Multiphoton 3D microprinting of protein microstructures and micropatterns for cell niche studies

Abstract

In native tissues, cells reside in a specialized niche, sustaining their lifelong maintenance and determining their pattern of differentiation. The major constituents of cell niche include neighboring cells, soluble factors, extracellular matrix, topological features and mechanical information. These cell niche factors interact among one another simultaneously and present important signals to living cells. Reconstituting a cell niche in a highly regulated manner in vitro is critical for understanding how cells sense and interact with their niches and rationalizing scaffold and protein chip design for future applications. We have established a multiphoton photochemical crosslinking-based platform technology, which is an emerging 3D printing-based biofabrication technology with numerous advantages such as submicron resolution, non-heating and non-contact procedure, free-form fabrication and spatial control ability. The non-contact laser-based crosslinking makes it possible to functionalize the surface of extremely soft 3D microstructures at selected locations with specific densities without damaging their topological features. Here, the ability of the two-photon microfabrication platform in fabricating complex protein micropatterns with precisely controlled voxels, topological structures and porosity, mechanical properties, extracellular matrix niche factors, the ability to decouple the mechanical and matrix niche factors and the recent effort to fabricate single cell 3D microniche will be reviewed. Moreover, a number of example applications of the protein microstructures and micropatterns such as single cell traction force measurement and screening of multiplex cell niche factors will be given. Finally, the outstanding challenges in developing a truly programmable cell niche platform with user-defined specifications and spatially controlled heterogeneity will be discussed.