Sedimentation Column Perfusion Systems

We have developed several new perfusion systems which do not use filtration methods for cell propagation. When the flow rate of the continuous supplying medium is minimized, for example, when it is 1 to 3 times its working volume per day, the system has the ability to separate the suspended cells from the supernatant fluid. This is accomplished by means of an internal cell-sedimentation column in which the cells settle by gravity. The shape and length of the column are sufficient to ensure complete separation of cells fi om the medium. Cells remain in culture whereas the effluent medium is continuously withdrawn at a rate less than that of the cell sedimentation velocity. We experimented with several shapes for the sedimentation column and found that the cone and two jacketed types work best. 

With the cone for a continuous flow rate of perfusion, the flow rate in the column is inversely proportional to the square of the radius of the cone at any given position. If the ratio of the radii of the inlet and outlet is 1 10 and the flow rate of the outlet is 1/100 of the inlet flow rate, then the separation efficiency of the supernatant fluid and suspended cells are improved. As shown in Fig. 13, the jacket type sedimentary system allows easy control of the temperature for separating the static supernatant from the cells. This jacket method was applied to an air-lift fermenter since it had not been done in an air-lift perfusion culture. According to Katinger et al., air-lift methods have smaller shear forces than impeller type agitation. However, in perfusion culture, comparable maximum cell densities were obtained using all three types of fermenters. 

---

To eliminate the use of a membrane and a filter, we have also tried to make a perfusion culture system using a sedimentation column,... 

High Density Culture Using a Perfusion Culture System with Sedimentation Column... 

The biofermenter BF-F500 system consisted of a 1.5 1 culture vessel, 2 1 medium reservoir and effluent bottle (2 1 glass vessels) for fresh and expended media which were connected to the perfusion (culture) vessel by a peristaltic pump. As shown in Fig. 14, the fermenter systems have a conical shape sedimentation column in the center of the fermenter, and an impeller on the bottom of the sedimentation column. The Namalwa cells, KJM-1, were cultivated by continuous cultivation in the biofermenter. In Fig. 15, the culture has been inoculated at 1 to 2 x 10 cells/ml with an initial flow rate of approximately 10 ml/h, sufficient to support the population growth. At densities of 7 x 10 -1.5 x 10" cells/ml, we have used a nutrient flow rate of 1340 ml/h using ITPSG and ITPSG-F68 serum-free media. The flow rate of fresh media was increased step-wise from 240 to 960 ml/d in proportion to the increase in cell density. This resulted in an increase of 4 to 10 fold in cell density compared to the conventional batch culture systems. This system was then scaled up to a 45 1 SUS316L unit mounted on an auto-sterilization sequence system with a medium reservoir and an effluent vessel of 901 each. 

---