Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Abstract

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

A General Strategy for Extrusion Bioprinting of Bio‐Macromolecular Bioinks through Alginate‐Templated Dual‐Stage Crosslinking

“…Meanwhile, as the crosslinked alginate was gradually dissociated and leached out of the bioprinted construct, the diameter of the remaining GelMA/alginate composite hydrogel microfibers increased due to swelling (Figure e). The diameters of the microfibers increased from 313 ± 25 µm at day 0 (immediately following bioprinting/photo‐crosslinking) to 437 ± 38 µm at day 1 and 509 ± 70 µm at day 3, consistent with the literature . Accompanying the swelling, the removal of the alginate could also induce the enlargement of the pores of the remaining hydrogel microfibers, potentially leading to improved behaviors of the encapsulated cells, as we previously reported …”

“…Of note, this microfluidic bioprinting procedure has been demonstrated benign to the encapsulated cells . As revealed in Figure f‐i, the viability of the HepG2/C3A cells was maintained at a level of >85% post‐bioprinting analyzed from semiquantitative calculations based on live/dead staining, suggesting the biocompatibility of the CaCl 2 crosslinker at the selected concentration, as well as the minimal damage exerted by the shear stress during the extrusion of the cell‐laden bioink.…”

“…It should be noted that this strategy is general, and can be possibly applied for bioprinting of a wide variety of bio‐macromolecular bioinks. Specifically, we first reproduced our previous reports in using such a strategy for bioprinting microfibrous GelMA‐based constructs to illustrate the entire microfluidic bioprintingenabled alginate‐templating process, and then extended it to the use of gelatin and collagen as the bio‐macromolecular bioinks demonstrated in this work.…”

“…Here, we have generalized a mechanism for extrusion bioprinting of bio‐macromolecular bioinks. Specifically, we adopted a templating strategy using a composite bioink containing both the desired bio‐macromolecular component and a nonhuman polysaccharide (i.e., alginate), generalized from our previously reported microfluidic bioprinting method . As such, the physically crosslinkable alginate component, achieved by co‐delivered calcium chloride (CaCl 2 ) solution in the sheath of the printhead, serves as the temporal structural support to stabilize the shape of the construct during the bioprinting procedure.…”

“…Figure 1 shows the general schematics of the microfluidic bioprinting‐based templating method, from both macroscopic and microscopic views, for extrusion of the composite bioink containing an ECM‐derived bio‐macromolecule and the polysaccharide alginate (Figure a). To perform the microfluidic bioprinting, a custom nozzle made of two concentric needles in a core–sheath structure was fabricated and fitted onto the printhead of a bioprinter (Figure b) . In a typical process, the bioink is first extruded from the inner needle of the nozzle to produce a microfibrous structure of any desired shape and architecture, where the alginate component of this composite bioink is immediately physically crosslinked by the sheath CaCl 2 solution co‐delivered from the outer needle, until a desired scaffold is bioprinted (Figure c).…”