Relative biological effectiveness of therapeutic proton beams for HSG cells at Japanese proton therapy facilities

摘要

A human salivary gland tumour (HSG) cell line (JCRB1070: HSGc-C5, Japanese Collection of Research Bioresources Cell Bank) was used in this study. The cell line is a standard reference cell line of RBE used at the NIRS (National Institute of Radiological Sciences)-HIMAC in carbon beam research. The cells were subcultured according to the method described below and were stored in liquid nitrogen at a concentration of 1 × 106 cells/ml in the complete culture medium described below with 10% DMSO. The cells were subcultured in a bottle twice a week until a final concentration of 1 × 104 cells/cm2 was obtained and were then used for experiments within 20 passages after purchase from JCRB to obtain stable results. All proton 6-cm spread-out Bragg peak (SOBP) beams were generated from 180–235 MeV beams. High-energy LINAC X-rays at 4 or 6 MeV were used as the reference at each facility, and the energy depended on the specific instrumentation of each facility. The samples were placed at the isocenter of the SOBP beam, and the depth was adjusted to be in the middle position of the SOBP beam. The cells were then irradiated at room temperature. Dosimetry was performed according to the standard protocol by IAEA [6] for both proton and X-ray beams. Briefly, either Type 30001 Farmer Chamber or Type 23343 Markus Chamber (PTW, Freiburg) was placed at sample position. Monitor chambers placed upstream of the sample were calibrated by output from the above-stated chambers. Cell samples were irradiated using the preset values of the monitor chamber. A summary of the proton and X-ray beams for each facility is given in Table 1. Irradiated cells were kept in the dark at a low temperature until they were moved to the laboratory. The cells were rinsed twice with PBS(-) and trypsinised (0.2% trypsin in PBS(-)) to harvest them and were then resuspended in the culture medium. After the cell concentration was determined with a particle analyzer (Coulter counter), cells were diluted adequately and plated onto triplicate 6-cm plastic dishes, aiming for 100 colonies per dish for the cell survival assay. After 13 days' incubation in the CO₂ incubator, the colonies were rinsed with PBS(-) once, fixed with 10% formalin solution for 10–15 min, and stained with 1% methylene blue. Any colony consisting of 50 cells was counted under a stereomicroscope as a surviving colony.