Revitalizing liver function in mice with liver failure through transplantation of 3D-bioprinted liver with expanded primary hepatocytes.

The utilization of three-dimensional (3D) bioprinting technology to create a transplantable bioartificial liver emerges as a promising remedy for the scarcity of liver donors. This study outlines our strategy for constructing a 3D-bioprinted liver, using in vitro-expanded primary hepatocytes recognized for their safety and enhanced functional robustness as hepatic cell sources for bioartificial liver construction. In addition, we have developed bioink biomaterials with mechanical and rheological properties, as well as printing capabilities, tailored for 3D bioprinting. Upon heterotopic transplantation into the mesentery of tyrosinemia or 90% hepatectomy mice, our 3D-bioprinted liver effectively restored lost liver functions, consequently extending the life span of mice afflicted with liver injuries. Notably, the inclusion of an artificial blood vessel in our 3D-bioprinted liver allowed for biomolecule exchange with host blood vessels, demonstrating, in principle, the rapid integration of the bioartificial liver into the host vascular system. This model underscores the therapeutic potential of transplantation for the treatment of liver failure diseases.

Resveralogues protect HepG2 cells against cellular senescence induced by hepatotoxic metabolites.

Progressive liver disease and dysfunction cause toxic metabolites including ammonia and unconjugated bilirubin to accumulate in plasma. As the population ages alternatives to liver transplantation become increasingly important. One approach for use as a bridge to transplant or recovery is the use of bioartificial liver systems (BALS) containing primary or immortalised hepatocytes as ex-vivo replacements or supports for endogenous liver function. However, exposure to the hepatotoxic metabolites present in plasma causes the rapid failure of these cells to carry out their primary metabolic functions despite remaining viable. Hypothesizing that this loss of core hepatocyte phenotypes was caused by cell senescence we exposed HepG2 cell populations, grown in both standard two-dimensional tissue culture systems and in three dimensional cultures on novel alginate modified HEMA-MBA cryogels, to physiologically reflective concentrations of hepatotoxic metabolites and cytokines. HepG2 cells are forced into senescence by the toxic metabolites in under six hours (as measured by loss of thymidine analog incorporation or detectable Ki67 staining) which is associated with a ten to twenty-fold reduction in the capacity of the cultures to synthesise albumin or urea. This state of senescence induced by liver toxins (SILT) can be prevented by preincubation with either 2-5 µM resveratrol, its major in vivo metabolite dihydroresveratrol or a series of novel resveralogues with differential capacities to scavenge radicals and activate SIRT1 (including V29 which does not interact with the protein). SILT appears to be a previously unrecognised barrier to the development of BALS which can now be overcome using small molecules that are safe for human use at concentrations readily achievable in vivo.

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an "off-the-shelf" bioartificial liver system.

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Rapid Self-Assembly Mini-Livers Protect Mice Against Severe Hepatectomy-Induced Liver Failure.

The construction of bioartificial livers, such as liver organoids, offers significant promise for disease modeling, drug development, and regenerative medicine. However, existing methods for generating liver organoids have limitations, including lengthy and complex processes (taking 6-8 weeks or longer), safety concerns associated with pluripotency, limited functionality of pluripotent stem cell-derived hepatocytes, and small, highly variable sizes (typically ≈50-500 µm in diameter). Prolonged culture also leads to the formation of necrotic cores, further restricting size and function. In this study, a straightforward and time-efficient approach is developed for creating rapid self-assembly mini-livers (RSALs) within 12 h. Additionally, primary hepatocytes are significantly expanded in vitro for use as seeding cells. RSALs exhibit consistent larger sizes (5.5 mm in diameter), improved cell viability (99%), and enhanced liver functionality. Notably, RSALs are functionally vascularized within 2 weeks post-transplantation into the mesentery of mice. These authentic hepatocyte-based RSALs effectively protect mice from 90%-hepatectomy-induced liver failure, demonstrating the potential of bioartificial liver-based therapy.

Assessing bioartificial organ function: the 3P model framework and its validation.

