Conditions of cultivation required for the formation of hemicysts in vitro by rat bladder carcinoma R-4909

Abstract

Rat urinary bladder carcinoma R-4909 grew readily in vitro. In areas where saturation density occurred in the cultures, occasional hemicysts were observed. A modification of technique producing "packed cultures" resulted in the appearance of greater numbers of hemicysts. Four clonal isolates of R-4909 were also studied in packed culture. Clone B formed hemicysts in abundance. Clone D produced occasional hemicysts similar to the parent stock line. No hemicysts were seen in cultures of clone A or clone C. The number of hemicysts formed by clone B in packed culture was responsive to the ratio between cell number and volume of medium, to serum concentration in the medium, and to pH of the medium. The last was of particular interest since a pH of 7.8 enhanced and a pH of 6.6 inhibited hemicyst formation. The effects were all reversible. On scanning electron microscopy, we found well-developed cell membrane structures between contiguous cells. In media with sufficient serum for hemicyst formation, the articulations between cells were prominent. With low serum concentrations, hemicysts did not form and the intercellular articulations were less distinct. We interpret the formation of hemicysts as an expression of fluid transport by epithelia, a function that requires a constellation of differentiated characteristics within cells and in their level of integrated association.