Page 289 - IJB-10-2

International Journal of Bioprinting Kidney hydrogel print for renal cancer model

Paraformaldehyde (PFA; P1110), 4,6-diamidino-2- concentrations were measured using a BCA Protein Assay
phenylindole (DAPI, C0060), phalloidin-FITC (CA1660), Kit I adherence with the manufacturer’s protocol. An
and phosphate-buffered saline (PBS; P1020) were all aliquot of total protein was separated by polyacrylamide
obtained from Beijing Solarbio (China). The primary gel electrophoresis under reducing conditions and was
antibodies used including GAPDH (5174S), CD44 (3578S), then transferred onto a polyvinylidene fluoride (PVDF)
CDH1 (3195S), vimentin (5741S), and CDH2 (13116S) membrane. The membrane was then blocked with
were obtained from Cell Signaling Technology (USA), and blocking buffer for 2 h before being incubated overnight at
the secondary antibody (ab150077) was obtained from 4°C with primary antibodies (GAPDH, 1:1000). Next, the
Abcam (UK). All primers were synthesized by Beijing membranes were incubated with the anti-rabbit secondary
Tsingke Biotech (China). Power SYBR Green PCR Master antibodies (1:5000) at room temperature for 1 h and finally
Mix (4367659) and TRIzol (15596018) were obtained from visualized with ChemiDoc MP Imaging System (BIO-
Thermo Fisher Scientific (USA). FastKing gDNA Dispelling RAD, USA).
RT SuperMix kits (KR118) and DNA extraction kits
(DP304) were obtained from Beijing TIANGEN (China). 2.6. Hematoxylin–eosin staining
Fetal bovine serum (FSP500) was obtained from Shanghai Fresh and decellularized samples were first fixed with 4%
ExCell Bio (China). BCA Protein Assay kits (23225) were PFA, dehydrated with ethanol, mounted in paraffin, and
obtained from Thermo Fisher Scientific (MA, USA). then cut into section with a thickness of 10 µm using a
microtome (Leica, Germany). Sections were then stained
2.2. Decellularization of kidney samples with hematoxylin and eosin (H&E). Briefly, the sections were
The kidney samples used in the experiments were derived first baked for 30 min at 60°C, deparaffinized with xylene,
from Chinese Middle White Pig, which was purchased and then washed with ethanol. Next, the sections were
from Central Fresh Market (Ningbo, China). The stained with hematoxylin solution for 10 min, differentiated
decellularization method was adapted from a previously with a differentiation solution for 3 min, and washed with
published method. Briefly, fresh kidney samples were first deionized water three times. The sections were then re-dyed
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cut into small pieces, washed with deionized water, stirred with eosin Y solution for 1 min, cleared with xylene, and
in 2% Triton X-100 solution for 24 h (changed every 12 sealed with resin. Finally, the samples were imaged using a
h), and soaked in 0.1% SDS for 48 h (changed every 12 h). microscope (DMi1, Leica Microsystems, Germany).
Finally, the samples were washed with deionized water for
24 h before being stored at −80°C. 2.7. GelMA synthesis
GelMA used in this study was synthesized based on a
2.3. DNA extraction previously published protocol. 32,33 Briefly, gelatin, Na CO
3,
2
The DNA from fresh and decellularized samples was and NaHCO were dissolved at 50°C for 3 h until complete
extracted using a DNA extraction kit with all procedures dissolution. Then, 2.5 wt% methacrylic anhydride was
3
stipulated in the product’s protocol. Briefly, the samples added to the dissolved gelatin solution for GelMA
were lysed with a tissue homogenizer (TISS-BAS1C24, synthesis to achieve a degree of methacryloyl substitution
Jingxin, China), then washed with buffers and extracted (DMS) of 30%. After continuous stirring in the dark for
using spin columns. The concentration of extracted DNA 3 h, five times of deionized water was used to dilute the
was determined using Nanodrop™ 2000 Spectrophotometer sample and dialyzed for 3 days with water change every 8
( Thermo Fisher Scientific, USA).
h. Finally, the synthesized GelMA were frozen overnight,
2.4. Scanning electron microscopy freeze-dried for 3 days, and stored at -20°C for the later
Decellularized kidney samples were ground to powder experimentation.
using a tissue homogenizer (TISS-BAS1C24, Jingxin, 2.8. Hydrogel preparation
China) and sputter-coated with gold on a sample holder The 10 wt% GelMA was prepared with a fully supplemented
for 1 min by a sputter coater (Supro Instrument, USA), and
finally imaged (n = 6 images at different spots per group) DMEM containing 0.5% LAP. Various amounts of
using a scanning electron microscope (SEM; Phenom Pro, kidney dECM powders were added separately to GelMA
Phenom, Netherlands). precursors to obtain 1 wt%, 2 wt%, and 3 wt% of 10%
GelMA-kidney solutions; hereafter, these are called the
2.5. Western blotting 10% GelMA-1% Kidney, 10% GelMA-2% Kidney, and 10%
For Western blotting, fresh and decellularized samples GelMA-3% Kidney solutions, respectively. Each precursor
were lysed with RIPA buffer and centrifuged at 12,000 solution was photo-crosslinked using 405 nm ultraviolet
rpm. The supernatant was then collected, and the protein (UV) light (10 W/cm ) for 1.5 min in a disc-like PDMS
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Volume 10 Issue 2 (2024) 281 doi: 10.36922/ijb.1413