A photolithographic method to create cellular micropatterns

Here we describe a simple and rapid system for creation of patterned cell culture substrates. This technique is based on (1) printing a mask on a standard overhead transparency, (2) coating a thin layer of a photocrosslinkable chitosan on a slide, (3) exposing the slide and mask to ultraviolet (UV) light, and (4) rinsing the uncrosslinked polymer to expose the underlying cell-repellent patterns. Photocross-linkable chitosan does not require photoinitiators, it is non-toxic and forms flexible, biocompatible hydrogel upon short (∼min) UV exposure. Patterns of various shapes (lanes, squares, triangles, circles) were created on two surfaces commonly used for cell culture: glass and tissue culture polystyrene. The pattern size could be varied with a μm resolution using a single mask and varying UV exposure time. Cardiac fibroblasts formed stable patterns for up to 18 days in culture. Cardiomyocytes, patterned in lanes 68–99μm wide, exhibited expression of cardiac Troponin I, well developed contractile apparatus and they contracted synchronously in response to electrical field stimulation. Osteoblasts (SAOS-2) localized in the exposed glass regions (squares, triangles, or circles; 0.063–0.5mm2). They proliferated to confluence in 5 days, expressed alkaline phosphatase and produced a mineralized matrix.