Poster O10 - Multifunctional Hydrogels with Cell Scale Features

Introduction: Native tissues feature complex extracellular microenvironments that have heterogenous physical, cellular, and chemical compositions, which affects cell behaviors, including migration, gene expression, and cytoskeletal organization. Traditional cell culture methods, such as tissue culture plastic, do not accurately reflect the complexity of the in vivo environment, and therefore, the complexity of cell behavior cannot be replicated. Hydrogels have been a promising cell culture substrate for achieving more accurate in vitro models, due to their biocompatibility and tunability. Photolithographic printing techniques and solvent exchange systems have been used to create hydrogels with spatial control over physical and chemical properties. Polyethylene glycol diacrylate (PEGDA) hydrogels, in particular, have been used to great effect to create hydrogels with complex characteristics, as they are easily modifiable and can incorporate different functionalities in their structures. Additionally, PEGDA hydrogels can incorporate DNA, which can be used to program mechanical, cellular, and chemical characteristics within the hydrogels. However, conventional PEGDA-DNA hydrogels do not possess spatial control that can be activated on-demand. Here, we have developed PEGDA hydrogels with physiologically relevant stiffnesses that incorporate coumarin photocaged oligonucleotides for the site-specific placement of cells. Furthermore, we used a solvent exchange system for the fabrication of modular hydrogels with distinct chemical functionalities, that share photocaged DNA that is orthogonal to the physical and chemical control. These hydrogels allow for the creation of more complex in vitro models that more accurately reflect the complexities of the in vivo environment.

Materials and

Methods: DNA was synthesized with standard bases along with coumarin protected guanine (5'-Dimethoxytrityl-N2-(4-isopropylphenoxyacetyl)-O6-[[7-(diethylamino)coumarin-4-yl]-methyl]-2'-deoxyGuanosine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; Glen Research) on an Expedite synthesizer following manufacturer recommendations. Deprotection was performed with ammonium hydroxide (28-30%; Fisher Chemicals; 24 h room temperature) and purified using a Glen-Pak DNA purification column (Glen Research). Thiol-terminated DNA for hydrogel incorporation was synthesized with 3'-Thiol-Modifier C3 S-S CPG beads (Glen Research) and then treated with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, EMD Millipore Corp) for 5 minutes for reduction of the disulfide bond. 7.2% PEGDA hydrogels were synthesized with 36 μL polyethylene glycol diacrylate (Average Mn 700, Sigma Aldrich), 60 μL water-soluble Diphenyl(2,4,6-trimethylbenzoyl)phosphine Oxide-Nanoparticles (TPO-nanoparticles, Sigma Aldrich, 100 mg/mL in H2O stock), 120 μL 1K PEG-SH (Creative Pegworks, 50 mg/mL in H2O), 22 μL DNA-SH (100 µM final concentration), and 262 μL DI H2O. The solution was placed on methacrylated coverslips, which had been plasma etched for 5 minutes and treated with 1% acetic acid (v/v), 2% 3-(Trimethoxysilyl)propyl methacrylate (TMSPMA) (v/v), and 97% ethanol (v/v) for 5 minutes. The coverslips with the solution were then selectively exposed to varying timepoints of 365 nm light using a digital micromirror device (DMD; Mightex) on an Olympus BX63 microscope. After the hydrogels were created, the gels were incubated with 5 µM of photocaged DNA. Sections of the gels were then patterned with 425 nm light using the DMD and then incubated with either cells functionalized with complementary DNA or 1 µM of fluorescent complementary DNA. Then, the hydrogel was rinsed with PBS and imaged.

Results, Conclusions, and Discussions: To achieve precise cell placement within hydrogels, we synthesized hydrogels containing coumarin photocaged oligonucleotides, whose ability to bind complementary DNA strands can be selectively activated by ultraviolet (UV) and non-UV light up to 500 nm. These hydrogels incorporated photocaged DNA by using a two-step process. First, PEGDA hydrogels were synthesized to incorporate DNA terminated with a thiol (DNA-SH). Next, we incubated the hydrogel with a photocaged DNA (PC-DNA) that is partially complementary to the DNA-SH while having a photocaged region that can be subsequently activated for capture of other DNA targets. Gel shift assays confirmed specific hybridization between DNA-SH and PC-DNA and that exposure to 425 nm light effectively uncages the PC-DNA for recognition of fluorophore-labeled DNA (Figure A).

By tuning PEGDA concentrations, we created hydrogels with tunable physiologically relevant stiffnesses (~1-15 kPa). Exposure to 425 nm light efficiently uncaged DNA, enabling repeated activation and spatially controlled integration of additional functionalities (Figure B).

Utilizing a solvent exchange system, we printed localized hydrogels with tunable stiffness and mixed chemical properties. By perfusing different polymerization solutions, with one solution containing rhodamine-PEG-thiol and the other containing biotin-PEG-thiol, and controlling exposure times, we generated multifunctional hydrogels with orthogonal light activation of DNA. Fluorescence imaging confirmed distinct functionalities and localized uncaging across gel interfaces (Figure C).

Finally, we validated the utility of photocaged DNA hydrogels for cell patterning. We attached complementary DNA to cell membranes. When the PC-DNA on the hydrogel surface was exposed to light, complementary binding could occur, allowing the cells to attach to the surface only in the locations of light exposure (Figure D).

These advancements in patterning cells on biomimetic hydrogel surfaces provide pathways to more accurate in vitro tissue models, allowing for a more accurate exploration of how cells behave. With the dynamic control afforded by photocaged DNA hydrogels, we can create microenvironments that closely mimic the complexities of native tissues, enabling the study of cell behavior in a more physiologically relevant context with high spatiotemporal control. These advancements can enhance our understanding of fundamental cellular processes, with great promise for applications in drug discovery, regenerative medicine, and disease modeling.

Acknowledgements (Optional): This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R21GM141563 and R35GM150577.