Void-free 3D Bioprinting for In-situ Endothelialization and Microfluidic Perfusion

Abstract

Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. We address both of these issues by introducing a versatile 3D bioprinting strategy, in which a templating bioink is deposited layer-by-layer alongside a matrix bioink to establish void-free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well-defined 3D network of interconnected tubular channels. This void-free 3D printing (VF-3DP) approach circumvents the traditional concerns of structural collapse, deformation and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered "unprintable". By pre-loading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in-situ endothelialization method can be used to produce endothelium with a far greater uniformity than can be achieved using the conventional post-seeding approach. This VF-3DP approach can also be extended beyond tissue fabrication and towards customized hydrogel-based microfluidics and self-supported perfusable hydrogel constructs.

Keywords: bioprinting; endothelialization; hydrogels; microfluidics; printability.

Figure 1

(A) Schematic of the VF-3DP process, where a biocompatible templating bioink (green) and a matrix bioink (yellow) were printed side-by-side, followed by photo-crosslinking of the matrix phase and 37 °C incubation to release the templating phase. Pre-loading endothelial cells in the templating bioink allowed in-situ endothelialization of the channels. Representative curves of the storage (G’) and loss (G”) components of the shear modulus (G) were measured for (B) 7.5 wt% gelatin and (C) 7.5 wt% GelMA during cooling and heating temperature sweeps (5 °C min^-1). (D) Representative photographs and (E) measured mass of the VF-3DP structure (10 × 10 × 3 mm, 7.5 wt% gelatin as templating bioink and 7.5 wt% GelMA as matrix bioink) before and after incubation. Arrows in the photographs indicate liquified gelatin, while the mass measurements showed a significant loss in mass due to the exuded templating phase. Representative images of (F) the final VF-3DP structure and (G) a directly-printed lattice comparison (7.5 wt% gelatin) showing both the top and cross-sectional views. Scale bars: 5 mm (D), 500 μm (F-G).

Figure 2. In-situ endothelization.

(A) Representative fluorescence micrographs comparing the conventional post-seeding method with in-situ seeding. RFP-labelled HUVECs (5 × 10⁶ ml^-1) were seeded in a single channel, with the white arrow indicating the direction of post-seeding. The white dotted lines denote the approximate width of the cellularized channel. (B) These images were used to assess the uniformity of cell seeding. Note that the metric of cellularized area refers to the observed cell fluorescence in the region-of-interest of the image (i.e. the channel) rather than a measure of absolute seeding density on the cylindrical channel walls. (C) A DNA assay was used to quantitatively assess the efficiency of post-seeding and in-situ seeding. Data shown as mean ± standard deviation from five samples (two-tailed Mann–Whitney test), p≤0.01 (**). VF-3DP was used to assemble HUVEC-laden structures with (D) 3D lattice channels or (E) 3D parallel channels. (i) Photographs were taken after printing, and bright field microscopy was performed (ii) before and (iii) after incubation at 37 °C. (i) Widefield fluorescence microscopy and (ii) confocal fluorescence microscopy was performed on (F) lattice and (G) parallel designs at day 7. These images indicated the widespread formation of endothelialized channels. (H) High-magnification confocal fluorescence microscopy images of immunostained constructs at day 8 indicated full occupation of CD31-positive HUVECs. (I) Metabolic activity of HUVECs in lattice channels (purple bars) and parallel channels (orange bars). Scale bars: 5 mm (D(i), E(i)), 500 μm (A, D(ii-iii), E(ii-iii), F-G), 100 μm (H).

Figure 3. VF-3DP hydrogel-based microfluidics.

(A) Schematic of hydrogel-based microfluidic system with customized perfusion patterns generated using VF-3DP, with a temporary bioink used to template well-defined channels. Representative (i) photographs and (ii) fluorescence microscopy images of (B) an S-shaped single channel, (C) an S-shaped channel with circular joints, and (D) a 3D lattice channel pattern. (E) Schematic of self-standing perfusable 3D constructs generated using VF-3DP, where both the matrix bioink (yellow) and templating bioink (green) were pre-loaded with Ca²⁺ (87 mM). These doped bioinks were used for (i) the VF-3DP of a lattice structure, which was (ii) dipped into a 1 wt% sodium alginate solution to rapidly form a calcium alginate hydrogel coating. (iii) The two extended ends of the construct could be cut so that the templated bioink could be liquified and released upon incubation. (F) Representative images of the self-standing perfusable construct (i) after alginate coating and (ii) after incubation and dye perfusion. (G-H) (i) Brightfield and (ii) fluorescence microscopy of the self-standing perfusable hydrogel, showing details of the 3D tubular porosity from either (G) top-down or (H) cross-sectional views. (I) Representative images of RFP-labeled HUVECs adhered to the walls of an S-shaped single channel after 12 d of perfusion culture, imaged at (i) low magnification and (ii-iii) high magnification. (J) Representative images of RFP-labeled HUVECs in structures with (i) a circular joint and (ii) lattice channels, imaged after 10 d of perfusion culture. (K) Confocal fluorescence microscopy images of HUVECs after 12 d of perfusion culture. The fluorescence channels show Calcein-AM live stain (green) and RFP-reporter fluorescence (red), with excellent co-localization (yellow). Scale bars: 5 mm (B(i), C(i), D, F), 500 μm (B(ii), C(ii), G, H, I, J), 200 μm (K).