Development of protein-production system by the use of immobilized animal cells

重组糖蛋白必须由动物细胞产生，因为原核细胞不是糖基化的。到目前为止，这些蛋白质都是由锚定依赖的细胞产生的，因此，发展一种能够高密度增殖这些细胞的方法对于高效生产这些有价值的蛋白质非常重要。在这个项目中，我们试图建立一种方法，将产生红血球因子的锚定依赖细胞固定在海藻酸凝胶中。固定化可以保护细胞免受有害物理力量的伤害，而海藻酸凝胶可以为细胞的生长提供广阔的空间。红细胞生成素(EPO)是一种生理刺激红细胞生成的糖蛋白，由3种锚定依赖的动物细胞固定在海藻酸凝胶颗粒中，连续生产超过32d。在含有细胞的藻酸盐溶液中加入二价阳离子引起的凝胶化，导致形成透明的空隙(这里称为通道)。每个通道起源于一个细胞，并向凝胶颗粒的中心延伸。动物细胞在通道内生长良好，但在通道外几乎没有增殖。凝胶颗粒中的细胞浓度达到1×10个/g凝胶以上。海藻酸盐固定化方法适用于高密度培养锚定依赖细胞。以海藻酸钙凝胶颗粒包裹的微载体高密度培养新生仓鼠肾细胞。在该系统中，BHK细胞不仅在微载体上增殖，而且在藻酸盐凝胶颗粒的空隙中增殖。这些空间有助于细胞的高密度培养和EPO的高生产率。

Recombinant glycoproteins have to be produced by animal cells, since procaryotic cells do not glycosylated. So far these proteins have been produced by anchorage-dependent cells and therefore, development of the method that makes it possible to proliferate these cells in a high density is important for production of these valuable proteins in a high efficiency. In this project, we attempted to establish the method in which the anchorage-dependent cells producing the erythropoietic factor were immobilized in alginate gels. Immobilization would protect the cells from harmful physical forces and alginate gels may provide the wide space for growth of the cells.Erythropoietin (EPO), a glycoprotein that is a physiological stimulator of erythrocyte production, was produced continuously for more than 32 days by three kinds of anchorage-dependent animal cells immobilized in alginate gel particles. Gelation caused by divalent cations added to an alginate solution containing cells resulted in the formation of clear vacant spaces (referred to here as channels). Each channels originated from a cell and extended towards the center of the gel particle. The animal cells grow well in the channels but proliferated little outside the channels. The cell concentration in the gel particles reached more than 1 x 10 cells/g gel. The alginate immobilization method was useful for high-concentration cultivation of the anchorage-dependent cells.Baby hamster kidney (BHK) cells engineered to produce recombinant human EPO were cultured at high density on microcarriers entrapped by calcium alginate gel particles. In this system, the BHK cells proliferated not only on the microcarriers but also in vacant spaces in the alginate gel particles. These spaces contributed to high-density cultivation of the cells and a high productivity of EPO.