Fig 2: BAL therapy prevents ALF progression by detoxification. A. Survival time in the ST+BAL, ST+Sham BAL and ST groups (n = 5 per group; log-rank test). B-H. Blood biochemistry parameters in the three groups during the experiment (ALT, AST, ammonia, Cr, TBIL, ALB, endotoxin; n = 5 per group; p < 0.05, p < 0.01, p < 0.001; n = 5 per group). I-L. Vital signs in the three groups during treatment (blood pressure, heart beat, blood oxygen and temperature; n = 5 per group). M-O. Ammonia and Urea metabolism during treatment (p < 0.05, p < 0.01; n = 5 per group). P-R. Blood biochemistry parameters in the three groups during treatment in the bioreactor and blood (Cr, ALT and AST levels; p < 0.05; n = 5 per group). S. PPH glucose consumption every 2 h during treatment (p < 0.05; p < 0.01; n = 5 per group). T. PPH live/dead staining at the beginning and end of treatment (p < 0.01; n = 5 per group).

Fig 3: Hepatocytes maintain cell viability and function under high-density, 3D culture in the bioreactor. A. The bioreactor contained fiber scaffolds made of PET. The pH, temperature and dissolved oxygen were monitored, and alkali, carbon dioxide (CO2) and oxygen (O2) were automatically added to the bioreactor to maintain a stable culture environment. The culture medium was vertically transported by a magnetic impeller (red arrow) and flowed as a thin film down the outer wall like a “waterfall” from the top, enabling O2/CO2 stripping without bubbles harming the cells. The medium volume ranged from 600 to 1000 mL depending on the cell number. The speed of the magnetic impeller was calculated using a medium speed and volume. B. Structural diagram of the FSB BAL therapy system. The red line identifies the route of blood circulation at a speed of 50 mL/min and pressure of 100 mmHg powered by a blood pump (BP). Heparin was injected at a baseline of 500 U/h to maintain the APTT between 175 s and 250 s (HP). The yellow and blue lines represent the plasma separated by a hollow fiber membrane plasma separator (EC-20W; 80% rejection rate at 100 kDa), which occurred at a speed of 15-25 mL/min and a pressure of 50-70 mmHg powered by pump P1. The nonbiological component represented by the yellow area contained active carbon and a bilirubin adsorption column (HA330-II; BS330; Jianfan, China). After the absorption step, the plasma entered the biological component and was detoxified by hepatocytes, followed by transport, powered by pump P2, to the second hollow fiber membrane plasma separator (OP-02; 330 µm) to block potentially exfoliated cells and fragments. Finally, purified plasma was returned to the animal at a speed of 50 mL/min and a pressure of 50-70 mmHg powered by pump P3. A bubble detector was used to monitor microbubbles (BD1, BD2 and BD3), which were removed by a venous chamber. The system pressure was monitored using a pressure detector (PD1, PD2, PD3 and PD4). Extranormal saline was injected at 10-15 mL/min as the baseline. To maintain the temperature of circulation, a heater (H1 and H2) provided additional heating. C. Live/dead detection of the viability of C3A cells (14 d) and PPHs (5 d), which remained more than 80% viable (Bar = 200 µm). Scanning electron microscopy (SEM) of C3A cells (14 d) and PPHs (5 d) revealed that the C3A cells and PPHs attached to the fibers and filled the space between the fibers (Bar = 100 µm, Bar = 20 µm, n = 3 per group). D. Cell number and viability of C3A cell culture from 0 to 15 d (n = 3 per time point). E. Oxygen supply and dissolved oxygen (PO2) of C3A cell culture from 0 to 15 d (n = 3 per time point). F. ALT and AST levels of C3A cell culture from 0 to 15 d (n = 3 per time point). G. Glucose and lactate levels of C3A cell culture from 0 to 15 d (n = 3 per time point). H. C3A cell functional gene expression after 15 d of culture (normalized to the corresponding levels of C3A cells cultured in flasks; p < 0.05; n = 3 per time point). I. Viability of PPH cells from 0 to 10 d (n = 3 per time point). J. Oxygen supply and dissolved oxygen (PO2) in PPH cells cultured from 0 to 10 d (n = 3 per time point). K. ALT and AST levels in PPH cells cultured from 0 to 10 d (n = 3 per time point). L. Glucose and lactate levels in PPH cells cultured from 0 to 10 d (n = 3 per time point). M. PPH functional gene expression after 10 d of culture (normalized to the corresponding levels of day-1 PPHs cultured in flasks, p < 0.05; n = 3 per time point). N. ALB secretion of day-1 PPHs cultured in the bioreactor (p = 0.0012; n = 3 per time point). O. Ammonia elimination of day 1 PPHs cultured in the bioreactor (p < 0.001; n = 3 per time point). P. Urea synthesis of day-1 PPHs cultured in the bioreactor (p < 0.001; n = 3 per time point). Q. CYP450 metabolic activity of day-1 PPHs cultured in the bioreactor (assayed by monoethylglycinexylidide (MEGX) synthesis; p < 0.001; n = 3 per group).

Fig 4: BAL therapy alleviates liver injury and enhances regeneration. A-B. H&E staining of liver tissue from the ST, ST+Sham BAL and ST+BAL groups (higher magnification images are shown in the insets below). Liver injury was scored (p < 0.01; Bar = 200 µm, Magnification Bar = 50 µm; n = 5 per group). C-D. TUNEL staining of liver tissue from all the groups (higher magnification images are shown in the insets below). The number of positive cells per 40× field was calculated for at least 4 separate tissue sections per pig (p < 0.01, p < 0.001; Bar = 200 µm; Magnification Bar = 50 µm; n = 5 per group). E-F. Ki-67 staining of liver tissue from all the groups (higher magnification images are shown in the insets below; red arrows represent bile ducts). The number of positive cells per 40× field was calculated for at least 4 separate tissue sections per pig (p < 0.001; Bar = 200 µm; Magnification Bar = 50 µm; n = 5 per group). G. Ki-67, SOX9, CK18, Epcam, YAP, AFP and ALB co-immunostaining of the liver from the ST+BAL group at 120 h (BD represents Bile Duct, PV represents Portal Vein area; Bar = 40 µm). H. Schematic of hepatocyte proliferation and regeneration.