Perfusion System for Replicate Mammalian Cell Cultures in T-60 Flasks

Abstract

Conventional, replicate culture flask techniques—viz., a series of stoppered T-flasks—for growth of mammalian cells in vitro are well adapted to intermediate metabolism studies associated with growth mechanisms. However, their disadvantages are that physical factors such as pH and gas tensions are not easily controlled and the nutrient solution is subject to extremes of enrichment and depletion by the usual change of medium regimes. This report describes a perfusion-type system for 8 T-60 flask cultures that permits control of these environmental factors. Each flask was fitted with influent and effluent gas and nutrient solution lines; gas tensions and composition were controlled with a flow rate meter and system of pinch clamps. The rate of gravity-flow of culture medium was regulated by a motorized clamp assembly attached across the influent lines leading to each culture flask; the assembly was actuated periodically by a variable rate, recycling timer. The proliferation curve of Jensen sarcoma cells of the rat in this system was characterized by an initial 2-day logarithmic rate of increase with as low as a 12-hour generation time; this was followed shortly with an equal period of a much slower (nearly stationary) phase, with generation times as high as 15 days before renewal of more rapid population increase. The pH. and glucose concentration were maintained at 7.0 ± 0.1 and above 0.2 g per 100 ml, respectively. Over 1.4 × 10⁸ cells were produced in 8 days in a single T-60 flask with 2.3 × 10⁶ cells in the initial inoculum.