Isolation and culture of hWJSCs
In present study, 10 human umbilical cords were received from pregnant women referred to Shahryar Hospital, Tabriz, Iran. The umbilical cords were transferred Biotechnology Research Center, Islamic Azad University, Tabriz branch, in proximity of the ice. Initially, the Wharton’s jelly tissues were primary cultured (explant culture) using complete Dulbecco’s modified eagle medium (DMEM) contains 1% penicillin-streptomycin antibiotic and 10% fetal bovine serum (FBS) at 37°C and 5% CO2 (9). It is noteworthy that all selected women were signed a consent form according to the ethical standards of Helsinki Declaration.
Preparation of conditioned medium and cell lysate of hWJSCs
The isolated hWJSCs were cultured using DMEM medium contains 1% penicillin-streptomycin (5000 units/mL-5000 mg/mL) antibiotics without FBS at 37°C and 5% CO2 for 72 hours. Next, supernatant medium was collected as conditioned medium, and sterilized using a 0.22 μm filter. Then, the cell lysis buffer (150 mM NaCl, 1.0% Nonyl Phenoxypolyethoxylethanol-NP40, 50 mM Tris-Cl, 1.0% sodium deoxycholate, 0.1% SDS) and protease inhibitor were added to the cultured cells. The obtained cell suspension was centrifuged and cells pellet was collected as cell lysate.
Cancer cells culture
The HT-29 cancer cells were purchased from cell bank of Immunology Research Center (IRC), Tabriz University of Medical Sciences. The obtained cancer cells were cultured using Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS and 1% penicillin-streptomycin at standard condition.
Cancer cells viability assay
The CC cells (1×104/well) were seeded in 96-well plate containing culture medium supplemented by FBS. After 24 hours, the cancer cells were treated with conditioned medium (38, 40, 42, 44, and 46%) and cell lysate (11, 12, 13, 14, and 15%), and incubated for 72 hours. The Tetrazolium Micro-culture (MTT) method was performed according to the manufacturer’s instruction. For this purpose, the old culture medium was replaced with RPMI medium containing MTT agent solution (50 µl) in phosphate-buffered saline (PBS), and incubated for 4 hours. Next, the old culture medium replaced with 50 µL Dimethyl sulfoxide (DMSO) and incubated for 30 minutes. The absorbance of wells was measured using enzyme-linked immunosorbent assay (ELISA) reader at 540 nm. Finally, the treated cancer cells viability was measured by following formula [(treated sample OD / untreated sample OD) × 100].
Transwell invasion assay
The CC cells were seeded in the upper chamber of transwell chamber in RPMI medium (serum-free), and treated by conditioned medium (44%) and cell lysate (14%). Moreover, the complete medium was added to the bottom chamber and incubated for 20 hours. Then the migrated cancer cells in bottom chamber were fixed by paraformaldehyde (4%) and stained by crystal violet (0.1%). Finally, the migrated cancer cells were evaluated by inverted phase contrast microscopy.
Wound‐healing migration assay
The CC cells (1×104/well) were seeded in 96-well plate containing culture medium supplemented by FBS. The monolayer cancer cells were scratched by pipette tips and washed with PBS. Then, cancer cells were treated with conditioned medium (44%) and cell lysate (14%), and incubated for 72 hours for 20 hours. Finally, the migrated cancer cells were evaluated by inverted phase contrast microscopy.
Cancer cells apoptosis assay
The CC cells (1×104/well) were seeded in 96-well plate containing culture medium supplemented by FBS. After 24 hours, the cancer cells were treated with conditioned medium (44%) and cell lysate (14%), and incubated for 72 hours. The CC cells were washed, trypsinized, and resuspended in binding buffer with propidium iodideand annexin V. Then, cancer cells suspension incubated at dark place and room temperature with Annexin V-FITC (5 mL), PI (10 mL), and binding buffer (400 µL) for 15 minutes. Finally, quantitative apoptosis was measured using fluorescein isothiocyanate (FITC)/PI by flowcytometry instrument.
Quantification of apoptosis-related genes expression
The CC cells (1×104/well) were seeded in 96-well plate containing culture medium supplemented by FBS. After 24 hours, the cancer cells were treated with conditioned medium (44%) and cell lysate (14%), and incubated for 48 hours. Extraction of total RNA was performed by Trizol reagent, and cDNA synthesized using reverse transcription. The mRNA expression of BAX, BCL2, SMAC, SURVIVIN, and Cas9 genes were evaluated using quantitative Real Time PCR and specific primers (Table 1). The Real Time PCR analysis was performed using SYBR green master mix and relative expression was evaluated using comparative CT (2-ΔΔCT) method. The β-actin gene was considered as endogenous control.
The statistical analysis between treatment and control cells were performed by one-way ANOVA and Tukey (post‑hoc) analysis. The results were presented as mean ± standard deviation (SD) from three replicates, and p<0.05 was considered statistically significant.