Hepa class 100

Human diploid fibroblast; Lep-3 cells from lung of 3-month old human embryo and cell culture established from bone explants of 2–3 months old ICR mice (Bone explant cells, BEC) as it was earlier described [39 (link)] were used as model systems in our study. Lep-3 and BEC cells were obtained from the Cell culture collection of the Institute of Experimental Morphology, Pathology and Anthropology with Museum—Bulgarian Academy of Sciences (IEMPAM-BAS).
Both the cultures were grown in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. The cultures were kept in a humidified incubator (Thermo Scientific, HEPA Class 100, Waltham, MA, USA) at 37 °C under 5% CO₂ in air. For routine passages, the cells were detached using a mixture of 0.05% trypsin and 0.02% EDTA. The cell cultures were passaged 2–3 times per week (1:2 to 1:3 split). The experiments were performed during the exponential phase of cell growth. During investigations performed the BEC cells were on their 44–50th passages.

Mettler Toledo AL104 analytical balance (Switzerland Mettler Toledo); BIOPAC mp150 type 16 channel physiological signal recording and analysis system (US BIOPAG); Delixi constant temperature operation (Delixi Group); AD113B08114 polarized light microscope (Olympus, Japan). Flow cytometry (FACS Calibur Becton-Dickinson, USA).5%CO2 incubator HEPA Class 100 was purchased from Thermo Fisher. Water bath termostat oscylator was bought from Taicang Laboratory Instrument Manufacture. CO2 incubator (5410-220) was made by Precision Scientific. Laminar flow hood was made by Suzhou Antai Air Technology Co., Ltd (BCM-1000A). Counter top centrifuge was purchased from Beijing Jingli Centrifuge Co., Ltd (LDZ 5-2). Electric Thermostat water bath (DK-450 B type) was purchased from Shanghai Senxin Experimental Instrument Co., Ltd. Liquid nitrogen tank was MVE CRYOSYSTEM750. Microplate reader was made by Thermo Fisher (3001-1249). UV spectrophotometer was made by Eppendorf. PCR gene amplifier was made by Bio-Rad Laboratories, Inc (US). iQ TM5 Real-Time quantitative PCR was made by Bio-Rad Laboratories, Inc (US).

ASCs were isolated from adipose tissues of macrodactyly and normal abdominal subcutaneous adipose tissues as described previously^{23 (link)}. Briefly, the adipose tissue was minced under sterile conditions, and digested with 0.05% Collagenase II (Sigma-Aldrich, St. Louis, USA) in serum-free DMEM (Hyclone, Logan, USA) at 37 °C for 2–3 h. After digestion, the fat tissue was filtered and centrifuged (1500 rpm at 37 °C for 5 min). The cells were re-suspended in low glucose DMEM (Hyclone, Logan, USA) containing 10% FBS (Gibco, Grand Island, USA) and 100 μg/mL penicillin-streptomycin (Gibco, Grand Island, USA), and cultured at 37 °C in a 5% CO₂ incubator (HEPA class 100, Thermo, Waltham, USA). When the cells became confluent, they were detached with 0.25% trypsin-EDTA (Gibco, Grand Island, USA) and subcultured at the same density for passage.

Three groups of permanent cell lines were used as model systems in our investigations. They were established from:

virus-induced transplantable tumours in chicken – LSCC-SF-Mc29 (hepatoma induced by the myelocytomatosis virus Mc29) and in rat – LSR-SF-SR (sarcoma induced by Rous sarcoma virus strain Schmidt-Rupin). Both cell lines were established, characterized and maintained in the Institute of Experimental Morphology, Pathology and Anthropology with Museum – Bulgarian Academy of Sciences (IEMPAM–BAS);[22, 23 ];

human cancers of the breast (MCF-7), uterine cervix (HeLa), lung (A549) and liver (HepG2);

three-month-old human embryo (Lep-3).

The human cell lines were obtained from the cell culture collection of IEMPAM–BAS.
The cell cultures were grown in the DMEM medium supplemented with 5%–10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cell number and viability were determined by a trypan blue dye exclusion test using a Countess® Automated Cell Counter (Invitrogen). The cultures were kept in a humidified incubator (Thermo Scientific, HEPA Class 100) at 37 °C with 5% CO₂ in the air. For routine passages, adherent cells were detached using a mixture of 0.05% trypsin and 0.02% etylenediaminetetraacetic acid (EDTA). The cell lines were passaged two or three times per week (1:2 to 1:3 split). The experiments were performed during the exponential phase of cell growth.

All cell lines were grown in D-MEM supplemented with 5–10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. They were seeded in 96-well microplates and kept in a humidified incubator (Thermo Scientific, HEPA Class 100, Waltham, MA, USA) at 37 °C under 5% CO₂ in the air. The cell cultures were treated with the designed pVEC-ASOs or with pVEC alone. They were cultured in the humidified incubator for 72 h at 37 °C. Next, MTT assays were performed as described by Mosmann et al. [44 (link)]. The assays were measured on an ELISA reader (TECAN, SunriseTM, Grodig/Salzburg, Austria).

Male C57BL/6-Tg (CH-EGFP) mice (age: 5 weeks) were anesthetized with a 8% chloral hydrate solution, and the femur and tibia bones were removed. The bone marrow was obtained by injecting phosphate-buffered saline (PBS) into the tibia and the femur using a 10-ml syringe. On a clean laboratory bench, the bone marrow was filtered through a 70-µm cell strainer (Corning Inc., Corning, NY, USA) and washed twice with PBS. The bone marrow was then cultured on a 60-mm culture dish (SPL Life Sciences Co., Ltd., Pocheon, Korea) containing 20% fetal bovine serum (FBS; GE Healthcare Life Sciences, Logan, UT, USA), 1% penicillin/streptomycin (P/S; Thermo Fisher Scientific Inc., Waltham, MA, USA), and α-minimal Eagle’s medium (MEM; Thermo Fisher Scientific Inc.), and stored in a CO₂ incubator (HEPA CLASS 100, Thermo Fisher Scientific Inc.) maintained at 37 °C. After 5 days, the medium was changed and the floating cells were discarded. The medium was replaced every 2–3 days, and the cells were subcultured using trypsin LE (Thermo Fisher Scientific Inc.) if the mBMSCs occupied approximately 80% of the dish.