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Biomanufacturing

Biomanufacturing - Poster Session C

Poster B3 - Investigating millimeter-sized hydrogel droplet formations

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Droplet microfluidics is a powerful approach to generate well-controlled emulsions (aqueous-in-oil) for tissue engineering and biomanufacturing applications. The field has previously focused on pico to nanoliter droplet volumes, emphasizing single-cell applications. However, these previously desirable small volumes limit long-term cell culture applications (e.g., organoids and spheroids). We have previously demonstrated simple co-flow microfluidics to generate mm-sized droplets. Our previous demonstrated the feasibility of water-in-oil and hydrogel-in-oil droplets. The prior hydrogel included Alginate and Matrigel. In this investigation, we expanded the library of materials to include Gelatin, Fibrin, Collagen peptides, and Collagen with PEGDMA. Cell loading and viability with 3T3 Fibroblast and HUVECs were also validated as proof of concept. Like water-in-oil droplets, the size of hydrogel-in-oil droplets could be tuned by adjusting the flow ratios. The system (see Figure 1) demonstrated high tunability and droplet uniformity. The investigation demonstrates a systematic characterization of hydrogel droplet formation under well-controlled conditions. The project's ultimate goal is to use cell-encapsulated droplets as therapeutics for the sustained release of factors emphasizing angiogenesis applications. Therefore, characterizing the role of materials in droplet formation and size tunability (to control for diffusion in or out of the encapsulation is an essential early step for the project's success.

Materials and

Methods:  The microfluidic chips were fabricated using laser-cut acrylic molds by casting PDMS replicas. PDMS parts were permanently attached to glass slides via plasma treatments. The chip was designed as a single channel with two inlets, creating a co-flow droplet generator. Two independent syringe pumps were used to transport the continuous phase (oil) and dispersed phase (material of interest) through the chip. Flow ratio (Φ) was defined as the ratio of the continuous phase's volumetric flow rate to the dispersed phase's volumetric flow rate. Figure 1 demonstrates the system. First, water and gelatin were used to calibrate the droplet fabrication. Droplet size was measured using NIH ImageJ. The droplet size distribution for each material as a function of flow ratio is presented (raincloud plot). T-tests were used to determine statistical significance. The coefficient of variance (COV) for each condition was calculated to determine the uniformity of droplets. Briefly, for all conditions, COV was < 10% (indicating good uniformity). For cell loading experiments, 3T3 Fibroblasts were encapsulated in the Collagen+PEGDMA microbeads, whereas HUVECs were encapsulated inside the fibrin microbeads. Droplets were generated and collected from the outlet tubing in a 96-well plate. The excess oil was removed from the well plates. Droplets were imaged via an EVOS fluorescent microscope under different conditions, including inside the chip, tubing, and 96-well plate with oil or cell media. The droplets were mechanically robust in these conditions. Calcein AM live stain were used for cell viability.

Results, Conclusions, and Discussions: The Water-In-Oil trials (Φ = 0.6, Φ = 0.75, Φ = 1.0, Φ = 1.5, Φ = 3.0), gelatin-in-oil (Φ = 0.75, Φ = 0.857, Φ = 1.0, Φ = 1.2, Φ = 1.5), collagen peptides-in-oil (Φ = 0.6, Φ = 0.75, Φ = 1.0, Φ = 1.5, Φ = 3.0), collagen peptides-in-oil with PEGDMA (Φ = 1.0, Φ = 1.5, Φ = 3.0), and HUVECs-in-fibrin (Φ = 1.0, Φ = 1.5, Φ = 3.0) microbeads were all uniform with coefficient of variance being below 10 for all different flow ratios. Figure 2 provides a summary of these results. Next, HUVECs-in-fibrin (Φ = 1.0, Φ = 1.5, Φ = 3.0) microbeads were generated, demonstrating uniformity with a coefficient of variance being below 10 (data for fibroblasts not presented). Results from the water-in-oil trials indicate that the system is tunable and uniform. The trials with gelatin and collagen peptides established the application of various hydrogels. Here, we sought to characterize the generation of millimeter-scale hydrogel droplets. These investigations are needed to enable predictive modeling of the droplet generations (particularly for non-Newtonian fluids) without iterative experimentations. In future steps, examining transport/diffusion (to or from droplets), cell migration, and co-culture experiments are needed to characterize the functional properties of cell-encapsulated droplets.

Acknowledgements (Optional):