Page 238 - IJB-10-3

International Journal of Bioprinting Increased ECM stiffness enhances chemoresistance

oscillatory tests were also conducted through cooling to each well of the 24-well plate. The culture medium was
process with the rate of 5°C/min. The intersection of the changed every two days.
G′ and G″ curves is generally considered the gel point
of the material. Different concentrations (6% and 8%) 2.5. Young’s modulus tests
of GelMA were incubated at 37°C before testing and the For each concentration of samples, the same 3D bioprinter
temperature-controlled testing plate was also set at 37°C was used to construct cuboids, which were 2 cm long, 1
as the initial temperature. The time dependence of G′ and cm wide and 0.6 cm high. Young’s modulus tests were also
G″ of GelMA hydrogels with different concentrations were performed on 3D models containing 6% GelMA and 8%
measured according to a set measurement temperature GelMA but without tumor cells, in addition to 3D tumor
(21.5°C), which corresponded to the setting of the printing models. Prior to the determination of Young’s modulus, all
temperature. samples were measured with vernier caliper to determine
their actual sizes, including length, width, and height. Three
2.3. Semi-quantification of printability samples of each condition were loaded and compressed
When GelMA is in the ideal gelation state, the extruded until rupture at the rate of 0.05 mm/s using a mechanical
filaments will show a clear morphology, marked by regular test instrument (Bose ElectroForce 3200, Bose Corp.). The
grids and square holes in the manufactured structure. elastic part (10% to 20% strain) of the stress-strain curve
We utilized the following formula to calculate GelMA was used to calculate the Young’s modulus.
printability (Pr) based on the square shape: 37
2.6. Cell survival assay
L 2 Cell survival in the 3D-bioprinted models was assessed
Pr= (I) on days 1, 3, 5, 7, and 10 after printing. Cell survival was
16 A measured using a live/dead fluorescence assay. Briefly,
a mixture of calcein-AM (C-AM, 1 µmol/L; Sigma-
where L means perimeter, and A means area. For an
acceptable printability state, the interconnected channels Aldrich, Burlington, USA) and propidium iodide (PI, 2
µmol/L; Sigma-Aldrich, Burlington, USA) was prepared
of the 3D-bioprinted model would be square shape, and and passed through a 0.22-µm filter prior to staining. The
the Pr value was 1. To determine the Pr value of each 3D-bioprinted model was gently washed with PBS and
concentration of GelMA at printing temperature, optical incubated in a C-AM/PI mixture at 37°C for 30 minutes
images of printed models were analyzed in ImageJ software in the dark. After incubation, the 3D-bioprinted model
(version 1.8.0) to calculate the perimeter and area of was gently washed with PBS and observed using a laser-
interconnected channels (n = 4).
scanning confocal microscope (C2/C2si; Nikon, Tokyo,
2.4. Construction of 3D-bioprinted ovarian cancer Japan). Five random fields were captured for each sample,
model and the cells in the five samples were counted using the
A 3D cellular bioprinter BIOMAKER (SUNP Biotech, ImageJ software (Version 1.8.0). Cell survival (%) was
Beijing, China) was used to construct in vitro ovarian cancer calculated using the following formula:
models according to previously reported procedures. 22,23,38
Briefly, 10% gelatin methacryloyl (GelMA) hydrogels Cellsurvival(%)= Numberof live cells ×100 (II)
(EFL-GM-30, Engineering for Life, China) were prepared Totalnumberofcells
with phosphate-buffered saline (PBS) containing 0.25%
(w/v) lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate 2.7. Cell proliferation assay
(EFL-GM-30, Engineering for Life, China). SKOV3 The 2D-Ov cells and 3D-bioprinted cells on days 1, 3, 5,
cells cultured in a 2D environment were collected and 7,10 after printing were incubated in a mixture of culture
prepared as a suspension. The cell suspension was then medium and Cell Counting Kit 8 (CCK8; Dojindo,
mixed with 10% GelMA in a volume ratio of 2:3 (6%- Kumamoto, Japan) at a volume ratio of 9:1. After 2 h of
3DP) or 1:4 (8%-3DP), with a final cell density of 5×10 / incubation at 37°C, the absorbance of the culture medium
6
mL. The cell/hydrogel mixture was pumped into a sterile at 450/620 nm was measured (AMR-100; ALLSHENG,
syringe with a 23G needle and placed in a 3D bioprinter Hangzhou, China).
at a controlled temperature. The temperature of nozzle
and molding chamber was 21.5°C and 10°C, respectively. 2.8. Evaluation of protein and mRNA expression
The cell/hydrogel mixture was squeezed at a rate of 1 The expression of ovarian cancer-associated proteins and
mm /s to fabricate the model and then exposed to a blue multidrug resistance genes was evaluated using several
3
laser (wavelength 405 nm) for 20 s to modify the GelMA. methods. Ki-67 and p53 protein expression were detected
Afterward, 1 mL SKOV3 cell-specific medium was added by immunofluorescence, and the mRNA expression of p53,

Volume 10 Issue 3 (2024) 230 doi: 10.36922/ijb.1673