Methods/Protocols

Cite This: ACS Biomater. Sci. Eng. 2019, 5, 234−243 pubs.acs.org/journal/abseba

3D Printing of Neural Tissues Derived from Human Induced

Pluripotent Stem Cells Using a Fibrin-Based Bioink
Emily Abelseth,† Laila Abelseth,† Laura De la Vega,‡ Simon T. Beyer,§ Samuel J. Wadsworth,§
and Stephanie M. Willerth*,‡,⊥
†
Biomedical Engineering Program, ‡Department of Mechanical Engineering, and ⊥Division of Medical Sciences, University of
Victoria, 3800 Finnerty Road, Victoria, British Columbia V8W 2Y2, Canada
§
Aspect Biosystems, 1781 W 75th Avenue, Vancouver, British Columbia V6P 6P2, Canada
*
S Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: 3D bioprinting offers the opportunity to automate

the process of tissue engineering, which combines biomaterial
Downloaded via CORNELL UNIV on September 3, 2020 at 13:22:59 (UTC).

scaffolds and cells to generate substitutes for diseased or damaged

tissues. These bioprinting methods construct tissue replacements
by positioning cells encapsulated in bioinks into specific locations
in the resulting constructs. Human induced pluripotent stem cells
(hiPSCs) serve as an important tool when engineering neural
tissues. These cells can be expanded indefinitely and differentiated
into the cell types found in the central nervous systems, including
neurons. One common method for differentiating hiPSCs into
neural tissue requires the formation of aggregates inside of defined
diameter microwells cultured in chemically defined media.
However, 3D bioprinting of such hiPSC-derived aggregates has
not been previously reported in the literature, as it requires the
development of specialized bioinks for supporting cell survival and differentiation into mature neural phenotypes. Here we detail
methods including preparing base material components of the bioink, producing the bioink, and the steps involved in printing
3D neural tissues derived from hiPSC-derived neural aggregates using Aspect Biosystems’ novel RX1 printer and their lab-on-a-
printer (LOP) technology.
KEYWORDS: biomaterials, regenerative medicine, additive manufacturing, 3D printing

■ INTRODUCTION
3D bioprinting offers an innovative method for engineering
tissues from stem cells.1 In particular, human induced
pluripotent stem cells (hiPSCs) serve as an important tool
for engineering tissue because of their ability to form any type
of tissue found in the human body.2 Development of printable
bioinks that support the differentiation of stem cells into the
appropriate phenotypes based on the target tissue remains
challenging.3,4 Popular forms of bioprinting include fused
deposition modeling (FDM), inkjet bioprinting, bioplotting,
and microfluidic extrusion.5 Table 1 details the benefits and
deficiencies of each of these printing methods. FDM deposits
melted thermoplastic in a layer-by-layer manner to form a
construct. However, this inexpensive method possesses many
limitations, including its accuracy and potential printable
geometries. It is often applied for 3D printing of bone and
cartilage tissues because of the nature of the thermoplastic inks
used.5 However, Hseih et al. printed a suspension of neural
stem cells using a polyurethane bioink.6 Inkjet bioprinting uses
a modified inkjet printer with a small nozzle size to generate
tissue constructs from bioinks, making it a simpler alternative
to FDM. Accordingly, the bioink must have low viscosity,
reducing the mechanical properties of the printed scaffold.5
Furthermore, inkjet bioprinting subjects cells to high amounts
of shear stress when they are expelled from the nozzle, which
can result in cell death.5 Inkjet printing of suspensions of
hippocampal and cortical neurons resulted in a cell viability of
74.2 ± 6.3% after 8 days in culture.7 Bioplotting utilizes
syringes, which contain high viscosity materials to maintain the
shape of the scaffold before cross-linking using one of the
following methods: radiation, chemical cross-linking, or
solidification.5 The need for high viscosity bioinks causes the
resulting bioplotted scaffolds to have mechanical properties
unsuitable for neural tissue.5 However, cortical neural stem
cells were printed in an alginate, carboxymethyl-chitosan,
agarose bioink and proliferated for 10 day with spontaneous
activity.8 Finally, microfluidic extrusion uses microscale
channels to extrude a bioink and cross-linker in tandem,
allowing the bioink to polymerize and form a printable
hydrogel while exiting the nozzle. A simple hand-held
Received: October 9, 2018
Accepted: November 26, 2018
Published: November 26, 2018

