Trypsin

Protein expression was assessed with flow cytometry using specific antibodies on a flow cytometer CyFlow Space (Partec, Meckenheim, Germany). Cells were washed with a Versene solution (Thermo Fisher Scientific, Waltham, MA, USA), treated with 0.25% trypsin (Paneco, Moscow, Russia), washed with the culture medium, and suspended in phosphate buffer solution (pH 7.4) (Paneco, Moscow, Russia). The cells were fixed with paraformaldehyde (PFA, Sigma-Aldrich, Saint Louis, MO, USA) at 37 C for 10 min, then washed three times with 0.5% BSA–PBS and permeabilized with 0.1% Triton X-100 (BSA and Triton X-100 were from Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 15 min at 20 °C or with 90% methanol (Sigma-Aldrich, Saint Louis, MO, USA) at 4 °C, then washed with 0.5% BSA–PBS (3 times). The cells were stained with conjugated antibodies (1 µg/mL) for 2 h at room temperature, washed with PBS, and analyzed by a flow cytometer (CytoFlex S, Beckman Coulter, Brea, CA, USA).

Poly(DL-lactide-co-glycolide) 50:50 (PLGA, Mw 45 kDa) was obtained from Boehringer Ingelheim (Belgium); oligo-(l)-lactide (OLL, Mw 5 kDa), oligo-(D)-lactide (OLD, Mw 5 kDa) were synthesized from the respective lactic acid isomers as described earlier [14 (link)]; Dextran sulphate sodium salt (DS) (Mw~9–15 kDa), Poly-l-arginine hydrochloride (PAr, Mw~15–70 kDa), Polyallylamine hydrochloride (PAH, Mw~58 kDa), Poly(sodium 4-styrenesulfonate) (PSS) (Mw~70 kDa), Poly vinyl alcohol (PVA, Mw 70 kDa), Fluorescein isothiocyanate (FITC), Rhodamine B isothiocyanate (RBITC), Resazurin sodium salt, Crystal Violet, EDTA, zimozan were all obtained from Sigma-Aldrich (Saint Louis, MO, USA); Phosphate buffered Saline (PBS), DMEM/F12, trypsin, penicillin and streptomycin were purchased from PanEco (Moscow, Russia); Fetal calf serum (FCS) was obtained from Invitrogen (Carlsbad, CA, USA); Chloroform was obtained from Chimmed (Moscow, Russia); DMSO was obtained from Panreac (Castellar del Vallès, Spain).

Cells were washed with a Versene solution (Thermo Fisher Scientific, Waltham, MA, USA), treated with 0.25% trypsin (Paneco, Moscow, Russia), washed with the culture medium and suspended in phosphate buffer solution (pH 7.4) (Paneco, Moscow, Russia). The cells were fixed with paraformaldehyde (PFA, Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C for 10 min, then washed three times with 0.5% BSA–PBS and permeabilized with 0.1% Triton X-100 (BSA and Triton X-100 were from Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 15 min at 20 °C or with 90% methanol (Sigma-Aldrich, Saint Louis, MO, USA) at 4 °C, then washed with 0.5% BSA–PBS (3 times). The cells were stained with conjugated antibodies (1 μg/mL) for 2 h at room temperature, washed with PBS, and analyzed by a flow cytometer (CytoFlex S, Beckman Coulter, Brea, CA, USA). To stain with unconjugated primary BAX antibodies, the cells were incubated with antibodies (1 μg/mL) overnight (+4 °C), washed with 0.5% BSA–PBS, then incubated for 1 hour (at room temperature) with secondary antibodies (1 μg/mL), washed three times with 0.5% BSA–PBS, and analyzed.
For flow cytometry ROS analysis, unfixed cells suspension were incubated with 10-μM solution of H2DCFHDA in PBS (Molecular Probes/Invitrogen, Carlsbad, CA, USA) 15 min in the dark, washed PBS, resuspended in PBS and analyzed by flow cytometer in FITC channel.

