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Multiresponsive Graphene-Oxide Embedded ECM Hydrogel for 3D Bioprinting
200 1/s at 1% strain and room temperature. The thermal negative marker cocktail. Quadrants have been set based
response of these hydrogels was evaluated between on isotype controls (Figure S2).
15°C and 37°C with a temperature ramp of 20°C/min,
at 1% strain and 10 rad/s frequency. Finally, to compare 2.12. Biofabrication of cell-laden SISMA-GO
the change in storage modulus upon photocrosslinking, constructs
time sweep assays were carried out before and after blue After sterile SISMA and SISMA-GO hydrogels were
light irradiation (5 min at 62 mW/cm constant intensity) successfully prepared, hAD-MSC in low glucose DMEM
2
under oscillatory mode, and constant strain of 1% and
10 rad/s. supplemented with 10% (v/v) FBS and 1% (v/v) P/S
was homogeneously mixed at a 1:10 volume ratio with
Shear-thinning properties of the hydrogels were
estimated by fitting the viscosity (η) versus shear rate each hydrogel formulation to a final cell density of 4.5
× 10 cells/mL. The prepared bioink (i.e., hydrogel with
6
(γ) plot to the power law regression model described by
Equation 2.9.1. embedded cells) was then loaded into a sterile printing
cartridge. Bioprinting experiments were performed on an
η = K γ n −1 (2.9.1) INKREDIBLE+ bioprinter (CELLINK AB, Gothenburg,
Sweden). Printing parameters were manually optimized
K and n were recovered for all samples to assess to achieve appropriate printing velocity and extrusion
shear thinning behavior . pressure according to the bioink’s characteristics (i.e.,
[24]
viscosity, printability, and shape fidelity), as well as
2.10. Scanning electron microscopy (SEM) nozzle diameter for the intended construct geometry. In
imaging of crosslinked structures this regard, SISMA and SISMA-GO hydrogels achieved
To evaluate the resulting hydrogel microstructure upon proper filament formation through a 25 G blunt needle
thermal- or photo-crosslinking, SISMA-GO samples nozzle at a constant pneumatic pressure of 10 kPa and
2
were imaged through SEM. Gelation temperature was 5 mm/s printing velocity. A 100 mm squared construct
achieved after incubation of the samples for 15 min at with a 0.4 mm height was bioprinted with 10% infill density
37°C. Photocrosslinking was achieved upon blue-light in a 6-well culture plate and was immediately crosslinked
2
irradiation for 5 min at a constant intensity of 62 mW/ with 405 nm blue-light irradiation (62 mW/cm ) for 1
cm . Samples were freeze dried and observed under min (Figure S3). After photocrosslinking, 4 mL of cell
2
vacuum conditions at 200× magnification with a 20 kV medium were added to each well and the constructs were
accelerating voltage (JSM 6490-LV, JEOL, Tokyo, Japan). incubated for up to 7 days. The cell medium was refreshed
every other day.
2.11. Isolation and in vitro culture of human
adipose tissue-derived mesenchymal stem cells 2.13. Cell viability, morphology and proliferation
(hAD-MSCs) assessment
For bioprinting experiments, hAD-MSCs were isolated To assess the viability of hAD-MSCs embedded in
following the protocol reported by Linero et al. SISMA and SISMA-GO constructs, nuclei and dead
[47]
according to which residual adipose tissue (obtained from cell staining was performed with Hoechst 3342 and
abdominoplasty procedures with the previous approval Propidium Iodide stains, respectively, at 2 h, 3 days,
from the research ethics committee at the Universidad de and 7 days after bioprinting. Accordingly, a working
Los Andes Act No. 942, 2018 and subsequent signing of solution of the fluorophores was prepared at a 1:1000
patient informed consent) was subjected to mechanical ratio with 1× PBS each, and subsequently added to
distortion combined with enzymatic digestion. The the culture medium at a 1:4 ratio. After 45 min, the
isolated cells were subcultured until passage four using medium was removed; the constructs were fixed with
low glucose DMEM, supplemented with 10% (v/v) 4% (v/v) paraformaldehyde for 5 min and stored in PBS
FBS and 1% (v/v) P/S. Their stem cell status was then until microscopic observation. Confocal microscopy
characterized through flow cytometry (BD FACSCanto™ (Olympus FV1000, Tokyo, Japan) was used to visualize
Flow Cytometer, BD Biosciences, San Jose, CA, nuclei and dead cell locations with a 358 nm and 559 nm
USA) with the aid of a human mesenchymal stem cell laser excitation, respectively. Emission channels were
validation kit (Human Mesenchymal Stem Cell Multi- overlapped to identify live and dead cells, where live
Color Flow Kit, R&D Systems, Minneapolis, MN, USA) cells comprised locations with only nuclei being stained
and data acquisition and analysis was done with the BD and dead cell locations with both nuclei and propidium
FACSDiva™ software version 6.1.3. Cells demonstrated iodide stained. Cell viability was assessed by stacking
positive expression of CD73, CD105, and CD90 as well 25 images, captured from adjacent Z-planes separated
as negative expression of all markers included in the by 2 µm each, and counting the live cells from the total

128 International Journal of Bioprinting (2021)–Volume 7, Issue 3