Page 239 - IJB-10-3

International Journal of Bioprinting Increased ECM stiffness enhances chemoresistance

IL-6, CD133, ALDH1, CD44, ABCA1, MRP-1, and MDR- analysis of two conditions/groups was performed using the
1 was detected by real-time quantitative polymerase chain DESeq R package (1.10.1). Genes with an adjusted p-value
reaction. The antibody details and primer sequences are <0.05 found by DESeq were assigned as “differentially
shown in Tables 1 and 2, respectively. expressed.” Gene Ontology (GO) enrichment analyses of
differentially expressed genes (DEGs) were implemented
2.9. Pharmacodynamic evaluation of antitumor using the cluster Profiler R package. GO terms with
drugs corrected p <0.05 were considered significantly enriched
On day 7 after printing, 3D-bioprinted cells were treated by DEGs.
with a range of concentrations of carboplatin (1 μM, 10μ
M, 30 μM, 50 μM, 70 μM, 100 μM), paclitaxel (0.1 nM, 2.11. Statistical analysis
1 nM, 10 nM, 1000 nM, 50000 nM) and olaparib (1 μM, Data are expressed as mean ± standard deviation.
10 μM, 30 μM, 50 μM, 100 μM, 150 μM). When 2D-Ov Student’s t-test was used to determine whether there
cells reached approximately 70–80% confluence, they were was a statistically significant difference between groups.
treated with different concentrations of the antitumor Statistical significance was defined as *p <0.05, **p <0.01,
drugs listed above. Cell growth was measured using the ***p <0.001, ****p <0.0001.
CCK8 assay, and dose-response curves were plotted using
GraphPad Prism (Version 9.0.0, San Diego, CA, USA). 3. Results

2.10. RNA sequencing and bioinformation analysis
RNA was extracted using the TRIzol method (Invitrogen, 3.1. Rheological evaluation
CA, USA) and treated with RNase-free DNase I (Takara, For assessment of printability, we conducted rheological
Kusatsu, Japan). A total amount of 1.5 μg RNA per evaluation for two concentrations of GelMA. As shown
sample was used as input material for the RNA sample in Figure 1A, temperature sweep oscillatory tests were
preparations. Sequencing libraries were generated using performed through cooling process, the temperature to
NEBNext® Ultra™ RNA Library Prep Kit for Illumina® reach G′ and G″ crossover of 6% GelMA and 8% GelMA
(NEB, USA) following recommendations of manufacturer were about 18.8°C and 20.7°C, respectively. When
and index codes were added to attribute sequences to each temperature is higher than crosslinking temperature, G″ is
sample. The library preparations were sequenced on an greater than G′, and the GelMA hydrogels would be in a
Illumina Novaseq 6000 platform by the Beijing Allwegene fluid or sol state. In addition, the results of time sweep tests
Technology Company Limited (Beijing, China), and paired- demonstrated that the different concentrations of GelMA
end 150 bp reads were generated. Differential expression showed time-dependence properties when operating from

Table 1. Antibodies used in immunofluorescence experiment

Antibodies Concentration Source
Rabbit anti--human Ki-67 1:100 Abcam, Cambridge, UK
Mouse anti-human p53 1:100 Abcam, Cambridge, UK
Goat anti-Rabbit (Alexa Fluor® 594) 1:300 Abcam, Cambridge, UK
Goat anti-Mouse (Alexa Fluor® 488) 1:300 Abcam, Cambridge, UK

Table 2. Primers used in quantitative polymerase chain reaction (qPCR)
Gene Forward (5’-3’) Reverse (5’-3’)
p53 ACTTGTCGCTCTTGAAGCTAC GATGCGGAGAATCTTTGGAACA
IL-6 GCCAGAGCTGTGCAGATGAG CAGTGGACAGGTTTCTGACC
CD133 AGTCGGAAACTGGCAGATAGC GGTAGTGTTGTACTGGGCCAAT
ALDH1 CCCTGGAGACGATGGATACAG TCTGAGGGTTCTAATACAGCCC
CD44 CTGCCGCTTTGCAGGTGTA CATTGTGGGCAAGGTGCTATT
ABCA1 TCTCACCACTTCGGTCTCCATG CCTCGCCAAACCAGTAGGACTT
MRP-1 TCTACCTCCTGTGGCTGAATCTG CCGATTGTCTTTGCTCTTCATG
MDR-1 TTGCTGCTTACATTCAGGTTTCA AGCCTATCTCCTGTCGCATTA
GAPDH CGAGATCCCTCCAAAATCAA TTCACACCCATGACGAACAT

Volume 10 Issue 3 (2024) 231 doi: 10.36922/ijb.1673