Cell behavior and differentiation are not only influenced by biochemical cues but also by physical properties like adhesive geometry, topography, and stiffness of the 3D extracellular environment. In this talk I will discuss how 3D laser nanoprinting can be applied to design 3D cellular microenvironments in the µm range with defined geometries and adjustable flexibility. To achieve a precise and patterned functionalization with biomolecules in 3D three approaches are chosen:

(i) By sequential printing of two different photoresists, composite-polymer scaffolds with distinct protein-binding properties can be fabricated and selectively bio-functionalised thereafter. Cells cultured in these scaffolds selectively form cell-adhesion sites with the functionalised parts, allowing for controlling cell adhesion and cell shape in 3D. Since the elastic modulus of the scaffold material varies between E=140-350 MPa, measurements of cell adhesion forces in relation to adhesion geometry are also feasible. In addition, these scaffolds can be used to mechanically stimulate cells at single defined adhesion sites.

(ii) By combining nanoprinting with an efficient surface photochemistry, also amenable to two-photon activation, it is possible to generate structurally complex 3D microstructures with 3D resolved chemical patterns. Microscaffolds with lattice constants of 10–20 microns can be patterned with protein ligands with a resolution close to one micron using a phototriggered cycloaddition. These techniques have been applied to guide cell attachment in 3D-microscaffolds selectively functionalized with two distinct adhesion proteins.

(iii) By using stimuli-responsive hydrogels, 3D scaffolds can be transferred from passive to dynamic systems. We have fabricated composite 3D scaffolds that allow for the micromanipulation of single cells. These scaffolds allow to directly correlate displacements to cellular forces and to quantify the effects with high throughput.

In summary, the above described 3D scaffolds enable to study the influence of spatial ligand-distribution on cellular differentiation, allow visualizing and measuring cell adhesion forces, and can be used to mechanically stimulate single cells at defined adhesion sites.