Three-dimensional Chemical Patterning of Transparent Hydrogels

Covalent modification of agarose with a 6-bromo-7-hydroxycoumarin (Bhc) sulfide derivative yields a hydrogel that generates bound thiol groups upon excitation with either UV light or a pulsed infrared laser. Using a multiphoton confocal microscope as the patterning platform, intricate internal chemically modified volumes of stable nucleophilic thiol groups are created within these hydrogel samples, which are in turn modified with biomolecules without causing hydrogel cross-linking or changes in its physical properties. The use of automated scripts and microscope stage control allows, for the first time, biologically relevant molecules of interest to be photochemically immobilized within the hydrogel in complex patterns with feature sizes comparable to those of mammalian cells. The resulting chemically patterned hydrogels have applications in tissue engineering, where they can be used to control cell behavior.