# A High-Throughput In Vitro Radiobiology Platform for Megavoltage Photon Linear Accelerator Studies
## 3 Results
### Dosimetric Verification of Accurate Delivery of Tissue Culture Plate IMRT Plans  
Radiochromic film dose distribution analyses showed good concordance between the predicted and the measured radiation dose profiles across both X and Y axes for target doses of 2, 4, 8, and 16 Gy for all three tissue culture plates IMRT plans. However, at 24 Gy, measured radiation doses were lower than the predicted doses for all three IMRT plans, and this effect was likely due to the saturation of radiochromic films at this dose level. The 2D ion chamber array analyses showed good matching between the predicted and measured doses. Gamma index analysis was used to evaluate the percentage of agreement for pre-set dose and distance differences [13]. EPID analysis confirmed accurate delivery of all IMRT plans with gamma index pass rates of 99.5%, 93.1%, and 89.1% (pre-set tolerance of 3% for dose and 1% for distance differences) for 96-, 24-, and 6-well plate IMRT plans, respectively (Figure 3). No Significant Differences in Cell Viability Signal, Clongogenic Potential, or \(\gamma\)-H2 AX Foci Signal Levels between IMRT and Open-Field, Homogeneous Irradiations  
For the A375 cell line, cell viability signal for 0, 2, 4, 8, 16, and 24 Gy with the IMRT plan were 2.08, 1.75, 1.42, 0.76, 0.55, and 0.55, respectively. With open-field, homogeneous irradiations, the corresponding values were 2.08, 1.65, 1.18, 0.65, 0.52, and 0.5. The differences between the techniques were not statistically significant (_p_ = 0.82; Figure 4A). The corresponding values for the LN18 cell line were 2.01, 1.7, 1.39, 0.77, 0.27, 0.25, and 1.91; and 1.65, 1.33, 0.66, 0.35, and 0.37, respectively. Again, these differences did not reach statistical significance (_p_ = 0.95; Figure 4B). \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \multicolumn{8}{c}{**96-Well Plate IMRT Plan**} & \multicolumn{4}{c}{**24-Well Plate IMRT Plan**} & \multicolumn{4}{c}{**6-Well Plate IMRT Plan**} \\ \hline Target & Mean & Maximum & Minimum & Mean & Maximum & Minimum & Mean & Maximum & Minimum \\ dose & dose & dose & dose & dose & dose & dose & dose & dose & dose \\
0 & 0.27 & 0.46 & 0.16 & 0.42 & 1.76 & 0.13 & 0.47 & 2.85 & 0.14 \\
2 & 2.41 & 2.7 & 2.07 & 2.4 & 3.59 & 1.77 & 2.41 & 3.6 & 2 \\
4 & 4.38 & 4.97 & 3.93 & 4.48 & 6.82 & 3.64 & 4.56 & 8.62 & 3.4 \\
8 & 8.57 & 9.84 & 7.76 & 8.71 & 12.27 & 7.41 & 8.74 & 14.14 & 6.84 \\
16 & 16.96 & 18.29 & 15.52 & 17.37 & 20.28 & 15.16 & 17.34 & 18.66 & 14.3 \\
24 & 25.2 & 27.11 & 22.74 & 25.54 & 27.59 & 21.46 & 26.08 & 27.72 & 22.86 \\ \hline \end{tabular}
\end{table} 
Table 1: Radiation dose statistics of multiwell tissue culture plate IMRT plans (radiation doses in Gy). Figure 3: _Cont._  
Figure 3: Cont. Figure 3: Dose distribution analyses of multiwell tissue culture plate IMRT plans. Screen shot images of radiochromic film (**A**); red line shows planned and green line shows predicted radiation doses across the X-axis), 2D ion chamber array (**B**); planned doses shown by orange line and measured doses by points), and EPID analyses (**C**); orange line shows predicted doses from the plan and green line shows measured doses). The clonogenic assays showed that the A375 cell line colony numbers for 0, 2, 4, 8, 16 and 24 Gy were 208, 141, 74, 0, 0, and 0, respectively, and for the open-field homogeneous irradiations, they were 194, 143, 55, 0, 0, and 0, respectively. These differences in colony numbers between the two techniques did not reach statistical significance (\(p\) = 0.92; Figure 5A,B). Figure 4: (**A**) Comparison of A375 cell viability signal between 96-well tissue culture plate IMRT and open-field, homogeneous irradiations (\(p\) = 0.82). (**B**) Comparison of LN18 cell viability signal between 96-well tissue culture plate IMRT and open-field, homogeneous irradiations (\(p\) = 0.95). Results are reported as the average from four independent biological experiments. Cell viability signal measured at 490 nm. Error bars indicate standard deviation.