Poster N19 - Spatially Controlling Activation of SynNotch receptors on a Gelma Hydrogel

Introduction: New approaches for controlling the spatial organization of cells are critically needed to recapitulate the complex architecture and function of human tissues. The synthetic Notch (synNotch) receptor is a synthetic transmembrane receptor modified to recognize a user-defined ligand and induce expression of user-defined transcription factors. For example, synNotch receptors have been engineered to recognize GFP and mCherry and induce MyoD and ETV2 expression, respectively. By spatially patterning the location of the activating ligands on a surface, a vascularized muscle tissue model can be constructed. However, current methods of spatially patterning ligands are somewhat limited. For example, microcontact printing has been used to micropattern GFP on PDMS, but this approach can only be used to reliably print a single ligand. Furthermore, PDMS substrates do not facilitate long term cell adhesion. Thus, our objective is to spatially conjugate GFP to the surface of a photoactive and biocompatible hydrogel using photomasks to direct exposure to UV light, resulting in patterned synNotch reporter activation. We then evaluate the stiffness of the photopatterned hydrogel, as well as the spatial resolution of the crosslinked ligands and synNotch activation.

Materials and

Methods: Gelatin methacryoyl (gelma) was synthesized by reacting gelatin with methacrylate anhydride (MA). GFP was reacted with MA to form GFP-MA. A gelma and Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate (LAP) solution was added to a glass cover slip. The cover slips were exposed to 300 seconds of 118 mw/cm2 UV light for an initial crosslinking (UV1). A GFP-MA and LAP solution was then added to the hydrogel surface and a photomask of 250 µm squares with either 500 or 100 µm spacing was placed on top of the hydrogel surface before being exposed to 3 seconds at 8.6 mw/cm2 of UV light (UV2).
AFM was conducted with a JPK NanoWizard 4 by determining the average elastic modulus within a 100-um scan size of the cover slips that had either been exposed to only UV1 with no GFP, or UV2 on either a uniformly exposed or photopatterned surface with GFP-MA and LAP.
L929 cells with an anti-GFP receptor that activated mCherry were seeded onto gelma hydrogels that had been exposed to UV light for either: .7, 3.2, 5.6, 8,6, or 10.5 mw/cm2 of UV light. 3 days post-seeding, the gelma was minced, digested with 4 mg/ml of collagenase IV, and strained. Flow cytometry analysis was performed to determine the percentage of activated synNotch cells by gating for mCherry expression. L929 synNotch cells were similarly seeded on cover slips that had been photopatterned with 250 µm squares either 500 µm or 250 µm apart and were imaged for GFP and mCherry signal.

Results, Conclusions, and Discussions: The GFP-MA was photopatterned with high fidelity to the corresponding photomask; in the areas of exposed light, there is a visibly distinct GFP signal (Fig1. A-B). There is subsequent mCherry activation of synnotch cells colocalized with the photopatterned GFP regions when a photomask with 250 µm squares with either 500 or 100 µm spacing is used (Fig.1 A-B). While both the gelma and the GFP-MA are photocrosslinkable, the AFM results of the elastic modulus of the surface demonstrate that exposure to additional UV light for the photopatterning step does not lead to a significant increase in stiffness between the photopatterned and unphotopatterned regions of the gelma substrate. Despite the visible corner of the GFP signal, the AFM topography map does not show a difference in elastic modulus of the surface (Fig. 1C). The average stiffness of the surface that had only been exposed to UV1 was not significantly different that a surface that had been exposed to UV2 as well (Fig. 1C). Additionally, light-induced conjugated of GFP-MA to the gelma successfully activates maximum synNotch expression; quantification of mCherry positive cells via flow cytometry demonstrate a UV light intensity-dependent response to GFP in L929 reporter cells up until 8.6 wm/cm (Fig. 1E). Thus, there is a minimum threshold of UV light required to ensure maximum crosslinking of GFP to the surface for peak synnotch cell activation.
The use of photomasking for gelatin-based hydrogels holds future potential for the spatial patterning of multiple ligands, allowing for cellular scale control over multiple synNotch pathways. The synNotch pathways have been expanded to engineer dual receptor cells, which activate either MyoD or EVT2 in the presence of GFP or mCherry, respectively. By spatially patterning both ligands using photomasking technology, muscle and endothelial transdifferentiation can be accomplished to engineer a multicellular muscle model. Thus, this technology can generate more sophisticated tissue models that incorporate multiple cell types with precise spatial control to better recapitulate human physiological conditions, allowing for improved insight into tissue development and disease and new approaches for regenerative medicine.

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