(G-242) Flow-Milli: Millimeter Sized Matrigel Droplets for 3D Cell Cultures

Introduction:: Millimeter-sized hydrogel droplets could enable well-controlled and tunable 3D cell culture. To this aim, droplet microfluidics could provide a suitable approach. Much of the droplet microfluidics has focused on developing tools for single-cell encapsulation, for example, sequencing applications. To enable 3D multi-cellular cultures, we developed and investigated mm-sized (0.5-2 mm diameter) droplets via co-flow microfluidics. We characterized the tunable generation of water and Matrigel droplets. Additionally, we used dimensionless number analysis to examine the characteristics of the millimeter-sized droplet generators. Finally, we used Matrigel droplets to culture fibroblast cells inside 3D environments for up to 7 days.

Materials and Methods:: Two syringe pumps independently controlled the continuous and dispersed phases (Fig. 1A). Oil (food-grade olive oil containing 1% surfactant Span 80) was used as the continuous phase. First, the tunable generation of water-in-oil droplets was investigated (Fig. 1B). The volumetric flow rate of Oil was kept constant at 5 mL/hr. In comparison, the volumetric flow rate of water was adjusted from 0.5 to 3 mL/hr. Next, the tunable generation of matrigel droplets (Fig. 1C) was investigated. Care was taken to keep the solutions and instruments cold. Fibroblast cells (200,000 ATCC cells) were premixed with the Matrigel solution (5mg/mL) to create a 2.2x10^5 cells/mL concentration for the 3D culture experiments. Droplets were collected and cultured inside a 96-well plate with sufficient media. In each time point (3, 5, and 7 days), two independent droplets were washed, fixed/permeabilized, and stained for cell nuclei (Fig. 1D).

Results, Conclusions, and Discussions:: Water-in-oil experiments provided an approach to systematically characterize the droplets. The Capillary numbers for the continuous phase were 3.96x10^-4, and the dispersed phase ranged from 1.48x10^-6 to 8.9x10^-6. The analysis suggested that droplets were generated in the desirable geometry-controlled regime [1]. The Weber number for the dispersed phase ranged from 1.37x10^-7 to 4.93x10^-6, suggesting that interfacial tension dominated the system. While tunable, Matrigel droplets were less uniform than the water droplets. Matrigel droplets sustained fibroblast culture within a 3D environment for up to seven days. In the future, we will work toward maintaining the stability/shape of the matrigel droplet were not well-maintained throughout the 7 days. Tunable generation of mm-sized hydrogel droplets is a versatile approach for scaling biomanufacturing applications. Co-flow microfluidics presented are a simple approach to accomplish this aim.

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References (Optional): : [1] Anna, Shelley Lynn. "Droplets and bubbles in microfluidic devices." Annual Review of Fluid Mechanics 48 (2016): 285-309.