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Using Spheroids to build 3D Bioprinted Tumor Microenvironment
Using the platform, laser direct-write, Kingsley et al. program used by outputting from slicing software and
had generated size and shape controllable chitosan- setting instructions for 3D printers to move the stage and
shelled alginate structures with human breast cancer printer nozzles in x-, y-, and z-axes along with bio-ink
cells and mouse embryonic stem cells encapsulated, extrusion.
respectively . Adjusting the beam diameter of the laser Finally, the embedded software that runs on the
[51]
enables control over the printed aggregate sizer ranging printer itself takes the G-code and turns it into electrical
from 200 µm and 400 µm. These obtained microbeads signals for running various motors. This is usually done
were further washed in chitosan solution, forming a in C/C++ but could also be written in anything that fits on
core-shell structure that could constrain the aggregate the printer’s hardware.
geometry. Over a 14-day culture period, both cells
showed high cell viability. Notably, both cell types self- 4.1. Direct extrusion-based printing
assembled into 3D aggregates to match the corresponding Swaminathan et al. investigated the bioprinting of pre-
geometry of their printed constructs. Similarly, with the formed breast epithelial spheroids in alginate-based bio-ink
laser-assisted bioprinting, Hakobyan et al. had created co-culture with endothelial cells [120,121] , and demonstrated
3D pancreatic cell spheroid arrays using the AR42J-B-13 that the printed pre-formed spheroids exhibited high cell
rat acinar cell line for studying the initial stages of viability and maintained their spheroid morphology after
pancreatic ductal adenocarcinoma development [118] . The bioprinting, either in monoculture or co-culture with
printed spheroids were observed with a diameter around HUVECs. Moreover, the 3D-printed spheroids were
30-40 µm. Taken together, this nozzle-free, laser-assisted shown to be more resistant to the paclitaxel treatment
method allows spheroid generation with a high resolution as compared to 3D-printed individual cells, highlighting
and density, but rather financially taxing in usage [115] . In that the spheroids have preserved their function after
addition, although extrusion-based bioprinting is not as being extruded. This study has validated the capability
capable in generating droplets, they have been utilized to of printing spheroids directly using extrusion-based
print 3D microtissue inserts [119] , or hanging drippers , bioprinting. To maintain the integrity and avoid spheroid
[40]
availing toward a high-throughput spheroid formation. aggregation in printing cartridge and nozzle clogging, the
4. 3D printing-assisted spheroid assembly size of the spheroids was confined to ~70 µm.
Recently, Horder et al. have interrogated the
Despite the great efforts that have been devoted to interaction between adipose-derived stromal cell (ASC)
3D-printed TME, the progress is limited by many reasons and breast cancer cells in a 3D-printed co-culture
including the incompliant mechanical stiffness of the model [122] . The model was composed of directly printed
bio-inks and thus compromised cell-to-cell, cell-to-ECM ASC spheroids in hyaluronic acid (HA)-rich hydrogel.
interactions. Spheroid is recognized as a physiologically The printability of ASC spheroids (228 ± 22 µm) was
relevant 3D model that could capture the key characteristics assessed using 2 different needle sizes, 250 and 330 µm,
of both healthy and disease tissues. Given the high cell with a corresponding pressure at 5 bar and 1 bar. Printing
density, increased deposition of ECM and accelerated with the 330 µm needle caused 9% damage on the
proliferation rate, spheroids-based model could greatly integrity of the printed spheroids, while the damage and
reduce the tissue maturation time. Such densely packed cell death was dramatically increased to 56% using the
spheroid is thus proposed as building blocks for either 250 µm needle. Over a 21-day differentiation culture, the
healthy or diseased tissue modeling. However, the spheroid printed ASC spheroids showed substantial and sustained
growth and fusion are highly disorganized, which would adipogenesis. Comparable levels of triglyceride, the
ultimately affect the consistency in therapeutic outcomes. expression of both early markers (PPAR and C/EBP)
To impose spatial control and guide the spheroid fusion and late marker (fatty acid-binding protein 4 (FABP4))
and arrangement, several strategies such as direct fusion of adipogenic differentiation, and the secretion of
and magnetic-driven assembly have been explored for adiponectin were demonstrated in both printed and non-
manipulating spheroids. However, major issues, such printed spheroids, indicating that the printing process
as poor positioning resolution, simple structures, and have negligible effects on the cellular differentiation.
the requirement of specialized instruments, are yet to be Evidenced by Oil Red O staining and quantitative analysis
addressed. Given the capability of bioprinting in spatial of intracellular triglycerides, a considerable reduction
control, several 3D printing strategies and its adaptions on lipid content in co-culture constructs was observed
have been investigated for their potentiality in spheroid as compared to ASCs monoculture model. Moreover,
assembly. The software programs for converting digital the immunostaining for the major ECM components
files to real 3D printing can be as complex as AutoCAD collagen I, IV, and VI, laminin, and fibronectin revealed
and SolidWorks or 3ds Max and Maya, or as simple as the ECM remodeling in the co-culture model. These
TinkecCAD or OpenSCAD. G-code is the standard features reflected what was observed in vivo, highlighting

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