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Using Spheroids to build 3D Bioprinted Tumor Microenvironment
on any commercially available cell culture plate. Among Evidenced by their successful fabrications of micro-
them, agarose is the most widely utilized material due to its molded non-adhesive agarose hydrogels composed of
cost-effectiveness and ease of handling. Agarose could be 822 concave recesses (800 µm deep × 400 µm wide) ,
[78]
simply prepared, sterilized by autoclave, and solidified in this technique is relatively high throughput with effortless
only a few minutes after coating. In addition, polyHEMA scale up capabilities. Spheroid growth becomes easily
is recognized as an effective alternative for spheroid accessible during culture. In contrast to the hanging drop
formation. Briefly, a homogeneous polyHEMA solution method, cell medium change and drug administration
can be obtained by dissolving polyHEMA powder in 95% are more convenient to accomplish without the risk of
ethanol at 65°C overnight, followed by a drying process at disturbing the spheroids using LOT and non-adhesive
room temperature and ethanol evaporation at temperature hydrogel microwells.
up to 37°C. The drying process could take days to weeks
according to different protocols [66-68] . Regardless of the 3.5. External force-driven methods
preparation time, polyHEMA solutions can be stored External forces, such as electromagnetic and acoustic
at 4°C for up to 2 months , whereas agarose solution forces, have been applied in generating spheroids
[68]
cannot be lengthily stored because repeated heating (Figure 2A-e). The utilization of dielectrophoresis
impairs the properties of agarose. (DEP), in the development of multicellular aggregation,
Like agitation-based techniques, spheroids generated was successfully demonstrated and optimized . DEP
[48]
by LOT on flat surfaces are revealed as irregularly shaped functions by producing an external force on a dielectric
and disorganized. Optimizations have been attempted in particle when it is subjected to a non-uniform electric
increasing amenability to high-throughput applications. field, analogous to piezoelectric materials. Cells could
Ivascu et al. had conducted a systematic parametric be guided by dielectrophoretic forces to form clusters;
study about optimal spheroid formation on polyHEMA- however, the clusters could be damaged by mechanical or
coated surface in terms of cell types, cell density, medium hydrodynamic forces after the removal of electric field.
additives, plate type (round bottom and conical shaped Hence, a range of biomaterials, including but not limit
bottom), and the presence of horizontal stirring . Twenty to collagen, PEGDA, agarose, pluronic, and PuraMatrix,
[69]
tumor cell lines of different lineages were examined. have been introduced for immobilizing and stabilizing
A series of medium additives with varied concentrations the cell aggregates [79-81] .
have been systematically screened for optimal spheroid Magnetized cells could be obtained by cell
formation, including reconstituted basement membrane internalization. Magnetic nanoparticles, including
(rBM), collagen type I and type IV, laminin, fibronectin, magnetoferritin, superparamagnetic iron oxide
heparan sulphate proteoglycan and chondroitin sulphate. nanoparticles and its cousin, gold, could be internalized
Interestingly, the results reported the compact spheroid into cell cytoplasm through endocytosis, or by
formation for all the cell lines with the addition of surface functionalization . As an example of surface
[82]
2.5% rBM. In addition to ECM-related components, functionalization, magneto-functionalized cell membrane
methylcellulose is also validated as an additive support was readily achieved with a combination of poly-L-lysine
for compact, unisized spheroid formation . mixed gold or iron oxide nanoparticles . Leveraging this
[70]
[83]
newly embedded magnetic capability, magnetized cells
3.4. Non-adhesive hydrogel microwell could be levitated and agglomerated to form spheroids
Micro-molded microwells using non-adhesive hydrogels when magnets are placed on the culture dish or using a
have been proven as an effective alternative for spheroid magnetic lid [46,82,84] . Spheroids formed with magnetized
formation. Similar to the LOT, non-adhesive materials cells demonstrated a negligible adverse effect on cell
are used in preventing cell adhesion to the bottom of viability while accelerating spheroidization time. Diverse
the microwell. A series of materials have been explored, structures and sizes of 3D cellular aggregates could
such as agarose , polyethylene glycol diacrylate be achieved through the adjustment a magnetic field
[71]
(PEGDA) , and PDMS . Among them, agarose is configuration . Urbanczyk et al. have investigated the
[73]
[85]
[72]
most prevalently used for micro-molding [74-76] . Briefly, a interaction of pancreatic β-cells with vascular endothelial
microfeature mold is fabricated through soft lithography cells in heterotypic pancreatic spheroid models using
or rapid prototyping, a PDMS-negative replicate could magnetic levitation in three different configurations. The
be achieved subsequently. Thereafter, the agarose micro- results identified that human umbilical vein endothelial
mold could be obtained by casting into the PDMS cells (HUVECs), which disassembles from the spheroids
replicate . Micro-molds with an array of various patterns, over time, spontaneously formed spheroids, highlighting
[75]
for instance, flat surface or a conical shape with a rounded this significant role of magnetic levitation. Magnetic
bottom and hemispherical shape, have been demonstrated levitation could enhance the stability of heterotypic
in promoting spheroid formation (Figure 2A-d) [75,77] . spheroids, facilitating HUVEC integration .
[83]
6 International Journal of Bioprinting (2021)–Volume 7, Issue 4