First Application of a Mixed Porcine-Human Repopulated Bioengineered Liver in a Preclinical Model of Post-Resection Liver Failure.

In this study, a mixed porcine-human bioengineered liver (MPH-BEL) was used in a preclinical setup of extracorporeal liver support devices as a treatment for a model of post-resection liver failure (PRLF). The potential for human clinical application is further illustrated by comparing the functional capacity of MPH-BEL grafts as assessed using this porcine PRLF model with fully human (FH-BEL) grafts which were perfused and assessed in vitro. BEL grafts were produced by reseeding liver scaffolds with HUVEC and primary porcine hepatocytes (MPH-BEL) or primary human hepatocytes (FH-BEL). PRLF was induced by performing an 85% liver resection in domestic white pigs and randomized into the following three groups 24 h after resection: standard medical therapy (SMT) alone, SMT + extracorporeal circuit (ECC), and SMT + MPH-BEL. The detoxification and metabolic functions of the MPH-BEL grafts were compared to FH-BEL grafts which were perfused in vitro. During the 24 h treatment interval, INR values normalized within 18 h in the MPH-BEL therapy group and urea synthesis increased as compared to the SMT and SMT + ECC control groups. The MPH-BEL treatment was associated with more rapid decline in hematocrit and platelet count compared to both control groups. Histological analysis demonstrated platelet sequestration in the MPH-BEL grafts, possibly related to immune activation. Significantly higher rates of ammonia clearance and metabolic function were observed in the FH-BEL grafts perfused in vitro than in the MPH-BEL grafts. The MPH-BEL treatment was associated with improved markers of liver function in PRLF. Further improvement in liver function in the BEL grafts was observed by seeding the biomatrix with human hepatocytes. Methods to reduce platelet sequestration within BEL grafts is an area of ongoing research.

Producing Human Livers From Human Stem Cells Via Blastocyst Complementation.

The need for organ transplants exceeds donor organ availability. In the quest to solve this shortage, the most remarkable area of advancement is organ production through the use of chimeric embryos, commonly known as blastocyst complementation. This technique involves the combination of different species to generate chimeras, where the extent of donor cell contribution to the desired tissue or organ can be regulated. However, ethical concerns arise with the use of brain tissue in such chimeras. Furthermore, the ratio of contributed cells to host animal cells in the chimeric system is low in the production of chimeras associated with cell apoptosis. This review discusses the latest innovations in blastocyst complementation and highlights the progress made in creating organs for transplant.

Comprehensive analysis of brain injury parameters in a preclinical porcine model of acute liver failure.

Introduction: Acute liver failure (ALF) is defined as acute loss of liver function leading to hepatic encephalopathy associated with a high risk of patient death. Brain injury markers in serum and tissue can help detect and monitor ALF-associated brain injury. This study compares different brain injury parameters in plasma and tissue along with the progression of ALF. Method: ALF was induced by performing an 85% liver resection. Following the resection, animals were recovered and monitored for up to 48 h or until reaching the predefined endpoint of receiving standard medical therapy (SMT). Blood and serum samples were taken at Tbaseline, T24, and upon reaching the endpoint (Tend). Control animals were euthanized by exsanguination following plasma sampling. Postmortem brain tissue samples were collected from the frontal cortex (FCTx) and cerebellum (Cb) of all animals. Glial fibrillary acidic protein (GFAP) and tau protein and mRNA levels were quantified using ELISA and qRT-PCR in all plasma and brain samples. Plasma neurofilament light (NFL) was also measured using ELISA. Results: All ALF animals (n = 4) were euthanized upon showing signs of brain herniation. Evaluation of brain injury biomarkers revealed that GFAP was elevated in ALF animals at T24h and Tend, while Tau and NFL concentrations were unchanged. Moreover, plasma glial fibrillary acidic protein (GFAP) levels were negatively correlated with total protein and positively correlated with both aspartate transaminase (AST) and alkaline phosphatase (AP). Additionally, lower GFAP and tau RNA expressions were observed in the FCTx of the ALF group but not in the CB tissue. Conclusion: The current large animal study has identified a strong correlation between GFAP concentration in the blood and markers of ALF. Additionally, the protein and gene expression analyses in the FCTx revealed that this area appears to be susceptible, while the CB is protected from the detrimental impacts of ALF-associated brain swelling. These results warrant further studies to investigate the mechanisms behind this process.

