Impact of various irradiation conditions on delivered dose and cell viability for in vitro cell irradiation

Introduction: To correlate dose from ionizing radiation with cell kill probability, viability and clonogenic assays are often performed following in vitro irradiation of cells in 96-well culture plates. The objective of this work was to examine how dose to adherent cells in 96-well culture plates depended on the conditions in which the irradiations were performed. The aim was to provide support for standardized irradiation setup conditions so that in vitro cell assays can be more precisely compared. Materials and Methods: 96-well culture plates were exposed to a 6 MV photon beam from a linear accelerator. Dose measurements were performed by securing a piece of EBT3 Gafchromic film to the underside of each plate. Each well contained either 200 μl of tap water or canine osteosarcoma cells (OSCA 40) in 200 μl of media. Exposures were performed to assess the dependence of dose to the depth of measurement, the thickness of the backscatter material, the field size, and the position of a plate within the treatment field. Results: Culture plate doses demonstrated a strong dependence on backscatter material thickness and field size. Providing less than 0.5 cm of water equivalent backscatter material resulted in a mean measured underdose of more than 3% for a 30 × 30 cm² field. Decreasing the field size to 13 × 9 cm² yielded a mean measured dose within 0.5% of the prescribed dose. At the central-Axis depth where maximum dose occurs (d_max = 1.5 cm), irradiation of four plates simultaneously resulted in a mean measured dose 3.5% higher than measurements of single plates placed in the center of the field. Conclusions: Inadequate consideration of in vitro irradiation setup conditions is likely to result in discrepancies between prescribed and delivered doses. Such discrepancies can be minimized by providing 5 cm of backscatter material and 10 cm of buildup material; and by exposing one centrally located plate at a time.