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Soman, et al.
method, live cells are distinguished by the presence 2.7. Immunocytochemistry of PN protein
of ubiquitous intracellular esterase activity, which marker
is determined by the enzymatic conversion of the
virtually non-fluorescent cell-permeant Calcein AM to The NSC seeded scaffolds or neural tissue constructs
the intensely fluorescent Calcein. The polyanionic dye were fixed with 4% paraformaldehyde (Sigma)
Calcein is well retained within live cells, producing an for 10 min at room temperature. Fixed cells were
permeabilized using 0.1% TritonX-100 in PBS (Sigma)
intense uniform fluorescence in live cells. Ethidium for 15 min, washed thrice with 0.05% Tween-20/PBS
homodimer-1 dye enters cells with damaged membranes (Sigma), and blocked with 1% bovine serum albumin
and undergoes a 40-fold enhancement of fluorescence for 1 h to avoid non-specific binding. Subsequently, the
on binding to nucleic acids, thereby producing a bright cells were incubated with Rabbit Anti-Neurofilament
red fluorescence in dead cells.
heavy polypeptide (NEFH) antibody (1:50 in 1% BSA
2.5. Cell proliferation on tunicate dECM in PBS, ab8135, Abcam) at room temperature for 1 h.
scaffolds The scaffolds or constructs were washed with PBS for
3 times and incubated with fluorescent labeled secondary
AlamarBlue assay (AlamarBlue HS Cell Viability antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor®
Reagent, Invitrogen, Catalogue number A50101) was used 488, ab150077, Abcam) for 1 h at room temperature,
as a measurement for the determination of cell viability then counterstained with 1 µg/mL of nuclear stain, DAPI
and proliferation. Cell growth was analyzed at different (4′,6-diamidino-2-phenylindole, Sigma). The images
time points: 3, 7, and 12 days. Scaffolds were incubated were taken with a Leica SP8 confocal laser scanning
with 10 µL of AlamarBlue solution per each 100 µL (1:10 microscope and analyzed.
ratio) of media and incubated for 4 h. The AlamarBlue
reaction mix was collected in a 96-well plate and the 2.8. Scanning electron microscopy (SEM)
absorbance was measured at a wavelength of 570 nm with After day 3 of culturing of NSCs on the tunicate dECM
a reference wavelength of 600 nm using a microplate scaffolds, the cell-loaded scaffolds appeared impermeable
reader (Epoch, BioTek). The percentage reduction of the to light; hence, SEM was carried out to get a clear picture
AlamarBlue reagent, which is linear measurement of the of the cell growth and differentiation. For SEM, the
viable cells in the culture was calculated using the online cultured scaffolds were fixed in 4% paraformaldehyde
AlamarBlue colorimetric calculator (Biorad). for 1 h at room temperature and dehydrated with serial
concentration of ethanol ranging from 50%, 70%, and
2.6. mRNA expression of PN markers 100%, then frozen in critical point dryer. The scaffolds

The RNA from the dECM cultured nerve cells and were, then, coated with gold and imaged using a SEM
bioprinted nerve tissues were isolated using Qiaquick (Qanta, Thermo Fisher Scientific).
RNA extraction kit (Qiagen) according to the 2.9. Design of tissue constructs
manufacturer’s instructions. The extracted RNA was
quantified using Nanodrop ND-1000 spectrophotometer Scaffolds were designed and fabricated using RegenHU
(Nanodrop technologies, Wilmington, DE). 1 µg of 3D Discovery printer BioCAD software (RegenHU,
mRNA was reverse transcribed into cDNA using Switzerland). They were designed in a square 8 mm ×
Superscript Vilo IV cDNA Synthesis Kit (Thermo Fisher 8 mm grid pattern with a line spacing of 2 mm and a total
Scientific). Real-time quantitative PCR reactions were thickness of 0.5 mm, with each layer being 0.5 mm thick
carried out in triplicates with 500 ng cDNA template comprising vertical and horizontal struts. Using a built-
per reaction using SYBR master mix (Thermo Fisher in software well plate editor, the toolpath was calibrated
Scientific) in a StepOnePlus Real-Time PCR System to print the constructs in a 24-well plate format. The
(Applied Biosystems). mRNA of neural markers – (i) a tool path was generated and saved as an.iso file in the
pan-neuronal marker which is TUBB3 (β3 tubulin), (ii) BioCAD software.
two PN specific markers, namely, peripherin (PRPH)
and neurofilament heavy polypeptide (NEFH), and (iii) 2.10. Bioink preparation
a stemness marker HNK1 were analyzed in the day 7 and For making the bioink, the decellularized tissue pieces were
day 12 induced samples. The sequences of the forward frozen at −80°C overnight and lyophilized using a Christ
and reverse primers of genes analyzed were adapted from Alpha 1-2 LD Lyophilizer for 48 h. The lyophilized scaffolds
Vijayavenkataraman et al. . The target gene expression were sterilized with ethanol and UV radiation before making
[25]
was normalized to the house keeping gene GAPDH. The the tunicate powder. For making the powder, the dECM
results were expressed as relative mRNA expression tissues were sliced into smaller sections and immersed
compared to the day 3 samples. in liquid nitrogen (~5 mL) in a mortar. The frozen tissue

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