Evaluation of photochemistry reaction kinetics to pattern bioactive proteins on hydrogels for biological applications

Bioactive signals play many important roles on cell function and behavior. In most biological studies, soluble biochemical cues such as growth factors or cytokines are added directly into the media to maintain and/or manipulate cell activities in vitro. However, these methods cannot accurately mimic certain in vivo biological signaling motifs, which are often immobilized to extracellular matrix and also display spatial gradients that are critical for tissue morphology. Besides biochemical cues, biophysical properties such as substrate stiffness can influence cell behavior but is not easy to manipulate under conventional cell culturing practices. Recent development in photocrosslinkable hydrogels provides new tools that allow precise control of spatial biochemical and biophysical cues for biological applications, but doing so requires a comprehensive study on various hydrogel photochemistry kinetics to allow thorough photocrosslink reaction while maintain protein bioactivities at the same time. In this paper, we studied several photochemistry reactions and evaluate key photochemical parameters, such as photoinitiators and ultra-violet (UV) exposure times, to understand their unique contributions to undesired protein damage and cell death. Our data illustrates the retention of protein function and minimize of cell health during photoreactions requires careful selection of photoinitiator type and concentration, and UV exposure times. We also developed a robust method based on thiol-norbornene chemistry for independent control of hydrogel stiffness and spatial bioactive patterns. Overall, we highlight a class of bioactive hydrogels to stiffness control and site specific immobilized bioactive proteins/peptides for the study of cellular behavior such as cellular attraction, repulsion and stem cell fate.