Co-culture Model of CCl4-Induced Hepatocyte Injury

In vitro injury was given for 6 h by treating cultured hepatocytes with a 5 mM concentration of CCl₄ in DMSO, as described previously [27 (link)]. The co-cultured model was established by culturing injured hepatocytes treated with MSCs and compounds (18), (14) and (10) for 24 h in a Transwell culture system with DMEM (sigma) medium containing 10% FBS, 100 μg/mL of streptomycin and 100 U/mL of penicillin. The co-culture was undertaken using a porous Transwell membrane, with a pore size of 0.4 mm (BD Biosciences). Hepatocytes were first seeded at a density of 1.5 × 10⁵/cm² on the collagen-coated 6-well plate. Once attached, the MSCs were seeded onto the Transwell membrane inserts at a density of 1.5 × 10⁴. The hepatocytes were divided into six groups: normal, CCl₄-treated injured, CCl₄-treated injured co-cultured with MSCs alone, CCl₄-treated injured co-cultured combined with MSCs + compound (18), CCl₄-treated injured co-cultured combined with MSCs + compound (Luciferin Generation Reagent) and CCl₄-treated injured co-cultured combined with MSCs + compound (10). After 24 h of co-culturing, the treated hepatocytes cells were then collected for the extraction of their RNA, LDH cytotoxicity assay, trypan blue assay, glutathione assay and immunostaining.