Polymer-mediated oligo delivery enables construction of spatially encoded 3D cultures for analysis by single-cell RNA sequencing

Spatial transcriptomics has been widely used to capture gene expression profiles, which are realised as a two-dimensional (2D) projection of RNA captured from tissue sections. Three-dimensional (3D) cultures such as spheroids and organoids are highly promising alternatives to overly simplistic and homogeneous 2D cell culture models, but existing spatial transcriptomic platforms do not have sufficient resolution and RNA capture efficiency for robust analysis of 3D cultures. We present a transfection-based method for fluorescent DNA barcoding of cell populations, and the subsequent construction of spheroidal cellular architectures using barcoded cells in a layer-by-layer approach. For the first time, changes in gene expression throughout this 3D culture architecture are interrogated using multiplex single-cell RNA sequencing in which DNA barcodes encode the spatial positioning of cells. We show that transfection with fluorophore-conjugated barcode oligonucleotides enables both imaging and sequencing at single-cell resolution, providing spatial maps of gene expression and drug response. Additionally, we show that fluorescently-tagged DNA barcodes support correlative imaging studies such as quantitative microelastography (QME) to capture information about mechanical heterogeneity in 3D cultures, also with spatial resolution. The ability to create customised, spatially encoded cellular assemblies is a general approach that can resolve spatial differences in gene expression in 3D cell culture models.