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Zhuang, et al.
the physiological relevance of the printed co-culture suitable for bio-inks with low viscosity. This method
model. Taken together, extrusion-based printing enables alleviates heat or mechanical damage exerted by the
the direct spheroid printing without any modification to bioprinting nozzle on the cells/spheroids, enabling
8
the printing setup. However, the resolution of extrusion- printing with high cell densities (>10 cells/mL).
based printing is needle size-dependent, thus limiting the
size and density of the spheroids. The integrity of large 4.3. Kenzan method
spheroids will be compromised, and the high spheroid Proposed by Prof. Koich Nakayama, Kenzan method has
density is liable to induce nozzle blockage. been frequently used in constructing tissue models with
Interestingly, utilizing a capillary micropipette with scaffold-free bio-inks. Kenzan, which is also referred to as
a defined diameter at 300 or 500 µm in bio-printer, Jakab a microneedle-based method, using stainless-steel needle
et al. have successfully delivered multicellular spheroids arrays that function as temporary support for spheroids
to collagen type I substrate and formed certain structures, and allow the in-situ fusion of the spheroids to form a
such as ring, sheets, and cylinders [123] . Interestingly, macro-tissue [127] . The spheroids are picked up by a mobile
the spheroids were formed by a rapid centrifugation, nozzle arm from well plates and moved on the top of the
incubation, and a cutting process to secure the size microneedle array. By switching the negative pneumatic
consistency in obtained spheroids. The spheroids were pressure to slightly positive, the spheroids are released
then aspirated into a capillary micropipette as a printing into the substrate. The process is repeated until the entire
cartridge and extruded from the cartridge through the construct is completed and left on the microneedle array
positive displacement of a piston within the micropipette. for continuous culture.
This printing technique was subsequently applied to Upon fusion, the needle arrays are retracted, and the
engineer vessels of distinct shapes and hierarchical obtained tissue could be perfused and cultured for further
trees with diameters spanning from 900 µm to 2.5 mm. maturation [128,129] . Kenzan method has found its application
Agarose was used as temporary support to facilitate the in many tissues, including blood vessel, tracheal, heart,
construction of hanging features. The deposited discrete liver, and urinary bladder [130] . In a seminal study, van Pel
spheroids underwent post-printing fusion and formed et al. investigated glioma cell invasion into neural-like
single-layer and double-layer tubular structures. Notably, tissues using Kenzan method [131] . Eight neurospheres
with the adapted capillary micropipette as printing formed from induced pluripotent stem cells (iPSC)-derived
cartridge, this scaffold-free approach circumvents some human neural progenitor cells were robotically placed in
shortcomings associated with exogeneous biomaterials the micro-needle arrays and cultured for 3 weeks for fusion
and provides much better control over the spheroid and maturation into a neural organoid. U118 human glioma
geometry and position, therefore greatly improving the cell spheroids were subsequently printed on the top of the
reproducibility and scalability as compared to the non- neural organoid and cultured for up to 4 weeks. Revealed
adapted extrusion-based printing. by cryosectioning and confocal imaging, GFP U118 cells
+
were found within the human neural organoid, which
4.2. Droplet-based bioprinting confirmed the glioma cell invasion. However, no gliosis was
Apart from extrusion-based printing, other bioprinting observed surrounding the tumor or invading cells, which
modalities such as microvalve-based printing [124] , laser- was different from the previous observations. In summary,
assisted printing [125] , and acoustic printing [126] have also the Kenzan method has greatly facilitated the scaffold-free
been explored for their capability in printing spheroid fabrication from various cell types into complex structures,
aggregates. By adopting an open cartridge, Chen et al. particular tubular constructs but the fixed distance between
introduced a nozzle-free, contact-free acoustic-driven needles (~400 µm) requires the size-consistent spheroids
bioprinting that allows both cell and spheroid ejection [126] . with a diameter approximate to ~600 µm to ensure their
Cell encapsulating GelMA droplets were ejected onto direct contact with one another [132] , which significantly
a receiving substrate in a pre-designed arrangement, restrains the size of usable spheroids and highly relys on
followed by UV crosslinking that stabilizes the structure. spheroid preparation, especially when a large quantity of
The authors had generated a co-culture TME with the spheroids is needed. Besides, the mechanical interruption
tumor spheroid in the central zone and the CAFs in the could induce the structural damage to the spheroids,
periphery. Over a 7-day culture period, increased tumor particularly the smaller ones.
spheroid invasion area and distance was observed in co- 4.4. Gripper and manipulator
culture model as compared to monoculture, suggesting
that CAFs may promote morphological changes within In addition, gripper or micromanipulator has also been
tumor cells. Such a nozzle-free printing approach holds introduced to assemble the spheroids. Notably, inspired
great potential for constructing tissue models with low by electronics manufacturing, an instrument named
cell damage, although the resolution is limited and only Bio-Pick, Place, and Perfuse (Bio-P3) was introduced

International Journal of Bioprinting (2021)–Volume 7, Issue 4 9