Mia paca 2

The PC cell lines BxPC-3 and MiaPaCa-2 were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Ibaraki, Japan), cultured, and stored based on the suppliers’ instructions. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 g/mL NaHCO₃, 1 mM sodium pyruvate, and 0.1 mM NEAA. All procedures were performed as described previously [26 (link),27 (link),28 (link),29 (link)].

Four pancreatic carcinoma cell lines, MiaPaCa-2, RWP-1, OCUP-AT, and Panc-1, were used. MiaPaCa-2, RWP-1, and Panc-1 were provided from JCRB Cell Bank (Osaka, Japan), and OCUP-AT was established at our department[27 (link)]. Each cancer cell line was cultured in 5% CO₂ and 95% air. The culture medium was Dulbecco's modified Eagle medium (DMEM; Wako, Osaka, Japan) with 10% fetal bovine serum (FBS; Nichirei, Tokyo, Japan), and 0.5 mM sodium pyruvate (Sigma-Aldrich, Steinheim, Germany). Two human pancreas CAF cell lines, pCaF-1 and pCaF-2, were respectively isolated from the tumoral pancreas specimens of two PDAC patients. The pathological diagnoses in both patients were classified as invasive ductal carcinoma. The CAFs were used in the third through twelfth passage in culture. The primary culture was initiated as follows: the primary tumor was minced with scissors, and was cultivated in the culture medium. When the experimental study was performed, cells were cultured at 37°C in 20% O₂ (normoxia) or 1% O₂ (hypoxia). Hypoxic conditions were maintained using a modular incubator chamber (Hirasawa, Tokyo, Japan) with 5% CO₂ and 1% O₂ balanced with N₂ gas. This study was approved by the Osaka City University ethics committee. Informed consent was obtained from the patients prior to the study.

MIA PaCa-2 and PANC1 human pancreatic cancer cell lines were purchased from the JCRB Cell Bank (Osaka, Japan). The original stocks of the cell lines were mycoplasma-, bacteria-, and fungi-free. MIA PaCa-2 and PANC-1 cells were cultured at 37 °C in 5% CO₂ in MEM (Sigma Aldrich, UK) and RPMI-1640 (Wako, Japan, respectively). Media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Immunodeficient male BALB/c nude mice at 6 weeks of age were obtained from CLEA Corporation (CLEA, Inc., Tokyo, Japan). They were maintained in a pathogen-free animal care system at 21–25 °C with 40–70% humidity. Food and water were provided ad libitum. All animal experiments were monitored, approved, and performed in accordance with the Kobe University Animal Experimentation Regulations (approval number: P160801).

EBC-1 (human lung adenocarcinoma cells), Suit-2 and Mia-Paca-2 cells (human PDAC cells) were obtained from Japanese Collection of Research Bio Resources Cell Bank (JCRB). AsPc-1 (human PDAC cells) were obtained from Iranian Biological Resource Centre, Tehran, Iran. EBC-1, AsPc-1 and Suit-2 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Mia-Paca-2 cells were grown in DMEM low glucose, containing 10% heat-inactivated FBS and 100 U/mL penicillin/streptomycin. All cells were grown in monolayer culture at 37 °C in a humidified incubator with 5% CO₂.