Results and Discussion
2.1 Microfluidic Device Used to Fabricate Cell Bead-Laden Struts
A schematic of the microfluidic process to fabricate cell bead-laden struts is shown in Figure 1a. The microfluidic system consisted of three inlets for three separate liquid materials: 1) a continuous phase of mineral oil, 2) cell-loaded GelMa bioink (MG63, cell density: 3 × 107 mL−1, and 4 wt% GelMa) with a crosslinking agent, and 3) alginate solution (3 wt%). All solutions were injected, and the flow rate was controlled using syringe pumps. The interfacial tension between the oil and cell-laden GelMa bioink was sufficient to fabricate spherical GelMa cell beads only with the support of shearing forces in the microchannel. The GelMa cell beads were then crosslinked using exposure to optimized UV conditions (500 mW cm−2) (Figure 1b). To generate stable cell bead-laden fibrous struts, we used alginate as structural support. The cell beads penetrated well in the alginate solution due to the low interfacial tension between the GelMa cell beads and the alginate solution, while the continuously flowing oil was fully separated from the alginate solution due to complete phase separation (Figure 1c). The space between the cell beads in the alginate strut was controlled by the alginate flow rate. The final outlet of the microfluidic system was connected to a CaCl2 bath (2 wt% CaCl2 in Dulbecco’s modified Eagle’s medium [DMEM]), and the cell bead-laden fibrous alginate was crosslinked in the bath. The fabricated GelMa cell beads were spherical or oval in shape, ranging from 250 to 350 µm in size, and the diameter of the fabricated alginate filament was ≈350–400 µm. Figure 1d shows optical, scanning electron microscope (SEM), and live (green)/dead (red) images of the cell-laden GelMa beads within the fabricated hybrid alginate strut. In the images, the cell beads survived well in the microscale alginate strut with high cell viability (>90%).
2.2 Fabrication of Cell Beads under Various Injected Solution Flow Rates
In the microfluidic device, homogeneous cell-laden beads were generating by manipulating the flow rates of continuously flowing oil and cell-loaded GelMa bioink. To determine the effect of the oil and GelMa bioink flow rates on the formation of cell beads with optimal geometrical size and cell viability, we used the microfluidic device shown in Figure 1b. Under various flow rates of GelMa bioink (0.025, 0.05, 0.1, and 0.15 mL min−1) and a fixed volume oil flow rate (0.2 mL min−1), the optical images shown in Figure 2a show that four distinct shapes of cell-aggregated beads were present under specific conditions: i) no bead formation due to an insufficient GelMa bioink flow, ii) stable spherical cell beads, iii) oval shape cell beads due to a higher GelMa bioink flow rate, and iv) continuous thread. The detailed effects of oil and GelMa flow rates on the resulting shapes of GelMa cell beads are shown in the process diagram in Figure 2b. The diameter of GelMa cell beads was quantified under various oil and GelMa flow rates. As expected, the diameter decreased from ≈350 to 280 µm with an increased oil flow rate (0.1–0.2 mL min−1) (Figure 2c). Additionally, under the GelMa bioink flow rate range of 0.05–0.1 mL min−1 and fixed oil flow rate of 0.2 mL min−1, the size of the spherical cell beads was 270–550 µm (Figure 2d). As expected, the size of the spherical cell beads gradually increased with increased volume flow rate and increased weight fraction of GelMa bioink (Figure 2e,f).