The rapid advancement in the fabrication and culture of in vitro organs has marked a new era in biomedical research. While strides have been made in creating structurally diverse bioartificial organs, such as the liver, which serves as the focal organ in our study, the field lacks a uniform approach for the predictive assessment of liver function. Our research bridges this gap with the introduction of a novel, machine-learning-based "3P model" framework. This model draws on a decade of experimental data across diverse culture platform studies, aiming to identify critical fabrication parameters affecting liver function, particularly in terms of albumin and urea secretion. Through meticulous statistical analysis, we evaluated the functional sustainability of the in vitro liver models. Despite the diversity of research methodologies and the consequent scarcity of standardized data, our regression model effectively captures the patterns observed in experimental findings. The insights gleaned from our study shed light on optimizing culture conditions and advance the evaluation of the functional maintenance capacity of bioartificial livers. This sets a precedent for future functional evaluations of bioartificial organs using machine learning.

Stem Cell-Based Strategies: The Future Direction of Bioartificial Liver Development.

Acute liver failure (ALF) results from severe liver damage or end-stage liver disease. It is extremely fatal and causes serious health and economic burdens worldwide. Once ALF occurs, liver transplantation (LT) is the only definitive and recommended treatment; however, LT is limited by the scarcity of liver grafts. Consequently, the clinical use of bioartificial liver (BAL) has been proposed as a treatment strategy for ALF. Human primary hepatocytes are an ideal cell source for these methods. However, their high demand and superior viability prevent their widespread use. Hence, finding alternatives that meet the seed cell quality and quantity requirements is imperative. Stem cells with self-renewing, immunogenic, and differentiative capacities are potential cell sources. MSCs and its secretomes encompass a spectrum of beneficial properties, such as anti-inflammatory, immunomodulatory, anti-ROS (reactive oxygen species), anti-apoptotic, pro-metabolomic, anti-fibrogenesis, and pro-regenerative attributes. This review focused on the recent status and future directions of stem cell-based strategies in BAL for ALF. Additionally, we discussed the opportunities and challenges associated with promoting such strategies for clinical applications.

Bioartificial liver system approved for clinical trials in China.

Bioartificial organ systems are becoming more attractive options for end-stage organ disease. Currently, end-stage liver disease has several palliative options but relies on transplantation for curative management. A new bioartificial liver has been approved for clinical trials in China and helps support the failed liver via stem cells.

Characteristics of circulating immune cells in HBV-related acute-on-chronic liver failure following artificial liver treatment.

BACKGROUND AND AIM: Liver failure, which is predominantly caused by hepatitis B (HBV) can be improved by an artificial liver support system (ALSS). This study investigated the phenotypic heterogeneity of immunocytes in patients with HBV-related acute-on-chronic liver failure (HBV-ACLF) before and after ALSS therapy. METHODS: A total of 22 patients with HBV-ACLF who received ALSS therapy were included in the study. Patients with Grade I according to the ACLF Research Consortium score were considered to have improved. Demographic and laboratory data were collected and analyzed during hospitalization. Immunological features of peripheral blood in the patients before and after ALSS were detected by mass cytometry analyses. RESULTS: In total, 12 patients improved and 10 patients did not. According to the immunological features data after ALSS, the proportion of circulating monocytes was significantly higher in non-improved patients, but there were fewer γδT cells compared with those in improved patients. Characterization of 37 cell clusters revealed that the frequency of effector CD8+ T (P = 0.003), CD4+ TCM (P = 0.033), CD4+ TEM (P = 0.039), and inhibitory natural killer (NK) cells (P = 0.029) decreased in HBV-ACLF patients after ALSS therapy. Sub group analyses after treatment showed that the improved patients had higher proportions of CD4+ TCM (P = 0.010), CD4+ TEM (P = 0.021), and γδT cells (P = 0.003) and a lower proportion of monocytes (P = 0.012) compared with the non-improved patients. CONCLUSIONS: Changes in effector CD8+ T cells, effector and memory CD4+ T cells, and inhibitory NK cells are associated with ALSS treatment of HBV-ACLF. Moreover, monocytes and γδT cells exhibited the main differences when patients obtained different prognoses. The phenotypic heterogeneity of lymphocytes and monocytes may contribute to the prognosis of ALSS and future immunotherapy strategies.

[Research progress of non-biological artificial liver support system therapy for paitents with liver failure].

