Growth of NS0 Cells in Protein-Free, Chemically Defined Medium

Abstract

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.

Cell Culture Processes in Monoclonal Antibody Production

“…[10][11][12] NS0 cells lack endogenous glutamine synthetase (GS) enzyme activity making them suitable for use with GS as a selectable marker for recombinant antibody expression. 13 High antibody productivity has been reported from non-GS NS0 cell lines as well.…”

Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

“…250× CCLC used in the present study is protein-free watersoluble cholesterol/methyl-β-CD complexes, using the CD as a vehicle (Gorfien et al 2000). The concentration of CD in the CLC is very low (about 10 −4 %), enabling us to load cholesterol to MIN6 cells without inducing significant cell toxicity.…”

The role of recombinant proteins in the development of serum-free media

“…Ex-cyte (Bayer) is a concentrate of cholesterol, lipoproteins and fatty acids that is used in SFM to enhance cell growth and protein production as well as glycosylation status in a variety of mammalian cells including: hybridomas, myelomas, CHO and HEK293 cells (Castro et al 1995;Gorfien et al 2000;Savonniere et al 1996). It is however bovine-derived and is recommended to be used in conjunction with BSA.…”

“…Plant-derived cyclodextrins (Gorfien et al 2000) and synthetic low density lipoprotein complexes (Hayari and Halbert 2005) offer an opportunity to replace animal-derived lipid transporters. For some commercial SFM such as VP-SFM (suitable for Vero, BHK-21, Cos, MDCK), the need for albumin is replaced by di and tri-peptides from plants.…”