Transferrin supplies iron to cells naturally in the body and as a supplement in cell culture media.
Transferrin in Cell Culture
Human serum transferrin, a bilobal ~75 kD glycoprotein that has the ability to reversibly bind Fe3+ with nanomolar affinity, represents one of the major vehicles for iron delivery to cells both in vivo and in vitro(1). Produced in the liver, transferrin is found in the blood plasma as a heterogeneous population at approximately 2.5 mg/mL consisting of diferric (holo or iron-saturated), monoferric N-lobe, monoferric C-lobe (partially iron saturated) or apo transferrin(2). At pH 7.4, the pH of human serum, holo transferrin has the highest affinity for the cell surface transferrin receptor (TFR) followed by the two monoferric forms(2). One of these three iron-bound forms of transferrin will bind the TFR and be internalized via clathrin-dependent endocytosis(3). Subsequent acidification of the endosome via ATP-dependent H+ pumps will trigger the receptor-stimulated release of iron from transferrin where the reduction of Fe3+ to Fe2+ ensues(2). The now iron free transferrin/TFR complex is subsequently redirected back to the cell surface where the weak association for the TFR at serum pH will trigger the dissociation of apo transferrin so that apo transferrin can bind additional Fe3+(4) (Figure 1). This process occurs quite rapidly so that each transferrin molecule delivers several payloads of iron per hour(2). Although the fully saturated diferric transferrin has the highest affinity for the TFR (Kd ~4 nM), the fact that monoferric forms also are capable of high affinity bonds with the TFR (Kd ~36 and 32 nM for the FeNhTF and FeChTF, respectively) would indicate that partially iron saturated transferrin would be functional in cell culture(2).
Figure 1. Internalization Mechanism of Human Serum Transferrin.
Given the central role of iron uptake in cell health, the inclusion of transferrin is absolutely critical in serum free cell culture media to ensure adequate cell proliferation and function ex vivo for most cell types. The natural abundance of transferrin in human serum combined with relatively straightforward purification has enabled the isolation and utilization of serum-derived transferrin for in vitro cell culture applications for years. However, given the inherent reliability issues and potential safety concerns of using serum-derived proteins, there have been extensive efforts to generate recombinant versions of human serum transferrin.
We have successfully expressed recombinant human transferrin in an animal component free (ACF) host(1). Interrogation of the biochemical and functional aspects of this recombinant transferrin indicated acceptable characteristics of the recombinant protein(1). We found that this recombinant transferrin, called Optiferrin, possessed the correct sequence and size and was functionally similar. Recombinant transferrin was able to compete with serum-derived transferrin for TFR binding sites on both CCL-2 and Caco-2 cells(1,6). As a result, cell proliferation was found to be identical in a hybridoma cell line across a range of relevant concentrations (Figure 2).
Animal Component Free, Recombinant Human Serum Transferrin Exhibits Broad Activity in Cell Culture
Substitution of serum-derived transferrin for Optiferrin in a serum free media is straightforward and can be expected to be nearly 1:1 if the concentration of native transferrin is known. If the concentration of native transferrin is not known, typical Optiferrin concentration ranges that have demonstrated adequate cell proliferation in the aforementioned cell types range from 10-400 mg/L and optimal concentration will have to be empirically determined.
Figure 2. Equivalence of Optiferrin in Inducing Hybridoma Cell Growth. Sp2/0 hybridoma (ATCC) were maintained in DMEM/F12 with GlutaMax + 10 mM HEPES and 10% FBS. To determine if bioactivity of either serum-derived transferrin, cells were washed extensively with basal DMEM/F12 and then seeded in the same base supplemented with 1 g/L rhalbumin, 10 mg/L rhinsulin, 6.7 µg/L selenite, and 2 mg/L ethanolamine with or without 0.1-10 mg/L serum derived or rhtransferrin (Optiferrin). Cells were incubated for 72 hrs and viable cell counts were subsequently determined. Optiferrin exhibited equivalent activity in inducing hybridoma cell growth to native human transferrin.
For more information on our animal-component free, recombinant Human Serum Transferrin, please visit the Optiferrin product page.
Recombinant human transferrin – Animal Free and defined source of transferrin for optimal cell growth and productivity.
Recombinant insulin and transferrin cell culture media supplement formulated with selenium and ethanolamine for improved cell growth and productivity. Animal component free.
- Steere A, B. C. (2012). Biochemical and Structural Characterization of Recombinant Human Serum Transferrin from Rice (Oryza Sativa L.). J Inorg Biochem, 37-44.
- Luck AN, M. A. (2012). Transferrin-Mediated Cellular Iron Delivery. Current Topics in Membranes, 69, 3-35. doi:10.1016/B978-0-12-394390-3.00001-X
- Morgan EH, A. T. (1969). Autoradiographic localization of 125-I-labelled transferrin rabbit reticulocytes. Nature, 1371-2
- Leverence R, M. A. (2010). Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry. Proc Natl Acad Sci USA, 8123-8.
- Zhang D, L. H. (2012). Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.). BMC Biotechnology, 12, 92. doi:10.1186/1472-6750-12-9