Engineering research on the production of useful biologicals by mass culture of animal and plant cells

1. 采用溶解氧电极、溶解氧转换器、PID控制器和质量流量控制器，以气体流速为被控变量，实现了介质溶解氧浓度（DO）的PID调节。通过对DO控制系统的研究，发现在带通风管的气升柱中，胡萝卜细胞生长的最佳DO水平为3.0ppm， β -胡萝卜素的最佳生成水平为0.05ppm。在DO-stat （0.15ppm）半连续培养中，胡萝卜细胞的产量最高，为0.24杯子/10^8个细胞/天，是常规分批培养的7.5倍。我们还通过调节聚氨酯泡沫（PUF）填充床培养系统的输出DO水平（超过5.8ppm），成功地实现了动物细胞的高密度培养（1.1X10^8个细胞/cm^3）。采用有限元方法对两叶桨叶搅拌容器中培养基的三维层流进行了数值模拟。采用careau模型作为本构方程。我们成功地得到了在一定搅拌速率（10- 60rpm）下的速度场和剪应力场。成年大鼠肝细胞在PUF孔中培养后形成球形多细胞聚集体（球形，直径约100mum）。球状体保持了高度分化的肝脏功能，这些球状体部分附着并固定在PUF的孔中。我们设计了一个PUF/球形填充床培养系统。在PUF填充床中固定的肝细胞/球体产生白蛋白和尿素的活性高于培养皿中通常的肝细胞/单层培养，并维持了26 d。这些结果表明，PUF/球形填充床培养系统为混合型人工肝支持系统提供了一个有希望的原型。

1. We could succeed in regulating a dissolved oxygen concentration (DO) of the medium in PID manner by using DO electrode, DO converter, PID controller and mass flow controller, in which the gas flow rate was used as the manipulated variable. By using the DO control system, it was found that there were optimum DO level in cell growth (3.0ppm) and beta-carotene production (0.05ppm) of carrot cells in an air-lift column with draft tube. Highest productivity (0.24mug/10^8cells/day) was obtained in DO-stat (0.15ppm) semi-continuous culture of carrot cells and this value was 7.5 time higher than that in the usual batch culture. We also succeeded in high density culture (1.1X10^8cells/cm^3) of animal cells by regulating a out-put DO level (more than 5.8ppm) of polyurethane foam (PUF) packed-bed culture system.2. The three-dimensional laminar flow of culture medium in an agitated vessel with 2-bladed paddle imoeller was simulated numerically by finite element method. The Carreau model was used as the constitutive equation. We succeeded in obtaining the fields of the velocity and the shear stress under a certain agitation rate (10-60 rpm).3. Adult rat hepatocytes formed spherical multicellular aggregates (spheroids, about 100mum in diameter) when they were cultured in the pores of PUF. Highly differentiated hepatic functions were maintained in the spheroid, These spheroids were partly attached and immobilized in the pores of PUF. We designed a PUF/spheroid packed-bed culture system. Albumin and urea production activities of hepatocyte/spheroid immobilized in the PUF packed-bed were higher than those of usual hepatocyte/monolayer culture in petri dish, and these activities were maintained for 26 days. These results indicated that the PUF/spheroid packed-bed culture system provided a promising prototype of hybrid type artificial liver support system.