# Development of an Artificial 3D Liver Phantom for Analysis of Radiotherapeutic Effects In Vitro
###### Abstract  
Over recent decades, stereotactic body radiotherapy has garnered increasing popularity. Unfortunately, conventional preclinical 2D in vitro models are often insufficient for studying radiotherapy effects. Therefore, in this study, we developed a novel anthropomorphic in vitro liver phantom, which simulates the relevant hepatocellular carcinoma (HCC) tumor microenvironment and spatial organization. The liver phantom was 3D printed, filled with tissue-mimicking agarose mixture, and designed to fit ten microfluidic chips (MCs), in which HepG2 cells were seeded. Airtight MCs induced hypoxic conditions, as verified by HiFi \(\alpha\) staining. Irradiation was conducted with 20 Gy in one fraction using a CyberKnife, in either a 2D setup, or by irradiating MCs arranged in the 3D-printed liver model using an individually calculated treatment plan. Post-irradiation cellular damage was determined via \(\gamma\)H2 AX staining. Here, we demonstrate a new physiologically relevant approach to model HCC pathology following radiotherapy. Comparing \(\gamma\)H2 AX staining in normoxic conditions to cells grown in MCs (hypoxic conditions) revealed a reduction in cellular damage of 30.24% (\(p=0.0001\)) in the hypoxic environment. Moreover, we compared the scattering effect of radiation on a conventional 2D in vitro model to our new 3D anthropomorphic liver phantom and observed a significant \(\gamma\)H2 AX intensity reduction of 9.6% (\(p=0.0294\)) in HepG2 cells irradiated in the phantom. Our approach of utilizing a liver phantom takes into account the hypoxic tumor microenvironment and 3D scattering effects of tissue irradiation, thereby modeling both physical and biological parameters of HCC tumors. The use of tissue phantoms lays the groundwork for future examination of other hypoxic tumors and offers a more comprehensive approach for screening and analysis of novel cancer therapeutics.  
15 September 2022 23 October 2022 23 October  
At present, in vitro 2D cell cultures are utilized preclinically to investigate new irradiation methods and analyze the effects of irradiation on tumors [7]. Although these methods provide important information on basic irradiation schemes and biological cell responses, they often fail to recapitulate complex scenarios present in vivo [8]. For example, one major limitation of 2D HCC tumor cell cultures is that cancer cells are grown in the presence of oxygen (i.e., a normoxic environment), while a hypoxic environment is characteristic for HCC in vivo. Moreover, oxygen deprivation in the microenvironment of a tumor leads to increased radioresistance and, therefore, a phenomenon which cannot be properly addressed in current 2D models [9]. Finally, human organs are 3D structures which do not resemble the 2D flat monolayer cell culture. Therefore, 2D cell cultures are solely irradiated at a 90\({}^{\circ}\) angle, without the calculation of an individualized 3D irradiation plan which is necessary for patients. In other words, 3D structures are normally irradiated from different angles utilizing a specific irradiation plan, which is not possible to simulate in 2D cultures [10].  
To overcome these obstacles, in this study, we designed an anthropomorphic liver phantom to simulate hypoxic and positioning effects of cells during high-dose radiotherapy. HepG2 cells were introduced into microfluidic chips (MCs) and completely sealed to reach hypoxic conditions. Furthermore, these chips were placed in the liver model to resemble the different positions of cells in the human liver. The aim was to show that the outcome of irradiation effects significantly changes if cells are irradiated in a 2D cell culture rather than in a 3D-printed phantom under hypoxic conditions.