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International Journal of Bioprinting Aflatoxin B1-induced cancer stem cells

Figure 1. (a) Schematic diagram that depicts the formation of cancer spheroids and drug-resistant cancer cells via 3D bioprinting. Daily measurement of
HepG2 liver tumor spheroids. (b) Numbered spheroids and bright-field images of the same region throughout a 7-day period. (c) Mean spheroid diameter
throughout the period. (d) Diameters of spheroids measured on day 0 (immediately after printing). (e) Diameters of spheroids measured on the 7th day.
(f) Growth percentage of all numbered spheroids.

2.7. Quantification analysis with fluorescence- dispersed in buffer and applied to a BD flow cytometer for
activated cell sorting (FACS) quantification analysis.
Initially, tumor spheroids were arranged randomly and
maintained in the gel structure. To dissociate tumor 2.8. Statistical analysis
spheroids from the hydrogel, 500 mg of collagenase NB All the quantitative measurements were performed in three
4G Proved Grade powder was dispersed in 5 mL double experimental replicates. The collected data are expressed as
distilled water (DDW) to form a solution. Then, the mean ± standard deviation (SD) from triplicate samples. A
collagenase solution was added to the hydrogel containing one-way ANOVA with Tukey’s post-hoc test was performed
tumor spheroids. By incubating the hydrogel in the to identify statistically significant differences.
collagenase solution for 30 min at 37°C, the hydrogel 3. Results
structure was totally dissociated. The broken and dissolved
hydrogels, along with the remaining spheroids, were 3.1. Development of tumor spheroids in hydrogel
collected and centrifuged at 1500 rpm for 1 min, and the structure
supernatant was removed. The residue was dispersed in To determine whether the bioprinting approach can
Accutase and incubated in 37°C; subsequently, the tumor facilitate proper spheroid formation, the diameters of the
spheroids were fully disintegrated into single cells. These spheroids were measured over a 7-day period using the
single cells are fixed with 4% PFA at 4°C in 10 min. After bright-field microscope. Spheroid images of the same
fixation and discarding PFA, the single cells were washed position were taken every 24 h. Figure 1 shows the growth
twice by dispersing in PBS, centrifuging and discarding the of spheroids and related measurement; an increase in the
supernatant. Subsequently, single cells were permeabilized mean diameter of 18 different spheroids was observed
and blocked with a mixture of 0.2% Triton-X and 1% (Figure 1c). The mean diameter started at an initial value of
FBS prepared in PBS for 10 min. In the next step, Anti- 18.5 μm and increased up to 39.2 μm on the 7th day, which
Alexa fluor 594 CD133 and Anti-Alexa fluor 647 ALDH1 corresponded to a relative growth of more than 210%. The
antibodies were diluted in a PBS buffer containing 1% FBS, growth rate was maintained quite steadily throughout the
at a rate of 1:100, and applied to the single cells for 1 h. study period, despite a slight decrease from the 4th day to
Finally, the antibody solution was discarded, and the single the 5th day. A fluctuation in growth of individual spheroids
cells were washed with PBS. The single cells were then was clearly observed (Figure 1d and e). Eighteen different

Volume 9 Issue 6 (2023) 364 https://doi.org/10.36922/ijb.0985