# Development of an Artificial 3D Liver Phantom for Analysis of Radiotherapeutic Effects In Vitro
## 4 Discussion  
The liver phantom proposed in this study enables 3D analysis and evaluation of the in vitro response to irradiation in an environment that closely resembles the relevant spatial organization and tissue environment in the human organ, as well as a relevant hypoxic tumor microenvironment. This was achieved by using a 1% agarose mixture, which adopts the attenuation coefficient of liver tissue. Additionally, agarose has been used before in phantoms for radiotherapy to simulate soft tissue equivalent absorption and scattering properties [13; 14; 15; 16]. Furthermore, growing cells in MCs resulted in hypoxic conditions, and thus provided a close simulation of the biological conditions of an anthropomorphic tumor environment comparable with the real HCC microenvironment. Of course, there are some limitations in this study that could be addressed in future research. First, in this study, only one cell line from hepatoblastoma was used. We selected HepG2 cells, which have been widely used as a model for HCC and hepatocyte-specific metabolic properties. Gene expression and proteomics studies show HCC-like expression patterns [17; 18] and hepatocyte-like metabolism when grown in MCs [11]. Furthermore, other studies used this hepatic cell line in hypoxia studies, demonstrating comparable hypoxic effects, as in other HCC cell lines [19; 20]. Depletion of oxygen concentration was measured in the MCs by showing \(\mathrm{Hf1}\alpha\) nuclear translocation. In future studies, advanced MCs could be used that enable oxygen measurement within the chip to provide more detailed information on the growth conditions in the MCs. Nevertheless, our experiments clearly showed the toxic effect of radiation on HepG2 cells, as previously published [21]. Indeed, as expected, irradiation of the liver phantom increased \(\gamma\)H2 AX, a marker of DNA damage, in HepG2 cells (MC #3). Additionally, cells in the MCs above the target zone (MCs #2 and #6) had significantly increased \(\gamma\)H2 AX expression rates. Interestingly, the MCs directly below the irradiated MC #3 (MCs #4 and #5) were less affected by the irradiation (Figure 4E). These results can be explained by the setup of the CyberKnife. The robotic arm with the irradiation source of the device can apply radiation from every angle. As the target was located further to the cranial side of the liver, radiation from the caudal side was reduced to spare tissue below the target. This also explains why \(\gamma\)H2 AX expression in MC #1, which is two MCs away from the target, was also significantly increased. Furthermore, it elucidates the high level of detected irradiation on MC #6, which is positioned at the cranial site in the posterior half of the liver (Figure 4E). When comparing these results with the 2D in vitro model, not only was DNA damage at the isocenter of the beam significantly reduced in the 3D model but also the radiation dose around the isocenter was significantly increased (Figure 4E). These findings emphasize the need for appropriate 3D anthropomorphic models for radiation intervention research and dose validation in preclinical models. This is especially necessary because single-shot radiotherapy will set a new standard of treatment that requires robust and reproducible verification. Furthermore, this study is of interest for in vitro research in the field of radiotherapy in order to not solely depend on Monte Carlo simulation, but actually introduce biological, living material in an anthropomorphic organ phantom. Although simple radiation understanding can be still achieved by using 2D cell culture, this method could ensure that the planned dose reaches the desired clinical outcome and spare healthy parts of the organ. Consequently, the next step would be to individualize this method by 3 Dprinting organs according to the patient's CT, and to introduce biopsy-derived samples of the patient in the phantom to analyze the individual, patient-specific outcome after irradiation.