# Development of an Artificial 3D Liver Phantom for Analysis of Radiotherapeutic Effects In Vitro
## 3 Results
### Analysis of 2D and 3D Radiotherapy on HepG2 Cells  
Two- and three-dimensional irradiation strategies were previously described above. Briefly, in the 2D model, four MCs were positioned next to each other and irradiated at a 90\({}^{\circ}\) angle, whereas in the 3D model, 10 MCs were strategically arranged in the anthropomorphic liver phantom (Figure 4A,B). An irradiation treatment plan of the 3D liver phantom which targets MC #3 was developed and can be visualized in Figure 4C and Supplementary Figure S1C.  
Figure 3: Fluorescence images of cells seeded in MCs, DAPI visualized in blue and \(\gamma\)H2 AX in red (**A**–**D**). \(\gamma\)H2 AX positive cells in control cells (**A**) and cells irradiated with 5 Gy (**B**), 10 Gy (**C**), and 20 Gy (**D**) merged with DAPI staining. Time lapse from 10 min to 24 h after irradiation of \(\gamma\)H2 AX intensity in cells irradiated with 5 Gy (**E**), 10 Gy (**F**), and 20 Gy (**G**). Comparison of \(\gamma\)H2 AX positive cell count seeded in MCs and 48-well plates after irradiation with 5, 10, and 20 Gy (**H**). Fluorescence images were acquired with a 10\(\times\) objective; the white scale bar corresponds to 100 \(\upmu\)m. Statistics were performed with one-way ANOVA (**E**–**G**) and two-way ANOVA (**H**) using Bonferroni for post-hoc test. \(n=5\), *** \(p<0.001\), ******* \(p<0.0001\).  
Figure 4: A 3D phantom with MCs #1 to #5 positioned in the anterior half of the liver (**A**), and MCs #6 to #10 located in the posterior half of the liver (**B**). Yellow circle indicates target position of the treatment plan (MC #3) (**A**). Irradiation plan from Monte Carlo simulation from the coronal plane with purple = 0.14 Gy, green = 2.86 Gy, and pink = 20 Gy (**C**). Measured \(\gamma\)H2 AX percentage of cells irradiated with 20 Gy in a 2D irradiation setup for 5 MCs with 4 MCs located at varying distances from the isocenter (0/orange bar) (**D**). Measured \(\gamma\)H2 AX percentage of cells for MCs #1-#10 in the 3D model irradiated at MC #3 (yellow bar/arrow) with 20 Gy (**E**). HepG2 cells irradiated in the 2D setup (20 Gy) were compared with cells irradiated in MC #3 in the phantom (20 Gy) (**F**). Representative images of non-irradiated control (**G**), MC #1 (**H**), #2 (**I**), and #3 (**J**) are presented with 10\(\times\) magnification. White scale bar corresponds to 100 \(\mu\)m. Significance was tested with a two-way ANOVA and Bonferroni post hoc test (**H**,**I**) and an unpaired \(t\)-test (**G**). To enhance readability, only significance compared to control is shown. \(n\) = 5, * \(p\) < 0.05, *** \(p\) < 0.001, *** \(p\) < 0.0001.  
controls (Figure 4E). Indeed, the \(\gamma\)H2 AX response in MC #3 increased 36.61% \(\pm\) 8.66% (\(p\) < 0.0001) (Figure 4E,J) compared to non-irradiated cells (Figure 4G). Additionally, a significant increase in the \(\gamma\)H2 AX-positive cell fraction was found cranial from the isocenter, with 24.38% \(\pm\) 7.20% (\(p\) < 0.0001) (MC #2, Figure 4E,I) and 15.25% \(\pm\) 2.57% (\(p\) < 0.0001) (MC #6). Additionally, in MC #1, 20 mm apart from the isocenter, a significant increase in \(\gamma\)H2 AX-positive cells was found (\(p\) = 0.0009, Figure 4E,H). However, cells caudal from the isocenter did not show an increased \(\gamma\)H2 AX expression (Figure 4E, MCs #7-#10). Comparing the irradiation effect in target MC #3 (36.61% \(\pm\) 8.66%) with the cell response in the 2D model (46.21% \(\pm\) 8.38%), DNA damage in MC #3 showed a significantly reduced \(\gamma\)H2 AX positive cell count of 9.6% (\(p\) = 0.0294) (Figure 4F). Therefore, we show that while the absolute estimated dose in the target region was significantly decreased in our 3D model, the dose area in the 2D model was greatly increased. Taken together, this model illustrates significant discrepancies between 2D and 3D model systems, which is an important step towards a better understanding of HCC response to radiotherapy treatment.