Development of mammalian cells capable of growing rapidly in serum-free media

细胞因子和生长因子对于因子依赖性哺乳动物细胞的增殖和维持是必不可少的。然而，细胞因子通常是如此昂贵，以至于将因子依赖性细胞用于工业应用如蛋白质生产是不实际的。在这里，我们试图建立一种方法来维持细胞生长与廉价的配体完全不同的细胞因子。基于抗鸡卵溶菌酶（HEL）抗体HyHEL-10的VH和VL区之间的相互作用仅在HEL存在时才变强的事实，将HyHEL-10的VH或VL区分别替换成促红细胞生成素受体（EpoR）的部分胞外区，分别形成Vn-EpoR或VL-EpoR。将V_H-EpoR和V_L-EpoR共转染IL-3依赖的前B细胞系Ba/F3，在无IL-3存在下，转染细胞呈剂量依赖性增殖。为了替代杂交瘤细胞的IL-6依赖性，将V_H-EpoR和V_L-EpoR的胞内结构域分别用gp 130的胞内结构域替换，以产生V_H-gp 130和V_L-gp 130。当V_H-gp 130和V_L-gp 130基因共转染Ba/F3细胞时，转染细胞在无IL-3的情况下以HEL剂量依赖的方式增殖。将V_H-gp 130和V_L-gp 130基因共转染IL-6依赖性杂交瘤细胞系7 TD 1，在无血清和含血清培养基中，转染细胞均表现出明显的HEL剂量依赖性生长和单克隆抗体的产生，从而显著降低了培养基成本。结果进一步表明，通过交换受体胞内结构域，应用该技术控制许多其他细胞功能的可能性。

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for the industrial applications such as protein production is not practical. Here we try to establish a method to maintain cell growth with an inexpensive ligand quite different from cytokines. Based on the fact that interaction between V_H and V_L regions of anti-hen egg lysozyme (HEL) antibody HyHEL-10 becomes strong only in the presence of HEL, part of the extracellular region of erythropoietin receptor (EpoR) was replaced with either V_H or V_L region of HyHEL-10 to create Vn-EpoR or V_L-EpoR, respectively. When V_H-EpoR and V_L-EpoR were cotransfected into an interleukin-3 (IL-3) dependent pro-B cell line Ba/F3, the transfectant proliferated in a HEL dose-dependent manner without IL-3. To substitute IL-6 dependency of hybridoma cells, the intracellular domain of V_H-EpoR and V_L-EpoR was replaced with that of gp130 to create V_H-gp130 and V_L-gp130, respectively. When V_H-gp130 and V_L-gp130 genes were co-transfected to Ba/F3 cells, the transfectant proliferated in a HEL dose-dependent manner without IL-3. When V_H-gp130 and V_L-gp130 genes were co-transfected to an IL-6 dependent hybridoma cell line 7TD1, the transfectant showed clear HEL dose-dependent cell growth and monoclonal antibody production both in serum-containing and serum-free media without IL-6, resulting in significant medium cost reduction. The results further suggest the possibility of applying this technology for the control of many other cell functions by exchanging the receptor intracellular domain.