Biological Research Division, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent, UK.
Cytotechnology. 1995 Oct;17(3):203-11. doi: 10.1007/BF00749658.
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 1.5×10(6) 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 2.4×10(6) cells ml(-1) was achieved in W38 medium compared with 1.41×10(6) 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 3.24×10(6) 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.
NS0 一直被用作生产杂交瘤的融合伙伴，最近已被设计用于生产重组蛋白。先前已经开发出一种无蛋白的培养基，称为 W38 培养基，该培养基支持大鼠骨髓瘤和杂交瘤细胞系的高密度生长。NS0 细胞无法在 W38 培养基以及其他支持其他骨髓瘤细胞系生长的几种无蛋白培养基中生长。NS0 细胞源自 NS-1 细胞系，已知该细胞系需要外源性胆固醇。研究发现，NS0 细胞在添加卵磷脂、胆固醇和白蛋白的 W38 培养基中生长，并且 NS0 细胞对胆固醇呈营养缺陷型。通过使用β-环糊精代替白蛋白作为脂质载体，可以实现 NS0 细胞的无蛋白生长。在这种无蛋白培养基中达到的最大细胞密度超过 1.5×10(6)细胞/ml(-1)。脂质补充剂在 +4°C 储存几天后会沉淀。为了解决这个问题，开发了一种方案，使 NS0 细胞能够适应 W38 培养基中胆固醇非依赖性生长。NS0.CF（胆固醇非依赖性 NS0 细胞）在 W38 培养基中连续培养数月。在摇瓶培养中，与含有 10%胎牛血清的 RPMI 1640 培养基中 1.41×10(6)细胞/ml(-1)相比，在 W38 培养基中实现了 2.4×10(6)细胞/ml(-1)的细胞密度。NS0.CF 细胞在使用 W38 培养基补充 Pluronic F68 的 1 升搅拌生物反应器中容易生长，达到 3.24×10(6)细胞/ml(-1)的密度。通过在 W38 培养基中进行有限稀释，NS0.CF 无蛋白克隆，在接种密度平均为每个 200 μl 0.32 个细胞的孔中形成菌落。这项研究首次证明了胆固醇依赖性小鼠骨髓瘤细胞系在完全定义的无蛋白培养基中的生长及其随后适应胆固醇非依赖性。
