Facile and Versatile Method for Micropatterning Poly(acrylamide) Hydrogels Using Photocleavable Comonomers

Abstract

We here present a micropatterning strategy to introduce small molecules and ligands on patterns of arbitrary shapes on the surface of poly­(acrylamide)-based hydrogels. The main advantages of the presented approach are the ease of use, the lack of need to prefabricate photomasks, the use of mild UV light and biocompatible bioconjugation chemistries, and the capacity to pattern low-molecular-weight ligands, such as peptides, peptidomimetics, or DNA fragments. To achieve the above, a monomer containing a caged amine (NVOC group) was co-polymerized in the hydrogel network; upon UV light illumination using a commercially available setup, primary amines were locally deprotected and served as reactive groups for further functionalization. Cell patterning on various cell adhesive ligands was demonstrated, with cells responding to a combination of pattern shape and substrate elasticity. The approach is compatible with standard traction force microscopy (TFM) experimentation and can further be extended to reference-free TFM.

Substrate stiffness facilitates improved induced pluripotent stem cell production through modulation of both early and late phases of cell reprogramming

“…[19] Improvements in reprogramming in our pAAm hydrogels compared to reports on 3D hydrogels was found to be slightly higher on the same comparators, being 4-fold versus 2.5-fold more Oct4 GFP+ mIPSC colonies generated (hiPSC colonies were measured using different markers). The 2D pAAm hydrogel format offers significant advantages over 3D alternatives, in being a low-cost, easily-made and scalable platform in itself, but also for further optimisation with the inclusion of other biophysical cues (e.g., microtopography [53] , light-induced patterned region [54] ) and small molecules. [55] In this study, we have identified a novel mechanosensitive molecule, Phactr3, which was upregulated in the pAAm gel condition at the earliest time point, without any influence from the exogenous TFs expression, and confirmed it to be crucial for Bmp2-mediated MET and reprogramming (Fig.…”

“…[19] Improvements in reprogramming in our pAAm hydrogels compared to reports on 3D hydrogels was found to be slightly higher on the same comparators, being 4-fold versus 2.5-fold more Oct4 GFP+ mIPSC colonies generated (hiPSC colonies were measured using different markers). The 2D pAAm hydrogel format offers significant advantages over 3D alternatives, in being a low-cost, easily-made and scalable platform in itself, but also for further optimisation with the inclusion of other biophysical cues (e.g., microtopography[53], light-induced patterned region[54]) and small molecules. [55]…”

Current strategies for ligand bioconjugation to poly(acrylamide) gels for 2D cell culture: Balancing chemo-selectivity, biofunctionality, and user-friendliness

“…Several methodologies allow ligand patterning using various chemical strategies as shown in previous sections. ( Daliri et al, 2021 ) Noteworthy, the introduction of photolabile comonomers into PAM has recently been exploited to dynamically and locally tune the mechanical ( Norris et al, 2021 ) and cell-adhesive ( Missirlis et al, 2022 ) properties of light-responsive PAM gels. In contrast, the realization of truly high throughput PAM systems is still in its infancy.…”

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

“… 19 Other photolithographic methods such as digital micromirror device projection or direct laser writing afford high spatial resolution and dynamic control over ECM patterning. 20 , 21 These techniques permit hierarchical cell co-patterning by dynamically introducing new ECM ligand availability, for example, through biotin-avidin interactions, 20 but remain limited by a lack of tools to engineer patterned cell deposition independently of ECM adhesion. Other approaches use non-uniform electric fields to selectively guide cell positioning into complex tissue patterns (such as liver lobules) onto an electrode-containing substrate.…”