© 2018 American Chemical Society 234 DOI: 10.1021/acsbiomaterials.8b01235

ACS Biomater. Sci. Eng. 2019, 5, 234−243
ACS Biomaterials Science & Engineering
Table 1. Comparison of Bioprinting Methods Summarizing Information from Ref
printing method benefits
fused deposition inexpensive
modeling
inkjet bioprinting simple process for printing multiple cell types and useful for printing
thin tissues
bioplotting can print cocultured scaffolds
microfluidic extrusion multiple cell types can be printed and offers protection from shear
stress when extruded
microfluidic device printed a single cell suspension of primary
cortical neurons in a gellan gum modified with RGD peptide,
with cells remaining viable after 5 days.9 Although many types
of bioprinters and bioinks have been used to generate neural
tissue,5 room remains to optimize these bioinks in terms of
promoting neuronal differentiation.
Fibrin, a protein found in circulating blood, becomes
activated during coagulation to enable the formation of
blood clots.10 It serves as a popular biomaterial for tissue
engineering applications, forming hydrogels that readily
support stem cell culture and differentiation.11−15 The Willerth
lab has done extensive work in optimizing fibrin formulations
for promoting pluripotent stem cells to form neural
tissues.16−20 As mentioned earlier, hiPSCs can become any
cell type found in the body, making it imperative to expose
them to a microenvironment conducive to neural differ-
entiation. In particular, protein concentration and hydrogel
stiffness play important roles in ensuring hiPSC survival and
differentiation inside of fibrin scaffolds.18−20 Also, using the
protein cross-linking reagent genipin improves the stability of
fibrin while promoting neuronal differentiation of hiPSCs.20
Here we translate our protocols for engineering 3D fibrin
scaffolds that generated neural tissue from hiPSCs into a
printable bioink formulation due to these desirable properties.
Our novel bioink consists of a fibrinogen base supplemented
with alginate, which then is cross-linked by a mixture of
chitosan, calcium chloride, thrombin, and genipin to ensure
printability and stability. Alginate, a low-cost and low-
cytotoxicity polymer, quickly polymerizes in the presence of
divalent cations. It has already been validated for encapsulating
neural stem cells.21 Our bioink is printed alongside a cross-
linker composed of calcium chloride (CaCl2), chitosan, and
thrombin to form a printable fibers. Thrombin cleaves
fibrinogen, initiating the polymerization process. Fibrin
monomers aggregate, forming protofibrils. Ca2+ ions interact
with binding sites, promoting stability and polymerization of
fibrin by increasing the rate and extent of lateral aggregation of
protofibrils.22 Ca2+ ions also polymerize alginate by binding to
guluronate (G) blocks, which in turn bind to surrounding G
blocks and create a cross-linked network. Chitosan and fibrin
are chemically cross-linked by genipin, which cross-links the
amine groups.19 See Figure 2 for graphical representation of
this process.
A variety of commercially available bioprinters exist, ranging
from low-end systems limited in the number of materials and
cells types for printing to high-end sophisticated systems for
printing complex systems.23 Aspect Biosystems’ novel RX1
bioprinter uses lab-on-a-printer (LOP) technology to engineer
tissues in a rapid and reproducible fashion24−26 in comparison
to other 3D methods such as organoids and traditional 2D
neural cell culture.27,28 LOP technology enables rapid
switching between different biomaterials during the production
235
Methods/Protocols
5
deficiencies
limitations with accuracy and potential geometries
reduced mechanical properties of the scaffold and small sized
nozzle damages cells
bioinks are either too hard or possess low elastic modulus for
neural tissue
limited to materials that are cross-linked chemically
process, allowing multiple cell types and scaffold components
to be precisely positioned in different regions within the same
3D tissue. Alternative approaches to bioprinting such as inkjet
and microfluidic extrusion, expose the cells to high levels of
shear stress when they are forced out of the printhead. Higher
print speeds and pressures, higher viscosities of bioink, as well
as smaller gauge needles exacerbate these stresses. Human
pluripotent stem cells, including hiPSCs, are fragile, being
particularly sensitive to high shear stress that can cause cell
death and premature differentiation.29,30 Thus, the RX1
bioprinter only exposes cells to low shear stresses during the
printing process, making it an ideal system for bioprinting. This
system provides versatility during the printing process and its
unique printhead enables separate channels for the bioink and
its associated cross-linker. The cross-linker meets the bioink on
either side of the mixing chamber before it is extruded.25This
process sheaths the material, protecting cells from high shear
stress and its associated effects of cell death and premature
differentiation, and ensures uniform polymerization as the
bioink is extruded.25 It also prints these structures in a rapid
fashion, taking minutes to produce each structure, providing a
distinct advantage in terms of throughput in comparison to
traditional neural tissue engineering methods.
Here we detail how to prepare a novel fibrin-based bioink
for printing hiPSC-derived neural aggregates using the RX1
bioprinter. Although other groups have bioprinted single cell
suspensions when generating neural tissue,6−9 no reports exist
of printing stem-cell-derived aggregates. Our detailed method-
ology covers the preparation of our printable bioink and the
associated work flow for using the RX1 bioprinter to produce
3D tissues derived from hiPSC-derived neural aggregates with
defined structures. This bioink formulation has also been used
with a syringe-pump-driven microfluidic system for testing in
house and is likely compatible with other printing systems that
allow for chemical cross-linking during printing. The cells
maintain viability throughout the printing process and we
could culture the resulting tissues for 41 days. These tissues
expressed neuronal markers and possessed high levels of
viability even after extended culture. We then provide relevant
examples of typical results along with videos of the printing
process.
■
METHOD 1: BIOINK PREPARATION
The bioink must be prepared the day of printing and used
within an hour, as genipin will cross-link the amine groups in
fibrin.19 The cells should be incorporated into the fibrin
solution. First, mix the genipin and alginate solutions, then add
this mixture to the cell/fibrin solution and gently pipet using a
wide bore or serological pipet to mix. It can be useful to make
stock solutions of alginate and genipin.
DOI: 10.1021/acsbiomaterials.8b01235
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Safety precautions: Appropriate personal protective equip-
ment (PPE) (gloves, lab coat, and eye protection) must be
worn when handling DMSO.
Materials. Reagents.
• Deionized Water
• Fibrinogen (EMD Millipore, Etobicoke, ON, cat no:
341578)
• Genipin (Sigma-Aldrich, St. Louis, MO, cat no: G4796)
• Potassium chloride (KCl) (ACP Chemicals, Montreal,
QC, cat no: P2946)
• Sodium alginate (e.g, Sigma-Aldrich, St. Louis, MO, cat
no: 180947)
• Sodium chloride (NaCl) (Caledon Laboratory Chem-
icals, Georgetown, ON, cat no: 756−1−80)
• Sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St.
Louis, MO, cat no: 276855)
• Tris hydrochloric acid (Tris HCl) (Biobasic Canada,
Markham, ON, cat no: TB 0103)
• Tris(hydroxymethyle)aminomethane (Tris Base)
(Sigma-Aldrich, St. Louis, MO, cat no: 252859)
Equipment.
• 15 mL conical tubes (VWR, Radnor, PA, cat no:
CA21008−940)
• 50 mL conical tubes (VWR, Radnor, PA, cat no:
CA62406−200)
• 4 L container (VWR, Radnor, PA, cat no: CA73560−
022)
• 10 mL syringe (Sigma-Aldrich, St. Louis, MO, cat no:
Z248029
• 0.2 μm syringe filter (Sarstedt, Nümbrecht, Germany,
cat no: 83.1826.001)
• Dialysis tubing and clamps (Thermo Fisher Scientific,
Waltham, MA, cat no: 68700 and 68011)
• Magnetic stir plate and stir bar (VWR, Radnor, PA, cat
no: 11−497−3A and 14−512−127)
• Spatula (Thermo Fisher Scientific, Waltham, MA, cat
no: 14−357Q)
• Spectrophotometer (NanoVue Plus, GE Life Sciences,
cat no: 289565965)
• General: 1 mL pipet tips, beaker, Petri dish, pH strips,
wide bore 1 mL pipet tips (tips cut off of 1 mL pipet
tips), Parafilm
Procedure. Overview.
Step 1: Fibrin preparation, sterilization, and concen-
tration measurement
Step 2: Alginate preparation and sterilization
Step 3: Genipin reconstitution
Step 4: Incorporating cells into the bioink
Step 1: Fibrin Preparation, Sterilization, And Concen-
tration Measurement. The preparation of fibrin was
