule ferritin into the transport and delivery molecule transferrin.
• Copper functions in a group of copper oxidases which include (not limited to) cytochrome c oxidase (E.C. 1.9.3.1), superoxide dismutase (E.C. 1.15.1.1), tyrosinase (E.C. 1.10.3.1), dopamine-β-hydroxylase (E.C. 1.14.17.1), amine oxidases (E.C. 1.4.3.4 and 1.4.3.6), lysyl oxidase and ceruloplasmin (E.C. 1.14.3.1).
• The copper oxidases; ceruloplasmin, ascorbate oxidase, and lactase are the only oxidases known that can reduce both atoms of di-molecular oxygen to water with the transfer of four electrons.
• Cu/Zn superoxide dismutase is a very important cytoplasmic enzyme that protects cells by transmutation of superoxide radicals into hydrogen peroxide. In reticulocytes, up to 60% of the copper is found in Cu/Zn SOD.
• Lysyl oxidase produces cross-linking required for formation of collagen and elastin. This protein has a relatively short half–life and needs to be regenerated. Copper and ascorbate both support the development of insoluble extra-cellular matricies necessary for the growth and differentiation of attached cells.

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

Copper is a transition metal that exists, in vitro, in an equilibrium as reduced (cuprous), Cu (I) and oxidized (cupric), Cu (II), copper. In its free form and in some chelates, it can participate actively in redox cycling. It oxidizes a number of important media components, such as cysteine and ascorbate.

Copper can cause the loss of the cysteine and cystine from cell culture media by oxidation and precipitation. In vitro, cysteine is freely soluble and exists almost exclusively as a neutral amino acid. It is unstable and undergoes non-enzymatic autoxidation in the presence of di-molecular oxygen to form cystine. Cupric copper accelerates the autoxidation of cysteine to cystine. Cupric copper can form chelate-precipitates with cystine. The depletion of cysteine from cell culture will stop the synthesis of proteins and glutathione, an important reducing agent. Reduced glutathione can complex with Cu (I) and inhibit its participation in the formation of hydroxyl free radicals. This interaction involves the cysteine sulfur atom. In vivo, Cu (1):glutathione complexes mediate the safe movement of Cu (1) that enters the cytoplasm, probably through the copper transporter 1 pore, to intra-cellular binding proteins such as metallothionein. The formation of Cu (1): glutathione complexes is spontaneous and non-enzymatic. In vitro, Cu (1) will spontaneously form complexes with reduced cysteine, glutathione and presumably organic sulfhydryls. In addition to forming cupri-cystine complexes, Cu (II) will form complexes with other amino acids through coordination of their alpha-amino nitrogen and carboxyl-oxygen groups. This is referred to as NNOO (glycine-like) bonding. Histidine binding of Cu (II) is unique. In the pH region 4.5-7.5, Cu (II) coordinates with the alpha-amino nitrogen, carboxyl-oxygen and the imidazole nitrogen of two histidines. The planar coordination is either NNNN (histamine-like) or mixed histamine-like and glycine-like. Binding of Cu (II) to histidine is important because this appears to be an intermediate involved in the movement of Cu (II) from albumin to the cell. Before the copper can cross the cell membrane it must be reduced to Cu (I).

Arguably the most important set of copper redox reactions in vitro involve Haber-Weiss driven Fenton chemistry that results in the formation of hydroxyl free radicals. This free radical species is one of the most reactive and destructive molecules found in vitro. Under physiological conditions, cupric copper can be reduced to cuprous copper by superoxides or ascorbate anion. Cuprous copper catalyzes the formation of hydroxyl free radicals. A hydroxyl free radical will attack the biomolecules near the site where the radical is created. Lipid peroxidation is initiated when the hydroxyl radical attacks a polyunsaturated fatty acid. Once initiated, lipid peroxidation will destabilize membranes and may result in cell death if the process is not terminated. Lipid peroxidation is often terminated by the formation of a relatively stable hydroperoxides. However, in the presence of copper this hydroperoxide can degrade into peroxyl and alkoxyl free radicals.

Serum copper is bound primarily to ceruloplasmin and albumin as cupric copper. Copper is incorporated into ceruloplasmin in the liver. In this bound form, it reacts poorly with other components of the medium. However, ceruloplasmin bound copper can be reduced and released at the cell’s surface in a controlled series of reactions that lead to its safe incorporation and utilization by the cell. This process is facilitated by ascorbate. Albumin binds cupric copper at multiple sites. Its primary N-terminal binding site has a high affinity for copper, Ka =1.1 to 1.6 x 1013 M-1. Neither superoxide radicals nor ascorbate can easily reduce copper when it is bound to this site. Hence, in these bound forms, copper is less available to participate in free radical chemistry. Superoxide radicals or ascorbate can reduce copper when it is bound to secondary sites on albumin. Copper bound to the secondary sites on albumin can catalyze formation of the hydroxyl radical. When this occurs, the radical will most likely attack the albumin molecule. This spares more important molecules and is one way that albumin acts as a sacrificial antioxidant.

Citations to specific statements made above can be found in our Media Expert.

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