Clinical characterization of a hypersensitivity mixed bacterial and fungal dermatitis in a translational model of porcine NASH.

Introduction: The development of animal models of chronic liver disease via diet modification is a promising avenue for translational research but can lead to unexpected side effects that impact model adoption. While these side effects are well characterized in rodent models of nonalcoholic steatohepatitis (NASH), limited knowledge of these effects exists for novel porcine models of NASH. To close this gap, the present study investigates the side effects of diet-based NASH induction in pigs, with a systematic analysis of the pathologic mechanisms underlying dermatitis development and evaluation of treatment approaches. Method: Twelve pigs (10 large domestic pigs, 2 Goettingen minipigs) were fed a methionine- and choline-deficient, high-fat diet for 8 weeks to induce NASH. A retrospective review of each animal's clinical record was performed to identify the side effects of the diet. Following the identification of diet-associated dermatitis, severity was judged by using a novel gradation system that characterized the individual lesions and body regions resulting in a cumulative evaluation. In addition to this clinical assessment, the etiology of the dermatitis was investigated via histopathologic and microbiologic testing. Furthermore, the success of prophylactic and therapeutic treatment approaches was evaluated by considering dermatitis development and clinical course. Results: All study animals demonstrated unexpected side effects of the methionine- and choline-deficient, high fat diet. In addition to marked dermatitis, study pigs showed impaired weight gain and developed steatorrhea and anemia. Based on the skin gradation system, five animals developed severe dermatitis, four animals moderate dermatitis, and three animals mild diet-associated dermatitis. Histological and microbiological evaluation of the affected skin showed signs of a hypersensitivity reaction with secondary infection by bacteria and fungi. The analysis showed that preemptive bathing extended the lesion-free duration by nearly 20 days. Furthermore, bathing in combination with a targeted antibiotic treatment represented a helpful treatment approach for diet-associated dermatitis. Conclusion: The provision of a methionine- and choline-deficient, high fat diet represents an effective approach for inducing NASH liver disease in pigs but predisposes study animals to multiple side effects. These side effects are universal to animals on study but can be adequately managed and do not represent a significant limitation of this model.

A non-human primate model of acute liver failure suitable for testing liver support systems.

Acute hepatic failure is associated with high morbidity and mortality for which the only definitive therapy is liver transplantation. Some fraction of those who undergo emergency transplantation have been shown to recover native liver function when transplanted with an auxiliary hepatic graft that leaves part of the native liver intact. Thus, transplantation could have been averted with the development and use of some form of hepatic support. The costs of developing and testing liver support systems could be dramatically reduced by the availability of a reliable large animal model of hepatic failure with a large therapeutic window that allows the assessment of efficacy and timing of intervention. Non-lethal forms of hepatic injury were examined in combination with liver-directed radiation in non-human primates (NHPs) to develop a model of acute hepatic failure that mimics the human condition. Porcine hepatocyte transplantation was then tested as a potential therapy for acute hepatic failure. After liver-directed radiation therapy, delivery of a non-lethal hepatic ischemia-reperfusion injury reliably and rapidly generated liver failure providing conditions that can enable pre-clinical testing of liver support or replacement therapies. Unfortunately, in preliminary studies, low hepatocyte engraftment and over-immune suppression interfered with the ability to assess the efficacy of transplanted porcine hepatocytes in the model. A model of acute liver failure in NHPs was created that recapitulates the pathophysiology and pathology of the clinical condition, does so with reasonably predictable kinetics, and results in 100% mortality. The model allowed preliminary testing of xenogeneic hepatocyte transplantation as a potential therapy.

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production.

