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Vessel-on-a-Chip for Antiangiogenic Drug Screening
staining was performed using 2-(4-amidinophenyl)-6- 2.5. Statistical analysis
indolecarbamidine dihydrochloride (DAPI) staining Unless otherwise stated, all characterizations were
solution (Yeasen Biological Technology Co., Ltd.). processed by data analysis software Origin 2018 and
Samples were firstly washed by PBS and fixed with image analysis software ImageJ, and all data are presented
4% paraformaldehyde for 30 min. After that, they were as mean ± standard deviation. Differences between
washed by PBS again and permeabilized with 0.5 % groups were conducted by one-way analysis of variance
Triton X-100 (Solarbio Co., Ltd., China) for 5 min. Then, followed by Student’s t-tests. Single asterisk (*), double
the samples were washed by PBS again, and stained asterisks (**), and triple asterisks (***) indicate P < 0.05,
with TRITC phalloidin (0.1 μM) for 30 min in the dark. P < 0.01, and P < 0.001, respectively.
Subsequently, they were washed by PBS again and
stained with DAPI (10 μg/ml) in the dark. Finally, the 3. Results
samples were washed by PBS and imaged by a confocal
fluorescence microscope (LSM880, ZEISS, Germany). 3.1. Fabrication of a perfusable vessel-on-a-chip
According to the bioprinting strategy we proposed , cell-
[38]
2.4.3. Cell activity analysis
laden tubular constructs could be efficiently produced by
Cell activity was measured using a cell counting kit-8 coaxial bioprinting of GelMA/gelatin prebioinks. Based on
(CCK-8; Dojindo Chemical Technology Co., Ltd., China). the thermosensitivity of GelMA/gelatin, gelled gelatin and
The cell-laden hydrogel tubes were separately cultured in EC-laden GelMA prebioinks were simultaneously printed
a 24-well plate for 1, 4, and 7 days. At first, tubes were through a coaxial nozzle to form a core-shell structure.
washed with PBS 3 times. Then, a mixture of 50 μl CCK- Then, it was exposed to 405 nm blue light for photocuring
8 reagent and 1450 μl ECM was added to each well. After of GelMA (Figure 2Ai). After that, the printed fibers were
3 h of culture, the solutions were transferred to a 96-well transferred into an incubator for 30 min, in which the
plate to test the optical density values using a microplate temperature of 37°C led to the liquification of gelatin and
absorbance reader (iMark, Bio-Rad, US). the formation of tubular constructs. These tubes were cut
into segments for better nutrient supply and subsequent
2.4.4. Immunostaining of HUVECs use, and they were cultured for 5–7 days, allowing
After 3 days of perfusion culture, vinculin antibody endothelial cells to proliferate and form an endothelialized
(Abcam, UK) and ZO-1 antibody (Invitrogen, US) vessel (Figure 2Aii). Inspired by coronary artery stents,
immunostaining was performed on the samples a PCL stent was adopted to support the hydrogel vessel.
to investigate the intercellular connection and It was achieved by fused deposition of PCL on a rotating
functionalization of HUVECs. The samples were fixed shaft (Figure 2Aiii). Stents were subsequently demolded
with paraformaldehyde for 30 min, and permeabilized and sterilized by soaking in ethanol (Figure 2Aiv). Next,
with 0.5% Triton X-100 for 5 min. Subsequently, samples the construction of the vessel-on-a-chip can be divided
were blocked in 5% bovine serum albumin (BSA; Sigma- into three steps: (i) The PCL stent was inserted into the
cell-laden vessel (Figure 2Av); (ii) GelMA containing
Aldrich) for 1 h and incubated overnight in vinculin VEGF was utilized for casting the hydrogel bulk restricted
primary antibody in 1:200 dilution and ZO-1 primary by a prepared polylactic acid (PLA) frame (Figure 2Avi);
antibody in 1:50 dilution according to instructions. (iii) the perfusion chip was established by assembling two
Then, samples were incubated in 1/500 dilution of Alexa cover plates, two polyester (PET) films, the hydrogel bulk,
Fluor 488 goat anti-rabbit immunoglobulin (Ig)G (H+L) a PLA subplate and a PDMS wall with four sets of bolts
secondary antibody (Beyotime, China) or Alexa Fluor and nuts (Figure 2Avii, 2B and C).
488 goat anti-mouse IgG (H+L) secondary antibody When biofabricating a large-scale hydrogel vessel,
(Beyotime) for 2 h at room temperature. Next, samples it is quite difficult to obtain excellent biological activities
were stained with TRITC phalloidin for 30 min and and adequate structural fidelity at the same time. The
DAPI for 10 min. They were finally imaged by a confocal GelMA (EFL-GM-30) used in this study is a soft and
fluorescence microscope (LSM880, ZEISS). All reagents biologically friendly hydrogel that has outstanding cell
were injected into the tubular lumen for better contact and viability but relatively poor mechanical properties. The
reaction. introduction of a PCL stent solved the problem of vessel
collapse or jamming during perfusion when liquid flowed
2.4.5. Real-time monitoring of HUVEC sprouting
through a soft hydrogel tube.
An incubation monitoring system (CM20, Olympus, Maintaining perfusion without leakage has always
Japan) was used for real-time observation of HUVEC been a challenge for vessel-on-a-chip. The employment
sprouting during perfusion culture in 48 h. Time-lapse of a PDMS wall and bolt-on mounting mode effectively
images of the sprouting area were taken every 20 min. achieved long-term perfusion culture. In addition, the

296 International Journal of Bioprinting (2022)–Volume 8, Issue 4