Fucoidan/Gelatin Microgel Formation with Droplet Microfluidics for Neural Spheroid Encapsulation

Abstract

Oxidized fucoidan (oFu) is a bioactive, sulfated polysaccharide that has demonstrated promising potential as a bio-inductive scaffold for neural engineering. Previous developments have characterized the anti-inflammatory properties of oFu; however, its capacity to direct the lineage commitment of neural progenitor cells (NPCs), which is critical for neuron regeneration, was not evaluated. The differentiation of NPCs desires a 3D culture that is more biomimetic to the natural in vivo microenvironment. Specifically, the structural similarity of fucoidan to heparan sulfate—a native extracellular matrix (ECM) component known for growth factor binding—suggests that fucoidan can similarly modulate the microenvironment to favor neuronal differentiation. Droplet microfluidics (DM), which can generate monodispersed emulsions of nanoliters at kHz rates by injecting fluid into another immiscible fluid in a microchannel network, offers a promising solution to precisely generate a high-throughput 3D culture environment when compared to conventional methods for differentiation culture, such as 2D surface coating. Therefore, this study was motivated by the implementation of an oFu-based matrix within DM architecture (Chapter 4). In line with enhancing neuronal differentiation, neural spheroid culture has shown to increase neuronal expression, thereby reducing the post-encapsulation duration needed. Therefore, we hypothesize that pre-formed neural spheroids within a droplet microfluidics-mediated oFu-based matrix would facilitate neuronal differentiation (Chapter 5). Following the introduction (Chapter 1) and materials and methods (Chapter 3), optimization of the operational parameters for incorporating oFu and other hydrogels into DM was performed through systematic experimental studies, where variables for gelation and degradation were identified and issues of spheroid-mediated channel adhesion, consistent droplet formation, and post-formation gelation were addressed (Chapter 4). Finally, the identified compatible gel systems were utilized to demonstrate neural spheroid encapsulation and evaluate their biological response (Chapter 5). Neural spheroids encapsulated in oxidized fucoidan (oFu)/gelatin methacrylate (GelMA) microgels were mostly viable and exhibited a significant upregulation of the early neuronal differentiation marker class III beta-tubulin. This study provides a foundational proof-of-concept for the integration of oFu/GelMA microgels with a droplet microfluidics platform.