2.5. Hypoxia Chamber as In Vitro Model of Ischemia and Reperfusion

Hepatocellular carcinoma (HepG2) cells (ATCC^®, Manassas, VA, USA) were seeded in complete medium—DMEM (Gibco™, Darmstadt, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS—ATCC^®, Manassas, VA, USA) and 1% (v/v) antibiotic–antimycotic solution (Merck, Darmstadt, Germany)—into a 96-well plate at 0.75 × 10⁵ cells/mL for MTS assays; into a 24-well plate at 2.5 × 10⁵ cells/mL for RNA extraction; or at 1.0 × 10⁵ cells/mL for ROS production assays. After 48 h, medium was replaced by fresh DMEM (supplemented or not with FBS) containing 10 µM or 500 µM quercetin liposomes or free drug (solubilized in 0.1% dimethyl sulfoxide—DMSO, ≥99.9%, Merck, Darmstadt, Germany). Immediately or 6 h after the incubation with liposomes, cells were placed in the hypoxia chamber BD BBL™ GasPak™ jar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with an anaerobic generator (bioMérieux SA, Craponne, France) and anaerobic indicator (bioMérieux SA, Craponne, France), at 37 °C. After 10 h, medium was removed, and cells incubated for an additional 4 h with fresh complete DMEM in an incubator at 37 °C with a humidified atmosphere of 5% CO₂.

For the HUVEC cell line (ATCC^®, Manassas, VA, USA), a cell density of 1.5 × 10⁵ cells/mL was seeded for MTS assays or 5 × 10⁵ cells/mL for RNA extraction assays. The assay was performed using Kaighn’s Modification of Ham’s F-12 Medium (F-12K—ATCC^®, Manassas, VA, USA), supplemented with 10% (v/v) FBS, 1% (v/v) endothelial cell growth supplement from bovine neural tissue (Merck, Darmstadt, Germany), and 0.1 mg/mL heparin sodium salt from porcine intestinal mucosa (Merck, Darmstadt, Germany)—F-12K complete medium. For co-culture of HepG2/HUVEC at 2:1 ratio, the same cell density previously described for HepG2 was used, with F-12K complete medium.

Controls with unloaded liposomes or without liposomes were also performed. A similar procedure was applied in normoxia conditions, where cells were placed 10 h in the incubator instead of the hypoxia chamber.