Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments

Click chemistry provides extremely selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions, the most common example being the copper(I)-catalysed reaction of azides with alkynes(1,2). While the versatility of click reactions has been broadly exploited(3-5), a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches into biological applications. This manuscript introduces a robust synthetic strategy where macromolecular precursors react through a copper-free click chemistry(6), allowing for the direct encapsulation of cells within click hydrogels for the first time. Subsequently, an orthogonal thiol-ene photocoupling chemistry is introduced that enables patterning of biological functionalities within the gel in real time and with micrometre-scale resolution. This material system enables us to tailor independently the biophysical and biochemical properties of the cell culture microenvironments in situ. This synthetic approach uniquely allows for the direct fabrication of biologically functionalized gels with ideal structures that can be photopatterned, and all in the presence of cells.