A reliable source of human hepatocytes and transplantable livers is needed. Interspecies embryo complementation, which involves implanting donor human stem cells into early morula/blastocyst stage animal embryos, is an emerging solution to the shortage of transplantable livers. We review proposed mutations in the recipient embryo to disable hepatogenesis, and discuss the advantages of using fumarylacetoacetate hydrolase knockouts and other genetic modifications to disable hepatogenesis. Interspecies blastocyst complementation using porcine recipients for primate donors has been achieved, although percentages of chimerism remain persistently low. Recent investigation into the dynamic transcriptomes of pigs and primates have created new opportunities to intimately match the stage of developing animal embryos with one of the many varieties of human induced pluripotent stem cell. We discuss techniques for decreasing donor cell apoptosis, targeting donor tissue to endodermal structures to avoid neural or germline chimerism, and decreasing the immunogenicity of chimeric organs by generating donor endothelium.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs.

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Autologous Adipose Tissue-Derived Mesenchymal Stem Cells Introduced by Biliary Stents or Local Immersion in Porcine Bile Duct Anastomoses.

Biliary complications (strictures and leaks) represent major limitations in living donor liver transplantation. Mesenchymal stem cells (MSCs) are a promising modality to prevent biliary complications because of immunosuppressive and angiogenic properties. Our goal was to evaluate the safety of adipose-derived MSC delivery to biliary anastomoses in a porcine model. Secondary objectives were defining the optimal method of delivery (intraluminal versus extraluminal) and to investigate MSC engraftment, angiogenesis, and fibrosis. Pigs were divided into 3 groups. Animals underwent adipose collection, MSC isolation, and expansion. Two weeks later, animals underwent bile duct transection, reanastomosis, and stent insertion. Group 1 received plastic stents wrapped in unseeded Vicryl mesh. Group 2 received stents wrapped in MSC-seeded mesh. Group 3 received unwrapped stents with the anastomosis immersed in an MSC suspension. Animals were killed 1 month after stent insertion when cholangiograms and biliary tissue were obtained. Serum was collected for liver biochemistries. Tissue was used for hematoxylin-eosin and trichrome staining and immunohistochemistry for MSC markers (CD44 and CD34) and for a marker of neoangiogenesis (CD31). There were no intraoperative complications. One pig died on postoperative day 3 due to acute cholangitis. All others recovered without complications. Cholangiography demonstrated no biliary leaks and minimal luminal narrowing. Surviving animals exhibited no symptoms, abnormal liver biochemistries, or clinically significant biliary stricturing. Group 3 showed significantly greater CD44 and CD34 staining, indicating MSC engraftment. Fibrosis was reduced at the anastomotic site in group 3 based on trichrome stain. CD31 staining of group 3 was more pronounced, supporting enhanced neoangiogenesis. In conclusion, adipose-derived MSCs were safely applied to biliary anastomoses. MSCs were locally engrafted within the bile duct and may have beneficial effects in terms of fibrosis and angiogenesis.

Stem Cell-Related Studies and Stem Cell-Based Therapies in Liver Diseases.

Owing to the increasing worldwide burden of liver diseases, the crucial need for safe and effective interventions for treating end-stage liver failure has been a very productive line of inquiry in the discipline of hepatology for many years. Liver transplantation is recognized as the most effective treatment for end-stage liver disease; however, the shortage of donor organs, high medical costs, and lifelong use of immunosuppressive agents represent major drawbacks and demand exploration for alternative treatments. Stem cell-based therapies have been widely studied in the field of liver diseases and are considered to be among the most promising therapies. Herein, we review recent advances in the application of stem cell-related therapies in liver disease with the aim of providing readers with relevant knowledge in this field and inspiration to spur further inquiry.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

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

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-1ß, 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.

Association between kidney intracapsular pressure and ultrasound elastography.

