Photopatterned biomolecule immobilization to guide three-dimensional cell fate in natural protein-based hydrogels Ivan Batalova,b, Kelly R. Stevensb,c,d, and Cole A. DeForesta,b,c,e,f,1 aDepartment of Chemical Engineering, University of Washington, Seattle, WA 98105; bDepartment of Bioengineering, University of Washington, Seattle, WA 98105; cInstitute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109; dDepartment of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98195; eMolecular Engineering & Sciences Institute, University of Washington, Seattle, WA 98105; and fDepartment of Chemistry, University of Washington, Seattle, WA 98105 Edited by Helen M. Blau, Stanford University, Stanford, CA, and approved December 6, 2020 (received for review July 6, 2020) Hydrogel biomaterials derived from natural biopolymers (e.g., fibrin, to gravitate toward natural protein-based systems, including those collagen, decellularized extracellular matrix) are regularly utilized in derived from fibrin, collagen I, gelatin, and decellularized extra- three-dimensional (3D) cell culture and tissue engineering. In contrast cellular matrix (dECM) (31). These materials generally exhibit to those based on synthetic polymers, natural materials permit en- higher biocompatibility and enhanced matrix remodeling com- hanced cytocompatibility, matrix remodeling, and biological integra- pared with synthetic alternatives. Moreover, natural biomaterials tion. Despite these advantages, natural protein-based gels have innately provide encapsulated cells with many of the same bio- lagged behind synthetic alternatives in their tunability; methods to chemical cues present in the native ECM, giving rise to greater selectively modulate the biochemical properties of these networks in cell–material integration and functional engineered tissue (9). With a user-defined and heterogeneous fashion that can drive encapsu- the goal of gaining 4D control over natural biomaterial properties, lated cell function have not yet been established. Here, we report a recent efforts have sought to functionalize protein-based materials generalizable strategy utilizing a photomediated oxime ligation to with photosensitive moieties. Installation of alkene functionality covalently decorate naturally derived hydrogels with bioactive pro- (e.g., methacrylate, acrylate) on reaction components has permitted teins including growth factors. This bioorthogonal photofunctionali- – photopolymerization of natural gels (32 34), just as chemical cross- APPLIED BIOLOGICAL SCIENCES zation is readily amenable to mask-based and laser-scanning linking with photoscissile moieties (e.g., nitrobenzyl) has enabled lithographic patterning, enabling full four-dimensional (4D) control their photodegradation (35, 36). Though existing methods offer over protein immobilization within virtually any natural protein- spatiotemporal control over the mechanical properties of natural based biomaterial. Such versatility affords exciting opportunities to biopolymer-based hydrogels, strategies to selectively modulate the probe and direct advanced cell fates inaccessible using purely syn- biochemical aspects of these systems in a user-defined and het- thetic approaches in response to anisotropic environmental signaling. erogeneous fashion remain largely unexplored. When properly de- veloped, these advances would further solidify natural gels as choice biomaterials | photochemistry | hydrogel | protein | 4D biology materials for fundamental cell studies and translational applications. A recent report from the Zenobi-Wong group was the first to ver the past several decades, hydrogels have found wide- demonstrate photoimmobilization of proteins within natural Ospread utility as biomaterial constructs for three-dimensional – (3D) cell culture and tissue engineering (1 5). Fulfilling a role Significance similar to the extracellular matrix (ECM) in native tissue, gel biomaterials can provide encapsulated cells with mechanical sup- port in a defined geometry, anchors for adhesion and migration, Over the past two decades, natural protein-based hydrogel biomaterials have catapulted cell biology into the third dimen- and bioactive chemical signals to guide complex cellular functions sion, becoming the modern workhorses of 3D cell and organoid (e.g., proliferation, differentiation) (6–8). Classically, such mate- culture. Although these systems mimic many important aspects rials are categorized as “synthetic” or “natural” based on the or- of native tissue while permitting matrix remodeling and bio- igin of their underlying components (9). Synthetic polymer-based logical integration, spatiotemporal control over cell fate within gels, including those composed of poly(ethylene