A new technique to register proliferation of clonogenic cells from brain tumors

The soft agar technique for culturing human clonogenic tumor cells has been usefully applied for predicting individual clinical responses to chemotherapy, for screening of new antineoplastic drugs, and in basic biological research. The counting of colonies formed by clonogenic cells is, however, a rather time consuming and inaccurate procedure. We here report a method to combine the easy and precise registration of DNA-synthesis by 3H-thymidine incorporation with the ability of soft agar to permit proliferation of clonogenic cells and inhibit proliferation of non-neoplastic cells. The glioma cell lines U 251 MG and T-MG 1, the benignant glia cells T-BG 1, T-BG 2, T-BG 3 and fibroblasts were cultured in Furcellaran gel. Twenty hours before harvesting 3H-thymidine was added. The Furcellaran gel was resolved by 50 mM LiI. The cells were trapped on glass fiber filters and incorporated radioactivity was measured. 3H-thymidine incorporation in malignant cells increased exponentially with time, while 3H-thymidine incorporation in the benignant glia cells and fibroblasts was inhibited. The correlation between number of colonies counted after 16 days and 3H-thymidine incorporation registered after different culture times was very good. The correlation was best when the cultures were harvested after 8 days (r = 0.95), indicating that it is possible to reduce the assay time. The five glioma biopsies tested grew well with a mean plating efficiency of 0.4% (range 0.02-1.8%). The most intense proliferation seemed to take place during the first week in culture. The good correlation between 3H-thymidine incorporation on day 7 and colony number on day 14 (r = 0.93), indicate that reduction of assay time is possible also for the glioma biopsies.