Page 352 - IJB-10-3

International Journal of Bioprinting hNVU chip for brain modeling and drug screening

15 were used for all experiments. Cells were cultured in a temperature was set to 18°C, and a 25G needle was used.
humidified incubator with 5% CO at 37°C. Subsequently, we placed the PET membrane containing
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the cells and gelatin in an incubator at 37°C. After 2 h,
2.2. Fabrication of the hNVU perfusion channel the gelatin turned into a sol state, and the cells began to
The perfusion channel structure was made by printing settle and aggregate on the porous PET membrane. In
SE1700. The SE1700 prepolymer (DOWSILTM SE1700 the printing process, we used human brain microvascular
Clear Base) was mixed with a curing agent (DOWSILTM pericytes (PCs) and human astrocytes (ACs), and their
SE1700 Catalyst) at a 10:0.6 (w/w) ratio before printing. cell densities were 4 × 10 cells/mL and 4 × 10 cells/mL,
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5
The mixed SE1700 silica gel was loaded into a 5 mL screw respectively. Finally, adequate volume of medium was
syringe (BD 302135), and then a 21G tapered needle was added for further cultivation.
installed. The syringe was loaded onto the nozzle of the
printer (SUNP Biotech, Biomaker2i), and the printing 2.4. Preparation of bioink for the brain regions in
temperature was set to 26°C. The perfused channel B the hNVU chip
structure was obtained by printing SE1700 silica gel on a Phosphate-buffered saline (PBS; VivaCell, catalog
160-μm-thick glass slide. Then, the samples were placed number C3580-0500) was used to dissolve biomaterials
in an oven at 80°C for 1 h to fully cure the material. including GelMA freeze-dried powder (stock solution
Ultraviolet (UV) irradiation and ozone sterilization concentration 12.5 wt. %), gelatin powder (stock solution
treatment of the device was performed. A 160-μm glass concentration 12.5 wt. %), fibrinogen (25 mg/mL),
was used for the convenience of confocal microscopy. thrombin powder (stock solution concentration 200
Perfusion channel A was printed on 1-mm-thick PMMA U/mL), and transglutaminase powder (stock solution
(printing parameters are the same as printing SE1700 on concentration 30 U/mL). GelMA was purchased from
160-μm-thick glass slide). SE1700 prepolymer was used to SunP (Beijing) Biotech Co., Ltd (SUNP-Gel-G1); gelatin
bond the Luer female connector (1.6 mm (1/16 inch)) to from MERCK (48722-100G-F); fibrinogen and thrombin
the PMMA board, which was then sterilized with UV light from Solarbio (F8050 and T8021, respectively); and
and ozone. The pagoda end of the Luer female connector transglutaminase from Shanghai Yuanye Biotechnology
was connected to a 0.5 × 0.8 mm sterile silicone hose. M2 Co., Ltd (S10156). GelMA ink comprising 5% GelMA +
+
screws were used to fix the PMMA board to the hNVU 2.5 mg/mL fibrinogen + 5% gelatin + 20 μg/mL laminin +
device. The Luer male connector was connected to a 3 U/mL transglutaminase was prepared by combining 0.4
stainless steel needle, and the pagoda end was connected mL GelMA (12.5 wt. %), 0.4 mL gelatin (12.5 wt. %), 0.1
to a 0.5 × 0.8 mm sterile silicone hose prior to assembly mL fibrinogen (25 mg/mL), 0.1 mL transglutaminase (30
and perfusion (Figure S1 in Supplementary File). U/mL), and 10 μL laminin (2 mg/mL). Printing ink was
prepared by mixing cells into the hydrogel; specifically,
2.3. Fabrication of the BBB structure for the hCMEC/D3 cells (12 × 10 cells), HBVPs (4 × 10 cells),
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hNVU chip astrocytes (4 × 10 cells), HMC3 cells (4 × 10 cells), and
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Cell printing technology and gravity-driven cell NPC cells (1 × 10 cells) were mixed into 1 mL GelMA ink.
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+
sedimentation technology were used for the fabrication
of the BBB structure (Figure S2, Step 1, in Supplementary 2.5. Fabrication of the hNVU chip system
File). To prepare the cell suspension, we first digested cells Sterile scalpels and scissors were used to cut out
from T75 cell culture flasks and prepared a cell suspension the constructed barrier structure (Figure S2, Step 1,
at a density of 4.67 × 10 cells/mL. Subsequently, we in Supplementary File), which was installed on the
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transferred 100 μL of cell suspension into 2 mL of cell NVU microenvironment device (Figure S2, Step 3,
culture medium using a pipette gun and mixed them by in Supplementary File). The outer frame structure
pipetting to prepare the cell suspension. Next, the cell of the hNVU device is installed (Figure S2, Step 4,
suspension was transferred into a container with a Corning in Supplementary File). The cell-containing GelMA
+
3450 chamber PET membrane and mixed through ink was loaded into a 3 mL screw-top syringe (BD
shaking. Afterward, the cell suspension was left to rest for 6 302113) installed with an autoclaved 25G stainless steel
h until the cells were fully attached to the PET membrane. needle. The 3 mL screw-top syringe was installed on
On the other side of the PET film, we uniformly printed the printing nozzle of the cell printer (SUNP Biotech,
7.5% gelatin ink material mixed with cells using extrusion Biomaker2i). A layer of cell ink material was printed at
technology (SUNP Biotech, Biomaker2i, Beijing, China) the temperature of the nozzle at 25°C (the temperature
(Figure S3 in Supplementary File). During printing, the of the bottom plate of the printing platform was set at
nozzle temperature was set to 21°C, the bottom plate 18°C). The stainless steel needle (Figure S2, Step 5, in

Volume 10 Issue 3 (2024) 344 doi: 10.36922/ijb.1684