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mL. The cell suspension was subsequently dispensed into was observed among the groups from both proteomics and
the chambers and exposed to 395 nm visible light for genomics perspectives through Western blot and reverse
2 min, enabling in situ crosslinking and solidification of transcription-quantitative (PCR RT-qPCR) experiments,
the GelMA matrix, which supported long-term perfusion respectively.
culture. The chip was equipped with transparent windows
on both the upper and lower sides to meet the requirements 2.5.1. Cell extraction
for real-time monitoring during the culture process. Briefly, 0.3 mg/mL working liquid (i.e., 1 mL working
solution + 6 μL lysis buffer stock) was prepared using a
2.4. Evaluation of the cartilage-on-chip complete cell culture medium. After adding sufficient
2.4.1. Equilibrium time for liquid permeation into working solution to submerge the hydrogel blocks in the
hydrogel via bottom flow channels well plate, the solution was pipetted repeatedly to detach the
Alcian blue-safranin dye was perfused into the bottom flow hydrogel blocks from the bottom of the plate and to ensure
channel at a constant flow rate of 0.5 mL/min. Changes in thorough fragmentation. The smaller the hydrogel blocks,
the hydrogel color were monitored at 0, 2, 4, 6, 12, 24, 36, the faster the lysis process. Sterile lysis was performed in
48, 72, and 96 h to assess the dynamics of dye permeation. a 37°C incubator, with the lysis condition observed under
a microscope every 15 min. Following complete lysis,
2.4.2. Biocompatibility of the cartilage-on-chip the mixture was centrifuged at 1000 rpm for 5 min, after
which the supernatant was discarded. The pellet was then
The biocompatibility of the chip was assessed through live/ resuspended in 5 mL of complete culture medium and
dead staining. The growth medium was loaded into 15 mL underwent an additional wash through another round of
centrifuge tubes and cultured for 10 days by perfusion. The centrifugation. The cells obtained could be further cultured
cells were divided into groups according to the different or used for subsequent analyses, including protein and
durations of culture (3, 6, and 10 days). The cells were nucleic acid extraction.
inoculated with hydrogel in the chamber and added with
200 μL live/dead staining solution (composition: 1 mL PBS, 2.5.2. Western blot
2 μL calcein acetoxymethyl ester, and 3 μL propyl iodide); After two gentle washes with PBS, the cells were lysed
the solution was incubated for 10 min, excess liquid using 300 μL of RIPA buffer containing protease inhibitors,
discarded, and excess staining washed away with PBS. Each phosphatase inhibitors, and EDTA, ensuring even coverage
gel was scanned in layers at intervals of 20 μm, and the of the culture dish bottom. The lysate was then incubated
collected images were analyzed and processed using ImageJ on ice for 30 min to facilitate complete lysis. Following
software. Cells exhibiting green fluorescence were identified lysis, cells were scraped uniformly in one direction and
as viable, whereas those displaying red fluorescence were transferred to a labeled 1.5 mL centrifuge tube. The sample
classified as non-viable. Cell viability was calculated using was subsequently centrifuged at 12,000 rpm for 15 min,
Equation I, and the average value was reported. and the supernatant was carefully collected. Protein

Cell viabilit y = concentration was quantified using an enhanced BCA
Live cell count ×100% protein assay kit, and the samples were adjusted to the
appropriate concentration for further analysis. Loading
Live cell count + dead cell count (I) buffer was added to the protein lysate in a 4:1 ratio, followed
by vortex-mixing. The samples were denatured in a metal
2.5. Cartilage maintenance of the cartilage-on-chip
bath at 100°C for 10 min, followed by centrifugation at
The extracted primary chondrocytes were evenly divided 12,000 rpm for 5 min to remove any insoluble impurities.
into three portions. One portion was placed in the cell SDS-PAGE gels were then prepared according to the
inoculation chamber and supplemented with a growth manufacturer’s instructions, and electrophoresis was
medium for adherent growth, designated as the “2D” performed at 80 V for 30 min, followed by an increase
group. The second part was grown adherently, such as the to 120 V for 1 h. Subsequently, the transfer process was
two-dimensional (2D) group, but the growth medium was carried out at a constant current of 300 mA for 100 min.
perfused through the bottom flow channel, which was the The membrane was carefully removed, washed with TBST
“2D + microfluidics” group. The third portion was spread to eliminate any residual transfer buffer, and then blocked
out into the hydrogel and placed in the chamber, with with 10 mL of protein-free rapid blocking solution on
a growth medium perfused through the flow channel, a shaker for 15 min. After blocking, the membrane was
designated as the “organ chip” or “3D” group. The control rinsed in TBST for 10 min. The membrane was cut into the
group consisted of untreated primary chondrocytes appropriate strips and incubated overnight at 4°C with the
(Figure 2A). The gene expression of type II collagen (Col II) diluted primary antibody solution. Afterward, the primary
and cartilage-specific proteoglycan core protein (ACAN) antibody solution was removed, and the membrane was

Volume 1 Issue 1 (2025) 4 doi: 10.36922/or.8461