Page 515 - IJB-10-5

International Journal of Bioprinting 3D model of neurogenesis in Alzheimer’s disease

2.10. Rheological characterization procedure was repeated on newly formed neurospheres.
The mechanical properties of the hydrogel were studied Half of the medium volume was replaced every 2–3 days.
through rheological assessment using a stress-controlled
rheometer with a parallel plate configuration (25 mm 2.14. Dissociation of neurospheres
diameter; 200 µm gap). To determine the hydrogel’s flow Once the neurospheres reached approximately 100–200
behavior and viscosity profile, samples were subjected to μm in diameter, they were dissociated as follows. Briefly,
shear rates ranging from 0.01 to 100 s and back with an cells were centrifuged at 200×g for 4 min and suspended
−1
interval of 30 s between curves. Next, oscillatory analyses in Accutase. Dissociation was performed mechanically
were conducted to evaluate the viscoelastic properties of and enzymatically with repeated up-and-down pipetting
the hydrogels and constructs. Through amplitude sweep (1 of neurospheres in Accutase. Then, the solution was
Hz frequency; 0.1–100% strain) and frequency sweep (1% incubated at 37°C for 10 min, and DMEM/F12 was added
strain; 0.1–10 Hz) measurements, the values for storage to the solution, which was then centrifuged at 200×g
modulus (G’), loss modulus (G”), and loss tangent (tan δ) for 5 min. After discarding the supernatant, dissociated
were obtained. All measurements were performed at 25°C neurospheres were resuspended in the complete medium.
(room temperature), and each experiment was conducted Four days after dissociation, neurospheres were immersed
in triplicate to ensure consistency. The rheograms are into the hydrogel and bioprinted as described below.
plotted based on the average of three replicates. 2.15. Bioprinting the NSC microenvironment
4
2.11. Electrical conductivity The bioink was composed of 2 × 10 neurospheres mixed
The electrical conductivity of 5 mL of the hydrogel was with 1 mL of the hydrogel with or without 1 µM Aβ
determined using a conductivity meter. oligomers. The bioink was transferred to a 5 mL syringe.
Bioprinting and crosslinking were performed as described
2.12. Animals in Section 2.4. Thereafter, the constructs were transferred
All experiments using animals and their respective to 24-well plates and cultured with the complete medium
extracted cells were conducted with the approval of at 37°C and 5% CO . The medium was changed every
2
the Ethics Committee on the Use of Animals from the 2–3 days. Constructs were evaluated 2, 3, or 8 days after
Universidade Federal de São Paulo (authorization number bioprinting.
CEUA 2428100423). C57BL/6 wild-type male mice were
dissected at six weeks of age. In detail, mice were divided 2.16. Cellular proliferation and viability
into two groups and housed in standard cages (hardwood The resazurin assay was used to assess cell proliferation at
bedding) in a conventional animal facility (12 h light/dark different time points (2, 3, and 8 days after bioprinting).
cycle). Mice were monitored for health and welfare for the Briefly, 24 h before the determined time point, the medium
whole duration of the experiments. Only mice without was substituted by 10% resazurin solution diluted in a
signs of stress or discomfort (including hair loss and complete medium. Samples were incubated at 37°C and 5%
stereotype behaviors) were included in the study. CO for 24 h. Subsequently, the solution of each sample was
2
transferred to a new plate for fluorescence measurement
2.13. Neural stem cell extraction and using a microplate reader (bottom-reading; excitation: 544
neurosphere culture nm; emission: 590 nm). As a negative control, cell-free
Briefly, six-week-old mice were euthanized by decapitation, 10% resazurin solution was incubated for the same period
and their brains were removed. The SVZ was dissected, and under the same conditions. The results are presented as a
cells were dissociated mechanically and enzymatically with percentage of the control.
0.5% trypsin for 5 min at 37°C. After dissociation, cells To observe the distribution and localization of live
were strained in a 40 μm mesh cell strainer and cultured and dead cells within the neurospheres cultured in the
in poly(2-hydroxyethylmethacrylate) (polyHEMA) pre- construct, viability was assessed using a Live/Dead assay
coated flasks, seeded in a complete medium (DMEM/ kit two days after bioprinting. Constructs were incubated
F12 supplemented with 2% B-27 without vitamin with a homogenized solution of calcein AM and ethidium
A supplement, 20 ng/mL epidermal growth factor homodimer-1 at room temperature for 15 min. Stained
(EGF), 20 ng/mL fibroblast growth factor 2 (FGF2), 1% cells were imaged using a confocal laser scanning
l-glutamine, 1% penicillin/streptomycin, and 5 μg/mL microscope.
heparin) and maintained in a humidified incubator at 37°C
and 5% CO for three weeks. For propagation, neurospheres 2.17. Neurosphere area
2
(100–200 μm in diameter) were subcultured by mechanical On a phase contrast microscope, constructs were imaged
and enzymatic dissociation (described below), and the on days 0, 1, 2, 3, 4, 7, and 8 after bioprinting. The area

Volume 10 Issue 5 (2024) 507 doi: 10.36922/ijb.3751