Adaptation of cholesterol-requiring NS0 mouse myeloma cells to high density growth in a fully defined protein-free and cholesterol-free culture medium

Abstract

NS0 has been used as a fusion partner for the production of hybridomas and has more recently been engineered to produce recombinant protein. A protein-free culture medium, designated W38 medium, has previously been developed which supported high density growth of rat myeloma and hybridoma cell lines. NS0 cells failed to grow in W38 medium and in a number of protein-free culture media which support the growth of other myeloma cell lines. NS0 cells are derived from the NS-1 cell line, which is known to require exogencus cholesterol. It was found that NS0 cells grew in W38 medium supplemented with phosphatidylcholine, cholesterol, and albumin and that NS0 were auxotrophic for cholesterol. Protein-free growth of NS0 cells was achieved by using β-cyclodextrin to replace albumin as a lipid carrier. The maximal cell density reached in this protein-free medium was in excess of 1.5×10⁶ cell ml⁻¹. The lipid supplements in the medium precipitated after a few days storage at +4°C. In order to overcome this problem a protocol was developed which allowed NS0 cells to be adapted to cholesterol-independent growth in W38 medium. NS0.CF (cholesterol-independent NS0 cells) were cultured continuously in W38 medium for several months. In shake flask culture a cell density of 2.4×10⁶ cells ml⁻¹ was achieved in W38 medium compared with 1.41×10⁶ cells ml⁻¹ in RPMI 1640 medium containing 10% foetal bovine serum. NS0.CF cells readily grew in a 1 litre stirred bioreactor using W38 medium supplemented with Pluronic F68 reaching a density of 3.24×10⁶ cells ml⁻¹. NS0.CF were cloned protein-free by limiting dilution in W38 medium, giving colonies in wells that were seeded at an average density of 0.32 cells per 200 μl. This study has demonstrated for the first time the growth of a cholesterol-requiring mouse myeloma cell line in a completely defined protein-free medium and its subsequent adaptation to cholesterol-independence.