An Intermittent Perfusion and Gas Exchange Method for Continuous Cultivation of Cells in the Rose Chamber

Abstract

A simple technique for providing gas exchange within the Rose tissue-culture chamber is described. The method involves initial introduction of a large gas phase within the chamber, and subsequent maintenance of communication between this gas phase and a gas reservoir by means of polyethylene tubing. The fluid phase is exchanged intermittently by means of a gravity flow system through polyethylene tubes. The gas bubble within the chamber is kept in constant position, away from the microscopic field. We accomplish this by placing the entire apparatus at a slight angle to the horizontal, which creates a spirit-level effect. With this technique it is possible to grow and maintain heavy populations of strains P388 D₁, and HeLa cells for periods of 4 and 5 months, respectively. After these intervals the condition of the cells was as good as at the outset and was comparable to that of the same strain carried in tube cultures. There is no apparent time limit for growing cell strains within such chambers. Preliminary trials demonstrated that in this system submandibular gland rudiments of mouse embryos undergo development comparable to that in tube or dish cultures. The advantages of the system are that it permits long-term, time-lapse cinematography, with phase or ordinary optics, at any magnification, of cultures under conditions approximating those in standard tubes or flasks. The technique may be applicable to studies of long-term phenomena such as “spontaneous” or induced morphologic transformation of cells in vitro.