Bioengineering mini-colons for ex vivo colorectal cancer research.

Tumor initiation remains one of the least understood events in cancer biology, largely due to the challenge of dissecting the intricacy of the tumorigenic process in laboratory settings. The insufficient biological complexity of conventional in vitro systems makes animal models the primary experimental approach to study tumorigenesis. Despite providing valuable insights, these in vivo models function as experimental black boxes with limited spatiotemporal resolution of cellular dynamics during oncogenesis. In addition, their use raises ethical concerns, further underscoring the need for alternative ex vivo systems. Here we provide a detailed protocol to integrate state-of-the-art microfabrication, tissue engineering and optogenetic approaches to generate topobiologically complex miniature colons ('mini-colons') capable of undergoing tumorigenesis in vitro. We describe the key methodology for the generation of blue light-inducible oncogenic cells, the establishment of hydrogel-based mini-colon scaffolds within microfluidic devices, the development of mini-colons and the induction of spatiotemporally controlled tumorigenesis. This protocol enables the formation and long-term culture of complex cancerous tissues that capture in vivo-like tumoral biology while offering real-time and single-cell resolution analyses. It can be implemented in 4-6 weeks by researchers with prior experience in 3D cell culture techniques. We anticipate that these methodological guidelines will have a broad impact on the cancer research community by opening new avenues for tumorigenesis studies.