Liver failure progresses quickly with high mortality. Non-biological artificial liver support system therapy is one of the important treatments for patients with liver failure. The basic techniques of non-biological artificial liver support system therapy include plasma exchange, plasma adsorption and continuous renal replacement therapy. In this paper, the effect and choice of these basic techniques, the treatment timing, the possible patients who may benefit, and the existing problems are summarized and discussed. We hope to provide a reference for the rational use of non-biological artificial liver support system therapy in clinical practice.

Mixed mode of artificial liver support in patients with acute-on-chronic liver failure: a retrospective cohort study.

BACKGROUND AND AIMS: Different modes of artificial liver support (ALS) therapy can improve the survival of patients with acute-on-chronic liver failure (ACLF). This study aimed to compare the effects of mixed using different modes of ALS (MALS) and single using one mode of ALS (SALS) on 28- and 90-day survival rates of ACLF. METHODS: Clinical data and survival times of patients with ACLF treated for ALS between January 1, 2018 and December 30, 2021 were retrospectively collected. Cox regression analysis was performed to identify risk factors of 28- and 90-day mortalities. RESULTS: Of the 462 eligible ACLF patients, 388 belonged to the SALS group (76.3% male, 74.2% cirrhosis) and 74 to the MALS group (86.5% male, 71.6% cirrhosis). Comparison of 28-day and 90-day crude mortality between the SALS and MALS groups showed no significant differences (28-day: 20.4% vs. 14.9%, p = 0.27; 90-day: 44.6% vs. 52.7%, p = 0.20). After adjusting for confounders, the 28-day mortality (adjusted hazard ratio [aHR]: 0.32, 95% confidence interval [CI] 0.16-0.65) and 90-day mortality (aHR: 0.65, 95% CI 0.44-0.95) in the MALS group were significantly lower than those in the SALS group. These associations were consistently observed across pre-specified subgroups according to age, sex, etiology, and Child-Pugh grade. However, positive interactions between MALS and 90-day mortality were found between MALS and 90-day mortality in those with MELD score ≥ 22 and international normalized ratio ≥ 1.9 (p for interaction < 0.05). CONCLUSION: MALS therapy significantly decreased 28- and 90-day mortalities of ACLF than SALS did, especially in advanced stages.

Screening and Validation Potential Therapeutic Marker of Early to Middle Stage ACLF Patients Treated by ALSSs.

BACKGROUND: Artificial liver support systems (ALSSs) are important approaches for treating acute-on-chronic liver failure (ACLF) patients. Few studies have investigated potential serum therapeutic markers of ACLF patients treated by ALSSs. METHODS: Serum samples were obtained from 57 early to middle stage ACLF patients before and after ALSSs treatment and analyzed by metabonomics. The diagnostic values were evaluated by the area under receiver-operating characteristic curve (AUROC). A retrospective cohort analysis was further employed. RESULTS: Metabonomic study showed that serum ratios of lactate: creatinine in ACLF patients is significantly altered and then restored to normal levels after ALSSs treatment. A retrospective cohort analysis (n = 47) validated that the lactate: creatinine ratio of ACLF patients in the one-month death group remained unchanged after ALSSs treatment, but fell markedly in the survival group with AUROC of 0.682 for diagnosis of survival group from death group, which is a more sensitive measure than measures of prothrombin time activity (PTA) to evaluate the therapeutic effect of ALSSs treatment. CONCLUSIONS: Our results demonstrated the greater the decline in the serum lactate: creatinine ratio with better effective treatments of ALSSs in the ACLF patients with early to middle stage, which presents a potential therapeutic biomarker of ALSSs treatment.

Bioartificial liver with reprogrammed hepatocytes ready for prime time.

In this issue, Wang et al.1 provide evidence of the pre-clinical as well as the clinical utility of in vitro-generated directly reprogrammed human hepatocytes in bioartificial liver. This approach will help offer patients a more curative surgical therapy for liver cancer and improve survival rates.

A multi-subgroup predictive model based on clinical parameters and laboratory biomarkers to predict in-hospital outcomes of plasma exchange-centered artificial liver treatment in patients with hepatitis B virus-related acute-on-chronic liver failure.

