Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-Viscosity Bioink

Abstract

A novel bioink and a dispensing technique for 3D tissue-engineering applications are presented. The technique incorporates a coaxial extrusion needle using a low-viscosity cell-laden bioink to produce highly defined 3D biostructures. The extrusion system is then coupled to a microfluidic device to control the bioink arrangement deposition, demonstrating the versatility of the bioprinting technique. This low-viscosity cell-responsive bioink promotes cell migration and alignment within each fiber organizing the encapsulated cells.

Keywords: 3D tissue engineering; bioinks; bioprinting; microfluidics; vascular tissue engineering.

Figure 1

(a) Schematic illustration of the layer-by-layer deposition based bioprinting technique consisting of two independent crosslinking steps. (b, c) The bioink contained GelMA (Red dashed lines), alginate (Green lines), photoinitiator and cells in the inner needle of the coaxial system. Simultaneously the CaCl₂ (blue dots) solution flows through the outer needle to induce the gelation of alginate chains. The construct was then UV crosslinked to solidify the GelMA prepolymer in the fiber. (d) Printability of different alginate concentrations and different CaCl₂ concentrations. GelMA concentration was kept constant (4.5% w/v). (e) Viscosities of alginate and GelMA and the combination of both at room temperature. (f) The fiber diameter varied with different deposition speeds, calculated for a bioink flow rate of 5µl/min. In the upper-right corner, photographs of the different fibers were obtained for deposition speeds of 6 mm/sec, 3 mm/sec, and 1mm/sec. (g) Photograph of the final construct (30 layers). (h) Top, lateral and 3D µCT reconstructions of the final bioprinted 3D structure.

Figure 2

(a) A microfluidic system was used to flow two separate bioinks containing red and green fluorescent beads that exited the device through a single extruder. Photograph (insert) of the coaxial needle system with a microfluidic chip with a “Y” shaped channel. The schematic diagram and fluorescence microscopy image of cross-section view of 3D construct with (b and c) alternative deposition, (d and e) alternative/simultaneous deposition, and (f–i) simultaneous deposition.

Figure 3

(a) Viability of bioprinted HUVECs at day 3 as a function of different UV exposure times. (b–d) Confocal live-dead staining of bioprinted HUVECs at day 5. (e) Cell viability was assessed in the top (b), middle (c) and bottom (d) of the construct. Confocal fluorescence F-actin and DAPI images of bioprinted HUVEC embedded construct with (f) 15 s and (g) 30s UV exposure time after 10 days in cell culture. (h) Elastic modulus of GelMA versus different UV exposure times.

Figure 4

(a) Schematic of the encapsulated HUVECs migrating to outer regions of the bioprinted fibers after 10 days of culture. Confocal microscopy images with (b) top view, (c) cross section view, and (d) fiber junctions showing interconnected structures. Confocal microscopy images of a 1mm thick construct that show a: (e) transversal cross-section, (f) longitudinal cross-section, (g) outer surface of the complete construct. (h) Top view of a single fiber immunostained for CD31 (red) and DAPI (blue). (i) Schematic illustration of the HUVEC structure before and after the cardiomyocyte seeding. (j) 3D surface plot of a microscopy image of the 3D HUVEC-cardiac tissue construct after two days of cardiomyocytes culture. (k) The beating rates of cardiomyocytes were monitored in four different zones (1, 2, 3, 4) of the construct which showed synchronous beating behavior.