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Microsphere-Based Bioink for Large Tissue with Angiogenesis
2.5. Observation of solation transferring process as 35%. The print environment temperature was set
of TSMs to 24°C and the temperature of receiving platform was
set to 10°C. In terms of 2D or 3D patterns printing, as
As an example, TSM-B was prepared with the TSMs above, a little GFP-GelMA or RFP-GelMA was added
electrosprayed under 3.5 kV voltage and the volume to the GelMA precursor solution. The extruding printing
proportion was set as 35%. A little GFP-labeled GelMA nozzle was 18G and the nozzle movement speed was set
(gelatin from the same batch with roughly the same to 120 mm/min. Other printing parameters were the same
molecular weight) was added to the electrospraying ink as above. 3D models of complex centimeter-scale 3D
as tracer molecules (The gelatin/GFP-GelMA mass ratio structures were built with SolidWorks software and sliced
was 100:1). The prepared TSM-B was poured into a with Repetier software to generate extruding printing path
cylindrical mold (∅9 mm × 6.3 mm) and photocrosslinked G-code. The layer height was set to 1 mm. The structures
for 30 s with a 405 nm blue flashlight. The structures were were captured with the confocal fluorescence microscope
then soaked in the PBS buffer and incubated in a 37°C with the functions of 2D stitching and Z-stack.
incubator, which was shot with confocal fluorescence
microscope after 0 h, 5 h, 10 h, and 24 h, respectively. 2.8. Bioprinting of cell-laden centimeter-scale

2.6. Observation of on-demand pores (nutrient structures
channels) distribution The TSMs with the diameter of 800 μm and 1500 μm

To initially observe the distribution of nutrient channels were prepared with 3.5 kV and 3.0 kV voltage,
respectively. The cell density of GFP-HUVECs
formed by TSM-B, the applied voltage in electrospraying (purchased from Zhongqiaoxinzhou Co, Ltd, Shanghai,
was set as 0 kV, 2 kV, 3 kV, and 3.5 kV (35%) and the China) in the gelatin electrospraying ink and MDA-
volume proportion was set as 50%, 35%, and 20% MB-231s (purchased from Zhongqiaoxinzhou Co, Ltd,
(3.5 kV), respectively. To facilitate the observation of Shanghai, China) in the GelMA precursor solution was
nutrient channel patterns under confocal fluorescence both set as 5 × 10 cells/mL. The volume proportion of
6
microscope, a little GFP-GelMA was added to the TSMs in TSM-B was set as 35%. The concentration of
GelMA precursor solution (The GelMA/GFP-GelMA vascular endothelial growth factor (VEGF) in the GelMA
mass ratio was 100:1). The prepared TSM-B was poured precursor solution was set as 200 ng/mL. The bioprinting
into the same cylindrical mold (∅9 mm × 6.3 mm), parameters were all the same with the ones in the printing
respectively, and photocrosslinked with 405 nm blue of complex structures above. The tissue model was set
flashlight for 30 s. The structures were captured with the as cube (1 cm × 1 cm × 1 cm). The photocrosslinked
confocal fluorescence microscope with the functions of tissues were further cultured with complete Dulbecco’s
two-dimensional (2D) stitching and Z-stack. In terms modified eagle medium supplemented with 10% v/v fetal
of scanning electron microscope (SEM) morphology, bovine serum and 1% v/v penicillin-streptomycin. In the
the casted centimeter-scale 3D structures were soaked experiments of growing state testing of MDA-MB-231s,
into PBS and incubated in a 37°C incubator. After 24 h, the TSMs with 800 μm were applied. The ones with
the samples were rapidly frozen with liquid nitrogen 1500 μm were applied in the bioprinting of breast tumor
and transferred to a vacuum freeze dryer in a vacuum tissue with angiogenesis.
environment at −80°C for 24 h. Then, the freeze-dried
samples were tear with scalpel along the cross sections, 3. Results and discussion
where were treated with a sputtering coating machine.
Finally, the cross sections of the samples were observed 3.1. Preparation and diameter regulation of TSMs
and captured with SEM. The received gelatin microdroplets in silicon oil were

2.7. Printability of TSM-B and centimeter-scale thermo-crosslinked at 4°C, followed by centrifugation
structural establishment to separate the TSMs from silicon oil, as shown in
Figure 2A. Because the microdroplets at the nozzle
The orthogonal experiments of printability were carried tip were affected by gravity, electric field force and
out with TSM-B and GelMA precursor solution. The surface tension as shown in Figure 2D, the diameters
tapered plastic nozzle types were set as 11G, 13G, 16G, of TSMs could be modified by nozzle size and applied
18G, and 20G, respectively. The movement speeds of voltage. To get uniform nutrient channels in the printed
printing nozzle were set as 40 mm/min, 60 mm/min, centimeter-scale structures, according to the previous
80 mm/min, 100 mm/min, 120 mm/min, 140 mm/min, research of our lab [31] , the electrospraying state was
160 mm/min, and 180 mm/min, respectively. The bioink selected as micro dripping state, which would occur in
flow rate was 150 μL/min. The TSMs were prepared relatively lower voltage. The electrospraying process
at 3.5 kV voltage and their volume proportion was set was observed with the high-speed camera, as shown

International Journal of Bioprinting (2022)–Volume 8, Issue 4 19