Title:

Hydrogel Synthesis via Beta-Thiolactone-Mediated Native Chemical Ligation for Cell Encapsulation: Imaging-Based Assessment of Cell Viability and Spreading

The demand for 3D cell cultures has led to an increase in cross-linked hydrogel methodologies. These systems mimic in vivo cellular processes better than their 2D analogs. However, researchers have struggled to develop nontoxic, inexpensive, and kinetically favorable hydrogels for cell encapsulation. Here we present our approach to hydrogel formation using native chemical ligation (NCL). Our use of beta-thiolactone enables hydrogel formation in less than two minutes, significantly faster than previous NCL-based systems forming at 10-20 minutes. Moreover, our NCL system eliminates the cytotoxic free thiols of previous work by sequestering the thiol within the PEG backbone. To accomplish this, we functionalized of tetra-armed PEG (Mw = 10,000) with beta-thiolactones and N-terminal cysteines. When dissolved in buffer, the two polymers combine, forming a network with favorable kinetics at 5 wt%. Additionally, our method also allows for post-gelation of attachment motifs and modifications. We have demonstrated the cell viability and adhesion properties of our gel through laser scanning confocal microscopy. We are currently optimizing our NCL system for 3D cell cultures and fiber-pulling platforms.

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