Page 371 - IJB-9-6

International Journal of Bioprinting Aflatoxin B1-induced cancer stem cells

by culturing it in Dulbecco’s modified Eagle’s medium 3D-printed cells were maintained at 37°C and 5% CO , with
2
(DMEM; Gibco, Grand Island, NY, USA) supplemented nutrients provided by DMEM supplemented with 10% FBS
with 10% fetal bovine serum (FBS; Gibco) and 1% and 1% antibiotic-antimycotic, which was exchanged every
antibiotic-antimycotic (Gibco) at 37°C under 5% CO . To 24 h. Their growths were monitored under the bright-field
2
maintain cell growth, media exchange was conducted every function of fluorescence microscope (Olympus BX51,
48 h after washing with phosphate-buffered saline (1× PBS, Japan), wherein the diameter measurement is possible.
pH 7.2, Gendepot, USA). When subculture was needed, 2.5. Aflatoxin B1 treatment
the cells were washed with 1× PBS before detachment with Aflatoxin B1 was purchased as a 5-mg powder in a
trypsin (TrypLE Express, Gibco, Denmark) for 5 min.
vial (Enzo, Farmingdale, NY, USA). It was dissolved in
2.2. Material preparation for 3D bioprinting dimethyl sulfoxide (DMSO) to give the stock solution with
To mimic the ECM in tumor microenvironment, gelatin a concentration of 0.5 mg/mL; subsequently, it was kept at
and alginate were purchased and employed in the -20°C. For the treatment on HepG2 tumor spheroids, this
lyophilized powder form. They were dispersed into 1× stock solution was diluted in serum-free DMEM to three
PBS at concentrations of 10% and 4% (w/v), respectively. concentrations of 1, 2.5, and 5 μM. After a 7-day period
The mixtures were heated up to 80°C in 7 h; to obtain a of tumor spheroids’ growth, DMEM supplemented with
homogenous gel, they were mixed every hour, before being 10% FBS and 1% antibiotic-antimycotic was discarded,
kept at 37°C. Finally, approximately 2.5 million HepG2 and the gel structures were washed twice with 1× PBS.
cells, prepared in 0.5 mL of DMEM supplemented with Subsequently, different plates were treated with different
10% FBS and 1% antibiotic-antimycotic, were added and concentrations of aflatoxin B1, along with the control,
mixed gently with the gel, which could be immediately which contained only serum-free DMEM. The carcinogen
applied to cell 3D printing. incubation was performed for 48 h under the condition of
37°C and 5% CO .
2.3. 3D printing of mini-well 2
The CAD program Rhino 6 was used to design 3D mini- 2.6. Immunostaining for the detection of surface
well structure. Subsequently, the design was converted marker on tumor spheroids
into stl files for application in the New Creator K V1.57.70 Two important markers are found in the HepG2 liver cancer
software. Once the file was uploaded and the 3D printer cell line. One is CD133, or prominin-1, a transmembrane
was connected to the software, the fabrication of the mini- glycoprotein that has been widely used to sort out liver
well was initiated automatically. Briefly, thermoplastic CSCs. The other is ALDH1, a cytosolic enzyme, which
[52]
polylactic acid (PLA) polymer was transformed into a is responsible for intracellular retinoic acid formation .
semisolid form by heating to 210°C, which flowed through Owing to their significant role, this study employed
the nozzle as semisolid fiber with a size of 0.2 mm. By these two markers to isolate and identify CSCs from the
optimizing the filling density and printing speed at 75% spheroids. After exposure to aflatoxin B1 at four different
and 10 mm/s, respectively, layer-by-layer printing was concentrations, the tumor spheroids were washed three
facilitated to form a mini-well that was 4 mm high and times with 1× PBS. Subsequently, fixation was conducted by
covered an area of 14×14 mm. Each mini-well dish included adding 4% paraformaldehyde (PFA) at 4°C to the samples
nine wells arranged in a 3 × 3 square. An individual well in 20 minutes, followed by three washes with PBS. In the
had a size of 3 × 3 mm. next step, permeabilization and blocking were done by
immersing the samples into a 1× PBS solution containing
2.4. 3D bioprinting of cell-laden hydrogel 0.2% Triton-X and 1% FBS for 15 min. Following this,
The 3D bioprinting of HepG2 cells in the hydrogel was the samples were stained for 1 h with Anti-Alexa fluor
conducted using the same 3D bioprinter and computer 594 CD133 and Anti-Alexa fluor 647 ALDH1 antibody
software as in the previous step. In particular, the prepared solutions at a dilution rate of 1:200, and then washed thrice
mixture of hydrogel and cells was loaded into a 10-mL with 1× PBS. Finally, counter-staining was required, using
syringe and fixed in the dispenser of the 3D bioprinter. Hoechst 33258 (Bloomington, MN 55431) at a dilution
Another stl file encoding the specialized design for gel rate of 1:500 for 15 min, followed by washing thrice using
printing was uploaded to the computer. Once printing PBS. The fluorescence imaging of tumor spheroids was
commander was activated, the printer automatically printed performed using confocal microscope (TCS SP8, Leica,
the cell-laden hydrogel with cross-shaped structures into Wetzlar, Germany) and LASX software after washing thrice
all nine individual PLA wells obtained from the previous with 1× PBS. The obtained images were analyzed with
step. After the cell-laden hydrogel was completely printed Imaris software (Bitplane, Zurich, Switzerland), whereas
into the wells, the hydrogel was crosslinked by a 160-mM the fluorescence intensity was measured by Metamorph
aqueous calcium chloride solution for 10 min. The software (Molecular Devices, San Jose, CA, USA).

Volume 9 Issue 6 (2023) 363 https://doi.org/10.36922/ijb.0985