previously described along with a video demonstration of the
process.16 This protocol describes the preparation of 9 mL of
∼35 mg/mL of fibrin. The protocol can be altered if additional
fibrin is required. Excess fibrin can be stored at −80 °C for
future use, and should only be stored at 4 °C for up to 1 week.
The fibrinogen is lyophilized in the presence of sodium citrate,
which prevents clotting. The fibrinogen solution must be
dialyzed in order to remove the citrates. The molecular weight
cut off of the dialysis tubing used is 7000 Å. All surfaces that
came in contact with the powdered fibrinogen and fibrinogen
236
Methods/Protocols
solution should be wiped down with 70% ethanol when
finished preparing the solution.
1. Prepare 4 L of tris buffered saline solution (for 4 L of
dH2O. use 17.44 g of tris HCL, 2.56 g of tris base, 32 g
of NaCl, and 0.8 g of KCl) and aliquot TBS for later use
(3 × 50 mL conical tubes should be enough).
2. Allow the lyophilized fibrinogen to come to room
temperature, which takes ∼20 min.
3. Add 3 mL of TBS to three 60 mm Petri dishes and
weigh out ∼105 mg of fibrinogen and sprinkle onto the
surface of a Petri dish, then wait 5 min for the fibrinogen
to absorb into solution.
4. Rinse covers of Petri dishes with TBS to remove static;
cover dish and parafilm.
5. Incubate Petri dishes at 37 °C for 1−2 h to allow
fibrinogen to fully dissolve in solution.
6. Wet dialysis tubing with TBS (dip it into container);
fold bottom end of the tubing and clamp shut using a
dialysis clamp.
7. Pipette the fibrinogen solutions from dishes into dialysis
tubing (hold tubing with one hand, and place the Petri
dish inside its lid at an angle, pipet fibrinogen into
tubing); fold over top end, leaving an air pocket, and
clamp shut.
8. Place dialysis tubing containing fibrinogen solution into
the 4 L container of TBS; mix using stir plate at low
speed (∼100 rpm)
9. Dialyze the solution overnight on stir plate for at least 12
h. TBS solution does not need to be changed over this
time period.
10. Remove the fibrinogen solution from dialysis tubing and
place into a 15 mL conical tube.
11. Using sterile technique in the biosafety cabinet (BSC),
sterile filter the fibrinogen solution with a 0.2 μm filter
into a 15 mL conical tube. You may need to use more
than one filter.
12. Aliquot 30 μL and measure the concentration using
spectroscopy.
13. If not using right away, seal fibrin container and wrap
with parafilm.
Fibrinogen solutions can be stored at 4 °C for up to 1 week,
or −80 °C for up to 6 months.
Step 2: Alginate Preparation and Sterilization. Alginate is
made using alginic acid sodium salt powder and distilled water.
This process takes 1 day. The desired concentration of alginate
is 25 mg/mL.
1. Measure distilled water for desired amount of alginate.
2. Weigh sodium alginate to obtain a solution concen-
tration of 25 mg/mL.
3. Pipette distilled water into the beaker and dissolve
alginate using magnetic stir plate and stir bar by stirring
at 600 rpm; allow alginate to fully dissolve overnight
before using.
4. Pipette alginate into a conical tube.
5. Using sterile technique in the BSC, sterile filter the
alginate solution with a 0.2 μm filter into a 15 mL
conical tube.
6. Close filtered alginate solution container and wrap with
parafilm, then turn on the UV light and leave in the BSC
for 45 min for further sterilization.
7. The resulting solution can be stored at 4 °C for 1 week.
Step 3: Genipin Reconstitution.
DOI: 10.1021/acsbiomaterials.8b01235
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1. Add sterile DMSO directly into the glass genipin vial to
make a 25 mg/mL solution from the powdered genipin
and pipet to mix.
2. Make 1 mL aliquots of solution into your preferred vial
after thorough mixing, close vials, parafilm, and store in
−20 °C freezer until use (stable for ∼2 years).
Step 4: Mixing the Bioink. Here, neural aggregates were
formed from hiPSCs as described previous.31 Briefly,
undifferentiated hiPSCs were cultured in Aggrewell 800 plates
in the presence of Neural Induction Media for 5 days before
bioprinting. There are approximately 10 000 cells per
aggregate.31
1. Prepare bioink with concentrations of: fibrin (20 mg/
mL) | alginate (5 mg/mL) | genipin (0.3 mg/mL).
2. Mix alginate and genipin, making sure to pipet alginate
slowly with a wide bore pipet.
3. Remove cells from the Aggrewell 800 plate as previously
described31 and place in a 15 mL conical tube.
4. After centrifuging, remove the supernatant, replace with
fibrinogen solution, and pipet gently to suspend the
aggregates.
5. Immediately before printing, mix the alginate/genipin
into the fibrinogen/aggregate solution.
Timing.
Step 1: Fibrin preparation, sterilization, and concen-
tration measurement
• TBS preparation, fibrinogen incubation and
dialysis = 15 h
• Filtering and protein measurement = 1 h
Step 2: Alginate preparation and sterilization
• Preparation = 12 h
• Filtering = 1 h
• UV exposure = 1 h
Step 3: Genipin reconstitution
• 15 min
Step 4: Mixing the bioink
• 30 min
■ METHOD 2: CROSSLINKER PREPARATION
Materials. Reagents.
• β-Glycerophosphate disodium salt hydrate (β-GP)
(Sigma-Aldrich, St. Louis, MO, cat no: G9422)
• Calcium chloride (Sigma-Aldrich, St. Louis, MO, cat no:
C1016)
• Chitosan (Sigma-Aldrich, St. Louis, MO, cat no: C3646)
• Distilled water (dH20)
• Glacial acetic acid (Caledon, Georgetown, ON, cat no:
1000−1−05)
• TBS (pH of 7.4, prepared in Method 1)
• Thrombin (e.g, Sigma-Aldrich, St. Louis, MO, cat no:
T7009)
Equipment.
• 15 mL conical tubes (VWR, Radnor, PA, cat no:
CA21008−940)
• 50 mL conical tubes (VWR, Radnor, PA, cat no:
CA62406−200)
• 125 mL Nalgene square laboratory bottle (VWR,
Radnor, PA, cat no: 16120−741)
• 10 mL syringe (Sigma-Aldrich, St. Louis, MO, cat no:
Z248029)
237
Methods/Protocols
• 50 mL syringe (Sigma-Aldrich, St. Louis, MO, cat no:
Z683698)
• 0.2 μm sterile syringe filter (Sarstedt, Nümbrecht,
Germany, cat no: 83.1826.001)
• Autoclavable container (VWR, Radnor, PA, cat no:
10754−816)
• Autoclave (Sterilmatic STM-EL Autoclave, Market
Forge, Essex Junction, VT)
• Glass pipet (VWR, Radnor, PA, cat no: 53223−005)
• Magnetic stir plate and stir bar (VWR, Radnor, PA, cat
no: 11−497−3A and 14−512−127)
• Steriflip vacuum driven filtration system (EMD Milli-
pore, Etobicoke, ON, cat no: SE1M003M00)
• General: 250 mL glass beaker, 100 mL graduated
cylinder, 1 mL pipet tips, wide bore pipet tips (tips cut
off of 1 mL pipet tips), Parafilm
Procedure. Overview.
Step 1: Chitosan preparation, pH regulation, and
sterilization
Step 2: Calcium chloride preparation and sterilization
Step 3: Thrombin reconstitution
Step 4: Mixing the cross-linker
Step 1: Chitosan Preparation, pH Regulation, and
Sterilization. Chitosan is made using acetic acid, dH2O, and
chitosan powder. The desired concentration for chitosan is 25
mg/mL. The protocol for preparing chitosan was based on the
procedure outlined by Q. He et al.32 Raise pH closer to
physiological value by adding β-Glycerolphosphate (β-GP).
This protocol will bring 3 mL of a 25 mg/mL chitosan solution
to a pH greater than 7 and it can be scaled for different
amounts. These methods were adapted from techniques
described by C. Jarry et al.33 The concentration of chitosan
solution should be 19 mg/mL after adding β-GP to chitosan.
Safety precautions: Appropriate personal protective equip-
ment (PPE) (gloves, lab coat, and eye protection) must be
worn when handling glacial acetic acid. Glacial acetic acid
should only be used in a fume hood.
1. Prepare a 1%v/v acetic acid solution with 98.89 mL
dH2O and 1.11 mL glacial acetic acid.
2. Place beaker on stir plate with large magnetic stir bar.
3. Weigh chitosan to obtain a solution concentration of 25
mg/mL (2.5 g of chitosan into 100 mL of 1% acetic
acid).
4. Slowly add chitosan to acetic acid while stir bar is
rotating at 600 rpm (may increase up to 1000 rpm if
needed) and then leave the solution for 16 h overnight.
5. Decrease stir bar rotation to 300 rpm and leave for
another 24 h.
6. Transfer chitosan to 50 mL conical tubes and filter the
chitosan solution using a vacuum-driven filtration system
(Steriflip, Millipore).
7. Transfer chitosan solution into an autoclave container
and steam sterilize at 121 °C for 30 min.
8. Prepare 0.9 mL of 560 mg/mL solution of β-GP with
dH2O and sterile filter using 0.2 μm filter.
9. Add 0.9 mL of β-GP solution to 3 mL of sterile chitosan
and then pipet with a wide bore 1 mL pipet to mix,
slowly, reducing bubble formation and solution loss.
10. Measure pH as necessary to determine if further
amounts of β-GP need to be added in order to achieve
physiological pH.
DOI: 10.1021/acsbiomaterials.8b01235
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11. Immediately seal sterile chitosan solution container and

