In this study, adult human ileum-derived organoids were used as a demonstration of the SPACECAT spatial omics technology. Organoids were cultured and passaged every 6-8 days in Matrigel domes with established media conditions meant to recapitulate the broad diversity of in vivo epithelial cell types (Fujii, M., et al., Cell Stem Cell. 2018). Organoid culture media contained recombinant Noggin, Rspondin-3, FGF2, IGF1, afamin-Wnt3A, in addition to Gastrin and TGF-b inhibitor A83-01 with and without recombinant EGF (E/NR3+F2I1Gi+Af-W3+A83). In the SPACECAT protocol, organoids were stained with photocaged fluorescent dyes (i.e., non-fluorescent due to the photocaging NVOC group). We targeted organoid crypts with spatially-controlled 405 nm illumination to uncage the dye and fluorescently stain specific cells. Organoids were then dissociated and sorted via flow cytometry to enrich for dye-positive cells and compare them to the rest of the organoid as well as a larger reference dataset.

Single cell RNA-seq was performed via Seq-Well S^3 on photoactivated samples, non-photoactivated control samples, and reference samples for increased sample size. Analysis was performed in Seurat and manually annotated based on marker gene expression.

Samples from this study were collected in collaboration with Vanessa Mitsialis, MD and Scott Snapper, MD PhD, Boston Children’s Hospital.