Radiotherapy is a commonly employed treatment for colorectal cancer, yet its radiotoxicity-related impact on healthy tissues raises significant health concerns. This highlights the need to use radioprotective agents to mitigate these side effects. This review presents the current landscape of human translational radiobiology, outlining the limitations of existing models and proposing engineering solutions. We delve into radiotherapy principles, encompassing mechanisms of radiation-induced cell death and its influence on normal and cancerous colorectal cells. Furthermore, we explore the engineering aspects of microphysiological systems to represent radiotherapy-induced gastrointestinal toxicity and how to include the gut microbiota to study its role in treatment failure and success. This review ultimately highlights the main challenges and future pathways in translational research for pelvic radiotherapy-induced toxicity. This is achieved by developing a humanized in vitro model that mimics radiotherapy treatment conditions. An in vitro model should provide in-depth analyses of host-gut microbiota interactions and a deeper understanding of the underlying biological mechanisms of radioprotective food supplements. Additionally, it would be of great value if these models could produce high-throughput data using patient-derived samples to address the lack of human representability to complete clinical trials and improve patients’ quality of life.

Patient-derived organoids offer greater human translational relevance than mouse models or immortalized cell lines, owing to their multicellular composition and patient-specific responses. Here, we present a protocol for establishing a human in vitro rectal tumor model for radiotherapy studies using patient-derived tumor organoids (PDTOs) and monolayer derivatives integrated into a microphysiological system (MPS). We describe steps for cultivating PDTOs, their transition into monolayers, and transfer into an MPS. This protocol enables radiotherapy modelling, toxicity testing, and adjuvant therapies screening.