Stereomask lithography (SML): a universal multi-object micro-patterning technique for biological applications.

The advent of biological micro-patterning techniques has given new impetus to many areas of biological research, including quantitative biochemical analysis, tissue engineering, biosensing, and regenerative medicine. Derived from photolithography or soft lithography, current bio-patterning approaches have yet to completely address the needs of out-of-cleanroom, universal applicability, high feature resolution, as well as multi-object placement, though many have shown great promise to precisely pattern one specific biomaterial. In this paper, we present a novel versatile biological lithography technique to achieve integrated multi-object patterning with high feature resolution and high adaptability to various biomaterials, referred to as stereomask lithography (SML). Successive patterning of multiple objects is enabled by using unique three-dimensional masks (i.e., the stereomasks), which lay out current micropatterns while protecting pre-existing biological features on the substrate. Furthermore, high-precision reversible alignment among multiple bio-objects is achieved by adopting a peg-in-hole design between the substrate and stereomasks. We demonstrate that the SML technique is capable of constructing a complex biological microenvironment with various bio-functional components at the single-cell resolution, which to the best of our knowledge has not been realized before.