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Mao, et al.
3D printing, mainly including the inkjet , extrusion , with calcium chloride (CaCl ) in the inner layer and the
[13]
[14]
2
and vat polymerization-based processes [13-15] , has evolved alginate solution in the outer layer was pulled out of the
as a prevalent technology for producing 3D living tissue coaxial nozzle by the electric force, instant crosslinking
constructs with excellent controllability of geometrical occurred when these bioink solutions met to form the core-
shapes and complex microscale architectures. Especially, sheath filaments. The width of the core and sheath hydrogel
the inkjet or extrusion-based bioprinting approaches have lines could be well modulated by changing the printing
been extensively explored over the past decade for creating parameters such as the feeding rate of collagen solution and
3D vascularized, heterogeneous cell-laden tissue constructs, alginate solution as well as the moving speed of the substrate
owing to their remarkable advantages in wide suitability to platform. Endothelialized lumen structure gradually
various cell-laden bioinks and controllable positioning of formed along with the interface between the core and
the multicellular organization on demand [16-18] . However, as sheath lines when the endothelial cells were encapsulated
a consequence of the jetting or extrusion process through into collagen solution as the inner-layer bioink. 3D porous
the printing nozzle, the cells would experience mechanical hydrogel constructs with a thickness of more than 3 mm
shear force inevitably, which has a negative effect on cell were successfully printed. As a concept of the study, we
viability [19,20] . As smaller nozzles were usually employed encapsulated endothelial cells and H9C2 cells into two
to improve the printing resolution, the cell viability would layers of the coaxial nozzle, respectively, and successfully
further be challenged and clogging might occur when the fabricated 3D pre-vascularized cardiac constructs.
viscous hydrogel was printed.
Electrohydrodynamic (EHD) printing, with inherent 2. Materials and methods
advantages in generating micro/nanoscale droplets or 2.1. Materials and bioinks
filaments, was recently explored to process cells-laden
hydrogel for fabricating functional tissue constructs with Type I collagen derived from rat tail was prepared
high resolution and cell viability [21,22] . It is a novel hybrid followed by previously described protocols and stored at
inkjet printing combined with the electrospinning technique. 4°C. CaCl and sodium hydroxide (NaOH) powder were
2
When the electric field was applied on the printing purchased from Aladdin (Shanghai, China). Agarose
nozzle, the bioink would be jetted out and the cells would powder with low melting temperature (87–89°C) was
experience a very low electrical current which would not bought from Biowest (Spain). Alginate solution with
cause notable damage to the cell integration, viability, and a viscosity of 2000 mPa·S was prepared by dissolving
proliferation [23-25] . Since it was the electric force that pulled alginate powder (Sigma, United Kingdom) into the cell
the bioink through the nozzle, the EHD cell printing process culture medium with 10% (w/v) fetal bovine serum and
enabled to reduce the damage of shear stress on the printed 1% (w/v) antibiotic/antimycotic. 0.1 mol/L NaOH solution
cells which contributed to relatively high cell viability . was prepared by dissolving NaOH powder into phosphate
[26]
However, the nozzle-to-collector distance in these studies buffer saline (PBS) at room temperature. 3% (w/v) CaCl
2
was over 600 μm to decrease the effect of high voltage solution was prepared by dissolving CaCl powder into
2
on cell viability, and the size of the EHD-printed hydrogel tris-buffered saline (TBS) at room temperature. 2%
filaments was larger than 200 μm [27,28] . To further improve (w/v) flat agarose hydrogel was prepared by casting the
the printing resolution, our group recently developed a novel boiling agarose solution with 3% (w/v) CaCl into a petri
2
EHD cell printing process by utilizing an insulating material dish at room temperature. Before the printing process,
to replace the conventional conductive or semiconductive the pH of the collagen solution was adjusted to 7.2-
material as the collecting substrate, which significantly 7.6. GFP expressing human umbilical vein endothelial
reduced the electrical current on the bioink to microamperes cells (GFP-HUVEC; ATCC, USA) and embryonic rat
(<10 μA) . The feature size of the resultant hydrogel cardiomyocytes (H9C2; ATCC, USA) labeled with red
[29]
filament could be smaller than 100 μm while the cell viability cell tracker (Invitrogen, cat. no. C34552) were cultured in
was as high as 95%, implicating a potential strategy to build Dulbecco’s Modified Eagle Medium (DMEM, Thermo)
high-resolution living tissues. One limitation of this EHD for printing cell-laden constructs.
cell printing process is that the height of the EHD-printed 2.2. EHD bioprinting setup
cell/hydrogel constructs was smaller than 145 μm, which
resulted from the limited diffusion of calcium ions from the The EHD bioprinting setup is mainly composed of three
collecting substrate of agarose hydrogel to the deposited key components: A high precision X-Y-Z moving stage
alginate filaments for crosslinking. (Xiamen Heidelstar, China), a voltage generator with high
In this study, we integrated a coaxial nozzle into voltage (ZGF-30/5, Welldone, Shanghai, China), and a
our house-made EHD cell printing system and explored precision syringe pump system (TJ-2A, Longer Pump,
its ability to fabricate thick pre-vascularized cell-laden Baoding, China). The coaxial printing nozzle (19G/26G)
constructs. When the collagen solution supplemented is mounted onto the Z-axis and connected with the high

International Journal of Bioprinting (2021)–Volume 7, Issue 3 87