Transferrin in Cell Culture

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

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

Transferrins make up an extensive micro-heterogeneous group of single chain glycoprotein isotypes with approximate molecular weights of 78 to 80 Kd. Transferrin is the physiologically appropriate method for providing iron to cells in culture. The delivery of iron using transferrin has historically been part of an 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 transferrin. These are generally iron chelators. Iron chelators should be used with great caution because many chelators do not control the redox cycling of iron that contributes to oxidative stress.

Transferrin is not a component of most commercially available classic basal media. Traditionally, it entered the cell culture system as a component of the sera supplement. More recently it has become popular to add transferrin to basal media as a component of the ITS, SPIT, and SPITE frozen supplements (see products below).

The chemistries and biochemistries of iron management using transferrin and iron chelators are extremely complex. Improperly managed iron storage and delivery in cell culture systems is a major contributor to oxidative stress and protein damage. For a more complete discussion of transferrin and iron chelators as a cell culture components go to our Media Expert.

Primary Functions of Transferrin in Cell Culture Systems:

• Transferrins facilitate extracellular iron storage, and transport.
• Transferrins are important extracellular antioxidants. They bind iron so tightly under physiological conditions that virtually no free iron exists to catalyze the production of free radicals.
• The delivery of iron to cells by transferrins is a receptor-mediated and controlled process. Cells regulate the amount of iron they receive from the extracellular environment by varying transferrin receptor expression

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

The transferrins make up an extensive micro-heterogeneous group of single chain glycoprotein isotypes with approximate molecular weights of 78 to 80 Kd. Isotypes of transferrins can be found in most biological fluids including serum, cerebrospinal fluid, synovial fluid and amniotic fluid. Transferrin micro-heterogeneity exists both within and between animal species. Primary contributors to micro-heterogeneity within a species are iron content, and the structure and sialic acid content of N-linked glycan chains. Across species the transferrins also differ in primary amino acid sequences.

The stability of transferrin is increased by conformational changes induced by iron binding. Iron binding increases the affinity of transferrin for cell transferrin receptors. The glycosylation pattern on transferrin does not appear to have a significant effect on its binding to the transferrin receptor. However, it may affect transferrin binding to general asialo receptors, and its half-life in solution.

Transferrins bind ferric iron with high affinity. Two atoms of ferric iron bind transferrin with distinctly different affinities. The first and second iron atom stability constant are approximately 10³⁰ and 10²⁷, respectively. Virtually no iron is free to participate in free radical chemistry when transferrin is present in cell culture medium with molar ratios of transferrin to iron greater than one. When the iron concentration in media exceeds the tranferrin’s total iron binding capacity (TIBC), free iron can exist in the medium. Unbound iron can participate in free radical chemistry and potentiate oxidative stress.

General mechanism of transferrin mediated iron delivery:

Two iron-loaded transferrin molecules bind to one transferrin receptor. Once bound, they are transferred into acidic endosomes within the cell’s interior. At reduced pH, the iron is released and incorporated into intracellular proteins and the storage molecule ferritin. The apo-transferrin (iron free) is recycled back to the exterior of the cell and released. Each transferrin receptor cycle can deliver up to four atoms of iron.

Reloading of transferrin:

The loading of ferric iron into transferrin can be relatively slow, taking minutes to complete. The ferroxidase activity of ceruloplasmin accelerates the rate of iron loading into transferrin to seconds. A number of agents in vitro appear to facilitate ferrous conversion into ferric iron making it available for uptake by transferrin. These include citrate, bicarbonate and phosphate. However, each of these oxidations is accompanied by the formation of free radicals. On the other hand, ceruloplasmin facilitates the oxidation of ferrous to ferric iron without the formation of free radicals. Ceruloplasmin mediated oxidation of iron is accompanied by the conversion of di-oxygen into water. Ceruloplasmin is the only circulating molecule that provides controlled and rapid oxidation of ferrous to ferric iron.