The Promotion of Cell Growth in Human Embryonic Kidney Cells with Recombinant Human Lactoferrin (Holo)

The use of transferrin in Cell Culture is a method for providing iron to the cells. The delivery of iron using transferrin has been well documented for cell culture systems and in biomanufacturing of therapeutic proteins, such as monoclonal antibodies. Transferrin is derived from both animal and human serum sources for use in these processes. Recently, a move is being made towards animal and human-free cell culture systems. The employment of a human recombinant alternative to transferrin would prove optimal for cell culture as well as for biomanufacturing.

Lacromin™

Recombinant human lactoferrin, Lacromin™ (InVitria) is a cell culture grade recombinant human lactoferrin (rhLF) that is produced from an animal- and human-free plant based expression system, ExpressTec (Nandi et al., Transgen. Res., 2005). Lacromin™ is a 80 kD glycoprotein belonging to the transferrin family of proteins and has an amino-acid homology and similar iron biding properties to human transferrin. Lacromin™ is a multi-functional protein, as such it is a cell growth factor, a source of iron, delays apoptosis and displays anti-microbial properties. Lacromin™ has been reported to have synergistic effects with both EGF (Kohno et al., Acta. Paedietr., 1993) and bFGF on cell growth (Shinoda et al., Adv. Exp. Med. Biol., 1994).

Human Embryonic Kidney Cells

Human embryonic kidney epithelial cells (HEK 293) were generated by transformation of cultures of normal human embryonic kidney cells. They are easy to grow, transfect very readily and have been widely-used in cell biology research. HEK 293 cells are also used by the biotechnology industry to produce therapeutic proteins and viruses for Gene therapy.

The Effect of Lacromin™ on Human Embryonic Kidney Cells

The human embryonic kidney cell line HEK293 was used to compare cell growth between holo human transferrin (holo-hTF) and holo-rhLF in a serum free cell culture media (250 mg/L). Holo-rhTF supplemented group displayed a maximum cell density that was 50% greater than the maximum cell density of the holo-hTF supplemented group after 6 passages (3-4 days in culture) and after the 7th Passage cells were allowed to grow for 8 days then the viable cells were counted. Furthermore, the cell doubling time for the Holo-rhTF supplemented group was shorter than the holo-hTF group. An additional study was performed to determine the optimal concentration of holo-rhLF for use with HEK293 cells. Concentrations ranged from 10-200 mg/L. HEK293 cells were cultured in a serum-free media supplemented with holo-rhLF and passaged 8 times (4 days in culture), and after the 9th passage cells were allowed to grow for 7 days. Viable cells were quantified at various intervals. The optimal holo-rhLF was determined for use with HEK293 cells was between 50 and 100 mg/L (Haung et al., In Vitro Cell. Dev. Biol.-Animal., 2008).

Conclusions

Holo-hTF, Lacromin™ (InVitria) outperformed hTF for the promotion of cell growth in human embryonic kidney cells. Additional advantages of Lacromin™ include high and stable expression of recombinant proteins, tissue specific expression in grain endosperm, rapid scalability to metric-ton quantities, prevention of gene flow with self pollinating crops, low capital investment and production costs, and efficient processing and recovery (Huang, BioProcess, 2004).

The use of Lacromin™ as a replacement for serum derived transferrin would provide higher cell density and reduction of cell doubling time under animal- and human-free conditions (InVitria Lacromin™ product brochure, www.InVitria.com). The employment of a human recombinant alternative to transferrin would prove optimal for cell culture as well as for biomanufacturing.

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