Multigas incubator

HIF-1 WT and KO MTECs were maintained as in [9 (link)]. All cells were cultured at normoxia (ambient air) or exposed to hypoxia (0.5% O₂) in a Sanyo multi-gas incubator (acutely, ≤6 h, or chronically, 16–24 h).

Adipose tissue samples were obtained in the frame of Scientific Agreement from multidisciplinary clinic “Souz” (Moscow, Russia) after elective liposuction procedures under local anesthesia from healthy patients with written informed consent. Adipose tissue was processed using guidelines specifically approved by Biomedicine Ethics Committee of Institute of Biomedical Problems, Russian Academy of Sciences (Physiology Section of the Russian Bioethics Committee, Russian Federation National Commission for UNESCO, Permit #314/МCK/09/03/13). Adipose stromal cells (ASCs) were isolated using standard method described by Zuk et al. with modifications by Buravkova et al. [20 (link), 21 (link)]. Cells were expanded in α-MEM (22561-021, Gibco, Invitrogen, UK) with 50 U/mL penicillin-streptomycin (PanEco, Russia) and 10% fetal bovine serum (FBS) (sv30160.03, HyClone, USA) at either ambient O₂ tension (20% CO₂, CO₂-incubator (Sanyo, Japan)) or under low O₂ (5% O₂) using a multigas incubator (Sanyo). Cells on the 2nd and 3rd passages were used in the experiments. ASCs expanded under different O₂ were characterised [22 (link)] before use.

Human breast cancer cells (MCF), human cervical cancer cells (HeLa), human colorectal cancer cells (HCT116, HCT15, LoVo, SW480, SW620, and WiDr), human fibrosarcoma cells (HT1080), human gastric cancer cells (NCI-N87, NUGC3, and SNU484), human hepatic cancer cells (HepG2, HT17, Huh7, SHJ1, and SK-Hep1), human lung cancer cells (A549 and H1299), human pancreatic cancer cells (AsPC1 and MIA-PaCa2) and normal lung fibroblast cells (CCD-34Lu and WI-38) were obtained from the American Type Culture Collection (Manassas, VA, USA) or the KRIBB cell line bank (Daejeon, Korea). The cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 5% (v/v) fetal bovine serum (WelGENE, Daegu, Korea), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). The cells were maintained at 37 °C in a humidified incubator with 5% CO₂, and hypoxia was induced in a multigas incubator (Sanyo, Osaka, Japan) adjusted to 1% O₂, 94% N₂, and 5% CO₂.

The human colorectal cancer HCT116, cervical carcinoma HeLa, hepatocellular carcinoma HepG2, and non-small cell lung cancer H1703 cells were obtained from the KRIBB cell line bank (Daejeon, Korea). HCT116 cells were cultured in a 5% CO₂ atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). Cells were seeded at a density of 5 × 10⁵ cells/ml/well in a 12-well tissue culture plate at 37°C for 20 h prior to subsequent experiments. Hypoxic conditions were achieved by incubating the cells in 1% O₂, 94% N₂, and 5% CO₂ in a multigas incubator (Sanyo, Osaka, Japan).

Two human PDAC (ASPC-1 and SUIT-2) and normal (HPDE and 293FT) cell lines were used in this study. Each cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY) and 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). To establish normoxic conditions, cells were cultured in 5% CO₂ and 95% air. To establish hypoxic conditions, cells were cultured in 1% O₂, 5% CO₂ and 94% N₂ in a multi-gas incubator (Sanyo, Tokyo, Japan).

To determine the sensitivity of the cells to stress including oxidative stress and hypoxia, AdMSCs cultured in 2D monolayer or in 3D aggregates were treated with 200 μM H₂O₂ or hypoxia followed reoxygenation. Briefly, cells were incubated with culture medium containing 200 μM H₂O₂ or vehicle for 2 hours to mimic oxidative stress. For the induction of hypoxia/reoxygenation, cells were subjected to hypoxia for 24 h followed by 2 hours of reoxygenation. Hypoxia (2% O₂) was achieved by using a multigas incubator (Sanyo, Pfaffenhofen, German) that maintained a gas mixture of 2% O₂, 5% CO₂, and 93% N₂ at 37°C. Cells cultured under regular culture conditions for the same period of time were set as control. Viability of cells was determined as described below.

ASCs were divided into two parts after isolation. The first was further expanded in a standard laboratory CO₂-incubator (Sanyo, Japan) with 5% CO₂ and 95% air (20% O₂) (normoxia); the other part was propagated in a multigas incubator (Sanyo) at 5% O₂, 5% CO₂, 90% N₂ (hypoxia). After reaching 70–80% confluence, cells were sub-cultured and 2–4 passages of ASCs were used in experiments.