Gradient to sectioning CUBE workflow for the generation and imaging of organoids with localized differentiation.

Advancements in organoid culture have led to various in vitro mini-organs that mimic native tissues in many ways. Yet, the bottleneck remains to generate complex organoids with body axis patterning, as well as keeping the orientation of organoids during post-experiment analysis processes. Here, we present a workflow for culturing organoids with morphogen gradient using a CUBE culture device, followed by sectioning samples with the CUBE to retain information on gradient direction. We show that hiPSC spheroids cultured with two separated differentiation media on opposing ends of the CUBE resulted in localized expressions of the respective differentiation markers, in contrast to homogeneous distribution of markers in controls. We also describe the processes for cryo and paraffin sectioning of spheroids in CUBE to retain gradient orientation information. This workflow from gradient culture to sectioning with CUBE can provide researchers with a convenient tool to generate increasingly complex organoids and study their developmental processes in vitro.