Paca 2

In view of the relevance of the clinical application of IRE in pancreatic cancers [29 (link),30 (link)], a pancreatic carcinoma cell line was selected for the validation of HyCHEED. The human pancreatic carcinoma Mia PaCa-2 (ATCC, Manassas, VA, USA) was grown in high glucose in Dulbecco’s Modified Eagle Medium (Life Technologies, Carlsbad, CA, USA) that was supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Rockville, MD, USA) and 2 mmol·L⁻¹ glutamine (Gibco, Rockville, MD, USA). The cells were cultured in a 37 °C incubator with humidified air supplemented with 5% CO₂. After reaching a confluence of 90%, the cells were harvested via trypsinization (5 time diluted 0.5% Trypsin-EDTA, Gibco, Rockville, MD, USA) and suspended in the culture medium at a concentration of 0.98 × 10⁶–1.40 × 10⁶ cells·mL⁻¹. The cell concentrations were determined using Trypan blue stain assay and an automated bright-field cell counter (LUNA^TM Automated Cell Counter, Aligned Genetics, Inc., Anyang-si, Korea). The protocol to determine the cell concentrations is described in Section 2.6.2. Please note that for the experiments in this study the culture medium was used in the cell suspension during the treatments. In Section 2.6 methods are discussed for the evaluation of the cells treated by HyCHEED.

The normal human pancreatic cell line HPDE6c7 (CL0317, Hunan Fenghui Biotechnology Co., Ltd., Changsha, China), PCa cell lines PANC-1 (CRL-1469), CFPAC-1 (CRL-1918), BxPC-3 (CRL-1687), ASPC-1 (CRL-1682), PACA-2 (CRM-CRL-1420), SW1990 (CRL-2172), and HEK293T (CRL-11268) from ATCC (Manassas, VA) were cultured in medium supplemented with 10% FBS and 1% penicillin/streptomycin with 5% CO₂ at 37°C. McCoy's 5a (Sigma-Aldrich, St Louis MO) medium was used for the following: HPDE6c7 cell culture; IMDM (Gibco) for CFPAC-1 cell culture; DMEM for PANC-1, PACA-2, and HEK293T cells; RPMI-1640 medium for BxPC-3 and ASPC-1 cells; Leibovitz's L-15 (Gibco) medium for SW1990 cells.

The pancreatic cancer cell lines AsPC-1 (ECACC nr. 96020930), BxPC-3 (ECACC nr. 93120816), CFPAC-1 (ATCC nr. CRL-1918), PaCa-2 (ATCC nr. CRL-1420), Capan-2 (ATCC nr. HTB-80), Capan-1(ATCC nr. HTB-79) and HPAF-II (ATCC nr. CRL-1997) (S1 Table) were obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK) and American Type Culture Collection (ATCC, Rockville, MD, USA). The pancreatic cell line PaCa-44 was a kind gift from Dr. Matthias Löhr (Karolinska University Hospital Huddinge, Stockholm, Sweden). Capan-1, Capan-2, PaCa-44 and BxPC-3 cells were cultured in DMEM/F12 medium (Gibco, Life Technologies, Karlsruhe, Germany), PaCa-2 cells were cultured in DMEM (high glucose) medium (Gibco) and AsPC-1, CFPAC-1 and HPAF-II cells were cultured in RPMI medium (Gibco) containing 10% FCS (Gibco), 2% glutamine (Biochrom KG, Berlin, Germany) and 100 ug/ml penicillin/ streptomycin (Biochrom KG) at 37°C in a humidified incubator with 5% CO2.