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Using Spheroids to build 3D Bioprinted Tumor Microenvironment
to assist the large-scale tissue block translocation [133,134] . and alginate microparticles were ~97%, 22%, 12%, and
Using polycarbonate track-etched membrane-integrated 34%, respectively. Notably, over a 3-day culture period,
cell culture inserts as the gripper and a peristaltic pump to spheroids cultured in 1.2% Carbopol showed a reduced
generate fluid suction through the nozzle’s membrane, the viability around 74%, while maintaining a 93% survival
nozzle could pick-up, transfer and release the spheroids/ rate in alginate microparticles. Similarly, Daly et al. have
microtissues with minimized damage to the living cells, also printed spheroids in a modified HA-enriched hydrogel
while perfusing the parts in an aqueous environment. As a supporting bath with a ~10% with respect to spheroid size
proof-of-concept, a series of features including spheroids, positional precision [137] . Conclusively, the AAB technique
toroids, and honeycombs with sizes range from 600 µm offered an effective alternative to position spheroids in a
to 3.4 mm was created to validate the capability of the highly reproducible and precise fashion, therefore giving
instrument. A stack of 16 donut ring constructs and 4 rise to reliable and robust 3D in vitro models for disease
honeycombs have been successfully assembled and fused modeling. The overall reported methods for generating
over a 48 h-period for culture to form a single tissue [134] . spheroids were summarized in below Table 1.
In a lateral study, the optimization on the system enabled
the stacking of 20 honeycomb-shaped structures with 5. Progress in establishing 3D tumor models
improved alignment accuracy [133] . Such strategies have via 3D bioprinting
offered an effective alternative for assembling spheroids Conventional 3D models, such as spheroids and
as building blocks, though with limited precision and scaffold-based constructs, offer limited control over
prolonged fabrication period.
cell organization and ascribe poor vascularization. Such
4.5. Aspiration-assisted bioprinting (AAB) shortfalls leave us with oversimplified tumor models,
which are incompetent for understanding tumor biology
Harnessing the strength of aspiration forces, Ayan et al. and fail to predict accurate therapeutic response. In
have developed an AAB technique that enables to pick recent years, 3D bioprinting technology has undergone
and print spheroids with a broader range of sizes from rapid development and evolution [141] . The outstanding
80 µm to 600 µm in a high precision manner. The printer spatial control over cells and materials, coupled with an
was adapted from a low-cost commercial printer and integration of vascular networks into the platform, could
equipped with a custom-made tapered pipette (diameter give rise to higher fidelity 3D tumor models with greatly
~80 µm) [135] . Coupling with conventional micro-valve increased complexity.
printing, the spheroids could be either printed on hydrogel- To date, bioprinting technologies can be categorized
based substrate or without scaffold. To demonstrate the into extrusion-based [142] , inkjet-based [143] , laser-based [144] ,
capability of the printing strategy, spheroids with different and stereolithography techniques [145] . Each technique has
viscoelastic surface tension properties and varied size its own merits and drawbacks and requires bio-inks with
ranges from 200-600 µm were prepared from a wide specific properties [146-148] . Indeed, the critical role of bio-
range of cell types, including HUVECs, mouse fibroblast inks is embedding a mechanical property that regulates
cell line (3T3), mouse mammary carcinoma line (4T1), the cellular response. Particularly, in extrusion-based
human mesenchymal stem cells (MSCs), HUVECs/ bioprinting, the major stumbling block is the imbalance
MSCs, and human dermal fibroblasts. A heterogeneous of printability and the mechanical property of the selected
pyramid construct was printed using spheroids with bio-ink [149] . With the development of FRESH printing,
different sizes and types, indicating that the technique where a semisolid suspension bath is used to print into, the
allows the printing of non-uniform spheroids. The printing resolution of printing intricate hierarchical features such
accuracy was reported to be ~11% with respect to the as vascular networks could be significantly improved.
spheroid size. The printed spheroids exhibited an overall Furthermore, this newfound capability of generating soft
moderate viability over 80%. Further, in combination matrices from low viscous bio-ink provides excitingly
with Freeform Reversible Embedding of Suspended tailorable elicitations of any desired cellular response,
Hydrogels (FRESH) printing, they have further extended thus boosting the cell proliferation [150] . Through the aid of
the versatility of AAB by precisely positioning spheroids bioprinting technologies, a variety of 3D printed models
in self-healing yield-stress hydrogels to achieve more have been created, producing significant advances toward
complicated tissue structures [136] . Both Carbopol with mimicking in vivo tumor structure and cell growth
varied concentrations at 0.8%, 1.2%, 1.6% and 0.5% behavior as summarized in Figure 3. Accumulating
alginate microparticles were investigated for their studies were reported, describing the use of 3D bioprinting
potentiality as supporting bath for spheroids printing using platforms with increased complexity and key features
AAB in terms of positional accuracy and cell viability for mimicking tumor progression in an architecturally
post printing. As demonstrated, the positional accuracy relevant manners, such as tumor heterogeneity [151-154] ,
for 0.8%, 1.2%, and 1.6% concentrations of Carbopol tumor angiogenesis [155-157] , metastasis [158-160] , and anti-

10 International Journal of Bioprinting (2021)–Volume 7, Issue 4