BACKGROUND: Kidney congestion is a common pathophysiologic pathway of acute kidney injury (AKI) in sepsis and heart failure. There is no noninvasive tool to measure kidney intracapsular pressure (KIP) directly. METHODS: We evaluated the correlation of KIP with kidney elasticity measured by ultrasound surface wave elastography (USWE). We directly measured transcatheter KIP in three pigs at baseline and after bolus infusion of normal saline, norepinephrine, vasopressin, dopamine, and fenoldopam; infiltration of 2-L peritoneal dialysis solution in the intra-abdominal space; and venous, arterial, and ureteral clamping. KIP was compared with USWE wave speed. RESULTS: Only intra-abdominal installation of peritoneal dialysis fluid was associated with significant change in KIP (mean (95% CI) increase, 3.7 (3.2-4.2)] mmHg; P < .001). Although intraperitoneal pressure and KIP did not differ under any experimental condition, bladder pressure was consistently and significantly greater than KIP under all circumstances (mean (95% CI) bladder pressure vs. KIP, 3.8 (2.9-4.) mmHg; P < .001). USWE wave speed significantly correlated with KIP (adjusted coefficient of determination, 0.71; P < .001). Estimate (95% CI) USWE speed for KIP prediction stayed significant after adjustment for KIP hypertension (-0.8 (- 1.4 to - 0.2) m/s; P = .008) whereas systolic and diastolic blood pressures were not significant predictors of KIP. CONCLUSIONS: In a pilot study of the swine model, we found ultrasound surface wave elastography speed is significantly correlated with transcatheter measurement of kidney intracapsular and intra-abdominal pressures, while bladder pressure overestimated kidney intracapsular pressure.

Cold storage of rat hepatocyte spheroids.

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 µM cyclosporin A (CsA); SFM + 1 mM Def + 1 µM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

Hepatocyte-like cells differentiated from human induced pluripotent stem cells: relevance to cellular therapies.

UNLABELLED: Maturation of induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs) has been proposed to address the shortage of human hepatocytes for therapeutic applications. The purpose of this study was to evaluate hiPSCs, HLCs and hepatocytes, all of human origin, in terms of performance metrics of relevance to cell therapies. hiPSCs were differentiated to HLCs in vitro using an established four-stage approach. We observed that hiPSCs had low oxygen consumption and possessed small, immature mitochondria located around the nucleus. With maturation to HLCs, mitochondria showed characteristic changes in morphology, ultrastructure, and gene expression. These changes in mitochondria included elongated morphology, swollen cristae, dense matrices, cytoplasmic migration, increased expression of mitochondrial DNA transcription and replication-related genes, and increased oxygen consumption. Following differentiation, HLCs expressed characteristic hepatocyte proteins including albumin and hepatocyte nuclear factor 4-alpha, and intrinsic functions including cytochrome P450 metabolism. But HLCs also expressed high levels of alpha fetoprotein, suggesting a persistent immature phenotype or inability to turn off early stage genes. Furthermore, the levels of albumin production, urea production, cytochrome P450 activity, and mitochondrial function of HLCs were significantly lower than primary human hepatocytes. CONCLUSION: - hiPSCs offer an unlimited source of human HLCs. However, reduced functionality of HLCs compared to primary human hepatocytes limits their usefulness in clinical practice. Novel techniques are needed to complete differentiation of hiPSCs to mature hepatocytes.

Gene expression and functional analyses of primary rat hepatocytes on nanofiber matrices.

Long term culture of primary hepatocytes is valuable for diagnostic and therapeutic applications. However, standard monolayer culture of primary hepatocytes on tissue culture plastic (TCP) - either uncoated or coated with a biological material such as collagen or laminin - is problematic. Thus, novel support matrices are under development to better maintain gene expression and differentiated function of primary hepatocytes in vitro. In this study, a fabricated nanofiber matrix was compared to control conditions of uncoated and laminin-coated TCP. Gene expression and biochemical analyses were performed to compare functional abilities of the hepatocytes in the different conditions. Hepatocytes cultured on nanofibers maintained higher cytochrome P450 1A activity (0.49 +/- 0.08 ng resorufin/ml/min) compared to hepatocytes on laminin (0.11 +/- 0.05 ng resorufin/ml/min). In addition, albumin production of hepatocytes on nanofibers was greater than twice the production of hepatocytes on laminin (day 14, 34.4 +/- 1.8 vs. 15.9 +/- 4.5 microg albumin/ml/day). Hepatocytes demonstrated the ability to generate urea from ammonia in all conditions; however, hepatocytes performed ureagenesis more effectively on nanofibers than on laminin (0.55 +/- 0.25 microM vs. 0.36 +/- 0.24 microM urea, day 14). Gene expression of hepatocytes cultured on nanofiber and laminin conditions were similar on a per cell basis determined by analysis using a custom microarray of 250 genes expressed in hepatocytes. Similar cell attachment data between conditions and similar numbers of cells expressing the hepatocyte marker hepatocyte nuclear factor 4alphaindicates that hepatocytes grown on nanofibers only marginally display improved hepatic functions compared to laminin control conditions.

