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. NOT PROVIDED; REQUESTED

空间转录组学（Spatial transcriptomics）已被广泛用于捕获基因表达谱，这类表达谱表现为从组织切片中捕获的RNA的二维（2D）投影。三维（3D）培养物（如球体（spheroids）与类器官（organoids））是过于简化且同质化的二维细胞培养模型的极具前景的替代方案，但现有空间转录组平台的分辨率与RNA捕获效率均不足以实现三维培养物的可靠分析。我们提出了一种基于转染的细胞群体荧光DNA条形码标记方法，并通过逐层组装策略，使用携带条形码的细胞构建球状细胞结构。我们首次借助多重单细胞RNA测序技术，对该三维培养结构中的基因表达变化进行解析——其中DNA条形码可编码细胞的空间位置信息。研究证实，使用荧光团偶联的条形码寡核苷酸进行转染，可实现单细胞分辨率下的成像与测序，从而获取基因表达与药物反应的空间图谱。此外，我们发现荧光标记的DNA条形码可支撑定量微弹性成像（QME）等相关成像研究，同样可在空间分辨率下捕获三维培养物中的机械异质性信息。构建定制化空间编码细胞组装体的方法具备通用性，可解析三维细胞培养模型中基因表达的空间差异。NOT PROVIDED; REQUESTED