Development of a 9L Rat Brain Tumor Cell Multicellular Spheroid System and Its Response to 1,3-Bis(2-chloroethyl)-1-nitrosourea and Radiation2

Abstract

Rat brain tumor cells (9L cells) were cultured in suspension as multicellular spheroids. When individual spheroids with diameters of approximately 200 μm were transferred to test wells, their diameters increased by about 25 μm/day; colony-forming efficiencies were routinely 40–50%. The durations of the S-phase (8.0–8.5 hr), the G₂ phase (3.0–4.0 hr), and the cell cycle time (20.1–20.5 hr) in spheroids with diameters from 200 to 500 μm were similar to those determined earlier for monolayer and intracerebral 9L cells. The growth fraction of spheroids was approximately 0.5, also similar to that of 9L intracerebral tumors. Autoradiographic studies showed that 30 minutes were sufficient for [³H]thymidine to penetrate to the center of the spheroids and that after prolonged periods of exposure, both labeled and unlabeled cells existed throughout the spheroid. Cells cultured and treated as spheroids with approximate diameters of 200–600 μm were more sensitive to 1,3-bls(2-chloroethyl)-1-nitrosourea (BCNU) than were cells grown as spheroids but treated as suspended single cells. The most prominent change between percent labeled mitoses curves of control and BCNU-treated spheroids was the absence of a prominent second wave in the treated groups. Flow cytometry studies showed a gradual increase in the G₂-M population 12 hours after BCNU treatment; a maximum was attained 30 hours after treatment. However, approximately 50% of the population remained in the 2C DNA position up to 56 hours after treatment. Radiation survival curves were obtained for spheroids having approximate diameters of 200–400 μm. In each case, intact spheroids were slightly more resistant to radiation than was the single cell suspension. Resistance was not displayed as a plateau region on the survival curves but rather as a shifting of the entire curve to the right. The amount of resistance over this size range was similar.