Page 353 - IJB-10-3

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

Supplementary File) used to construct the perfusion method can be used in studies assessing cell viability and
channel into the chip device was inserted and centered toxicity. GFP and RFP (Hanheng Technology Corporation,
axially (a 250 μm diameter silver needle was inserted Shanghai, China) were transfected as tracer markers into
into the inner tube of the stainless steel needle). The cells to observe their positions.
print cell ink material fills the area where the stainless
steel needle was located at a nozzle temperature of 21°C. 2.7. Immunohistochemistry
The cell ink material was printed at a nozzle temperature The cells in the hNVU chips were fixed with 4%
of 25°C to fill the entire “brain area” chamber. Then, UV paraformaldehyde (Beyotime, Shanghai, China) for 30 min
crosslinking (wavelength = 405 nm) was performed with and permeabilized with 0.05% Triton X-100 (Beyotime,
50 mW irradiation for 30 s. Medium containing 100 U/ Shanghai, China) for 30 min. After washing and blocking
mL thrombin and transglutaminase was sprayed on the with 5% bovine serum albumin (Leagene), the samples
ink material, and the constructed barrier structure was were incubated with primary antibodies at 4°C for 1 day,
cut out with a sterile scalpel and scissors after 10 min and followed by washing with PBS (BIOIND) for more than 5
installed in the hNVU microenvironment device (Figure h. The samples were then incubated with Alexa Fluor 488
S2, Step 6, in Supplementary File). Perfusion channel A (Thermo Fisher Scientific) or Alexa Fluor 594 (Thermo
was installed on the hNVU device (Figure S2, Step 7, in Fisher Scientific)-tagged secondary antibodies at 4°C for 1
Supplementary File), and then the hNVU device was day, followed by washing with PBS (BIOIND) for more than
placed in a 37°C incubator and incubated for 30 min 5 h. The nuclei were counterstained with 4′,6-diamidino-2-
(during this period, the hNVU device was placed on a phenylindole (DAPI, Thermo Fisher Scientific #D1306).
homemade rotating device so that the cells in the model The primary and secondary antibodies used in this study
would not settle rapidly due to gravity). The needle was were listed in the Table S1 (Supplementary File).
pulled out to form a hollow hydrogel channel in the chip 2.8. TEER measurement
device. It should be noted that no spatial control of cells The trans-epithelial electrical resistance (TEER) of
was achieved within each layer of the printing process. the endothelial monolayer formed in the device was
An exploded view of the hNVU chip is shown in measured using a Millicell ERS-2. We cut and fixed the
Figure S1 (Supplementary File). For the perfusion constructed BBB structure on a homemade jig (Figure
culture of the hNVU chip device, a 100 mL glass bottle S5A in Supplementary File) and inserted the jig with the
(Shu cattle, high borosilicate material) was chosen as the barrier structure into the corresponding test slot to ensure
storage chamber for the perfusion culture (Figure S4B in good contact. After 1 min of stabilization, three multiple
Supplementary File). A sterile silicone hose of 0.5 × 0.8 readings were averaged for each device. To calculate TEER,
mm (inner diameter × wall thickness) connects the liquid the measurements from the chips in the absence of the cells
storage chamber and the microenvironment device of the were subtracted from the resistance of each device. The
neurovascular unit (Figure S4A in Supplementary File). transmembrane impedance of the barrier was measured
Using a rotation speed of 16 rpm, the BT100-2J driver using the Millicell ERS-2 instrument, the measured values
(Rongbai Tech, Baoding, China) and the DG10 pump head of each sample were recorded, and the data were processed.
could achieve a shear rate of approximately 0.02 dyne/cm . The measured value multiplied by the area is the displayed
2
The connection method of the perfusion channel interface TEER value.
and the peristaltic pump tube is shown in Figure S4C
(Supplementary File). Continuous perfusion culture was 2.9. Quantification of barrier permeability
carried out for 10 days, and half of the medium (20 mL) To evaluate BBB permeability, the BBB structures of
was renewed on the 5th day. the hNVU chips were placed horizontally on a custom
device (Figure S5B in Supplementary File), and FITC–
2.6. Cell viability assay and cell tracking in the 3D dextran (10 kDa, 40 kDa, and 70 kDa) (Sigma-Aldrich)
hNVU chip was administered into the device. A cell-free PET
Cell viability was determined using the Calcein-AM/PI membrane was used as the control group. The detection
Cell Double Staining Kit (C542, Dojindo, Japan). Briefly, reagent was extracted, and the fluorescence intensity
calcein-AM stock solution and propidium iodide (PI) was measured with a fluorescence spectrophotometer
stock solution were added to PBS to generate working (excitation wavelength 485 nm, emission wavelength
solutions with concentrations of 2 μmol/L and 4.5 μmol/L, 535 nm). The permeability coefficient P = F/F0 was
respectively. The working solution was added to the model calculated, where F represents the fluorescence intensity
and incubated at 37°C for 15 min prior to observing value of the transmembrane liquid (reservoir B) and F0
living cells at an excitation wavelength of 490 ± 10 nm is the fluorescence intensity of the initial FITC–dextran
and dead cells at an excitation wavelength of 545 nm. This solution (reservoir A).

Volume 10 Issue 3 (2024) 345 doi: 10.36922/ijb.1684