Activated B lymphocytes induce hepatocyte injury in autologous co-culture system

Hepatic B cells are increasingly implicated in liver inflammation and fibrosis, but whether activated hepatic B cells aggravate primary hepatocyte injury remains insufficiently defined. We established a paired same-donor co-culture system using primary hepatocytes and hepatic B cells isolated from the same mouse liver. Primary hepatocytes were obtained from perfused liver lobes by blood clearance, collagenase I digestion, filtration, and Percoll gradient purification. Hepatic B cells were isolated from paired liver lobes after rapid cold phosphate-buffered saline (PBS)-based washing to remove residual blood, tissue mincing, collagenase IV digestion, red blood cell lysis, Percoll gradient purification, and negative magnetic B-cell enrichment. The workflow yielded viable primary hepatocytes and hepatic B cells with CD19-positive purity above 90% after magnetic sorting and 72-h culture. Under optimized co-culture conditions, lipopolysaccharide (LPS)-activated hepatic B cells secreted TNF-α, IL-6, and IL-1β in culture supernatants and induced hepatocyte morphological damage, increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity, and upregulation of cleaved caspase-3. These findings support activated hepatic B cells as inflammatory amplifier cells that can promote hepatocyte injury and apoptosis through soluble inflammatory mediators in a same-donor primary-cell system.