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3D Bioprinting of Human Neural Tissues
and nerve differentiation studies (Figure 3M). NSCs 3D neural outgrowth . Many different factors influence
[1]
were added to the optimized hydrogel formulation cell differentiation and axon protrusion in vitro. Here, we
to make the bioink for neural tissue bioprinting. The modified the neural tissue environment using both physical
formulation followed the same seamless pattern as that and chemical stimuli. The physical environment was
of the cell-free printing and we could bioprint quickly at modified by optimizing the bioink composition by adding
room temperature. specific concentration of Matrigel. Initial experiments
Cross-linking of the printed constructs was of NSC differentiation on dECM scaffolds (Figure 2)
optimized using the combinations of cell-free hydrogel did not require Matrigel coating for the cell growth
controls. Alcian blue was added to the hydrogel to give and differentiation. However, when the bioinks were
clear visibility in the liquid interphase of the cross-linking formulated without Matrigel for bioprinting, the NSCs did
solution. The optimized formulation showed quick and not grow well, contrary to the expectations (Figure 4D-
stable crosslinking while treated with the ionic cross- F). To provide a more cell-friendly environment, Matrigel
linker CaCl . The filament formation of the hydrogel was was then added to the dECM bioink. Matrigel is a
2
consistent on printing within a 250 mM CaCl solution reconstituted basement membrane derived from extracts
2
when compared to the PBS (Figure 3A-F). On addition of Engelbreth-Holm-Swarm mouse tumors. The tumor
of Matrigel, hydrogel with Matrigel showed consistent basement membrane consists of a thin layer of ECM sheets
droplet formation and downward flow compared to the that are primarily made up of type IV collagen, entactin,
hydrogel without Matrigel (Figure 3G and H). heparan sulfate proteoglycans, laminin, and growth factors
For the bioprinting experiments, the tissue constructs to support cell growth [28,29] . Matrigel closely resembles
were designed and fabricated using RegenHU 3D the complex extracellular environment of the basement
Discovery printer (Figure 3I). The toolpath for the tissue membrane, where cells adhere during tissue formation.
construct was also generated (Figure 3J). The design Both the live-dead staining (Figure 4A-C) and AlamarBlue
facilitated the deposition of the bioink in a layer-by-layer cell proliferation assay (Figure 4K) showed highly
fashion with the dimensions of the tissue construct being significant cell growth by day 7 and day 12 post-printing in
8 mm × 8 mm × 1 mm. The pore size of the lattice was the Matrigel-containing bioink. Figure 4J shows the live-
measured as ~900 µm using the digital camera images dead staining image of the whole construct.Figure 4G-I
(Figure 3K). The bioprinted lattice showed uniformity shows the live-dead staining of control hydrogels (without
in the strut size and the pore size as measured in the any cells). From these results, it can be inferred that the
bioprinted tissue constructs (Figure 3L). This study used addition of Matrigel to the tunicate dECM bioink aids better
0.51 mm diameter needle and the number of layers was cell encapsulation and favor enhanced cell adhesion and
set as two, which gives an approximate height of the growth . While the dECM scaffold, without any matrix
[19]
construct as 1 mm. The chosen dimensions facilitated the coating, favored cell adhesion, growth, and differentiation,
mounting of the tissues in a glass slide for imaging and it could be probably due to its inherent porous structure
for the scaling up of the tissue production using specific as the NSCs were directly seeded onto the scaffold,
quantities of the bioink. without subjecting the cells to any undue stress (as with
Cell-laden constructs were printed after adding bioprinting). We hypothesize that the addition of Matrigel
the NSCs to the optimized hydrogel, cross-linked post- might have help to resist the cellular stress generated by the
printing and cultured in vitro. The cross-linked tissue bioprinting procedure, which was evident from the better
constructs stayed soft enough to allow the cellular cell proliferation in the Matrigel containing bioink. The
activities such as adherence, migration, proliferation, and bioprinted tissue showed more cell proliferation compared
differentiation and at the same time possessed enough to the dECM cultured cells due to the presence of Matrigel
post-printing structural stability and stiffness to form a and also due to better exchange of nutrients in all parts of
nerve tissue throughout the in vitro culture period. The the construct than the dECM scaffolds.
method was scaled-up to automate the printing process With 3D bioprinting, it is a difficult task to find the
of printing tissue constructs in 24-well culture plates, bioink formulations that are printable with good post-
expanding the scope of bioprinting to develop disease- printing structural stability and at the same time provide
in-dish models and for making human tissues for the physicochemical cues to meet the biological needs
regenerative medicine applications (Figure 3N). of the cells for differentiation, as these characters of the
3.3. NSC proliferation and PN differentiation bioinks are mutually exclusive with many hydrogels [30-32] .
Most of the high shape fidelity bioinks are highly viscous
The neural growth and building of neural network from and pose difficulty in printing due to nozzle clogging.
the stem cells in vitro require the guidance of axons in an There were difficulties in extruding our bioink with a high
efficient and long-lasting manner. Our experiments proved percentage of Matrigel as a bioink component. Matrigel
that the formulated bioink provide ideal conditions for the containing bioink required more care and optimization

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