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

SummaryA rocker mammalian cell culture system is described. The basic mechanical component is a special incubator with contained rocker culture trays. The system provides for precise environmental temperature regulation with intermittent rocking of contained cultures in such a manner as to permit thin fluid overlay of cells with maximum accessibility to oxygen while circumventing the problems of evaporation, substrate depletion, etc. IntroductionSeveral reports have delineated the critical role of gas diffusion in cell cultureOn the basis of experimental* considerations it has been demonstrated that good growth of human embryonic cells can be achieved if the culture fluid overlay is restricted to 0.5 mm or less. The fluid overlay depth of 1.5-5.0 mm utilized in mmt conventional cell culture systems severely limits access of oxygen to primary cells and in time creates an anoxic environment.3 Via the described rocker system, thin-layer culture waa achieved and demonstrated to be an important parameter in culture of cells representative of the tissue of origin.3The Rocker Culture SystemThe rocker culture system design is such as to provide a very thin layer of culture fluid over the mammalian cells. This establishes optimum conditions for rapid diffusion of gsses and thus ready access of oxygen to the cells. Intermittently, the thin fluid layer is replaced by a gentle rocking motion that permits reservoir fluid to bathe the cells. This effectively circumvents the problems of evaporation, exhaustion of substrate, and loss of pH control as anticipated in thin-layer culture systems incubated in the horizontal position. The Rocker IncubatorThe rocker incubator was designed on the basis of the following criteria: to provide a maximum number of cultures per unit space with minimum contact of the culture flask surface with the holding device; to provide ready access t o an individual culture flask ; to provide temperature-control circuits compatible with rocker incubator box providing a capability of temperature adjustment from ambient to 45°C and to control at the selected value within fO.1"C; to provide for rapid temperature recovery following biologic manipulative procedures ; to provide maximum reliability over long periods of time with minimum adjustment and maintenance ; to provide an electronically controlled drive mechanism BIOTECHNOLOGY AND BIOENGINEERING, VOL. X, ISSUE 5 X (1968) capable of rocking contained culture trays ~-2 5 ' off the level position with a smooth movement at a constant rate such as to prevent "sloshing" of culture fluid and, finally, to provide ready accesa for connections to auxiliary unita such as monitor flasks,' gas monitor,5 etc. COMMUNICATIONS TO THE EDITORSThree small incubators were constructed to allow concurrent investigations at different environmental temperatures as well as to decrease the temperature gradients usually encountered in large incubators.The incubators were designed to fit in a Par-Metal rack S o . PR-ii27-B vertically (Fig. 1). Plexiglas was chosen for favorable...

SummaryThe Meta-Stat gas monitor, il component instrument which automatically monitors and controls the pH and gas content of the liquid phase in suspension cell-culture systems, has been developed to provide continuous pH control within 3~0.015 units, as well as continuous oxygen control within 0.5yo of the preset level in the range of 0-40% 0 2 .