A Dynamic-Stiffness Hydrogel Platform Utilizing Phytochrome B and Phytochrome Interacting Factor 6 as a Light-Inducible Crosslinker
Traditional cell culturing methods are generally static with respect to mechanical properties unlike in vivo conditions, suggesting that the results from in vitro experiments form an incomplete picture for downstream studies. My cell culturing platform utilizes phytochrome B (PhyB) and phytochrome interacting factor 6 (PIF6), plant proteins derived from A. thaliana which associate or dissociate based on two distinct wavelengths of light, as a controllable, reversible crosslinker within a hydrogel platform.
Both PhyB and PIF6 were created in bulk within E. coli and purified via Ni-NTA, ion exchange, and size exclusion columns. The ex vivo activity of my synthesized proteins was tested via total internal reflection fluorescence (TIRF) microscopy. A hyaluronic acid (HyA) polymer gel was chosen as the base of the cell culturing platform for both its biological compatibility and its ability to be modified for protein conjugation. A semi-interpenetrating polymer network (semi-IPN) matrix was made by creating a base HyA hydrogel through thiol-ene click chemistry and incorporating a free HyA polymer stand which was modified to have methacrylate groups for protein conjugation. The modification of the HyA polymers was confirmed via proton nuclear magnetic resonance (1H NMR) spectroscopy. Confocal microscopy confirmed the covalent conjugation of fluorescently-labeled PhyB and PIF6 to the cell culturing platform. To confirm compatibility of the platform for cell culture, human mesenchymal stem cells were cultured for six days on the platform.
Atomic force microscopy (AFM) was used to measure the stiffness changes in the hydrogel platform when exposed to different wavelength light and preliminary data shows promising stiffness changes based on light cues. Future work on the platform will include establishing and characterizing the material properties of the hydrogel platform after variations are made on polymer lengths, protein density, and light intensity or duration. Upon optimization, this dynamic hydrogel platform could be utilized to mimic a variety of mechanical conditions found in vivo and provide insight to mechanobiological phenomena.