TECHNOLOGY
Mammalian cell culture methods improved Monsanto scientists have developed ways to grow rat tumor and human cells in larger batches and at higher densities than before
vessel specifically designed to provide optimal growing conditions for mammalian cells. In a conventional cell culture system, cells are exposed to a constantly changing environment that starts out rich in nutrients and gradually becomes depleted in them. At the same time, the concentrations of cell waste products steadily increase. Continuous perfusion of fresh culture medium through the culture system is one way to overcome this problem. The Monsanto team has been able to do this in moderate-scale (4- and 44-L) reaction vessels. The perfusion is accomplished by attaching a satellite vessel that contains a cylindrical filter. The filter retains the cells, which are pumped back into the culture vessel. The system works very well, Feder
New techniques that substantially increase the scale on which mammalian cells can be grown in culture have been developed by a team of researchers at Monsanto. Because cell culture is the best, and in some cases the only, way to synthesize some potentially important mammalian proteins like interferon and certain monoclonal antibodies, the techniques may have important implications for research on these substances and their applications. The three related techniques were developed by Joseph Feder, a senior fellow, and William R. Tolbert, a cell-culture group leader, at Monsanto's St. Louis laboratories. The scientists describe the techniques in the current issue of Scientific American [248,36(1983)]. Although microbiologists can grow bacterial cells in batches of 50,000 gal or more, mammalian cells are much harder to grow on a large scale. For one thing, mammals are not single-celled organisms, as bacteria are. Most animal cells will not grow at all in suspension, as bacterial cells will; they grow only when attached to a surface. The Monsanto scientists began with the special case of those types of mammalian cells that will grow in suspension, such as blood and lymphatic system cells and many tumor cells. They decided that rather than try to adapt bacterial fermenters to Monsanto technician Billie accommodate mammalian cells, they Reddick samples mammalian cells would try to develop an inexpensive grown in 100-L steel vessel 26
January 10, 1983 C&EN
and Tolbert say. When rat tumor cells were grown in the perfused vessel and in a conventional one, the rate of growth of the cells was about the same in each system, but cell density was 25 times higher in the perfusion reactor. Moreover, all the cells remained alive in that reactor—even at maximum density—compared with a 30% loss due to cell death in the conventional reactor. For the majority of mammalian cells that cannot be grown in suspension, Feder and Tolbert have developed two modifications of their perfusion reactor system. One grows cells on the outside of hollow fibers and the other on the surface of microcarrier beads. In the first of these, a shallow core bed of fibers, only three to six layers deep, forms the surface on which the cells are grown. The bed is perfused with fresh medium, which flows past the fibers in a direction parallel to their axes. The researchers built two prototype reactors of this type, with surface areas of 930 sq cm and 9300 sq cm. The smaller of these prototypes grew human lung cells to a density of about 1 million cells per sq cm— about 10 times greater than can be achieved by conventional roller bottle culture. ' T h e system does not just grow large numbers of cells; in effect it builds an artificial tissue as billions of cells proliferate to encrust the fibers and even penetrate them/' the researchers say. The cells can be maintained for as long as two months, continuously producing cell products that can be harvested from the spent medium without disturbing the culture. The second modification uses polymeric beads as the surface on which the cells are grown. The principal improvement on this method made by Feder and Tolbert was the addition of a settling bottle between the main reactor vessel and the satellite vessel housing the filter. In one experimental run of a 4-L microcarrier perfusion reactor, the scientists grew 40 billion human fibroblasts, a harvest that would require 1300 roller bottles. When those cells were transferred to a 44-L reactor, they produced 340 billion cells, the equivalent of production from 11,000 roller bottles. D
