From Gradient to Sectioning in CUBE: Workflow for Generating and Imaging Organoid with Localized Differentiation

Advancements in organoid culture methods have led to a widespread availability of 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 organoid samples during post-experiment analysis processes. In this paper, we present a workflow for culturing organoids with gradient using a previously developed CUBE culture device, then sectioning samples with the CUBE to retain information on the gradient direction of the sample. We show that hiPSC spheroids cultured with separate differentiation media on two opposing ends of the CUBE resulted in localized expressions of the respective differentiation markers, in contrast to homogeneous distribution of markers in controls with the same media on both ends. We also describe the processes for cryo and paraffin sectioning of gradient-cultured 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 .