2.1 Microfluidic Device Used to Fabricate Cell Bead-Laden Struts<\/h3>\n
A schematic of the microfluidic process to fabricate cell bead-laden struts is shown in\u00a0Figure<\/b>\u00a01a<\/a>. 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 \u00d7 107<\/sup>\u00a0mL\u22121<\/sup>, 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\u00a0mW cm\u22122<\/sup>) (Figure\u00a01b<\/a>). 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\u00a01c<\/a>). 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<\/sub>\u00a0bath (2 wt% CaCl2<\/sub>\u00a0in 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\u00a0\u00b5m in size, and the diameter of the fabricated alginate filament was \u2248350\u2013400\u00a0\u00b5m. Figure\u00a01d<\/a>\u00a0shows 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%).<\/p>\n Figure 1<\/strong><\/p>\n 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\u00a01b<\/a>. Under various flow rates of GelMa bioink (0.025, 0.05, 0.1, and 0.15\u00a0mL min\u22121<\/sup>) and a fixed volume oil flow rate (0.2\u00a0mL min\u22121<\/sup>), the optical images shown in\u00a0Figure<\/b>\u00a02a<\/a>\u00a0show 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\u00a02b<\/a>. The diameter of GelMa cell beads was quantified under various oil and GelMa flow rates. As expected, the diameter decreased from \u2248350 to 280\u00a0\u00b5m with an increased oil flow rate (0.1\u20130.2\u00a0mL min\u22121<\/sup>) (Figure\u00a02c<\/a>). Additionally, under the GelMa bioink flow rate range of 0.05\u20130.1\u00a0mL min\u22121<\/sup>\u00a0and fixed oil flow rate of 0.2\u00a0mL min\u22121<\/sup>, the size of the spherical cell beads was 270\u2013550\u00a0\u00b5m (Figure\u00a02d<\/a>). As expected, the size of the spherical cell beads gradually increased with increased volume flow rate and increased weight fraction of GelMa bioink (Figure\u00a02e,f<\/a>).<\/p>\n Figure 2<\/strong><\/p>\n![\"image\"](\"\/cms\/asset\/dbb7ca2f-01c7-430a-baac-d74c0c867436\/smll202106487-fig-0001-m.jpg\" "\\"image\\"")
<\/picture><\/a>2.2 Fabrication of Cell Beads under Various Injected Solution Flow Rates<\/h3>\n
![\"image\"](\"\/cms\/asset\/189c5be1-63c7-49eb-ab1c-0604b2330bae\/smll202106487-fig-0002-m.jpg\" "\\"image\\"")
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