Engineering analysis and development of the spheroid reservoir bioartificial liver.

A significant demand exists for a liver support device such as a Bioartifical Liver (BAL) to treat patients experiencing acute liver failure. This descriptive paper outlines the design and development of two of the key components of the Mayo Spheroid Reservoir Bioartificial Liver (SRBAL) system. One of the components is the multifunctional Spheroid Reservoir and the other is Multi-shelf Rocker. The Spheroid Reservoir provides an environment to support the viability and functionality of the hepatocyte spheroids at very high cell densities. The Spheroid Reservoir is the biologically active component of this extracorporeal liver support device. Since the Spheroid Reservoir is designed to support 200-400 grams of hepatocyte spheroids, a method to quickly produce large quantities of spheroids is required. The Multi-Shelf Rocker fulfills the production requirement by allowing the culturing of up to six liters of hepatocyte suspension in a conventional laboratory incubator. The SRBAL is designed to provide life sustaining liver-like function to patients in acute liver failure.

E-cadherin protects primary hepatocyte spheroids from cell death by a caspase-independent mechanism.

Cultivation of primary hepatocytes as spheroids creates an efficient three-dimensional model system for hepatic studies in vitro and as a cell source for a spheroid reservoir bioartificial liver. The mechanism of spheroid formation is poorly understood, as is an explanation for why normal, anchorage-dependent hepatocytes remain viable and do not undergo detachment-induced apoptosis, known as anoikis, when placed in suspension spheroid culture. The purpose of this study was to investigate the role of E-cadherin, a calcium-dependent cell adhesion molecule, in the formation and maintenance of hepatocyte spheroids. Hepatocyte spheroids were formed by a novel rocker technique and cultured in suspension for up to 24 h. The dependence of spheroid formation on E-cadherin and calcium was established using an E-cadherin blocking antibody and a calcium chelator. We found that inhibiting E-cadherin prevented cell-cell attachment and spheroid formation, and, surprisingly, E-cadherin inhibition led to hepatocyte death through a caspase-independent mechanism. In conclusion, E-cadherin is required for hepatocyte spheroid formation and may be responsible for protecting hepatocytes from a novel form of caspase-independent cell death.

Optimization of mass transfer for toxin removal and immunoprotection of hepatocytes in a bioartificial liver.

This study was designed to determine optimal operating conditions of a bioartificial liver (BAL) based on mass transfer of representative hepatotoxins and mediators of immune damage. A microprocessor-controlled BAL was used to study mass transfer between patient and cell compartments separated by a hollow fiber membrane. Membrane permeability (70, 150, or 400 kDa molecular weight cut-off-MWCO), membrane convection (high: 50 mL/min; medium: 25 mL/min; low: 10 mL/min; diffusion: 0 mL/min), and albumin concentration in the cell compartment (0.5 or 5 g%) were considered for a total of 24 test conditions. Initially, the patient compartment contained pig plasma supplemented with ammonia (0.017 kDa), unconjugated bilirubin (0.585 kDa), conjugated bilirubin (0.760 kDa), TNF-alpha (17 kDa), pig albumin (67 kDa), pig IgG (147 kDa), and pig IgM (900 kDa). Mass transfer of each substance was determined by its rate of appearance in the cell compartment. Membrane fouling was assessed by dextran polymer technique. Of the three tested variables (membrane pore size, convection, and albumin concentration), membrane permeability had the greatest impact on mass transfer (P < 0.001). Mass transfer of all toxins was greatest under high convection with a 400 kDa membrane. Transfer of IgG and IgM was insignificant under all conditions. Bilirubin transfer was increased under high albumin conditions (P = 0.055). Fouling of membranes ranged from 7% (400 kDa), 24% (150 kDa) to 62% (70 kDa) during a 2-h test interval. In conclusion, optimal toxin removal was achieved under high convection with a 400-kDa membrane, a condition which should provide adequate immunoprotection of hepatocytes in the BAL.

Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function.

UNLABELLED: The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. CONCLUSION: Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker-formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct.