Novel spheroid reservoir bioartificial liver improves survival of nonhuman primates in a toxin-induced model of acute liver failure

Abstract

This study aims to evaluate the effectiveness and safety of the spheroid reservoir bioartificial liver (SRBAL) with porcine hepatocyte organoids in a preclinical nonhuman primate model of acute liver failure (ALF). Methods: Thirty healthy rhesus monkeys were infused with α-amanitin and lipopolysaccharide and randomized into five groups (ALF alone control group; sham no-cell SRBAL treatment group; groups A, B and C with SRBAL treatment started at 12 h, 24 h and 36 h after induction of ALF, respectively). Animals were continuously treated with the SRBAL device for 6 h and followed for up to 336 h. Results: Survival of ALF monkeys improved with hepatocyte SRBAL treatment compared to control groups. Blood ammonia and total bilirubin were lower, and albumin levels were higher in all hepatocyte SRBAL treatment groups. No evidence of porcine endogenous retrovirus was identified in monkey liver or blood after SRBAL treatment. Titers of monkey antibody (IgG, IgM) did not rise after SRBAL treatment. In survival cases, the proportion of necrotic and apoptotic hepatocytes was lower in SRBAL-treated groups, with earlier liver regeneration leading to recovery. Cytokines TNF-α, IL-6, IL-12, IL-1β, IL-8, IFN-γ and IL-2 were ameliorated by the SRBAL treatment, while levels of M-CSF; HGF, EGF and VEGF; IL-1RA and MIF rose on priming, proliferation and the late phase of liver regeneration. Conclusions: The benefit of SRBAL therapy included preventive effects and therapeutic effects. SRBAL improved survival rate and prolonged median survival time in a nonhuman primate model of drug-induced ALF, and these benefits declined with a delay in the initiation of therapy. Improved survival and recovery of ALF monkeys was associated with a reduction in blood ammonia levels, inhibition of the pro-inflammatory response of ALF, and provided a microenvironment more suitable for regeneration of the injured liver.

Keywords: Macaca mulatta; acute liver failure; bioartificial liver; hepatocyte; organoid.

Figure 1

Experimental design. (A) Schematic of the extracorporeal circuit including the SRBAL apparatus. The red line indicates the blood compartment, while the yellow/blue lines indicate the acellular albumin dialysate (AD) compartment. Pressures, temperature and oxygen pressure were detected by respective sensors. Flow rates were determined by pumps. The semipermeable membrane of hollow fiber cartridge 1 was used to diffuse and convect waste molecules from blood to the treatment medium. An oxygenator was used to maintain the inlet oxygen tension above 500 mmHg. The spheroid reservoir functioned as a suspension bioreactor containing hepatocyte organoids with fluid entering its bottom and exiting its top. Hollow fiber cartridge 2 was used to maximize removal of detoxification products and redundant fluid in the AD circuit. (B) Experimental timeline showing the sequence of events of pig donors and monkey receptors and initial time of treatments of groups A, B and C.

Figure 2

Morphology of isolated porcine hepatocytes. (A)Freshlyisolated hepatocytes. (B)Hepatocyte spheroids formed after 24hrocker culture. (C)Viability Fluoroquench staining of hepatocyte spheroids after 24h rocker culture (green:live cells; red:dead cells). (D)Diameter measurement of hepatocyte organoids using Multisizer after 24hrocker culture. (E)Expressions of liver-specific genes including alb, hnf4, g6pc and cyp3a7. (F)Oxygen consumption, (G)albumin production and (H)urea synthesis of newly-formed hepatocyte organoids in SRBAL reservoir for the first half hour of adaptiveculture (2.41±0.38 ×1010cells). *p < 0.05. Scale bar = 100 μm.

Figure 3

Evaluation of SRBAL therapy. (A) Survival curves of all five conditions. Survival rate and median survival time (MST) of monkeys treated with SRBAL were improved in group A (100%, MST = 336 h, p < 0.001), group B (50%, MST = 248 h, p < 0.001) and group C (17%, MST = 131.5 h, p < 0.001) compared to those of the two control groups, sham (0%, MST = 90 h) and ALF (0%, MST = 60.5 h). (B) IgM and (C) IgG antibody levels in monkey blood before and after SRBAL treatments. (D)Electrophoresis results of the PERV DNA extracted from porcine hepatocytes and monkey PBMCs.Sample 1, 3, 5: porcine liver; Sample 2, 4, 6: rhesus monkey blood. (E) Droplet digitalPCR(ddPCR) results of the PERV DNA from porcine hepatocytes and monkey PBMCs and liver. Sample 1: pig liver; Sample 2, 3: monkey liver; Sample 4, 5: monkey blood. (F)RT-PCR results of the PERV mRNA extracted from porcine hepatocytes and monkey PBMCs. (G)Hematological parameters including red blood cell (RBC), hemoglobin (HGB), platelet (PLT) and prothrombin time (PT). (H)Biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin (TB), ammonia, blood urea nitrogen (BUN) and S-100 β. ◇p < 0.05 groupsA, B, C versus ALF control group; *p < 0.05 sham group,groupsA, B, C versus ALF control group; ▽p < 0.05 sham group, group A versus ALF control group; &p < 0.05 group A versus ALF control group; #p < 0.05 groupsA, B versus ALF

Figure 4

Histology and immunohistochemistry of monkey liver biopsies. (A-A”)No changes in organizational structure and cell morphology were observed in all groups before ALF. Extensive parenchymal hemorrhagic necrosis and steatosis was observed at 48 h after toxin infusion in the control group. The livers were still extensively necrotic with obvious bleeding across the entire lobule after sham treatment with a no-cell device at 48 h after toxin infusion. The numbers of hepatocytes and hepatic parenchymal cells increased significantly after SRBAL treatment at 48, 168 and 336 h after toxin infusion (H&E, ×20). (B-B”)Active caspase-3 staining for hepatocyte apoptosis was scarcely observed in healthy liver. The percentage of caspase-3-positive cellswasincreased in the ALF and sham groups at 48 h after drug infusion. In the experimental groups, the caspase-3 expression levels were lower after SRBAL treatment at the same timepoint. Caspase-3-positivestainingcan still be occasionally observed in some inflammatory cells after the repair at 168 h and 336 h (cleaved caspase-3, ×20). (C-C”) CD68 staining showed KCs liningthe walls of the sinusoids in the liver. CD68-positive cells were intensively recruited and activated after ALF induction. SRBAL treatment alleviated the overwhelming recruitment of CD68-positive cells. In the experimental groups, the levels of CD68-positive cells were lower after the treatments at 48 h after drug infusion. After the vigorous process of phagocytosis, the dead cells were cleared, and the numbers of KCs dropped back to normal (CD68, ×20).(D-D”)Staining of the regenerative marker Ki-67 wasrare in normal livers and a small numberof proliferating hepatocytes wasobserved in the remaining cells in the ALF and sham groups. Remarkable liver regeneration was observed after SRBAL treatment at 48, 168 and 336 h after toxin infusion. The SRBAL treatments with hepatocyte organoids increased significantly the percentage of proliferating cells in group A at 48 h after drug administration. The proliferation completed before 168 h. Most of the proliferated cells were locatedin the hepatocytes that had normal morphology in groupsB and C at 48 h. There were still hepatocytes proliferating at 168 h and 336 h in groupsB and group C after drug administration (Ki-67 staining, ×20and ×40).

Figure 5

Dynamic changes in inflammatory parameters in monkey blood. (A)Pro-inflammatory cytokines including TNF-α, IL-6, IL-12, IL-1β, IL-8, IFN-γ, IL-12, and anti-inflammatory cytokine IL-10. (B)Growth factors including HGF, EGF, VEGFand colony-stimulating factor M-CSF. (C)Other cytokines including IL-1RA and MIF in all groups.(D)Correlation curves ofserum ammonia, IL-6andTNF-α levelversusS-100 βlevel. (E)Serum M-CSF levels in ALF monkeys (survive, n = 10; died, n= 20).*p < 0.05 sham group, groupsA, B, C versus ALF control group;

Figure 6

Intercellular interactions and priming, proliferation and termination in liver regeneration. Kupffercells (KCs) prime T cells, hepatocytes and stellate cells via pro-inflammatory cytokines such as TNF-α and IL-6 (priming phase). Growth factors such as HGF and EGF are released from hepatic stellate cells (HSCs) and extracellular matrix, leading to proliferation of hepatocytes (proliferation phase). Other cytokines such as IL-1RA are released from KCs, and MIF is released from stellate cells, terminatingthe proliferation process after liver regeneration (terminationphase).