parafilm and store at 4 °C if not being used right away
(solution will slowly gel at room temperature).
Step 2: Calcium Chloride Preparation and Sterilization.
Calculate how much thrombin and chitosan are required for
the total cross-linker solution volume, then calculate remaining
volume of cross-linker solution, this is how much CaCl2 is
required (Vtotal − Vthrombin − Vchitosan = Vcalcium chloride). Calculate
the required concentration for this added volume of CaCl2
solution so that the final concentration will be 20 mg/mL in
the final volume of cross-linker.
1. Prepare calculated volume of CaCl2 solution at
calculated concentration.
2. In the BSC, sterile filter the CaCl2 with 0.2 μm sterile
syringe filter.
3. Parafilm solution and store at 4 °C if not being used
right away.
Step 3: Thrombin Reconstitution.
1. Add sterile TBS to make 1000 U/mL solution from the
lyophilized thrombin and pipet to mix in the BSC.
2. Close vial, parafilm, and store in −20 °C freezer until
use, for up to 5 years.
Step 4: Mixing the Cross-Linker.
1. In a sterile 125 mL bottle, compatible with the RX1
printer, prepare cross-linker with concentrations of
CaCl2 (20 mg/mL) | chitosan (0.75 mg/mL) | thrombin
(10 U/mL).
2. Pipette the CaCl2 and thrombin solutions into the bottle
and mix by pipetting up and down with a serological Figure 1. Suggested biosafety cabinet set up for the RX1 bioprinter.
pipet.
3. Add chitosan by slowly pipetting with a wide bore pipet,
ensuring the full required amount of the high viscosity transferred into the tissue culture dishes using a sterile spatula.
solution is taken up and deposited. Each sheet takes ∼2 min to print. Such a short print time
allows for high-throughput production and ensures cells can be
4. Mix cross-linker solution by pipetting up and down with
quickly transferred into culture medium. This procedure can
a serological pipet.
be altered, depending on the desired properties of the target
Timing. tissue. Figure 2 details the reaction between the bioink and
Step 1: Chitosan preparation, sterilization, and pH cross-linker through Aspect Biosystems’ DUO-1 Printhead in
regulation order to make a printable hydrogel. In this protocol, “reservoir”
• Dissolving chitosan into acetic acid = 48 h refers to the 15 mL conical tube holding the bioink and the
• Autoclaving = 45 min 125 mL bottles holding the buffer and cross-linker solutions.
• BGP preparation and pH regulation of chitosan = All materials listed below must be properly sterilized
30 min (autoclave, 70% ethanol, or UV exposure) prior to entering
Step 2: Calcium Chloride preparation and sterilization the BSC. The tubing and DUO-1 printhead provided by
• Preparation = 30 min Aspect Biosystems are supplied sterile thus do not need to be
• Filtering = 30 min sterilized. If there are issues during the printing process, refer
Step 3: Thrombin reconstitution to Table 2 for common troubleshooting techniques.
• 15 min Materials. Reagents.
Step 4: Mixing the cross-linker • Anti-adherence rinsing solution (optional) (STEM-
• 30 min CELL, Vancouver, BC, cat no: 07010)

