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International Journal of Bioprinting Biomimetic 3D printed glioma model

any cells in the outer layer (G) and a 3D model without removed and the cortex tissue was collected and digested
any cells in the inner layer and with neurons in the outer with trypsin for 20 min at 37°C. After incubation, the
layer (N). The morphology and number of both types of reaction was inhibited by the addition of DMEM containing
cells were respectively studied in these three models to 10% FBS. The supernatant was discarded by aspiration. The
analyze the interactions between neurons and glioma cells digested tissue was soaked with fresh medium containing
in in vitro glioma tissue. We advance with the novel design DMEM added with 1% L-glutamine, 2% B27 neural
and in vitro 3D model fabricated by extrusion-based 3D supplement, and 1% penicillin/streptomycin. The tissue was
bioprinting to mimic the natural interactions between mechanically dissociated with a pipette and then filtered
neurons and glioma cells, which provides a potential way through a 40-µm cell strainer. The volume of 20 µL of cell
to study the pathological process and treatment of glioma. suspension was added to 180 µL of DMEM, and 20 µL of
the diluted cell solution was used to perform cell counting
2. Materials and methods by cellometer (Nexcelom Biosciences). Finally, the cell
2.1. Materials suspension was ready for further bioink preparation.
Sprague Dawley (SD) rats and Kunming mice were The GL261 cells, the murine glioblastoma multiforme
provided by the experimental animal center of Fourth (GBM) cells, were cultured in DMEM supplemented with
Military Medical University (FMMU) (Xi’an, China). 10% FBS and 1% penicillin/streptomycin in advance for a
GL261 cell lines were purchased from Shanghai Zhongqiao period of 2 days.
Xinzhou Biotechnology Co., Ltd (Shanghai, China).
Trypsin, Dulbecco’s modified eagle medium (DMEM), 2.4. Bioink preparation
penicillin/streptomycin, and phosphate-buffered solution The collagen solution (4 mg/mL) was harvested from the
(PBS) were purchased from Hyclone (South Logan, UT, tail of SD rats and filter-sterilized prior to use. Collagen
USA). Fetal bovine serum (FBS), L-glutamine, and B27 solution was mixed with DMEM with a blending ratio of
neural supplement were purchased from Gibco (Grand 1:1 (v/v) to prepare collagen solution (2 mg/mL) at 0°C.
Island, NY, USA). Hank’s balanced salt solution (HBSS) and A 0.5 M NaOH solution was added dropwise in order to
LIVE/DEAD Viability/Cytotoxicity Kit were purchased adjust the pH value to about 7.4. An appropriate amount
from Thermo Fisher Scientific (Waltham, MA, USA). of cell suspension was taken according to the amount of
6
Fluo-4, AM was purchased from Invitrogen (Camarillo, bioink and required cell density (neuron: 6 × 10 cells/mL
6
CA, USA). and GL261 cell: 1 × 10 cells/mL). After centrifuging the
cell suspension and removing the supernatant, the collagen
2.2. Structure design of the bioprinted glioma solution (2 mg/mL, pH 7.4) was added gradually and
in vitro 3D model blown gently in order to obtain the neuron bioink and the
In clinical cases, glioma cells are found to be wrapped with GL261 cell bioink (Figure 2A). The prepared bioink was
neurons to form a double-layer spherical glioma tissue placed at 0°C for bioprinting.
(Figure 1A ). Bearing such architecture in mind, the
[29]
design of a novel bioprinted glioma in vitro 3D model (G/N) 2.5. Bioprinting of the glioma in vitro 3D model
was advanced as depicted in Figure 1B. The simplified The bioprinting was performed by an in-house developed 3D
bilayer hemispherical model was established as an artificial printer (Figure 2B). Bioinks were loaded in printing barrels in
3D microenvironment to represent the spherical tissue an ice bath and extruded by air pressure. Some main printing
and replicate the spatial relationship between neurons and parameters are given as follows: needle travel speed of 15 mm/s,
glioma cells in actual tissues. In this model, the radius of air pressure of 320 mbar, needle diameter of 0.2 mm, printing
the inner hemisphere loaded with glioma cells is 4 mm, and temperature of 0°C, initial layer height of 0.3 mm, and layer
the thickness of the outer layer with neurons is 2 mm. To height of 0.2 mm. The bioprinting of the glioma 3D model
probe the interactions between neurons and glioma cells in was achieved in a two-step manner as depicted in Figure 2B–
the designed model, two controls were set up, which only G. Firstly, the neuron tissue in the outer layer was formed (a
contains glioma cells in the inner layer (G, Figure 1C) and hemispherical shell with 12 mm external diameter and 8 mm
neurons in the outer layer (N, Figure 1D), respectively. internal diameter). Then, the glioma tissues in the inner layer
were shaped (a hemisphere with 8 mm diameter). Prior to
2.3. Cell culture bioprinting, the equipment was sterilized with overnight
Mouse primary cortical neurons were isolated from E14 exposure to ultraviolet (UV) light. The neuron bioink and the
mouse embryos. The optimized methods were carried GL261 cell bioink were transferred aseptically into separate
out under sterile conditions as fast as possible in order to printing barrels with attached needles, and then connected
avoid obtaining damaged neurons. After decapitation, the to the pneumatic hoses of the 3D printer. The G-code was
brain was exposed, the meninges of the cortex region were generated using the printer software and loaded into the 3D

Volume 9 Issue 4 (2023) 3 https://doi.org/10.18063/ijb.715