Figure 12. Static maintenance culture systems. Static maintenance type: hollow fiber,1381 ceramic opticell, membroferm, static maintenance systems. Suspension culture type: membrane dialysis, rotating filter,t44) membrane agitator, sedimentation column systems.'461
The most important technique for perfusion culture methods is to separate the concentrated cells and conditioned medium from the suspended culture broth. As noted above, the separation methods chiefly used are filtration with tubular and flat membranes as well as ceramic macroporous filters. These membrane reactors can be employed for both anchorage-dependent and suspension growing cells. Static maintenance type systems are commercially available for disposable reactors, and small size unit reactors from 80 ml to 1 liter are used for continuous production of monoclonal antibodies with hybridoma cells. The maintainable cell densities are about 107-108 cells/ ml which is essentially mouse ascites level. However, in these systems, the cell numbers cannot be counted directly because the cells adhere to membranes or hollow fibers. Therefore, the measurement of cell density must use indirect methods. Such indirect methods include the assaying of the quantities of glucose consumption and the accumulation of lactate. The parameters of scale-up have not yet been established for these static methods.
Tolbert et al. and Himmelfarb et al.l44] have obtained high density cell growth using a rotating filter perfusion culture system. Lehmann et al.t45] used an agitator of hollow fiber unit for both perfusion and aeration. In our laboratory, we[47J constructed a membrane dialysis fermenter using a flat dialysis membrane. The small size system is well-suited for the cultivation of normal lymphocytes (Lymphokine actived killer cells). These cells are employed in adoptive immunotherapy due to their high activities for thirty or more days and their acceptance by the reactor cells.
To eliminate the use of a membrane and a filter, we have also tried to make a perfusion culture system using a sedimentation column.
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 from 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.
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