High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices

Objective: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). Methods: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 x 10(6) cells/cm(2) on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. Results: The optimal cell density in this three-dimensional culture configuration was 1 x 10(6) cells/cm(2). The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 +/- 5.6 ng/h/mug DNA and of 1.8 +/- 0.14 mug/h/mug DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/mug DNA (28.7, 42.8) and 32.7 ng/h/mug DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively, Conclusion: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems. Copyright (C) 2001 S.Karger AG, Basel

Characterization of a functional C3A liver spheroid model

More predictive in vitro liver models are a critical requirement for preclinical screening of compounds demonstrating hepatotoxic liability. 3D liver spheroids have been shown to have an enhanced functional lifespan compared to 2D monocultures; however a detailed characterisation of spatiotemporal function and structure of spheroids still needs further attention before widespread use in industry. We have developed and characterized the structure and function of a 3D liver spheroid model formed from C3A hepatoma cells. Spheroids were viable and maintained a compact in vivo-like structure with zonation features for up to 32 days. MRP2 and Pgp transporters had polarised expression on the canalicular membrane of cells in the spheroids and were able to functionally transport CMFDA substrate into these canalicular structures. Spheroids expressed CYP2E1 and were able to synthesise and secrete albumin and urea to a higher degree than monolayer C3A cultures. Penetration of doxorubicin throughout the spheroid core was demonstrated. Spheroids showed increased susceptibility to hepatotoxins when compared to 2D cultures, with acetaminophen having an IC50 of 7.2 mM in spheroids compared to 33.8 mM in monolayer culture. To conclude, we developed an alternative method for creating C3A liver spheroids and demonstrated cellular polarisation and zonation, as well as superior liver-specific functionality and more sensitive toxicological response compared to standard 2D liver models, confirming a more in vivo-like liver model

Modeling human liver biology using stem cell-derived hepatocytes

Stem cell-derived hepatocytes represent promising models to study human liver biology and disease. This concise review discusses the recent progresses in the field, with a focus on human liver disease, drug metabolism and virus infection

A metabolomics cell-based approach for anticipating and investigating drug-induced liver injury

In preclinical stages of drug development, anticipating potential adverse drug effects such as toxicity is an important issue for both saving resources and preventing public health risks. Current in vitro cytotoxicity tests are restricted by their predictive potential and their ability to provide mechanistic information. This study aimed to develop a metabolomic mass spectrometry-based approach for the detection and classification of drug-induced hepatotoxicity. To this end, the metabolite profiles of human derived hepatic cells (i.e., HepG2) exposed to different well-known hepatotoxic compounds acting through different mechanisms (i.e., oxidative stress, steatosis, phospholipidosis, and controls) were compared by multivariate data analysis, thus allowing us to decipher both common and mechanism-specific altered biochemical pathways. Briefly, oxidative stress damage markers were found in the three mechanisms, mainly showing altered levels of metabolites associated with glutathione and γ-glutamyl cycle. Phospholipidosis was characterized by a decreased lysophospholipids to phospholipids ratio, suggestive of phospholipid degradation inhibition. Whereas, steatosis led to impaired fatty acids β-oxidation and a subsequent increase in triacylglycerides synthesis. The characteristic metabolomic profiles were used to develop a predictive model aimed not only to discriminate between non-toxic and hepatotoxic drugs, but also to propose potential drug toxicity mechanism(s)

Synergistic interaction between lipid-loading and doxorubicin exposure in Huh7 hepatoma cells results in enhanced cytotoxicity and cellular oxidative stress: implications for acute and chronic care of obese cancer patients

© The Royal Society of Chemistry 2015.There has been a dramatic increase in the number of clinically obese individuals in the last twenty years. This has resulted in an increasingly common scenario where obese individuals are treated for other diseases, including cancer. Here, we examine interactions between lipid-induced steatosis and doxorubicin treatment in the human hepatoma cell line Huh7. The response of cells to either doxorubicin, lipid-loading or a combination were examined at the global level by DNA microarray, and for specific endpoints of cytotoxicity, lipid-loading, reactive oxygen species, anti-oxidant response systems, and apoptosis. Both doxorubicin and lipid-loading caused a significant accumulation of lipid within Huh7 cells, with the combination resulting in an additive accumulation. In contrast, cytotoxicity was synergistic for the combination compared to the individual components, suggesting an enhanced sensitivity of lipid-loaded cells to the acute hepatotoxic effects of doxorubicin. We demonstrate that a synergistic increase in reactive oxygen species and deregulation of protective anti-oxidant systems, most notably metallothionein expression, underlies this effect. Transcriptome analysis confirms synergistic changes at the global level, and is consistent with enhanced pro-inflammatory signalling in steatotic cells challenged with doxorubicin. Such effects are consistent with a potentiation of progression along the fatty liver disease spectrum. This suggests that treatment of obese individuals with doxorubicin may increase the risk of both acute (i.e. hepatotoxicity) and chronic (i.e. progress of fatty liver disease) adverse effects. This work highlights the need for more study in the growing therapeutic area to develop risk mitigation strategies

