Trypsin edta solution

Based on the data compiled and reported earlier [60 (link),61 (link)], umbilical-cord-derived mesenchymal stem cells (UC-MSCs) were isolated using the mixed enzymatic-explant method from Wharton’s jelly of umbilical cord. Collection of umbilical cords was approved by the Commission of Biomedical Ethics at National Medical Research Center for Obstetrics, Gynecology, and Perinatology of the Ministry of Healthcare of Russian Federation, Moscow (Ethic’s committee approval protocol No. 12, 17 November 2016). Written informed consent was obtained from all participants prior to the study.
UC-MSCs were cultured in Dulbecco’s modified Eagle medium/F-12 (DMEM-F12) (PanEco, Moscow, Russian Federation) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (PanEco, Moscow, Russian Federation) at 37 °C under 5% CO₂ humidified atmosphere. UC-MSCs were detached from the culture substrate with a trypsin-EDTA solution (PanEco, Moscow, Russian Federation), then the cell count and viability were estimated using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA USA).

The intracellular localization of BgDV1 capsid proteins was performed using the HeLa cell line. In certain experiments, the African green monkey kidney fibroblast-like cell line (Cos-1), suitable for transfection with the vectors requiring the expression of the SV40 T-antigen, was used. Cells were grown in flasks in Dulbecco’s modified Eagle medium (DMEM) medium supplemented with 10% bovine serum (Gibco, Gaithersburg, MD, USA) at 37 °C in 5% CO₂ atmosphere. Cells were grown till they covered 70–80% of the growing area and were passaged approximately every five days. Cells were removed from the culture surface using trypsin/EDTA solution (PanEco, Moscow, Russia). Prior to transfection, cells were counted and then transferred to the Petri dishes containing culture medium to a final cell density of 1 × 10⁵. The bottom of the Petri dishes was laid with cover slips.

Vesicles were isolated from MSCs (MSC CIMVs) and SNB-19 cells (SNB-19 CIMVs) using cytochalasin B (cytochalasin B from Drechslera dematioidea, #C6762-5MG, Sigma-Aldrich, St. Louis, MO, USA) as previously described [17 (link)]. When the cell culture reached a monolayer density of 90%, the medium was removed, the culture was washed twice with PBS and the cells were detached with a 0.25% trypsin-EDTA solution (PanEco, Moscow, Russia). Then the cells were washed with PBS and incubated in modified serum-free DMEM containing 10 µg/mL cytochalasin B (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C in a humidified atmosphere with 5% CO₂. After incubation, the cells were vortexed for 60 s. Next, a series of subsequent centrifugations were carried out. Firstly, the cells were centrifuged at 700 rpm for 10 min, then the supernatant was collected and centrifuged at 1400 rpm for 10 min, after which the supernatant was passed through a filter (1 µm) and centrifuged at 12,000 rpm for 15 min. The precipitate containing CIMVs was washed with PBS. After centrifugation, the supernatant was removed, and the resulting CIMV pellet was dissolved in the culture medium, PBS, or another buffer, depending on the purpose of further experiments.

Flow cytometry was used to analyze the ability of the drugs to reactivate the expression of epigenetically repressed GFP. Cells were seeded in 24-well plates at 2.5 × 10⁴ cells per well and incubated with LCS-1208 (concentration range 2–0.25 μM) and LCS-1269 (concentration range 4–0.5 μM) for 72 h. The medium in the wells was replaced with fresh one 24 h after treatment and then the cells were incubated for an additional 48 h. Next, the cells were detached from the culture plates using 0.25% trypsin-EDTA solution (PanEco, Russia) and washed with PBS. To maintain high cell viability, PBS solution with 2% fetal bovine serum was used as a cell-storing buffer. The relative number of GFP-positive cells was assessed using BD FACSCanto™ II flow cytometer. Histone deacetylase inhibitor Trichostatin A (TSA) was used as a positive control. Maximum concentration of DMSO for TSA, LCS-1208, and LCS-1269 in media was 0.01%. Experiments were performed three times.

B16F0 mouse melanoma tumor cells were cultured with the standard method on RPMI nutrient medium (Roswell Park Memorial Institute medium; Gibco, USA) added with 0.06% L-glutamine, 10% fetal bovine serum (Gibco, USA), 50 U/ml of penicillin, and 50 μg/ml of streptomycin sulfate (PanEco, Russia) in culture flasks (25 cm³) (Corning, USA) in an incubator at 5% CO₂ at 37°C and 85% humidity. Cells were removed using a 25% trypsin–EDTA solution (PanEco, Russia) during 5 min.

The cytocompatibility of the synthesized and commercial GelMA was studied in comparison with unmodified gelatin and DMEM medium (PanEco, Moscow, Russia) in terms of such indicators as the ability to adhere and the proliferative activity of ADSCs. Hydrogels, 10% concentration, were prepared in accordance with the described method and were poured into the wells of 24-well culture plates (Nunc, Roskilde, Denmark) at 350 µL per well. Synthesized and commercial GelMA was irradiated with UV (365 nm) for 10 min in the chamber of a Rokit Invivo 3D printer. The temperature of the 3D printer stage on which the tablet was placed was 0 ℃. The unmodified gelatin was not irradiated with UV but kept on the printer stage at the same temperature until solidified. After polymerization, the height of the hydrogel layer in each well, 1.5 cm in diameter, was 0.2 cm. ADSCs were seeded on top of hydrogels and in a plate without gelatin in the amount of 2.2 × 10⁴ cells per well. The cells were incubated in a 1 g/L glucose content DMEM medium (PanEco, Moscow, Russia) supplemented with 10% fetal bovine serum (Biosera, Nuaille, France). After 3 and 7 days, the medium was removed, and the cells were removed with a trypsin-EDTA solution (PanEco, Moscow, Russia) and counted.

Human pancreatic PANC-1, ovarian HeLa tumor cell lines, immortalized embryonic kidney HEK293 cells and murine colon CT26 cell line were used in the study. HEK293 were grown in DMEM supplemented with 10% fetal calf serum (FCS), pen-strep-glut (all from PanEco, Moscow, Russia). All other cell lines were grown in RPMI-1640 with the same supplements. Cells were passaged by trypsinization using Trypsin/EDTA solution (PanEco, Moscow, Russia) twice a week. Twenty-four hours before the assays, cells were seeded in 96 well plates at 10⁴ cells/well and incubated overnight to achieve standardized growth conditions.