Budget-Friendly Generation, Biochemical Analyses, and Lentiviral Transduction of Patient-Derived Colon Organoids

For the past decade, three-dimensional (3D) culture models have been emerging as powerful tools in translational research to overcome the limitations of two-dimensional cell culture models. Thanks to their ability to recapitulate the phenotypic and molecular heterogeneity found in numerous organs, organoids have been used to model a broad range of tumors, such as colorectal cancer. Several approaches to generate organoids exist, with protocols using either pluripotent stem cells, embryonic stem cells, or organ-restricted adult stem cells found in primary tissues, such as surgical resections as starting material. The latter, so-called patient-derived organoids (PDOs), have shown their robustness in predicting patient drug responses compared to other models. Because of their origin, PDOs are natural offspring of the patient tumor or healthy surrounding tissue, and therefore, have been increasingly used to develop targeted drugs and personalized therapies. Here, we present a new protocol to generate patient-derived colon organoids (PDCOs) from tumor and healthy tissue biopsies. We emphasize budget-friendly and reproducible techniques, which are often limiting factors in this line of research that restrict the development of this 3D-culture model to a small number of laboratories worldwide. Accordingly, we describe efficient and cost-effective techniques to achieve immunoblot and high-resolution microscopy on PDCOs. Finally, a novel strategy of lentiviral transduction of PDCOs, which could be applied to all organoid models, is detailed in this article. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of PDCOs from biopsies Basic Protocol 2: Long-term maintenance and expansion of PDCOs in BME domes Basic Protocol 3: Cryopreservation and thawing of PDCOs Basic Protocol 4: Lentiviral transduction of PDCOs Basic Protocol 5: Immunoblot and evaluation of variability between donors Basic Protocol 6: Immunofluorescence labeling and high-resolution microscopy of PDCOs Basic Protocol 7: Transcriptomic analyses of PDCOs by RT-qPCR.