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Soman, et al.
as well as to support specific cell populations . Induced ideal bioink provides smooth flow through the nozzles
[4]
pluripotent stem cells (iPSCs) and iPSC-derived stem without any clogging that will reduce the total printing
cells are important cell sources for tissue bioprinting, time and cellular stress.
as these cells can be differentiated to cells of choice The use of cellulose-rich sea squirts (other
when cultured in specific media. The printed tissue can species of tunicates) for developing cardiac patches
be used for regenerative medicine applications to make was explored recently, mainly due to its conductive
tissue transplants such as peripheral nerve conduits, brain nature and fiber orientation [21] . We hypothesized that the
patches, and for neurodegenerative disease modeling . tunicate dECM would support neural tissue engineering
[5]
Specific genetic line iPSCs derived from patients, are as they also are conductive tissues like cardiac
a powerful tool to study diseases such as Parkinson’s patches [22] . Previously, our laboratory has reported
disease, Alzheimer’s disease, and cancer. In the 3D the biocompatibility of tunicate-derived hydrogels
bioprinting field, it has been presumed that the soft tissues for bioprinting mouse embryonic fibroblasts (MEFs)
such as brain and nerves require much optimization as and as a wound dressing material [23] . This work is an
they are difficult to bioprint, compared to the hard tissues, advancement to use the marine tunicate-based bioink
due to the finer variations in the viscoelastic properties of to 3D bioprint NSCs and its differentiation into PNs.
the hydrogels. Many recent research papers have reported The cytocompatibility of the marine tunicate dECM
the necessary conditions for 3D bioprinting neural scaffolds was evaluated by culturing and differentiation
tissues using soft hydrogel-based bioinks . Researchers of the human iPSC-derived NSCs into PN. Further, a
[6]
successfully bioprinted brain-mimicking tissues using bioink using the tunicate dECM powder and Matrigel
primary cortical neurons mixed in a gellan gum-based was formulated and optimized for bioprinting of
bioink modified with the RGD peptide . A recent work NSCs that differentiated in vitro into PNs. The bioink
[7]
attempted to bioprint a model spinal cord using human formulation and bioprinting parameters were optimized
iPSC-derived neural stem cells (NSCs) suspended in an for bioprinting NSCs that proved to be efficient in
alginate-based bioink . However, most of these studies providing a conducive tissue microenvironment
[8]
brought up the difficulty in proliferation of NSCs in for the PN differentiation. Lattice-shaped neural
traditionally-used hydrogels . tissue constructs were bioprinted in a dish and their
[9]
The advent of 3D bioprinting and tissue engineering cellular properties and cold storage potential were
has opened up a new discipline to precisely develop characterized. The neural tissue cultures and constructs
human organ systems in vitro. Essentially, 3D bioprinting were analyzed for cell viability, cell proliferation, and
helps to biofabricate compatible biomaterials into cell differentiation as PNs (Figure 1). The current work
desirable shapes designed with a software. Most of the expands the scope of bioprinting by adopting a novel
bioprinted neural tissues have been generated using sustainable bioink for bioprinting of human NSCs and
extrusion-based methods, laser-assisted printing, inkjet its differentiation into PN for regenerative medicine
printing, drop-on-demand method, microfluidic printing applications and disease modeling.
technology, and point-dispensing printing method [10-12] . 2. Materials and methods
The most common method used for bioprinting neural
tissue is extrusion bioprinting. In this type of bioprinting, 2.1. Cell culture
one or more types of neural cells were mixed and
suspended in a compatible hydrogel, and extruded in iPSC-derived normal human NSCs were purchased
a layer-by-layer fashion according to a digital design, from AddexBio, San Diego, USA (Catalogue number
assisted by pressure, to form a tissue construct [13-15] . The P0005048). The cell culture plates were coated with
6
choice of cells, the formulation of cell-specific bioinks, Matrigel and 1 × 10 cells were seeded onto one well of
and optimized printing parameters are the most important a six-well plate. The cells attached on the plates in 24
topics in bioprinting . It is considered difficult to – 48 h. NSCs were cultured in 5% CO at 37°C with
[16]
2
optimize printing conditions for the soft tissues, due alternate day media changes using NSC Growth Medium
to their mechanosensitive nature . Compared to other (Catalogue number C0013-09, AddexBio).
[7]
types of cells, stem cells are more sensitive to sheer 2.2. Decellularization of tunicate extracellular
stress generated by the bioprinting process . Hence, it matrix (dECM) scaffold
[17]
is essential to formulate bioinks and optimize printing
methods that can protect the cells from the sheer stress Fresh tunicates (Polyclinum constellatum, NCBI
and provide an ideal tissue microenvironment for the cell Accession number MW990087) were collected from
growth and differentiation . When it comes to peripheral the Zayed Port, Abu Dhabi, United Arab Emirates. The
[18]
neurons (PNs), the bioink should allow outgrowth of samples were thoroughly washed with deionized water.
neurites and axons within the printed construct [19,20] . An The outer rough layer of the tunicates was removed

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