Background: Postoperative risk stratification is challenging in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) who undergo artificial liver treatment. This study characterizes patients' clinical parameters and laboratory biomarkers with different in-hospital outcomes. The purpose was to establish a multi-subgroup combined predictive model and analyze its predictive capability. Methods: We enrolled HBV-ACLF patients who received plasma exchange (PE)-centered artificial liver support system (ALSS) therapy from May 6, 2017, to April 6, 2022. There were 110 patients who died (the death group) and 110 propensity score-matched patients who achieved satisfactory outcomes (the survivor group). We compared baseline, before ALSS, after ALSS, and change ratios of laboratory biomarkers. Outcome prediction models were established by generalized estimating equations (GEE). The discrimination was assessed using receiver operating characteristic analyses. Calibration plots compared the mean predicted probability and the mean observed outcome. Results: We built a multi-subgroup predictive model (at admission; before ALSS; after ALSS; change ratio) to predict in-hospital outcomes of HBV-ACLF patients who received PE-centered ALSS. There were 110 patients with 363 ALSS sessions who survived and 110 who did not, and 363 ALSS sessions were analyzed. The univariate GEE models revealed that several parameters were independent risk factors. Clinical parameters and laboratory biomarkers were entered into the multivariate GEE model. The discriminative power of the multivariate GEE models was excellent, and calibration showed better agreement between the predicted and observed probabilities than the univariate models. Conclusions: The multi-subgroup combined predictive model generated accurate prognostic information for patients undergoing HBV-ACLF patients who received PE-centered ALSS.

Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes.

Liver resection is the first-line treatment for primary liver cancers, providing the potential for a cure. However, concerns about post-hepatectomy liver failure (PHLF), a leading cause of death following extended liver resection, have restricted the population of eligible patients. Here, we engineered a clinical-grade bioartificial liver (BAL) device employing human-induced hepatocytes (hiHeps) manufactured under GMP conditions. In a porcine PHLF model, the hiHep-BAL treatment showed a remarkable survival benefit. On top of the supportive function, hiHep-BAL treatment restored functions, specifically ammonia detoxification, of the remnant liver and facilitated liver regeneration. Notably, an investigator-initiated study in seven patients with extended liver resection demonstrated that hiHep-BAL treatment was well tolerated and associated with improved liver function and liver regeneration, meeting the primary outcome of safety and feasibility. These encouraging results warrant further testing of hiHep-BAL for PHLF, the success of which would broaden the population of patients eligible for liver resection.

Risk factors of nosocomial infection in patients underwent artificial liver support system: a retrospective case-control study.

BACKGROUND: Although nosocomial infection is one of the most discussed problems in patients undergoing artificial liver support system (ALSS) treatment, only few solutions have been proposed so far. This study aimed to explore the risk factors of nosocomial infection in patients treated with ALSS in order to aid in the development of future preventive measures. METHODS: This retrospective case-control study included patients treated with ALSS at the Department of Infectious Diseases, First Affiliated Hospital of xxx Medical University between January 2016 and December 2021. RESULTS: One hundred seventy-four patients were included. There were 57 patients in the nosocomial infection group and 117 patients in the non-nosocomial infection group, of them 127 males (72.99%) and 47 females (27.01%) with an average age of 48.15 ±â€…14.19 years old. Multivariate logistic regression analysis revealed that total bilirubin [odds ratio (OR) = 1.004; 95% confidence interval (CI), 1.001-1.007; P  = 0.020], number of invasive procedures (OR = 2.161; 95% CI, 1.500-3.313; P  < 0.001), blood transfusion (OR = 2.526; 95% CI, 1.312-4.864; P  = 0.006) were independent risk factors and haemoglobin (Hb) (OR = 0.973; 95% CI, 0.953-0.994; P  = 0.011) was a protective factor for nosocomial infection in patients treated with ALSS. CONCLUSION: The total bilirubin, transfusion of blood products and higher number of invasive operations were independent risk factors for nosocomial infection in patients treated with ALSS, while higher Hb was a protective factor.

A bioartificial transgenic porcine whole liver expressing human proteins alleviates acute liver failure in pigs.