■
• Bioink & cross-linker (prepared above)
METHOD 3. BIOPRINTING WITH THE RX1 PRINTER • Buffer solution (Aspect Biosystems, Vancouver, BC)
• Cell culture medium (STEMdiff Neural Induction
The RX1 printer should be placed inside a BSC to ensure
Medium) (e.g, STEMCELL, Vancouver, BC, cat no:
sterility of the printed constructs. Movement in and out of the
05835)
BSC during printing should be avoided to maintain the air
• General: 70% ethanol, sterile water
flow. Set up the BSC to maintain a clear path for the airflow
around the culture dish, and when transferring the scaffold to Equipment.
the culture dish try to use lateral movement only. Everything • Autoclave safe rinse bottle (VWR, Radnor, PA, cat no:
must be sterilized before bringing it into the BSC. A suggested 16651−904)
set up of the BSC is shown in Figure 1. This procedure will • Cell culture plate (ThermoFisher Scientific, Waltham,
print scaffolds for 6-well culture dishes. Scaffolds are MA, cat no: 142475)
238 DOI: 10.1021/acsbiomaterials.8b01235
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ACS Biomaterials Science & Engineering Methods/Protocols

Figure 2. Reaction between the bioink and cross-linker in Aspect Biosystems’ DUO-1 Printhead. (A) Thrombin cleaves fibrinogen to form fibrin
monomers which aggregate to form protofibrils. (B) Calcium chloride stabilizes fibrin and promotes polymerization. (C) Calcium chloride cross-
links alginate. (D) Genipin cross-links fibrin. (E) Genipin cross-links chitosan. (F) Alginate and chitosan interact due to polarity. (G) Printable gel
enters the nozzle of the printhead and is subsequently deposited on the print bed.

