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International Journal of Bioprinting Bioprinted vascular tumor model

from Cell Signaling Technology (USA), and Alexa Fluor ink needs to be pre-dissolved at 4 °C in advance. The entire
568 donkey anti-rabbit IgG (H+L) (2 mg/mL) was obtained operation process is strictly carried out in compliance with
from Thermo Fisher (USA). aseptic operation specifications.

2.2. Cell culture 2.5.2. Printing and post-processing
The cells (HFL1, HUVECs, HA-VSMCs, and HepG2) were Coaxial extrusion-based printing was performed using a
maintained in high-glucose DMEM medium supplemented 20G/25G nozzle, with flow rates of 2.5 and 3.0 mL/min
with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin for the outer and inner inks, respectively. The printed
at 37 °C in a humidified incubator (STIK, China) with 5% tubular structures were immersed in 2% CaCl₂ solution
CO₂. When the cells reached 90% confluence, they were for 2 min to induce ionic crosslinking, rinsed with PBS,
passaged at an appropriate ratio using 0.25% Trypsin- and then photocrosslinked under 405 nm light for 30 s.
EDTA. The culture medium was refreshed every 2–3 days. Subsequently, fibronectin (1 mg/mL) was infused into the
lumen via a sterile syringe and incubated dynamically at
2.3. Preparation of type I collagen 37 °C for 15 min, with inversion every 5 min. HUVECs
The skin of the rat tail was removed to extract the silver- suspended in 2 mg/mL collagen (1 × 10⁶ cells/mL) were
colored tendons, which were washed thoroughly and then loaded into the lumen in two steps, 12 h apart, with
blotted dry. After cutting the tendons into small pieces, rotation to ensure uniform endothelial coverage. All
they were immersed in 0.1% acetic acid for 7 days. The procedures were conducted under sterile conditions.
solution was centrifuged at 8000 rpm (6738 × g) for 30 min,
and the supernatant was collected and freeze-dried. The 2.6. Tumor spheroid formation
lyophilized collagen was weighed and dissolved in 0.1% Tumor spheroids were fabricated via inkjet bioprinting.
acetic acid supplemented with 1% penicillin-streptomycin A bioink was prepared by mixing a cell suspension
to prepare a 10% collagen solution, which was stored at 4°C. containing HepG2 (6 × 10⁷ cells/mL) and HFL1 (2 × 10⁷
cells/mL) at a 3:1 ratio of HepG2/HFL1, with a base ink
4,9
2.4. Preparation of GelMA containing 10% (w/v) GelMA and 0.5% (w/v) LAP at a 1:1
Gelatin was dissolved in PBS and heated at 50 °C for 2 h ratio. Optimized printing parameters (e.g., pressure, valve
until fully dissolved. The solution was stirred on a magnetic timing, and viscosity) were applied to generate uniform
stirrer, and methacrylic anhydride (MA) was added droplets at 37°C. The printed microspheres were deposited
dropwise for a complete reaction. The reaction mixture onto low-attachment culture dishes and crosslinked under
was centrifuged at 4500 rpm (2132 × g) for 10 min, after 405 nm light for 30 s to obtain tumor spheroids with
which the supernatant was dialyzed in distilled water at controlled diameters.
37°C for 7 days, and the dialyzed solution was subsequently
freeze-dried. Both GelMA and the original gelatin 2.7. Chip design and fabrication
were characterized by 1H-NMR (Figure S1, Supporting A custom-designed microfluidic culture chip was
Information), demonstrating the successful synthesis of fabricated using polylactic acid (PLA) via FDM. The chip
GelMA (30–50% substitution rate). The resulting GelMA featured a central channel (diameter: 2 mm) for embedding
37
was dissolved in sterile complete medium to prepare a 14% the bilayered vascular construct, and four radial culture
solution, which was stored at 4°C. chambers for establishing a vascularized TME. The PLA chip
was bonded to a glass slide using medical-grade adhesive
2.5. Fabrication of bilayered vessels and sealed with a coverslip to form a closed culture system
(Figure 1). Regarding the disinfection of the culture system,
2.5.1. Bioink preparation it is worth noting that all autoclave-tolerant components
The outer shell bioink consisted of HA-VSMCs embedded (silicone tubing, stainless-steel connectors) were sterilized
in a matrix containing 8 mg/mL rat tail collagen, 1% (w/v) via autoclaving at 121°C for 30 min. For non-autoclavable
sodium alginate, 0.25% (w/v) LAP, and 3% (w/v) GelMA. FDM-printed chips (disposable products), a sequential
The inner sacrificial ink was a thermosensitive composite protocol was implemented as follows: immersion in 75%
composed of 3% (w/v) CaCl₂ and 4% (w/v) Pluronic ethanol for 30 min, thorough rinsing with PBS, and UV
F-127. Crosslinking of the outer shell bioink was achieved treatment for 30 min.
through ionic gelation by Ca² , while Pluronic F-127
+
served as a temporary scaffold to maintain the hollow 2.8. Construction of the multiscale vascularized
structure during printing. Notably, rat tail collagen must tumor model
be neutralized to physiological pH with NaOH under ice- The bilayered vessel was positioned in the central channel
bath conditions, and the mixing of the outer shell bioink and connected to a perfusion loop using silicone tubing
should also be performed at 4 °C. The internal sacrificial (inner diameter: 1 mm). A pre-chilled matrix, which was

Volume 11 Issue 4 (2025) 380 doi: 10.36922/IJB025180180