A Single-Cell Bioprinting Method to Reconstruct Native Cellular Microenvironments with Subcellular Resolution

Abstract

Tissue development and function are determined by the spatial organization of individual cells and their interactions. Yet experimental platforms capable of reconstructing cellular organization with single-cell precision for systematic interrogation remain lacking. Here we introduce a single-cell bioprinting platform that enables programmable reconstruction of cellular microenvironments through controlled single-cell placement with down to subcellular spatial control. We demonstrate intercellular spacing down to 1.3 {micro}m, multiplexed deposition of up to eight cell types within a single construct, and reconstruction of biopsy-derived native cellular organization with 99% accuracy for single-cell placement relative to their native tissue coordinates. Using this framework, we show that manipulation of cellular arrangement allows for controlled interrogation of spatial transcriptional programs, cell-cell signaling networks, and migration dynamics in complex tissue microenvironments. This platform provides a generalizable experimental framework for causal interrogation of spatially defined cell-cell interactions at single-cell resolution.