Analysis of the Physician Variable in Pain Management

The role of physician variability in pain management is unknown. Objective.  To assess the role of physician variability in the management of pain and provide quantitative data regarding the status of pain management in Michigan. Design.  A multi-item mail survey was used to determine the physician's perceived knowledge of pain management modalities, goals, satisfaction, and confidence with pain treatment. Participants. The focus of this report was a group of 368 licensed Michigan physicians who provide clinical care. Results.  Overall, 30% of the study group reported no formal education in pain management, although younger physicians reported more education (correlation coefficient = −0.252, P < .001). The physicians reported greater confidence in their knowledge of meperidine than other Schedule II opioids ( P < .001 ). In regards to the opinion that prescribing strong opioids would attract a medical review, the physician responses ranged from 1 (strongly disagree) to 5 (strongly agree). The median score for this scale was 4, accounting for 46% of the responses. The study group expressed less satisfaction with their treatment of chronic pain as well as lower goals for relief (mean: 3.8; 95% confidence interval: 3.7–3.9). Conclusions.  Lower expectations for relief and less satisfaction in its management may contribute to the undertreatment of chronic pain. Perceptions of regulatory scrutiny may contribute to suboptimal pain management. These preliminary data highlight physician variability in pain decision making while providing insights into educational needs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74700/1/j.1526-4637.2001.01045.x.pd

Vasoactive intestinal peptide induces proliferation of human hepatocytes.

OBJECTIVES: Proliferation of hepatocytes in vitro can be stimulated by growth factors such as epidermal growth factor (EGF), but the role of vasoactive intestinal peptide (VIP) remains unclear. We have investigated the effect of VIP on maintenance and proliferation of human hepatocytes. MATERIALS AND METHODS: Human hepatocytes were isolated from liver specimens obtained from patients undergoing liver surgery. Treatment with VIP or EGF was started 24 h after plating and continued for 3 or 5 d. DNA replication was investigated by Bromodeoxyuridine (BrdU) incorporation and cell viability detected by MTT assay. Cell lysate was analysed by western blotting and RT-PCR. Urea and albumin secretion into the culture supernatants were measured. RESULTS: VIP increased DNA replication in hepatocytes in a dose-dependant manner, with a peak response at day 3 of treatment. VIP treatment was associated with an increase in mRNA expression of antigen identified by monoclonal antibody Ki-67 (MKI-67) and Histone Cluster 3 (H3) genes. Western blotting analysis showed that VIP can induce a PKA/B-Raf dependant phosphorylation of extracellular signal-regulated kinases (ERK). Although EGF can maintain hepatocyte functions up to day 5, no marked efffect was found with VIP. CONCLUSIONS: VIP induces proliferation of human hepatocytes with little or no effect on hepatocyte differentiation. Further investigation of the role of VIP is required to determine if it may ultimately support therapeutic approaches of liver disease

The carcinogenic potential of tacrolimus ointment beyond immune suppression: a hypothesis creating case report

BACKGROUND: Since tacrolimus ointment was approved by the U.S. Food and Drug Administration (FDA) as a promising treatment for atopic dermatitis, it has been approved in more than 30 additional countries, including numerous European Union member nations. Moreover, in the current clinical routine the use of this drug is no longer restricted to the approved indication, but has been extended to a wide variety of inflammatory skin diseases including some with the potential of malignant transformation. So far, the side-effects reported from the topical use of tacrolimus have been relatively minor (e.g. burning, pruritus, erythema). Recently, however, the FDA reviewed the safety of topical tacrolimus, which resulted in a warning that the use of calcineurin inhibitors may be associated with an increased risk of cancer. CASE PRESENTATION: Oral lichen planus (OLP) was diagnosed in a 56-year-old women in February 1999. After several ineffective local and systemic therapeutic measures an off-label treatment of this recalcitrant condition using Tacrolimus 0.1% ointment was initiated in May 2002. After a few weeks of treatment most of the lesions ameliorated, with the exception of the plaques on the sides of the tongue. Nevertheless, the patient became free of symptoms which, however, reoccurred once tacrolimus was weaned, as a consequence treatment was maintained. In April 2005, the plaques on the left side of the tongue appeared increasingly compact and a biopsy specimen confirmed the suspected diagnosis of an oral squamous cell carcinoma. CONCLUSION: The suspected causal relationship between topical use of tacrolimus and the development of a squamous cell carcinoma prompted us to test the notion that the carcinogenicity of tacrolimus may go beyond mere immune suppression. To this end, tacrolimus has been shown to have an impact on cancer signalling pathways such as the MAPK and the p53 pathway. In the given case, we were able to demonstrate that these pathways had also been altered subsequent to tacrolimus therapy

Characterisation of a functional rat hepatocyte spheroid model.

Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo-like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research

Elevated Expression of Osteopontin May Be Related to Adipose Tissue Macrophage Accumulation and Liver Steatosis in Morbid Obesity

OBJECTIVE—Osteopontin (OPN) plays an important role in the development of insulin resistance and liver complications in dietary murine models. We aimed to determine the expression pattern of OPN and its receptor CD44 in obese patients and mice according to insulin resistance and liver steatosis