The effect of platelet-rich fibrin on the biological properties of urothelial cells – Scientific Reports

Institutional review board statement

The animal study protocol was approved by the Institutional Review Board of Hainan Medical College (No. HYLL-2022-403, 16 November 2022). All methods were performed in accordance with the relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.

Cultivation of iPSCs

In this study, mouse iPSCs were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Mouse iPSCs were cultured in a high-glucose DMEM medium. When the cells were fused to 90%, the medium was discarded, washed with PBS, and added with trypsin-EDTA digestion solution, and observed under the microscope for about 30 s. When the cells became rounded, a complete medium was added to terminate the di-gestion quickly, and the cells were collected. The cells were resuspended with a complete medium and cultured in separate flasks, and the solution was changed every other day.

Identification of the iPSCs

Alkaline phosphatase stain

Cells are discarded from the old medium and washed with pre-warmed DMEM/F-12 medium for 2–3 min, the medium is aspirated discarded, and repeated once. Prepare 1× AP live staining working solution by diluting 500× stock solution in DMEM/F-12. Appropriate amount of 1× AP live staining solution was applied directly onto the adherent cell cultures and incubated for 20–30 min. The DMEM/F-12 medium is washed again and then the fluorescent-labeled colonies are displayed on a fluorescence microscope (200×) using a standard FITC filter. Images can be captured within 30–90 min after staining, and after visualization, the DMEM/F-12 medium is replaced with fresh medium and the cells can be returned to normal culture.

Observation of cell morphology

The morphology of iPSCs was observed under a light microscope (100×).

Cell immunofluorescence

Cells were inoculated into 6-well plates at 5 × 105 cells/well respectively, observed under an inverted microscope, and the supernatant was aspirated and discarded when the cell proliferation reached 80%. The cells were fixed with 500 µL 4% paraformaldehyde for 15 min, washed with PBS added with 0.1% Triton X-100, and incubated at room temperature for 10 min, washed with PBS. The cells were blocked with a blocking solution for 30 min at room temperature, the blocking solution was discarded, and a primary antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488)) was added, and the cells were incubated at 4 °C overnight. At the end of incubation, PBS solution was washed, and then the corresponding Alexa Fluor 488 labeled secondary antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)) was added and incubated for 1 h in the dark, and washed three times with PBS solution, each time for 5 min. The plate was sealed by adding DAPI dropwise, and then the excess liquid was drained off, the plates were observed under a laser confocal microscope. The expression of Oct4, Sox2, and SSEA-1 was observed under a laser confocal microscope (Zeiss LSM), and the field of view was randomly selected at 200× magnification for photographing (Antibodies and dilution are shown in Table 1).

Table 1 Antibodies and dilution.

Western blot

Extraction of total cellular proteins: Cell samples were lysed with RIPA containing PMSF, and the supernatant was collected in a pre-cooled Eppendorf tube after centrifu-gation. The cellular proteins obtained from the extraction were quantified using the BCA method. Gel electrophoresis and membrane transfer: To measure proteins with different molecular weights, 10% separating gel and 5% concentrating gel were prepared for SDS-PAGE. The gel was filled, and 1× electrophoresis buffer was added. Sampling began with the volume of samples adjusted to 60 µL. Proteins on the membrane were observed after the completion of membrane transfer. Developing: The membrane was wetted and transferred to a dish with a sealing solution, and closed by shaking on a decolourising shaker for 1 h at room temperature to seal the immunoglobulin binding site of the PVDF membrane. The primary antibody was added overnight. The membrane was washed with TBST, placed in a closed pouch, and a secondary antibody was added (the primary antibody, secondary antibody and dilution ratio are as shown in Table 1). The pouch was closed and incubated at room temperature for 1 h. The membrane was washed with TBST, and equal volumes of chemiluminescent reagents, liquid A and liquid B, were mixed. The mem-brane proteins were placed face down in full contact with this mixture for 5 min, then detected by Tanon-6600 Luminescence Imaging Workstation. Protein expression was analyzed using Image Pro Plus 6.0 software for optical density values.