Table 2. Common Printing Issues and Troubleshooting Techniques

printing issue
low system pressure
the material is not traveling up
the tubes during the priming
sequence
no material is being printed
the structure is being dragged
along the print bed
the vacuum is not aspirating
underlying problem
a leak in the system due to
poor insertion of the
pressure tubes
a loose material reservoir
cap(s)
the tube is inserted too far
into the inlet
there is too low a pressure
the brown cap fitting is
screwed in too tight
the print head is clogged at the
bioink inlet
the print head is clogged at
bioink/cross-linker
intersection
low system pressure
the print head is too low
there is a buildup at print head
outlet
there is a block in the vacuum
chuck
there is a block at the tube
outlet in the waste reservoir
there is a block inside the tube
external to the printer
there is a block inside the tube
internal to the printer
solution
ensure all of the pressure tube are inserted correctly to both the printer and the material reservoirs
tighten the cap(s)
pull the tube out slightly
ensure the proper insertion of the pressure tube and/or raise the pressure
loosen the fitting, it should be finger tight
if gelled bioink is blocking the channel, flush the print head
if multiple cell aggregates are collecting at the inlet, ensure the aggregates are evenly dispersed in the
bioink
use another chip or material channel if the blockage cannot be removed
the cross-linker pressure is too low, raise the pressure, flush the print head to remove the clog before
resuming
see above
raise the print head so it does not make contact with the printed structure but not so high that the cross-
linker falls in droplets
stop the print, remove the buildup with an ethanol-soaked Kimwipe, restart the print, and watch the print
head height
if possible stop printing, clean the vacuum chuck, and sterilize the vacuum chuck before future use
turn off the vacuum, remove the reservoir from the BSC and move to a vacant fume hood, open the
reservoir, remove the blockage with an ethanol-soaked Kimwipe, close the reservoir, and return to the
BSC after sterilizing
remove the tubing and flush completely
bypass the internal tubing and inform Aspect Biosystems for cleaning

• Conical rack (VWR, Radnor, PA, cat no: 82010−750) (3) Material tubing + nylon sheet
• Metal spatula × 2 (VWR, Radnor, PA, cat no: 82027− (4) Vacuum chuck
528) Procedure. Overview.
• Petri dish (VWR, Radnor, PA, cat no: 470210−568)
• From Aspect Biosystems: Step 1: Creating a tissue design
(1) RX1 Printer + accessories Step 2: Material and cross-linker setup and priming
(2) DUO-1 Printhead cartridge Step 3: Printing
239 DOI: 10.1021/acsbiomaterials.8b01235
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ACS Biomaterials Science & Engineering
Step 4: Finishing the print session
Step 1: Creating a Tissue Design.
1. Select New STL and input parameters for the structure
you would like, then select Save STL and name your
STL file.
○ For example, our engineered tissue constructs
consist of a 15 × 15 × 1 mm sheet.
2. Select Infill Editor and add infills to your design.
○ Here we add a repeating infill that uses Material 1
in order to control how the printhead deposits the
fiber. Select rectilinear infill type (crosshatch
pattern), 10% infill, 0 perimeters, and a fiber
diameter of 0.1 mm.
3. Select Build Design to convert the STL file into a
Design file readable by the printer and name your design
file.
Step 2: Material and Cross-Linker Setup and Priming.
Precision is essential during the printing process, especially
when handling the print head and material tubing. The
optional Steps 12−16 describe how to rinse the printhead with
anti-adherence rinsing solution to prevent clogging. The BSC
and printer should be sterilized before beginning the print
session and all materials except the bioink should be in place in
the BSC before starting the printing process. Expose the
cabinet to UV light for 30 min then wipe down both the BSC
and the printer with 70% ethanol. Everything must be properly
sterilized before moving it into the BSC. To begin, move
everything into the BSC except the bioink. Place and open
three sterile 15 mL conical tubes with the stainless-steel
spatulas and tweezers being placed into their own conical tube.
1. Turn on the printer using the power switch located on
the right side at the rear.
2. Open Aspect Studio software, and connect the printer
by pressing Connect in the Printer Controller tab.
3. Load a design file.
4. Place the Petri dish on the print bed, to the right of
where the vacuum chuck will be for waste collection
(Figure 2).
5. Connect the buffer and cross-linker reservoirs to the
printer and parafilm the green fittings.
6. Open the printhead bottle and remove the printhead
using sterile tweezers.
7. Attach the printhead to the printer, and connect the
valve tubing to the printhead.
8. Open the tubing package and remove one tube, then
connect one end directly into the printhead and the
other into the buffer reservoir through the green fitting.
9. Take another material tube from the packaging, connect
one end to the printhead and one end to the reservoir
through the green fitting.
10. Using the motion controls move the printhead so it is
above the Petri dish and press Set Waste.
11. Press the Pressure source button.
12. If necessary, select Manual on the upper toolbar and
load a 10 mL syringe with anti-adherence rinsing
solution using an 18 gauge blunt needle. Then flush
the printhead by inserting the syringe into the Material 1
channel, manually opening the channel, and flushing
solution through the channel. Then repeat these steps
with the Material 2 channel.
13. Soak a kimwipe with 70% ethanol and wipe the surface
of the printhead to remove excess rinsing solution.
240
Methods/Protocols
14. Incorporate the cells into the bioink and move into the
BSC, open the container holding the material caps,
remove one cap, close the container, and connect the
cap to the bioink reservoir.
15. Take another material tube from the packaging, connect
one end to the printhead and one end to the reservoir
through the brown fitting, then connect the reservoir to
the printer using the Material 1 pneumatic channel.
16. For priming the printhead, set the pressure of the bioink
and cross-linker to 60 mbar and set the buffer to 100
mbar.
17. Prime the printhead either manually or using the
priming sequence in the software.
Step 3. Printing. The pressure required for printing must be
high enough to extrude the polymerized fiber of bioink but
cannot be too high, as it will damage cells. Furthermore, the
bioink pressure should not be higher that the cross-linker
pressure to avoid back flow into the cross-linker channels that
can clog the microfluidic printhead. The buffer pressure must
be high enough to clear the nozzle after printing to prevent
build-up and clogging. We use a bioink pressure of 20 mbar, a
cross-linker pressure of 40 mbar, and buffer pressure of 100
mbar during our printing process.
1. Set the bioink pressure to 20 mbar and the cross-linker
pressure to 40 mbar.
2. Open the container holding the vacuum chuck, remove
the chuck, close the container, and connect the chuck to
the printer.
3. Remove the nylon sheet from its packaging and place
onto the vacuum chuck.
4. Press Vacuum in the software and use the rinse bottle to
soak the sheet.
5. Use the motion controls to move the printhead to the
center of the vacuum chuck, and lower it to be 0.3 mm
away from the surface of the chuck, then press Set
Home.
6. In the Distance box, set the distance to 0.1 mm.
7. Press the Print button and watch the height of the
construct and the height of the nozzle. You may need to
move it up or down throughout the print session.
8. Press the Waste button once the construct is finished.
9. Use the spatula to lift the construct off of the sheet, then
transfer it to the cell culture plate using the rinse bottle
to remove it from the spatula.
10. Check the bioink and cross-linker levels to continue
printing, then press Home and repeat Steps 7 through 9.
a. If you run out of bioink or cross-linker, do the
following:
i. Set the material pressure to 0 mbar.
ii. Disconnect the material reservoir from the
printer and loosen the cap on the new
material reservoir.
iii. Unscrew the stainless-steel material cap,
remove the empty reservoir and replace
with the new material reservoir and
reconnect to the printer.
iv. Reset to the pressure described in Step 1.
b. Repeat Steps 7 through 9.
11. Once the printing process is finished, remove the media
from the constructs and replace with fresh media.
Parafilm the plate if moving from one BSC to another.
Step 4. Finishing the Print Session.
DOI: 10.1021/acsbiomaterials.8b01235
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ACS Biomaterials Science & Engineering Methods/Protocols