BACKGROUND: Preventing heterologous protein influx in patients is important when using xenogeneic bioartificial livers (BALs) to treat liver failure. The development of transgenic porcine livers synthesizing human proteins is a promising approach in this regard. Here, we evaluated the safety and efficacy of a transgenic porcine liver synthesizing human albumin (hALB) and coagulation factor VII (hFVII) within a bioartificial system. METHODS: Tibetan miniature pigs were randomly subjected to different interventions after surgery-induced partially ischemic liver failure. Group A (n = 4) was subjected to basic treatment; group B (n = 4) was to standard medical treatment and wild-type porcine BAL perfusion, and group C (n = 2) was to standard medical treatment and transgenic BAL perfusion. Biochemical parameters, coagulation status, survival time, and pathological changes were determined. Expressions of hALB and hFVII were detected using immunohistochemistry and enzyme-linked immunosorbent assays. RESULTS: The survival time in group A was 9.75 ± 1.26 days; this was shorter than that in both perfused groups, in which all animals reached an endpoint of 12 days (P = 0.006). Ammonia, bilirubin, and lactate levels were significantly decreased, whereas albumin and fibrinogen levels were increased after perfusion (all P < 0.05). hALB and hFVII were detected in transgenic BAL-perfused pig serum and ex vivo in the liver tissues. CONCLUSIONS: The humanized transgenic pig livers could synthesize and secrete hALB and hFVII ex vivo in a whole organ-based bioartificial system, while maintaining their metabolism, detoxification, transformation, and excretion functions, which were comparable to those observed in wild-type porcine livers. Therefore, the use of transgenic bioartificial whole livers is expected to become a new approach in treating acute liver failure.

Artificial liver support systems for hepatitis B virus-associated acute-on-chronic liver failure: A meta-analysis of the clinical literature.

To evaluate the short-term and long-term survival efficacy of an artificial liver support system (ALSS) in patients with acute-on-chronic liver failure (ACLF). A systematic search was performed for relevant published data in PubMed, Web of Science and Cochrane Library databases. Studies that evaluated the efficacy of ALSS in patients with ACLF and provided the short-term or long-term survival rate were included. A total of 10 studies involving 3685 patients were included in this analysis. The pooled 28-day survival rate and 90-day survival rate were 68.7% (95% CI: 64.5%-72.9%) and 53.4% (95% CI: 45.5%-61.4%), respectively. The pooled estimates of the OR for the 28-day and 90-day survival rates between the ALSS group and the control group were 1.91 (95% CI: 1.21-3.04) and 1.41 (95% CI: 1.17-1.70), respectively. Subgroup analysis showed that patients treated with lower levels of TBIL and MELD scores had a higher 28-day survival rate (χ2  = 15.75, p < 0.01; χ2  = 13.80, p < 0.01). The present meta-analysis suggests that ALSS treatment could remarkably improve short-term survival rates in HBV-ACLF patients, which implies that treatment with ALSS may help to reduce high mortality. Further prospective randomized trials are needed to validate these findings.

Analysis of Efficacy and Safety of Small-Volume-Plasma Artificial Liver Model in the Treatment of Acute-On-Chronic Liver Failure.

To explore the efficacy and safety of a small-volume-plasma artificial liver support system (ALSS) in the treatment of acute-on-chronic liver failure (ACLF). A retrospective analysis was performed. All ACLF patients received ALSS of plasma exchange & double plasma molecular absorb system (PE+DPMAS) treatment, and successfully completed this treatment. Patients were divided into small-volume and half-volume plasma groups. We compared the changes of the indicators on liver function, kidney function, blood coagulation function, and blood ammonia level before and after PE+DPMAS treatment; we compared the short-term and long-term curative effects between small-volume and half-volume plasma groups; and the factors influencing Week 4 and Week 12 mortality of ACLF patients were analyzed. The Week 4 improvement rates were 63.96 % and 66.86 % in the small-volume and half-volume plasma groups, respectively. The Week 12 survival rates in the small-volume-plasma and half-volume plasma groups were 66.72 % and 64.61 %, respectively. We found several risk factors affecting Week 4 and Week 12 mortality. Kaplan-Meier survival curves suggested no significant difference in Week 4 and Week 12 survival rates between the small-volume and half-volume plasma groups (P=0.34). The small-volume-plasma PE+DPMAS treatment could effectively reduce bilirubin and bile acids, and this was an approach with high safety and few complications, similar to the half-volume-plasma PE+DPMAS treatment. The small-volume-plasma PE+DPMAS has the advantage of greatly reducing the need for intraoperative plasma, which is especially of importance in times of shortage of plasma.