Single-cell patterning is the ability to precisely position individual cells in spatially defined locations. In contrast to conventional cell culture techniques which typically involve culturing millions of cells together at random, single-cell patterning considers the cellular heterogeneity and provides more statistical power. This technology has the potential to revolutionize biomedical research and applications by enabling researchers to study individual cells and their interactions with their microenvironment in more detail, create complex tissue and organ models that more accurately mimic the structure and function of real tissues and organs, and develop new diagnostic and therapeutic approaches. A crucial step in cell patterning is to regulate cell adhesion on a substrate by adapting several physical and biochemical strategies for substrate modification and patterning.

In the current work we present a high-throughput single-cell patterning method with 97-99% efficiency that is universal to all cell types, using microcontact printing. We created a micro-pillar stamp made of polydimethylsiloxane (PDMS) with different pillar diameters (40-100 μm) and a patterning area of 1 cm x 1 cm. These stamps were utilized to imprint distinct proteins onto a surface, and then pattern single cells to small clusters of cells on the surface, depending on the pillar diameter. This single-cell patterned platform is further utilized for massively parallel optoporation-mediated intracellular delivery of small to very large biomolecules such as PI dye (668 Da), Dextran (3000 MW), SiRNA (20-24 bp), and enzyme (464 kDa). The near infrared mediated optoporation process is facilitated by a two-dimensional array of titanium micro-dish (TMD) device that is simple to setup and operate, allowing biomolecules to enter cells with high efficiency (96%) and cell viability (98%) by disrupting the cell plasma membrane. Overall, this platform is compact, robust, and easy to use and has the potential to be used for a variety of applications, including single-cell analysis, drug discovery, and regenerative medicine.