Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells

Biopatterning, which enables regulation of cell–material interaction, is usually achieved by techniques that rely on physical contact, which can seriously damage cells. A simple and efficient non-contact technique is now demonstrated using an aqueous two-phase polymer system. Microscale biopatterning enables 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 carried out 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 enable 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 that enables patterning nanolitres of a reagent-containing aqueous phase, in arbitrary shapes, within a second aqueous phase covering a cell monolayer. With the appropriate medium formulation, reagents of interest remain confined to the patterned phase without significant diffusion. The fully aqueous environment ensures high reagent activity and cell viability. The utility of this strategy is demonstrated with patterned delivery of genetic materials to mammalian cells for phenotypic screening of gene expression and gene silencing.