Cellular vector preparation
glioblastoma
VESICULAR STOMATITIS, VIRUS, MYCOPLASMA INFECTION, STERILE, MYCOPLASMA, GLIOBLASTOMA
For ADSC isolation, 100 mL of subcutaneous adipose tissue was obtained from a healthy donor through lipoaspiration techniques, under sterile conditions. The isolated adipose tissue was then washed with a solution containing warm PBS and antibiotics. After that, using a blade, adipose tissue was cut into 1 mm pieces and mixed with equal volumes of 0.2% collagenase enzyme for 15–30 min in the incubator (95% humidity and 5% COFor lentivirus production, the human embryonic kidney (HEK) 293 cell line was cultured in a high-glucose DMEM medium, 10% FBS, 1% nonessential amino acids, and L-glutamine under the standard condition. The gain of function study was performed using a plasmid (pCDH-CMV-MCS-EF1-copGFP), which had been previously digested by BamHI and EcoRI endonuclease enzymes. The TK gene was cloned into the plasmid and the protocol was tested in vitro using the GFP product. After that, by using the calcium phosphate transfection method, the HEK 293 cells (packaging cell line) with 70% confluency were transfected with 21 µg PCDH-TK, 21 µg PsPAX2 plasmid (containing Gag, Pol, Rev, and Tat), and 10.5 µg PMD2 plasmid (containing vesicular stomatitis virus glycol protein) in T75 flask using four optimized reagents containing HBSS 2 × (0.05 M HEPES, 0.28 M NaCl, 0.75 mM Na2HPO4, 0.75 mM NaH2PO4 PH = 7:05,), TE1 × (10 mM Tris-HCL, 1 mM EDTA; PH = 8), Cacl2 (2.5 Mm) and Buffered Water ( 2.5 mM HEPES PH = 7.5) on a vortex [
Flow cytometry results demonstrating positive CD73, CD90, CD105, and negative CD34 and CD45 in mesenchymal stem cell (MSC) populationAfter confirming the high quality of transfection, the supernatant was isolated every 12 h for 3 days and concentrated using ultracentrifuge at 47,000 x g for 2 h at 4 °C. The TK expression in ADSCs was induced through their culture in DMEM-F12 medium and transduction with pCDH-TK lentivirus. A total of 3 × 10
HEK-293T, as packaging host, were co-transfected by psPAX2, pMD2.G, and PCDH-TK with the ratio of 2:1:2 and calcium phosphate reagent (
The high efficiency transduced U-251/PCDH (transfected with the plasmid without the HSV-TK gene) and U-251/HSV-TK cells (more than 80% of population) observed using fluorescent microscopy (To investigate the HSV-TK expression, the total RNA was extracted from 5 × 10The effect of HSV-TK gene expression on the U-251 glioblastoma cell line in the presence of ganciclovir was measured by MTT assay. First, the resistance of the U-251 cell line against ganciclovir was determined by exposing this cell line to different concentrations of ganciclovir (0, 5, 10, and 20 µM) and investigating cell viability with MTT assay. Second, 5 × 10To prepare transduced MSCs for injection, following the purification using puromycin, cells were washed three times with normal saline and prepared for injection. Potential endotoxin contamination was prevented by using EndoFree Plasmid Qiagen Maxi Kit (Cat No./ID: 12,362) for plasmid extraction. Potential contamination with mycoplasma was evaluated using PCR-based methods with universal mycoplasma primers (forward primer: 5´-GGC GAA TGG GTG AGT AAC ACG-3´ and reverse primer: 5´-CGG ATA ACGC TTG CGA CCT ATG-3´) for detecting all mycoplasma species before and after the antibiotic usage. No mycoplasma infection was observed.