1. Set the material, cross-linker, and buffer pressures to 0

mbar and wait for them to reach zero before proceeding.
2. Turn off the Pressure and the Vacuum source.
3. Close the software.
4. Disconnect the bioink and cross-linker reservoirs from
the printer.
5. Remove the material tubing from the printhead, then
remove the tubing from the bioink and cross-linker
reservoirs and discard.
6. The tubing from the buffer can be removed in one of
two ways:
a. If you are printing again in the near future and
would like to leave the buffer connected to the
printer, cut a small piece of parafilm to fit over the
green fitting on the buffer cap, remove the tubing
from the buffer cap, then quickly parafilm the
green fitting, remove the tubing from the
printhead and discard. Figure 3. Neural aggregates in the microfluidic print head during
b. If you are not printing in the near future and printing.
would like to disconnect the buffer, disconnect the
buffer reservoir from the printer, remove the
material tubing from the green fitting, then
unscrew the cap and replace with the original
buffer cap and parafilm, remove the material
tubing from the printhead and discard.
7. Remove the valve tubing from the printhead, then
discard the printhead from the printer. Figure 4. Neural aggregates during culture at (A) day 5, (B) day 19,
8. Remove everything from the BSC, then spray paper (C) day 36. Scale bar indicates 200 μm.
towel with 70% ethanol and wipe down the entire
surface of the BSC and the printer. degradation. The media was gradually changed from neural
9. Clean the vacuum chuck and the material caps. induction media to neural basal media, and 0.5um puromorph-
Timing. amine (puro) and 0.25 um retinoic acid (RA) were
Step 1: Creating a tissue design incorporated into the media at Day 17 to direct differentiation
of the NAs into motor neurons. A viability assessment was
• 15 min conducted with both live/dead assay and flow cytometry.
Step 2: Material and cross-linker set up and priming
Viability Assessment. Live/dead viability using calcein-
• Set up = 20 min
AM (Life Technologies, C1430), which fluoresces live cells
• Priming = 5 min green and ethidium homodimer-III (EthD-III) (Invitrogen,
Step 3: Printing
E1169), which fluoresces dead cells red, was conducted for
• 2 h dependent on sample size qualitative images of cell viability. Live/dead staining (Figure
Step 4: Finishing the print session
5) was done on Day 10 (5 days after printing) and Day 15, and
• Turning off the printer = 20 min showed high cell viability in the scaffolds. Quantification of
• Cleaning material caps and vacuum chuck = 10 live/dead stains was performed in ImageJ by calculating the
min

■
average area of each stain, then using the equation:
ANTICIPATED RESULTS
3D-Printed Tissues Containing Neural Aggregates.
3D printed tissues can offer a valuable and high-throughput
way of modeling neural degenerative disorders for drug testing.
3D printed hiPSC-derived neural aggregates (NAs) allows for
patient specific tissue to be engineered, which is crucial for
making tissues with physiological relevance. Here we present
how to prepare a novel bioink for printing neural tissues that
consists of a mixture of naturally derived materials, all
incorporated for improving printability, integrity, and cell
viability within the scaffold. We used this bioink to successfully
print 3D scaffolds containing hiPSC-derived NAs.
Figure 3 shows the NAs in the DUO-1 printhead, which was
rinsed with AggreWell rinsing solution before printing to
prevent clogging. 3D-printed NAs were kept in culture for 41
days (Figure 4) (5 days on the AggreWell 800 culture plate, 36 Figure 5. 3D printed neural aggregates. (A) Day 10 phase contrast.
days in the scaffolds). The scaffolds maintained their integrity (B) Day 10 live/dead stain. (C) Day 15 phase contrast. (D) Day 15
for the entire culture period and showed few signs of visible live/dead stain. Scale bar indicates 200 μm.

