Quantitative cloning of malignant human glioma cells by conditioned medium

The use of conditioned medium (CM) obtained from monolayer cultures of human glioma cells induced colony formation from single glioma cells in culture. In contrast, no colonies were observed in cultures incubated with nonconditioned standard Eagle's basal medium. The number of colonies formed closely depended on the concentration of CM. The glioma CM not only stimulated colony formation but also induced the formation of fibrillary cell extensions. Culture conditions influencing the production of colony-stimulating factors included cell density and duration of culture medium contact with glioma cells. The colony-stimulating activity (CSA) was stable after freezing and thawing, but decreased 30-40% when CM was exposed to temperatures over 66 degrees C for 30 minutes. In addition, the CSA was filtratable (0.45 mu), dialyzable, and passable through an Amicon PM10 filter, which indicated a molecular weight less than 10,000. The use of CM provided an improved method for quantitative assays of neural tumor cells, based on their colony formation in culture.