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Microsphere-Based Bioink for Large Tissue with Angiogenesis
confirmed the potential of the bioink in further building ones in the structures without nutrient channels showed
centimeter-scale tissue with rich nutrient channels and to be spherical shape or beginning to crack, as shown in
angiogenesis. Figure 6C. The results above verified the capability the
TSM-B to provide promising growing environment and
3.6. Growth of porous centimeter-scale breast sufficient nutrient channels for the encapsulated subject
tumor tissue printed by TSM-B cells.
During the gradual deterioration process of tumors 3.7. Vascularization of endothelial cells in a
in organisms, their size will gradually increase to the centimeter-scale 3D structure
centimeter scale. Meanwhile, in such a large tissue,
rich 3D blood vessels will spontaneously form under To demonstrate the ability of TSM-B to form 3D
appropriate induction condition such as VEGF [37,38] to vessels spontaneously, GFP-HUVECs-loaded TSMs
provide adequate nutrients and oxygen for the growth of were electrosprayed under the voltage of 3.5 kV and
tumor cells. Therefore, in the related research of tumor 3 kV, respectively, with diameters of approximately
tissue, the large scale tumor tissue with 3D vascular 800 μm and 1500 μm. With the prepared TSMs, two
networks constructed in vitro owns much application kinds of TSM-B were prepared, respectively, with
value. The validity of the established centimeter-scale GelMA precursor solution (containing VEGF), with
tumor tissue with angiogenesis is mainly reflected by which cubic tissue at centimeter scale (1 cm × 1 cm ×
two factors: the normal growth of the subject tumor cells 1 cm) was printed to examine the effect of angiogenesis.
and the vascularization of vessel cells. Firstly, GelMA From the images captured by fluorescence microscope,
precursor solution (containing VEGF) mixed with at the start of culture, GFP-HUVECs were initially
human breast tumor cells (MDA-MB-231s) was applied distributed in the area of TSMs, as shown in Figure 7A.
to prepare TSM-B, with which cubic tumor tissue at After culture, from the images shown in Figure 7C,
centimeter scale (1 cm × 1 cm × 1 cm) was printed to GFP-HUVECs gradually settled and adhered to the
examine the effect of nutrient channels on the growth of inner surfaces of the nutrient channels with the solation
tumor cells. As a control, the GelMA precursor solution transferring of TSMs. On the 2 day of culture, the
nd
(containing VEGF) without TSMs was printed in the attached GFP-HUVECs in the structures with 1500 μm
same way as the centimeter-scale tumor tissue without nutrient channels began to show the sign of 3D sprout
nutrient channel. under the induction of VEGF. On the 3 day of
rd
On the 1 , 3 , and 5 days of culture, the survival culture, it could be found that GFP-HUVECs in porous
rd
st
th
of MDA-MB-231s in the centimeter-scale 3D structures structures with the two nutrient channel sizes began
were tested with Live/Dead cell staining kits. It is worth to form 3D sprout into the GelMA hydrogel. On the
emphasizing that because nutrient/oxygen supply at the 5 day of culture, the fluorescence microscope images
th
center cross section of the centimeter-scale 3D structure showed that GFP-HUVECs further formed longer 3D
would be the least adequate, the cell viability in the sprout structures into the GelMA hydrogel and began to
region can directly reflect the effectiveness of the printed exhibit angiogenesis in the 3D environment, as shown
tissue. Thus, the stained cells there were observed and in Figure 7B.
captured with confocal fluorescence microscope and the Besides, β-tubulin proteins of the loaded GFP-
viability was analyzed with ImageJ software, as shown HUVECs were stained and observed by confocal
in Figure 6A. The results showed that MDA-MB-231s fluorescence microscope. As shown in Figure 8C,
in the centimeter-scale 3D structure without nutrient GFP-HUVECs showed obvious 3D sprout in the
channels died from lack of nutrient and oxygen after centimeter-scale 3D structures with both nutrient
1-day culture, while the ones in the structures printed with channel sizes. It is worth noting that, since the GFP-
TSM-B maintained the viability of more than 95% during HUVECs density in the electrospraying ink was the
5-day culture benefiting from the rich nutrient channels, same, the cell attaching density on the inner surface
as shown in Figure 6B and D. Furthermore, the cell would be different. The attaching density obtained
counting kit-8 (CCK-8) results of the loaded MDA-MB- by 800 μm and 1500 μm TSMs was found to be
231s showed that TSM-B could ensure the proliferation of 1333.33 cells/mm and 2500 cells/mm respectively
2
2
the encapsulated cell, as shown in Figure 6E. In addition, (refers to the additional discussion in Supplementary
the loaded F-actin and nucleus of MDA-MB-231s were File). Thus, more immense angiogenesis would
stained by phalloidin and DAPI (4d,6-diamidino-2- happen at higher attaching density of endothelial
phenylindole) to observe the cell morphology. The results cells. What is more, CD31, which is a kind of marker
showed that during the 5-day culture, most of the MDA- protein of HUVECs, could normally express, as
MB-231s in the centimeter-scale 3D structures with shown in Figure 8A. The connection between GelMA
nutrient channels were spreading and migrating, while the hydrogel and cell membrane was established, which

International Journal of Bioprinting (2022)–Volume 8, Issue 4 25