Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.

Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.