Page 257 - IJB-10-3

International Journal of Bioprinting Bioprinting organoids for toxicity testing

in 4% formaldehyde for 30 min, washed, and stained with The following antibodies and dilution ratios were
red neutral lipid dye (Invitrogen, H34476) according to employed: HNF4α (1:200, Abcam, ab92378), FOXA2 (1:200,
the manufacturer’s instructions. The nucleus was stained Abcam, ab256493), ALB (1:100, Abcam, ab83465), CD31
with DAPI. The DOs were visualized by means of confocal (1:100, Cell Signaling Technology, 89C2), MRP2 (1:100,
microscopy. The fluorescence intensity of neutral lipids was Abcam, ab3373), αSMA (1:100), COL1A1 (1:100, Cell
analyzed using Nikon NIS-Elements AR. The fluorescence Signaling Technology, 72026), F-actin (Invitrogen, A12379),
intensity of neutral lipid dye in each slide was normalized Alexa Fluor® 594-conjugated secondary antibody (1:1000,
to the DAPI area. Then, the fluorescence intensity of Abcam, ab150080), and Alexa Fluor® 488-conjugated
neutral lipid staining was counted by finding the average of secondary antibody (1:1000, Abcam, ab150113). The nuclei
all the slides in three independent experiments. were stained with DAPI (Sigma, D9542).

2.7.2. Indocyanine green uptake assay 2.9. Total RNA isolation and qRT-PCR analysis
The indocyanine green (ICG) uptake assay was performed The DOs were harvested from microspheres with a de-
by adding 1 mg/mL ICG to the medium for 30 min. ICG crosslinking solution. The DOs were resolved in TRIzol
was wholly released from the cells after 7 h. ICG uptake and (Life Technologies, USA, 15596018). Total RNA samples
release images were captured from over 20 microspheres were isolated according to the TRIzol manufacturer’s
using an inverted optical microscope. Image-Pro software specifications. RNA (2 µg) was reverse-transcribed into
was used to calculate the uptake ratio as the Integrated cDNA with the RT Reagent Kit with gDNA Eraser (Takara,
Optical Density (IOD) of the microsphere divided by the Japan, RR047A). Quantitative polymerase chain reaction
area of the microsphere. (qPCR) was performed using a SYBR Premix Ex Taq kit
2.8. Immunofluorescence staining (Takara, Japan, RR420A) on a QuantStudio 6 Flex System
2.8.1. Staining of cultured planner cells (Thermo, USA). The PCR primers are listed in Table S1
hiPSC-derived HE and EPC were fixed with 4% (Supplementary File).
paraformaldehyde for 15 min and blocked with Dulbecco’s 2.10. RNA-seq
phosphate-buffered saline (DPBS; Solarbio, D1040) The samples for RNA-seq were collected in TRIzol. RNA
containing 5% goat serum (Solarbio, China, SL038) and isolation, cDNA library construction, sequencing, and
0.1% Triton X-100 (Amresco, USA, 0694-1 L) for 1 h at primary analysis were performed at Novogene (Tianjin,
room temperature. Then, the cells were incubated with China). Briefly, 1 µg of RNA per sample was used as input
primary antibodies for 1 h and secondary antibodies for 30 material for the RNA sample preparations. Following the
min at room temperature. manufacturer’s recommendations, sequencing libraries
2.8.2. Staining of DOs were generated using the NEBNext® UltraTM RNA Library
The DOs-laden microspheres were incubated in a de- Prep Kit for Illumina® (NEB, USA), and index codes were
crosslinking solution (150 mM sodium chloride containing added to attribute sequences to each sample. After being
55 mM sodium citrate and 20 mM EDTA), and DOs were analyzed with an Agilent 2100 Bioanalyzer, the RNA was
harvested by gravitational settlement. found to be of good standard quality.
The harvested DOs or DOs-laden microspheres Sequencing was performed with an Illumina NovaSeq
were fixed with 4% paraformaldehyde for 45 min at platform, and 150 bp paired-end reads were generated.
4°C, washed three times with 0.1% v/v Hanks (Solarbio, Reads were filtered and mapped to the reference genome
H1025)-Tween-20, and blocked with Hanks containing sequence with HISAT2 v2.0.5. Feature Counts v1.5.0-p3
2% BSA and 0.1% Triton X-100 for 15 min at 4°C. Then, was used to count the read numbers mapped to each gene.
the samples were incubated with primary antibodies The fragments per kilobase of exon per million fragments
overnight at 4°C. After being washed three times, the DOs mapped (FPKM) method was used to calculate gene
were continuously incubated with secondary antibodies expression levels. Differentially expressed genes (DEGs)
overnight at 4°C. Fluorescent images were taken with an with fold change ≥ 1.25 and FDR q-value <0.05 were
LSCM (Nikon, Z2). calculated using the DESeq2 R package (1.16.1).
The fluorescent intensities of COL1A1, αSMA, and Gene Ontology (GO), Kyoto Encyclopedia of Genes
F-actin were analyzed by Nikon NIS-Elements AR. The and Genomes (KEGG), Reactome, and DisGeNET
fluorescence intensity in each slide was normalized to enrichment analyses of DEGs were implemented using the
the DAPI area. The fluorescence intensity of each target clusterProfiler R package. Significantly enriched GO and
protein was calculated by determining the average of all KEGG terms were determined based on a corrected P <0.05.
the slides in three independent experiments. Based on KEGG, Reactome, and DisGeNET datasets, gene

Volume 10 Issue 3 (2024) 249 doi: 10.36922/ijb.1403