241 DOI: 10.1021/acsbiomaterials.8b01235

ACS Biomater. Sci. Eng. 2019, 5, 234−243
ACS Biomaterials Science & Engineering Methods/Protocols

live area
live area + dead area
= %live area . Single cells could not be
100%
counted, as the embryoid body stained as one big mass. Cell
viability in the scaffold on Day 10 was 94.72 ± 3.48%, and on
Day 15 it was 64.12 ± 21.27%. As this method of capturing
viability did not capture viability of cells in 3D, it does not
account for cells inside the NA that may be viable. As seen in
Figure 5, the highly viable areas are more concentrated to the
center of the aggregate. This significant drop in viability may
be due to increased number of cells on the outer regions of the
aggregate dying, which would impact the calculated cell
viability.
Cell viability was quantified on Day 6 by flow cytometry
using the Guava ViaCount assay (Millipore). The scaffolds
were first degraded with a solution of 50 mM sodium citrate Figure 7. RX1 bioprinted neural tissues demonstrate neurite
and 0.125% trypsin for 30 min, then the cells were harvested extension and express the neuronal marker TUJ1 (green) and
nucleated cell marker (DAPI). Scale bar indicates 500 μm.
and passed through a cell strainer to ensure a single cell
suspension. On Day 6, NAs in the fibrin-based scaffolds
showed high cell viability (91.65 ± 6.85%).
Immunocytochemistry and Morphology. The NAs
viability throughout the culture period (91.65 ± 6.85% on Day
underwent visible physical changes (Figure 6). At Day 30,
6 as indicated by flow cytometry, and 64.12 ± 21.27% on Day
15 as indicated by quantification of live/dead staining) and
underwent no visible signs of degradation. In conclusion, we
described here how to prepare our novel fibrin-based bioink
and how to print human tissues using Aspect Biosystems’ RX1
printer. Together, our novel bioink and Aspect Biosystems’
RX1 printer allow for high-throughput fabrication of 3D
human tissues, offering a valuable solution for drug-screening
models
Figure 6. Portion of a neural aggregate at Day 41. (A) Phase contrast.
(B) Fluorescent stain DAPI (blue), TUJ1 (green), and GFAP (red).
Scale bar indicates 200 μm.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsbiomater-
there was visible extension from multiple NAs into the bioink ials.8b01235.
scaffold. Immunocytochemistry was performed as previously
Movie S1, bioprinting with the RX1 printer step 1
described34 to visualize neurite extension into the scaffold and
(MPG)
show that the NAs could successfully be differentiated into
motor neurons. The primary antibodies used were TUJ1 Movie S2, bioprinting with the RX1 printer step 2
(mouse, Millipore) and GFAP (rabbit, Millipore). The (MPG)
secondary antibodies used were Alexa Fluor goat antimouse Movie S3, bioprinting with the RX1 printer step 3
488 and Alexa Fluor goat antirabbit 568, and the samples were (MPG)
counterstained with DAPI (Invitrogen). Images were taken
using a Leica DMI3000B inverted microscope, Lumen
Dynamics X-Cite 120Q LED fluorescence light source, and
■ AUTHOR INFORMATION
Corresponding Author
QImaging camera and software. Staining of the NAs on Day 41 *Email: willerth@uvic.ca.
showed positive for the early neuronal marker, TUJ1, and
ORCID
neurite extension into the scaffold (Figure 7). Staining for
GFAP was negative; therefore, the NAs did not undergo Stephanie M. Willerth: 0000-0002-1665-7723
astrocytic differentiation. Aspect Biosystems’ LOP technology Author Contributions
protects the cells as they are being printed, preventing them The manuscript was written through contributions of all
from differentiating prematurely because of shear stress as they authors. E.A., L.D.L.V., and L.A. developed the experimental
exit the nozzle. The NAs were able to survive and differentiate methods detailed here with guidance from S.B., S.W., and
over the 41-day culture period, confirming that the bioink S.M.W. E.A. drafted the initial manuscript with L.D.L.V. and
scaffold can support a favorable environment for neuronal L.A. providing revisions. S.W. and S.M.W. critically read and
differentiation. edited the manuscript. All authors have given approval to the

■ SUMMARY
3D-printed fibrin-based scaffolds provide a suitable environ-
ment for NAs to survive and differentiate. hiPSC-derived NAs
in the presence of puro and RA differentiated into neurons as
indicated by early neuronal expression marker TUJ1 and the
long neurite extensions into the scaffold. They maintained cell
242
final version of the manuscript.
Funding
This work was funded by the Stem Cell Network (S.M.W. and
Aspect Biosystems), British Columbia Innovation Council
Ignite grant program (S.M.W. and Aspect Biosystems), the
Canada Research Chairs program (S.M.W), and the NSERC
Discovery Grant program (S.M.W.)
DOI: 10.1021/acsbiomaterials.8b01235
ACS Biomater. Sci. Eng. 2019, 5, 234−243
ACS Biomaterials Science & Engineering
Notes
The authors declare the following competing financial
interest(s): Dr. Willerth and Aspect Biosystems have a
commercialization agreement together.
■
ACKNOWLEDGMENTS
Dr. Willerth and Aspect Biosystems have ongoing collaborative
research agreement. This work was supported by funding from
the Stem Cell Network, the B.C.I.C.’s Ignite grant program,
the NSERC Discovery Grant program, and the Canada
Research Chairs program.
■
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