A Single-Cell Bioprinting Approach with Subcellular Resolution to Reconstruct Native Cellular Microenvironments and Interrogate Spatial Biology

Tissue development, function, and disease are largely driven by the spatial organization of individual cells and their interactions within a complex and heterogeneous cellular microenvironment. Engineered tissue models provide a controllable platform to dissect these spatially driven processes. However, strategies to fabricate tissues that replicate near-exact spatial organization and cellular composition of native microenvironments, necessary to faithfully recapitulate in vivo spatial relationships, have remained elusive to date. Here we present a precision bioprinting approach capable of generating high-fidelity replicas of patient biopsies that preserve complex cellular organization and heterogeneity with subcellular resolution. We demonstrate spatial placement of individual cells with down to ~3 µm spatial precision and up to eight cell types per bioprint. Using this approach, we demonstrate for the first time that altering the microscale spatial organization of tumor microenvironments of similar cellular composition can significantly alter transcriptomic programs of cell response. The ability to engineer and manipulate native cellular microenvironments with single-cell resolution marks a significant advancement toward understanding the critical influence of spatial biology on complex biological systems, such as the tumor microenvironment and organogenesis. Overall design: A combination of MCF10A, MCF7, and primary human fibroblasts cells were single-cell bioprinted onto a collagen coated glass microscope slide either in a patterened or randomly deposited arrangement. The cells were cultured in M87 media for 24 hours before being fixed with 4% PFA and permeabilized with 100% methanol. The Visium HD spatial transcriptomics protocol was then followed to generate the data deposited here. Complete protocol for sample preparation through library preparation can be found at: Helms HR and Bertassoni LE. Single-cell spatial transcriptomic profiling of cultured cells and engineered tissues without embedding or sectioning. STAR Protocols. 2025. DOI: 10.1016/j.xpro.2025.104074