Poster P5 - GELAIN METHACRYLATE MACROPOROUS CELL SCAFFOLD FABRICATION VIA ONE- POT AQUEOUS TWO-PHASE SEPARATION

Introduction: Microgels are increasingly recognized as versatile building blocks for cell culture scaffolds, offering advantages over hydrogels for certain applications, such as drug delivery and wound healing. Their enhanced porosity facilitates nutrient transport and cell migration, while their concentrated polymer phase improves mechanical properties and cell adhesivity. Moreover, the modularity of microgels permits the construction of customized scaffolds through selective integration. Existing methods for scaffold fabrication often require multistep processes involving separate microgel formation and assembly. Here, we introduce a streamlined, one-pot approach that achieves microgel formation and scaffold assembly in just a few minutes. This method is robust, reproducible, user-friendly, and requires no specialized equipment, making it broadly accessible.

Materials and

Methods: Aqueous two-phase separation (ATPS) was utilized to form ~2 μm gelatin methacrylate (GelMA) microgels in sodium sulfate salt and assemble them into scaffolds under ultraviolet (UV) light (365 nm, 10 min exposure time) in the presence of cells (Figure 1A). U-87 glioblastoma or NIH 3T3 fibroblast cells were introduced to a precursor GelMA (30% w/v) and photoinitiator (Irgacure, 1% w/v) solution, to which salt solution (0.5 M) was added simultaneously with UV exposure. As microgels were forming, they rapidly “clicked”, encapsulating cells, and forming a macroporous scaffold. The scaffold swelling was tested (swollen mass/dry mass), and porosity was examined by scanning electron and confocal microscopy (Leica Confocal SP8, Leica Microsystems Inc.). To visualize via microscopy, GelMA was fluorescently labeled with fluorescein isothiocyanate (FITC), and cells were stained with cell tracker to assess morphology or with live/dead staining to assess viability from confocal imaging.

Results, Conclusions, and Discussions: Here we used an ATPS set up to form GelMA microgels of ~2 µm in diameter, which then assembled into a bulk scaffold. Microgels formed within 30 sec and complete scaffold formed in 10 min, at which point ~100% of the GelMA precursor solution was integrated into the scaffold. The microporosity of the scaffold enabled by the ‘clicking’ or individual microgels was confirmed by SEM and microscopy imaging (Figure 1B, D). Our results indicated a mechanically stable scaffold able to quickly imbibe water due to its interconnected macropores and showed a swelling ratio of ~4.4 (compared to a swelling ratio of 10 for a hydrogel), the scaffold was able to support a suture (Figure 1C) which might be particularly valuable in complex tissue repairs, surgeries that involve delicate or soft tissues, and situations where precise tissue regeneration is necessary. Cellsencapsulated in the scaffold exhibited an elongated morphology at 24 hours and >90% viability over 14 days of culture (Figure 2). Cell viability was high despite the high solution osmolality, probably due to the short duration of salt exposure (10 min during gelation) and perhaps a protective effect of GelMA (data not shown). The ability to produce microgel scaffolds containing living cells in one step opens new routes to the production of cell-laden porous scaffolds.

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