PCR

mRNA extraction: cell suspension was inoculated in a 6-well plate with 5 × 105 cells per well, when the cell confluence reached 90%, the cells were collected and centrifuged. Trizol, chloroform, and isopropanol were added in sequence for shaking, and centrifugation respectively to precipitate the RNA. Mixing the dissolved RNA to get the total solution, and 2 µL of the sample was taken, the concentration and purity of cDNA were determined by Nanodrop-2000. cDNA synthesis: take 2 µL of total RNA as a template, configure the reverse transcription reaction system according to the kit manual, place the reaction system in the PCR instrument, select the set program, and complete the cDNA. Real-time quantitative PCR: Carry out the reaction system configuration according to the kit manual, add the premixed reagents into the 8-link tube, and set the reaction parameters to amplification. The data were processed by the 2-ΔΔCt formula, and the expression level of the target mRNA was obtained using the internal reference gene β-actin as a reference (PCR reaction condition and the primers used are shown in Tables 2 and 3).

Table 2 PCR reaction condition.
Table 3 Primers used in quantitative real-time PCR.

Transformation of iPSCs into urothelial cells

The iPSCs were continued to be cultured in a medium containing 50 µg/L activin A for 14 days for transformation to directed endoderm. The expression levels of SOX17 and FOXA2 were detected by flow cytometry. DE cells were cultured in DMEM medium to continue the culture for 14 days, and they were transformed into urothelial cells. The morphology of the cells was observed by light microscopy, and the expression levels of UP1a, UP1b, and UP2 were detected in each group by the same immunofluorescence and flow cytometry as previously (the antibodies used in immunofluorescence and the dilution ratios are as shown in Table 4).

Table 4 Antibodies and dilution.

Preparation of PRF and HE staining

10 mL of healthy rat whole blood was collected into an anticoagulant-free test tube and immediately centrifuged at 3000 r/min for 10 min. After centrifugation, the blood in the centrifuge tube was divided into three layers, with a yellowish platelet-poor plasma (PPP) layer on the top, a red blood cell debris layer on the bottom, and a platelet-rich fibrin gel layer in between, the PRF gel was removed and left to stand for 10 min to be slowly dehydrated to get the PRF.

Fresh PRF was prepared, fixed using 4% paraformaldehyde for 48 h, and dehydrated. The tissue wax blocks were loaded on the clamping table of the slicer and the thickness adjuster was adjusted to 5 microns for slicing. The prepared paraffin sections of PRF membranes were baked in an electrically heated oven for 3 h, and the dried paraffin sections were subjected to conventional xylene dewaxing, ethanol hydration, distilled water washing, hematoxy-lin-stained nuclei, and eosin-stained solution staining and then sealed. A field of view was randomly selected using a phase contrast microscope to be photographed using a 1000× magnification field of view.

Transmission electron microscopy observation of PRF

The prepared PRF was placed into a 6-well plate, take 1 mL of cell suspension and drop it on 1 piece of PRF membrane (each 1 mL of PRF prepared from arterial blood is 1 piece of PRF) for culture, the PRF membrane inoculated with cells was fixed after the culture was completed. The simple PRF membrane and the PRF membrane containing cells were fixed and dehydrated after osmium acid, and the dehydrated specimens were infiltrated and embedded. The embedded specimens were cut into sections with a thickness of 70 nm on an ultrathin sectioning machine. The sections were stained with lead citrate and uranyl acetate, after the ultra-thin sections were dried, they were observed by transmission electron microscopy and photographed.

Effect of PRF on the proliferation and migration of urothelial cells

Urothelial cells derived from iPSC in the logarithmic growth phase were taken and made into cell suspension and inoculated into 96-well plates at a concentration of 3 × 103/mL, 100 µL per well. DMEM and PRF medium were added 24 h later, respectively, and incubated for 24, 48, and 72 h, respectively. 10 µL of CCK-8 solution was added to each well, and incubation was continued for 1 h. The absorbance was detected at 450 nm by an enzyme counter-clockwise.

Cells were routinely digested and centrifuged, then resuspended using serum-free medium and counted, the cell density was adjusted to 2.5 × 105 cells/mL. Incubated chambers were removed, and cell suspension was added to the upper chamber for in-cubation, after incubation, the chambers were removed for staining. After staining, cells passing through the chambers were counted using an inverted microscope with randomly selected counting fields.

Statistical analysis

Data were analyzed and graphed using GraphPad Prism 9 (Version 9.4.0) and collated using Adobe Illustrator 2022 (Version 2022). All data were expressed as mean ± SD, and statistical differences between groups were determined by 2-way ANOVA, one-way ANOVA, and Tukey’s test. P values < 0.05 were considered significant.