Glioblastoma cells (U-251 MG and DBTRG-05MG) and osteosarcoma cells (HOS) were purchased from ATCC and maintained in DMEM medium with 4.5 g/L glucose, 10% bovine serum (BioSera, France), and 2 mM glutamine (PanEco, Russia). In addition, VSV (Indiana strain), kindly gifted by Oleg Zhirnov (D.I. Ivanovsky Institute of Virology, a division of N.F. Gamaleya National Research Center of Epidemiology and Microbiology, Moscow, Russia), was used for virus infections.
For primary cultures obtaining tumor, fragments were disintegrated mechanically with a scalpel and washed with phosphate-buffered saline within 24 h after excision. Then further homogenization of the tumor was carried out using a strainer with a pore diameter of 200 μm. The resulting suspension was sown on culture plates with DMEM medium (PanEco, Russia) with 10% fetal bovine serum (Gibco, USA) and the addition of an antibiotic and antimycotic (Anti-anti, Invitrogen, Waltham, MA, USA). Then, for the next 14 days, the medium was changed every 2 days; the cells attached to the plastic formed colonies. Finally, the cells were passaged using standard methods: they were detached by washing with a versene solution, then treated with trypsin 0.025% (PanEco, Russia). In the experiments, 15–25 passages of cells were used, the morphology of which remained unchanged during the entire study.

l-Glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, HEPES, Hanks’ salts solution, trypsin, DMEM, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. HEPES, KCl, CaCl₂, MgCl₂, DMSO, Triton X-100, d-glucose, non-essential amino acids, Hoechst 33258, human erythrocyte AChE, equine serum BChE, acetylthiocholine iodide, CHAPS, EDTA, dithiothreitol, PNU 120596, protease inhibitor cocktail, SCP0139, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were from Sigma-Aldrich, St. Louis, MO USA. Arachidonic acid and Z-VAD-FMK were purchased from Cayman Europe, Hamburg, Germany. Ac-DEVD-AFC and Ac-LEHD-AFC were from Tocris Bioscience, Bristol, UK. Fluo-4AM and probenecid were from Thermo Fisher Scientific, Waltham, MA, USA.

Cytotoxicity of SA scaffolds has been evaluated by a colorimetric method using MTT. MSCs were detached from the surface of Petri dishes using a Versene solution (PanEco, Russia) with the addition of 0.25% trypsin (PanEco, Russia) and seeded in 24-well plates (Corning, NY, USA) with a density of 5 × 10⁴ cells per well. Non-cross-linked SA, control scaffolds cross-linked with 2 wt.% or 10 wt.% CC solutions, as well as gene-activated scaffolds, were placed in Transwell system with a pore size of 8 µm (Corning Transwell). The wells without scaffolds (cells only) were used as controls. After 1 and 7 days, MTT (tetrazolium (3-(4,5-dimethyl thiazolyl−2)−2,5-diphenyltetrazolium bromide, PanEco, Russia) at a concentration of 0.5 mg/mL was added to the wells and incubated for 2 h at 37 °C. Formazan crystals were extracted from cells using dimethylsulfoxide (DMSO, PanEco, Russia) stirring on a shaker for 20 min. The formazan absorption was evaluated by measuring the optical density of the eluate at a wavelength of 570 nm subtracting the background value at 620 nm on a BioRad Reader XMark tablet reader (Bio-Rad Laboratories, Hercules, CA, USA).

To assess the viability, gene expression profile, and immunophenotype of hAMSCs adhered to the plastic around the CaP-coated samples, cells were preliminarily harvested with 0.05% trypsin (PanEco, Moscow, Russia) in 0.53 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) and washed twice with phosphate-buffered saline.
The surface markers on viable hAMSCs were analyzed with a Human MSC Phenotyping Kit (cat. no. 130-095-198, Miltenyi Biotec, Bergisch Gladbach, Germany), which detects the markers CD14, CD20, CD34, CD45, CD73, CD90, and CD105. After a 10-min incubation with the labeled monoclonal antibodies (mAbs), the cells were assayed using a MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. The in vitro viability of hAMSCs was also estimated with the MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) after staining with a solution of Annexin V: FITC (Abcam, Cambridge, UK) and propidium iodide (Abcam, Cambridge, UK) according to the manufacturer’s protocol. The flow cytometric data were analyzed using KALUZA analysis software (Beckman Coulter, Brea, CA, USA).