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3D Bioprinting of Human Neural Tissues
sections were pulverized into fine powder using a mortar The printer and the biosafety cabinets were sterilized
and a pestle. After lyophilization, the ECM powder was under UV light for 1 h before printing. Bioink containing
mixed with pepsin enzyme in a ratio of 10:1 w/w/100 mL NSCs were loaded in a 3 mL sterile syringe and connected
0.01 N HCl. The solution was digested for 48 h at room to the air pressure supply. A needle with 0.51 mm inner
temperature under constant stirring using a magnetic stir bar diameter was used for the printing (Needle DD-135N
and plate until the solution becomes viscous with no visibly ID=0.51/G21 L=25.4, RegenHU, Switzerland). Print
undigested granules. Then, 10 mg/mL of digested ECM parameters were adjusted to obtain continuous flow rate
solution was aliquoted and frozen at −80°C to terminate and smooth hydrogel fibers with minimal spreading.
pepsin digestion. Further, the digested ECM solution was A feed rate of 2 mm/s and pressure of 0.3-0.4 MPa were
mixed well and dialyzed against water at 4°C for 72 h. used, the total print time was under 30 min per one 24-
Finally, the obtained ECM powder was freeze-dried and well plate. The printability of the bioink was assessed
lyophilized for further use. All the buffer components and by switching on the pressure and the filament formation
chemicals used for ECM powder preparation were from at the tip of the needle. The needle diameter, pneumatic
Sigma-Aldrich, USA. The tunicate powder was sterilized pressure, and nozzle moving speed were optimized
using UV irradiation for 2 h before preparing the base to deliver continuous extrusion of the bioink in the
hydrogel. The base hydrogel for bioprinting was prepared designated well of the well plate.
by slowly adding NSC media to 100 mg of tunicate powder
to make a final volume of 1 mL. The hydrogel concentration 2.13. Cross-linking of the printed tissue
was optimized for NSC bioprinting by adding different constructs and tissue culture
concentrations of Matrigel (Matrigel hESC-qualified
Matrix, Catalogue number 354277, Corning) starting from For optimization of printing and crosslinking, the cell-
50%, 35%, 31%, and 26%. The higher concentrations of free control hydrogel filaments were immersed in a cross-
Matrigel made the hydrogel more viscous and did not allow linking solution and PBS to check the strength of the
printing. About 26% of the Matrigel in the base hydrogel filament formation. Immersion in 250 mM of CaCl could
2
make a smooth filament of cross-linked hydrogel at room
was found to be optimal and facilitated smooth printing of
the cell-free scaffolds. Therefore, this formulation was used temperature. We have added the blue stain Alcian blue
to make the bioink for cell printing. A bioink containing to the control hydrogels for better visibility. The same
26% Matrigel, 10% tunicate powder, and 0.1 mL of the NSC concentration of the CaCl solution was used to crosslink
2
suspension containing 7.58 × 10 cells were formulated (a the cell containing tissue constructs after bioprinting. The
6
total volume of 1 mL), which could print 20 tissue constructs cross-linker solution was removed after 5 – 10 min of
in a 24-well plate. Each of the constructs consumed ~50 µL incubation at room temperature and the constructs were
of the bioink, with ~4 × 10 NSCs. Matrigel was kept at washed with prewarmed PBS. After washing with PBS,
5
4°C before being added to the media, as the pure Matrigel the cell-laden constructs were incubated in nutrient-rich
solidifies in higher temperatures. The preparation of bioinks NSC media containing 10% fetal bovine serum at 37°C
was carried out in a biosafety cabinet at room temperature in 5% CO . The nutrient rich media was changed to NSC
2
within 15 min before printing. medium after 15 min.
2.11. Rheology and printability 2.14. PN differentiation of bioprinted tissue
ElastoSens Bio 2 (Rheometer) was used to justify constructs
the rheology properties of the hydrogel as well as the Bioprinted constructs were initially cultured in the NSC
printability of layered structures under room temperature. media for 5 days; once the cells adapted to the new 3D
A minimum volume of 4ml of hydrogel was used for environment, they were induced with PN media for
the test. The test lasted a total of 120 min. The test was differentiation to PN. The tissue constructs were analyzed
performed under room temperature (22°C) to mimic the for cell viability and cell proliferation as described for the
3D bioprinting condition. About 10% tunicate hydrogel dECM scaffolds. The tissue constructs were analyzed for
and 26% Matrigel in NSC media were used to make the PN-specific marker expression by immunofluorescence
hydrogel. Shear storage modulus and shear loss modulus and qPCR with the same procedure used for the dECM
over time were obtained. The characterized hydrogel was scaffolds. SEM was done to see the tissue construct
used to print lattice structures with the layers ranging morphology after culturing and differentiation.
from one to ten.
2.15. Viability and proliferation of the freeze-
2.12. 3D bioprinting thawed dECM-grown and bioprinted PN

RegenHU 3D discovery bioprinter inside a biosafety On day 12 of induction, the differentiated PN grown on
cabinet at room temperature were used for bioprinting. the dECM scaffolds and bioprinted tissues were washed

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