Page 63 - IJB-9-3

International Journal of Bioprinting Peritoneal scaffolds for the peritoneal adhesion prevention

interval, three mice were killed, and the peritoneal tissues solubilized peritoneal resident macrophages (6 × 10 )
4
were harvested at the scaffold suture site for hematoxylin– were inoculated into the upper chamber of the Transwell
eosin (HE) staining. at 100 μL per well. Peritoneal resident macrophages were
allowed to migrate across the Transwell membrane for 12 h.
2.7. Preparation and identification of peritoneal The remaining cells in the upper chamber were scraped
scaffolds out with a cotton swab, and the migrating macrophages
Primary peritoneal mesothelial cells were seeded onto the at the bottom of the upper chamber were detected by
scaffolds and inoculated with the mesothelial cell culture immunofluorescence.
medium (CM-M170; Procell Life Science and Technology
Co. Ltd.) in 24-well low-adhesion plates (3473; Corning, 2.10. Fluorescent staining
Corning, NY, USA). Freshly isolated primary mesothelial To detect the morphology of peritoneal mesothelial cells
cells (0.75 × 10 ) were suspended in 50 µL of each sample seeded on the scaffolds, the staining of CK-18 (ab24561;
6
in tissue culture-treated 24-well plates. To promote cell Abcam, Cambridge, MA, USA) and 4′,6-diamidino-2-
adhesion, cells were incubated at 37°C with 5% CO for phenylindole (DAPI; ab104139; Abcam) was performed
2
0.5 h, followed by adding 1 mL of cell culture medium. as described previously . The slides with cells on them
[23]
were then washed three times in PBS, fixed for 15 min in
2.8. Isolation of peritoneal resident macrophages 4% paraformaldehyde, permeabilized for 15 min in 0.5%
After the mice were killed, they were soaked in 75% alcohol Triton X-100, and blocked for 30 min in 1% bovine serum
for 10 s. The mice were removed from alcohol, drained, albumin. Samples were incubated in CK-18 solution at 1:200
and placed in the supine position on the ultraclean bench. for 45 min, and DAPI solution at 1:1000 for 6 min, both
The abdominal cavity was gently rubbed for 2 min to allow in the dark at 37°C. Samples were imaged using confocal
the physiological saline to flow in the cavity after 6 mL of laser scanning microscopy. Cross-sectional photographs
saline was added into the cavity with a syringe. The lower of the scaffolds were chosen from Z-Stack images that
abdominal skin was lifted with ophthalmic forceps so that were prepared using an FV1000 Viewer (Olympus, Tokyo,
the animal tilted to one side. After cutting the abdominal Japan) and collected every 5 μm to examine cell migration.
skin, a small incision was made in the muscle layer, and
the abdominal fluid was aspirated with a rubber-tipped The procedure of staining for vimentin (60330-
dropper and transferred into a centrifuge tube. The 1-Ig; Wuhan Sanying, China), phosphoenolpyruvate
aspiration volume was 4–5 mL per mouse. The collected carboxykinase (PCK; 16754-1-AP; Wuhan Sanying, China)
peritoneal lavage fluid was centrifuged at 1000 r/min for and DAPI (C1002; Beyotime Biotechnology, China) is the
10 min at 4°C. The supernatant was removed, and 10% same as described above. The tissue immunofluorescent
[24]
high-sugar Dulbecco’s modified Eagle’s medium (DMEM) staining procedure is essentially the same for the cells .
was added before cell counting using a cell counting plate.
Macrophages were counted under a microscope. The cell 2.11. Ischemic buttons model (IBM)
concentration was adjusted to the desired level. Cells were The Jinling Hospital’s Animal Investigation Ethics
inoculated into culture flasks and incubated at 37°C for 4 h. Committee authorized all of the animal care and
After full wall attachment, the supernatant was discarded, experimental protocols, which were carried out in strict
and the nonadherent cells were removed. Afterward, accordance with the Chinese Guidelines for the Care
high-sugar DMEM was added, and the culture flasks were and Use of Laboratory Animals (Ministry of Science
returned to the incubator. and Technology [2006] file no. 398). Pain was kept to
a minimum by doing all procedures under anesthesia.
2.9. Cell migration assay Adhesion induction procedures were performed on wild-
Migration assays of the peritoneal resident macrophages type B6 (C57BL/6J; GemPharmatech Co. Ltd., China)
were performed using an 8-μm pore size polyester mice at 6–8 weeks. On the median line, a skin incision
membrane Transwell (Corning). The scaffolds were was made along the length of the abdomen. Based on
trimmed to completely cover the bottom layer of the upper the length of the peritoneum, a comparable midline
chamber of the Transwell. The control group was not abdominal incision was created in the peritoneum. The
covered with any object; the blank group was covered with peritoneum was gently folded to the right and compressed
a scaffold that did not carry cells; and the experimental with a hemostat. An ischemia button was inserted into
group was covered with a peritoneal scaffold that carried the right side of the peritoneal wall after a tiny section
mesothelial cells. Five hundred microliters of 20% fetal of peritoneum (about 5 mm in diameter) was clamped
bovine serum-DMEM was dispensed per well in the lower with hemostatic forceps, the bottom of which was ligated
chamber of the Transwell. Cell-tracker Green (C2925; with 4-0 silk (Suzhou Medical Co. Ltd., China), and the
Thermo Scientific, USA) -labeled, serum-free DMEM- forceps were then released. Optionally, a light scrubbing of

Volume 9 Issue 3 (2023) 55 https://doi.org/10.18063/ijb.682