glycol) (PEG) these materials has not been demonstrated. Here, we introduce and polyacrylamide, have gained significant traction within the a generalizable strategy to covalently decorate cell-laden natural biomaterials community, owing to tunable control over their initial hydrogels with bioactive proteins including growth factors and network mechanics, chemical composition, and biodegradability – then use these methods to anisotropically govern cell fates in (10 12). Since these systems are synthetically defined, stimuli- ways currently unobtainable in synthetic biomaterials. Enabling responsive functional handles can be installed during formulation 4D biochemical tunability within natural protein-based gels, to afford constructs with exogenously modifiable properties. these approaches are likely to find great utility in probing and Though synthetic gels have been engineered to be responsive to directing biological functions and in engineering heterogenous – many external stimuli (e.g., pH, temperature, enzyme) (13 15), functional tissues. those sensitive to light can be uniquely modulated in four dimen- sions (4D, comprising 3D space and time) through lithographically Author contributions: I.B., K.R.S., and C.A.D. designed research; I.B. performed research; defined irradiation (16, 17). In addition to regulating viscoelastic I.B. and C.A.D. contributed new reagents/analytic tools; I.B. and C.A.D. analyzed data; and properties of these materials (18–22), photochemical methodolo- I.B., K.R.S., and C.A.D. wrote the paper. gies have been developed to immobilize bioactive peptides and The authors declare no competing interest. proteins in user-defined patterns within cell-laden gels (23–30). This article is a PNAS Direct Submission. Such spatiotemporal control over network biofunctionalization of- Published under the PNAS license. fers exciting opportunities in recapitulating the dynamic biochemi- 1To whom correspondence may be addressed. Email: [email protected]. cal heterogeneity characteristic to native tissues. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Despite the historically superior tunability of synthetic hydrogel doi:10.1073/pnas.2014194118/-/DCSupplemental. materials, many cell biologists and tissue engineers alike continue Published January 18, 2021. PNAS 2021 Vol. 118 No. 4 e2014194118 https://doi.org/10.1073/pnas.2014194118 | 1of7 Downloaded by guest on September 25, 2021 hydrogels (30). Relying on a sortase transpeptidase-mediated cou- permitting localized condensation with aldehyde-functionalized pling of a chemically tagged streptavidin onto a photouncaged proteins (Fig. 1 B and C). Through mask-based and laser- polyglycine peptide grafted to a backbone material, biotinylated scanning lithographic activation of this photomediated oxime li- proteins of interest could be noncovalently immobilized with spatial gation, we achieve full 4D control over protein immobilization control within natural gels. While the group beautifully demon- within three distinct natural biopolymer-based hydrogels. High- strated controlled axon guidance using nerve growth factor within lighting the versatility of the approach, we pattern two responses an engineered hyaluronan-based matrix (37), photopatterned cell that—despite considerable effort by our laboratory and others— function on or within protein-based biomaterials was not demon- have not been achieved using purely synthetic materials, namely, strated. Seeking to fill this important void, we postulated that the two-dimensional (2D) primary rat hepatocyte proliferation on photomediated oxime ligation—a chemistry recently developed and collagen gels using immobilized epidermal growth factor (EGF) reported by our group for photopatterning of synthetic polymer- and 3D U2OS Notch signaling activation within fibrin gels deco- based materials (26, 38)—could be used to spatially modify natu- rated with tethered Delta-1. ral gels, but with additional benefits associated with covalent protein immobilization, chemical accessibility, design simplicity, and overall Results and Discussion ease of use. As this chemistry represents one of exceptionally few To test our workflow and verify its compatibility with different bioorthogonal reactions that can be photochemically triggered, natural biomaterials, we biochemically decorated gels based on − − thereby enabling user-defined control over when and where ligation collagen I (4 mg mL 1) or fibrin (10 mg mL 1) with full-length occurs with high specificity in the presence of living systems, we proteins. Prior to gelation via conventional methodologies, col- identified the photomediated oxime ligation as uniquely capable of lagen I and fibrin gel precursors were incubated