Ceruloplasm in Cell Culture

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

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

Ceruloplasmin is a 134 kDa, single-chain, alpha2-globulin that tightly binds up to 6 copper atoms. Ceruloplasmin is the physiologically appropriate method for providing copper to cells in culture. It is not a component of most commercially available classic basal media. Traditionally, ceruloplasmin has entered the cell culture system as a component of the serum supplement. The secondary method of delivering copper to cells in culture has been as a complex with albumin. The delivery of copper using albumin has been part of the iron management program in biomanufacturing systems for production of therapeutic proteins, such as monoclonal antibodies. Efforts to move to animal protein-free and protein-free cell culture systems have motivated developers to seek small molecule alternatives to ceruloplasm and albumin. These are generally copper chelators. Copper chelators should be used with great caution because many copper chelators do not control the redox cycling of copper that contributes to oxidative stress.

The chemistries and biochemistries of copper management using ceruloplasm, albumin and copper chelators are extremely complex. Improperly managed copper storage and delivery in cell culture systems is a major contributor to oxidative stress and protein damage. For a more complete discussion of ceruloplasm, albumin and small molecule copper chelators as cell culture components go to Sigma's Media Expert.

Primary Functions of Ceruloplasm in Cell Culture Systems:

Ceruloplasmin protects cells from oxidative stress in a number of ways. Iron and copper are important elements for cell growth.  As free ions or redox active chelates, these metals can catalyze the production of hydroxyl free radicals via the Haber-Weiss-Fenton reactions. In appropriate physiologically bound states, they generally do not participate in free radical formation. Some important roles of ceruloplasmin in vivo are to bind copper in the liver, transport and deliver it to other cells, facilitate the incorporation and retention of iron by ferritin, and the mobilize iron into transferrin for transport and delivery to cells.

Copper Transport and Delivery:

Ceruloplasmin is the preferred delivery mode for copper to most cell types including cancer cells. Ceruloplasmin receptors have been reported on a wide range of cell types including endothelial cells, blood cells such as lymphocytes, monocytes, and granulocytes, and erythroleukemic cells. One model of copper delivery to cells by ceruloplasmin involves ascorbate. In this model, ceruloplasmin containing 6 or 7 atoms of copper in the cupric state binds to the cell membrane where ascorbate reduces the cupric copper to cuprous copper and is itself oxidized to dehydroascorbate. The cuprous copper and the dehydroascorbate cross the cell membrane and enter the cell.

Antioxidant:

In serum and in cells, a significant amount of iron is stored in ferritin as ferric oxyhydroxide and ferric oxyphosphate. Strong reductants, such as superoxides, can reduce and release iron from ferritin. If this iron is not oxidized and reincorporated into ferritin, it can promote oxidative stress.  Ceruloplasmin protects cells by oxidizing iron back to the ferric state and facilitating its re-incorporation into ferritin.

Iron Mobilization and Fixation: Ferroxidase activity:

Loading iron into transferrin can be relatively slow, taking minutes to occur. A number of agents in vitro may facilitate ferrous conversion into ferric iron, making it available for uptake by transferrin. These include citrate, bicarbonate and phosphate. However, these oxidations are accompanied by the formation of free radicals. Ceruloplasmin facilitates the oxidation of iron without the formation of free radicals and shortens the loading time to seconds.

Chemical Attributes of Ceruloplasm that make it a useful Serum-Free Medium Supplement:

The glycoprotein ceruloplasmin is a blue colored alpha2-globulin. Its molecular weight is approximately 134 Kd and it is a single chain polypeptide containing 6 tightly bound copper atoms. The protein is soluble under physiological conditions. The bound copper does not undergo exchange with the surrounding milieu and the release of copper to cells appears to be mediated by specific events at the cell surface.

Ceruloplasm 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.