The design and application of a system permitting the active proliferation of mammalian cells, as individual units, in the submerged state, would seem to be of value to individuals interested in investigations relating to the overall kinetics of the host/cell virus interaction and to studies requiring high concentrations of uniform tissue cells or virus. This report presents data establishing the feasibility of obtaining uniform proliferation of mammalian cells in the submerged state in volumes up to 3 L in a stationary, impeller-agitated fermentor. Various types of agitated systems have been suggested as satisfactory for permitting growth of tissue cells in the submerged state, i. e., the rotary shaker of Earle et al. (1954), the tumble tube of Owens et al. (1953), the roller tube of Graham and Siminovitch (1955), the suspended stirrer in Erlenmeyer flasks of Cherry and Hull (1956), Powell's (1954) hexagonal roller tube, Brown and Hardy's (1957) wrist shaker, as well as the glass stirrer of Danes (1957). Inherent difficulties have been encountered with certain of the submerged culture systems mentioned. Thus, in shake flask systems there is a marked tendency toward the formation of large granules (McLimans et al., 1957). Additionally, proteinaceous precipitates are frequently formed. Certainly, the extrapolation to large vessels, 3 to 16 L, would be difficult in the instance of roller-tube type units or flask systems. We have reported (McLimans et al., 1957), l Contribution No. 12 from Microbiology in Medicine, Wistar. The investigations herein reported were conducted under the auspices of a grant (Contract no. DA-18-064-404-CML-74) from the U. S. Army Chemical Corps, Fort Detrick, Maryland.

summaryIt has been demonstrated experimentally that the thickness of fluid overlay in conventional tissue culture systems limits the oxygen available to mammalian cells growing as a submerged monolayer. A rocker culture system is described which circumvents critical problems associated with thin f i l m culture while permitting nearly unlimited access of oxygen to the cell monolayer. Good growth of primary hepatic cells as isolated sheets has been obtained.

Mathematical models have been constructed which relate the depth of the culture fluid overlay to the oxygen available to mammalian cells cultured under static conditions. These models suggest that the maintenance of a given rate of oxygen utilization by some culture systems may be critically depended on this fluid depth and on the solubility and rate of diffusion of oxygen in the culture fluid. The importance of these concepts as applied to the isolation and growth of differentiated cells representative of the tissue of origin are noted. 741 0.21 mm. This hypothesis was quickly confirmed experimentally by Minami5Hill6 extensively surveyed the kinetics of gas diffusion in tissue. He developed formulas for the mathematical expression of the events occurring while approaching and under conditions of equilibrium or steady state. He defined the diffusion constant ( K ) as the number of BIOTECHNOLOGY AND BIOENGINEERING, VOL. X, ISSUE 6

No abstract