Page 354 - IJB-10-3

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

2.10. Detection of chemotherapeutic fibrinogen were added, facilitating rapid cell attachment
drug concentration to the PET membrane. On the inner side, human brain
Samples (50 μL) were extracted and accurately added to vascular pericytes (HBVPs; PC is also used in this article
5-fluorouracil (5-FU). The solution (or vorinota) was to denote HBVPs) and astrocytes (ACs) with hydrogel
placed in a clean 1.5 mL centrifuge tube, and acetonitrile/ were uniformly deposited by extrusion bioprinting
methanol (1:1, v/v) solution containing 0.1% formic acid, (Figure 2A). The BBB cells of hCMEC/D3 (EC), HBVPs
four times the volume of the samples, was added to the (PC), and astrocytes (ACs) were recovered to high
centrifuge tube and then vortexed for 5 min. The mixture viability after 5 days of culture (Figure 2C and D), and the
was centrifuged for 10 min at 13,000 rpm at 4°C, and then endothelial cells were observed to form a dense endothelial
the mixture in the centrifuge tube was transferred into a barrier after seven days of culture (Figure 2E), confirming
sample bottle for LC-MS/MS analysis. At the same time, the long-term structural stability of the co-cultured BBB.
the standard curve for the preparation of blank 5-FU (or
vorinota) solution without drugs was used for calculation. 3.1.1. The BBB structure closely resembles the
Detailed instrument parameters are provided in the in vivo BBB
Supplementary File. A 3D reconstruction of the fluorescence image revealed
a dense distribution of hCMEC/D3 cells on one side and
2.11. Rheological measurements HBVP cells/astrocytes on the other side of the structure. The
GelMA and hydrogels were prepared in accordance with thickness of the BBB model structure was approximately
the procedures described in section 2.4. To determine 34 μm (Figure 2F). Furthermore, green-labeled endothelial
the thermal responsivity of the hydrogels, an oscillation cells intersected with red-labeled PC+AC cells, suggesting
temperature sweep was performed using a rheometer potential interactions between astrocytes and endothelial
(MCR302, Anton Paar) employing a cylindrical cells through the voids of the PET membrane (Figure 2F).
geometry (NO. PP25/TG-SN32620, diameter 25 mm), Immunofluorescence staining of tight junction protein
which corresponded to a sample volume of 350 μL. The ZO-1 and adherens junction protein CD144 on day 7
temperature was set for a ramp change from 37°C to 4°C revealed the formation of a tight arrangement of the
and then back to 37°C at a rate of 5°C/min. To determine endothelial cells (Figure 3A and B). Furthermore, we
the UV illumination responsivity of the hydrogels, the comprehensively investigated the dynamic expression
temperature was set at 25°C. The hydrogels were maintained of genes related to BBB integrity, nutrient transporters,
at 25°C for 5 min before the rheological experiment begins. and cell–matrix interactions (Figure 3C; Figure S6 in
Five minutes after the start of the experiment, the UV light Supplementary File). qRT-PCR analysis demonstrated
(405 nm, 30 mW, 60 s) was turned on to crosslink the an upregulation of genes associated with BBB integrity
hydrogels. A consistent strain (1%) and frequency (1.5 Hz) (occludin, claudins, ZOs, CD144) (Figure 3C) and cell‒
were used throughout the temperature ramp. cell interactions in the BBB (CX30, CX43) (Figure S6 in

2.12. Statistical analysis Supplementary File), as well as genes controlling amino
All data are presented as the mean ± standard deviation acid transport (LAT1) and excitatory neurotransmission
(S.D.). Unpaired Student’s t-test was applied to calculate termination in the central nervous system (EAAT1)
statistical probability. Unless otherwise stated, at least three (Figure 3D). These genes exhibited significant upregulation
biological replicates were performed for each experiment. on day 7, indicating an enhancement in the tightness of
Statistical analysis was performed using GraphPad Prism 8 the BBB and improved cellular communication between
software (GraphPad Software, San Diego, CA, USA). The barrier cells.
statistical significance of the data was determined as *p < Moreover, no significant changes were observed in the
0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. expression of P-gp (also known as ABCB1/MDR1), an
Other experimental procedures and methods are given important protein associated with multidrug resistance,
in the Supplementary File. suggesting that the model’s drug resistance was unlikely to
be altered during the culturing process (Figure 3D).
3. Results and discussion 3.1.2. Barrier function of the reconstituted BBB in the
3.1. The BBB in the hNVU chip mimics the hNVU chip
characteristics of the BBB in brain We subsequently assessed the integrity and barrier function
We selected a semipermeable PET porous membrane with of the BBB by evaluating permeability and transmembrane
a pore size of 0.4 μm to serve as the physical support for impedance. Permeability analysis demonstrated that
the BBB model. On the outside of the PET membrane, after 3 days of BBB membrane formation, dextran
hCMEC/D3 (EC) cells and vascular extracellular matrix macromolecules with molecular weights of 10 kDa, 40 kDa,

Volume 10 Issue 3 (2024) 346 doi: 10.36922/ijb.1684