Microscale biopatterning allows regulation of cell-material interactions1,2 and cell shape3, and enables multiplexed high throughput studies4,5,6,7,8 in a cell and reagent efficient manner. The majority of available techniques rely on physical contact of a stamp3, pin8, or mask9,10 with mainly a dry surface. Inkjet and piezoelectric printing11 is performed in a non-contact manner but still requires a substantially dry substrate to ensure fidelity of printed patterns. These existing methods, therefore, are limited for patterning onto delicate surfaces of living cells because physical contact or substantially dry conditions are damaging to them. Microfluidic patterning with laminar streams12,13 does allow non-contact patterning in fully aqueous environments but with limited throughput and reagent diffusion across interfacial flows. Here, we describe a polymeric aqueous two-phase system (ATPS) that enables patterning nanoliters of a reagent-containing aqueous phase, in arbitrary shapes, within a second aqueous phase covering a cell monolayer. With the appropriate media formulation, reagents of interest remain confined to the patterned phase without significant diffusion. The fully aqueous environment ensures high reagent activity and cell viability. Utility of this strategy is demonstrated with patterned delivery of genetic materials to mammalian cells for phenotypic screening of gene expression and gene silencing.

An optimized polymeric aqueous two‐phase system allows direct and non‐contact printing of cells onto a monolayer of living cells in arbitrary shapes as well as in a high‐density microarray format to create heterocellular microenvironments and study the effect of direct cell–cell interactions on cell fate. The entire process is performed in aqueous media to support full cell viability and functionality.