Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels

Abstract

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Figure 1

Bioprinter setup for direct-write printing of cell-laden GelMA hydrogels. A) Photograph of NovoGen MMX Bioprinte™ (Organovo) showing the gel and cell-dispensing capillaries mounted on an X-Z motorized stage. B) To print the hydrogel fibers, a metallic piston fitted inside a glass capillary is immersed in a vial containing the cells and the hydrogel precursor. C) The upward movement of the metallic piston aspirates the cell-laden hydrogel precursor, which is subsequently crosslinked by exposure to light. D) Next, the coordinated motion of the motorized stage enables precise printing of cell-laden GelMA hydrogel fibers.

Figure 2

Printability of GelMA hydrogels as a function of concentration, UV light exposure time, and cell density. A) Printability of cell-free GelMA hydrogels at concentrations ranging from 5 to 15%, photocrosslinked from 10 to 60 s. B) Printability of 10% HepG2-laden GelMA, photocrosslinked from 10 to 60 s (n=9).

Figure 3

Mechanical properties of GelMA hydrogels as a function of concentration and UV light exposure time. A) Representative stress vs. strain curves for 5% GelMA hydrogels at different UV light exposure times. B) Elastic modulus of GelMA hydrogels increased proportionally with an increase in polymer concentration and UV light exposure time (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001). Results suggest that printability is improved for hydrogels presenting higher stiffness, as illustrated by the dashed line representing the lower threshold for successful printing (n=6). (Statistical analyses comparing hydrogels of different concentrations are show in Figure S1).

Figure 4

Interfacial properties of GelMA hydrogels as extruded from a glass capillary during the bioprinting process. A) Representative load vs. displacement curves for 15% GelMA hydrogels extruded at a rate of 2 mm/s. B) Maximum load for debonding hydrogels from the glass capillary, representative of force required to initiate bioprinting (n=6). Stars indicate significant difference against the control group (*p<0.05, **p<0.001, ***p<0.001, ****p<0.0001). The dashed line represents the lower threshold for successful printing. (Statistical analyses comparing the effect of UV light exposure time within hydrogel concentrations are shown in Figure S2)

Figure 5

Interfacial properties of cell-laden GelMA hydrogels as extruded from a glass capillary during the bioprinting process. A) Representative load vs. displacement curve for cell-laden 10% GelMA hydrogels extruded at a rate of 2 mm/s. B) Maximum load to debond cell-laden hydrogels from the glass capillary (n=6).

Figure 6

Elastic modulus of GelMA hydrogels as a function of maximum load at debond (irrespective of gel concentration and UV light exposure times) representing the respective threshold for consistent printing. Gels with elastic modulus values above 2.6 kPa and maximum load at debond above 0.53 N were reproducibly printed.

Figure 7

Different architectures bioprinted with cell-laden GelMA hydrogels. A) Fluorescence image of F-actin/DAPI stained 2-layered lattice architecture bioprinted with HepG2-laden GelMA hydrogels. B) Representative brightfield image of lattice architecture shown in (A). C) Cross-section images of 5-layered stacked lines of NIS3T3 cell-laden hydrogels containing 0, 1 and 5 microchannels (left to right). D) Photograph of hydrogel array bioprinted with a HepG2-laden GelMA. E) Photograph of MIT logo bioprinted with fluorescent microbead-laden GelMA hydrogel fibers and actual MIT logo, for comparison (inset). F-G) Photograph o bioprinted agarose hydrogel fibers replicating 3D branching networks embedded in GelMA hydrogel blocks. H) Cross-section fluorescence image of microbead-laden hollow GelMA hydrogel fibers. I) Longitudinal view of hollow fibers perfused with a red fluorescent dy. J-L) Higher magnification of cross-sectional view of constructs shown in (C) stained for live and dead cells with 0 (J), 1 (K) and 5 (L) microchannels, respectively. The viability data for figures supplementary information (Figure S3).

Figure 8

Viability of bioprinted HepG2-laden 10% GelMA hydrogels at different exposure times. Representative Live/Dead images from day 8 illustrating high HepG2 viability following (A) 15, (B) 30 and (C) 60 s of UV light exposure. D) Quantitative data for cell viability in bioprinted dell-laden hydrogels at different UV light exposure times (**p<0.01; ****p<0.0001).