A novel high-throughput irradiator forin vitroradiation sensitivity bioassays

Abstract

This paper describes the development and characterization of a fully automated in vitro cell irradiator using an electronic brachytherapy source to perform radiation sensitivity bioassays. This novel irradiator allows complex variable dose and dose rate schemes to be delivered to multiple wells of 96-well culture plates used in standard biological assays. The Xoft Axxent® eBx™ was chosen as the x-ray source due to its ability to vary tube current up to 300 μA for a 50 kVp spectrum using clinical surface applicators. Translation of the multiwell plate across the fixed radiation field is achieved using a precision motor driven computer controlled positioning system. A series of measurements were performed to characterize dosimetric performance of the system. Measurements have shown that the radiation output measured with an end window ionization chamber is stable between operating currents of 50 μA to 300 μA. In addition, radiochromic film was used to characterize the field flatness and symmetry. The average field flatness in the in-plane and cross-plane direction was 2.9 ±1.0% and 4.0 ±1.7%, respectively. The average symmetry in the in-plane and cross-plane direction was 1.8 ±0.9% and 1.6 ±0.5%, respectively. The optimal focal spot resolution at the cellular plane was determined by measuring sequential irradiations on radiochromic film for three different well spacing schemes. It was determined that the current system can irradiate every-other well with negligible impact on the radiation field characteristics. Finally, a performance comparison between this system and a common cabinet irradiator is presented.

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

“…We thus examined if AXL inhibition could augment the sensitivity of these cell lines to radiotherapy. Using a high-throughput x-ray radiation system that delivers the same absorbed dose of ionizing radiation to cells plated in a 96-well format (development and characterization (33)), SCC1, SCC47, SCC147T, and SCC2 were irradiated with 4 Gy after 24 hour pre-treatment with either vehicle or R428 (1 uM). Then, the induction of DNA double strand breaks (DSBs) was examined via gamma-H2AX (γ-H2AX), which is phosphorylated and recruited to sites of DNA damage in response to radiation (Figure 4A).…”

“…A novel high-throughput irradiator utilizing a 50 kVp x-ray beam spectrum was used to deliver 4 gray (Gy) as previously described (33, 34). Cells were fixed in 4% paraformaldehyde 4 hours later, permeabilized in 90% methanol, blocked, and incubated with γ-H2AX primary antibody (1:500) overnight.…”

Biological characterization of a novel in vitro cell irradiator

“…We have previously described the detailed physical dosimetry and specifications of our irradiator [ 3 ]. We used our previously described 96 well plate phantom to assess the contribution of scatter radiation when using the 20-mm surface applicator.…”

“…Over the last several years, we have developed a micro-irradiator capable of delivering multiple dose and dose-rates within a single cell-culture microplate for use in high-throughput assays. We have previously described the physical characteristics of this system [ 3 ]. The design of any high throughput technology for in vitro biology research must limit the impact of mechanical and environmental stresses on the cells being tested.…”

High-Throughput Detection of DNA Double-Strand Breaks Using Image Cytometry

“…Cells are first cultured in flat-bottom 96-well plates and differentially irradiated in groups of 4 wells (for sample replicates) using a high-throughput variable dose-rate irradiator we described previously (6). Following radiation, cells are either immediately assayed to assess maximal γH2AX response or assayed at later time points to assess DNA repair kinetics.…”