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Coaxial nozzle-assisted electrohydrodynamic printing for microscale 3D cell-laden constructs

assisted electrohydrodynamic cell printing process was Alginate solution and calcium chloride solution were
further presented aiming to fabricate 3D cell-laden loaded into two 1 mL syringes which were separately
constructs with high resolution and high cell viability. controlled by the syringe pump. A coaxial printing
The printed alginate filament can be instantly crosslinked nozzle was mounted onto the z-axis and connected with
by the calcium chloride solution flowed from the coaxial the positive terminal of a high-voltage generator. The
nozzle during the 3D electrohydrodynamic printing core inlet of the nozzle was connected with the syringe
process. loaded with alginate solution while the sheath inlet of
2. Materials and Methods the nozzle was connected with the syringe loaded with
calcium chloride solution respectively via soft tubes
as shown in Figure 1. Insulating petri dish and agarose
2.1 Materials hydrogel with calcium ions were sequentially placed
Alginate with medium viscosity (2000 mPa·s) was on the grounded x-y moving stage as the collecting
purchased from Sigma (United Kingdom). Calcium substrate. The distance between the coaxial nozzle and
chloride powder was bought from Aladdin (Shanghai, the collecting substrate was fixed at 200 μm.
China). Agarose powder with low melting temperature To initialize the electrohydrodynamic printing process,
(87–89 °C) was bought from Biowest (Spain). 3% (w/ high voltage was applied and the syringe pump was
v) alginate solution was prepared by dissolving alginate opened to simultaneously feed alginate solution and
powder into phosphate buffer saline (PBS). 2% (w/v) calcium chloride solution into the coaxial nozzle. The
agarose solution with 3% (w/v) calcium chloride was electrohydrodynamically printed alginate solution was
prepared by dissolving agarose and calcium chloride instantly crosslinked in contact with calcium chloride
powders in tris-buffered saline (TBS) at 100 °C. 1% (w/ solution to form hydrogel filaments and deposit onto the
v) calcium chloride solution was prepared by dissolving collecting substrate. The deposition of alginate hydrogel
calcium chloride powder into TBS at room temperature. filaments could be flexibly controlled to fabricate
Flat agarose hydrogel with a thickness of 3 mm was complex patterns by directing the movement of x-y
prepared by casting agarose solution in a petri dish. For stage according to a user-specific design. A 3D hydrogel
electrohydrodynamic cell printing, rat myocardial cell structure can be further electrohydrodynamically printed
lines (H9C2, ATCC) were added into alginate solution by precisely stacking the alginate filaments in a layer-by-
-1
6
with a density of 1×10 cells mL . layer manner.
2.2 Coaxial Nozzle-Assisted Elec tro hy dro dy-
nam ic Cell Printing Platform 2.3 Effect of Applied Voltage on the Width of
A house-made electrohydrodynamic printing platform the Printed Filaments
was used which mainly consists of three components: a To tightly attach the printed hydrogel filament to the
high-voltage generator (ZGF-30/5, Welldone, Shanghai, collecting substrate, calcium chloride solution was not
China), a multi-channel syringe pump system (TJ-2A, supplied in the printing of the first three layers. The
Longer Pump, Baoding, China) and a high-resolution electrohydrodynamically printed alginate filament was
x-y-z movement stage (Xiamen Heidelstar Co., China). crosslinked by the calcium ions in agarose hydrogel.

Figure 1. Schematic of coaxial nozzle-assisted electrohydrodynamic printing for microscale cell-laden constructs

2 International Journal of Bioprinting (2018)–Volume 4, Issue 1