Functional Assessment of Cardiac Arrest Hepatocytes and Effect of Mechanical Perfusion on Function in a Rat Model.

BACKGROUND: Hepatocyte transplantation has been reported to be useful for metabolic diseases and acute liver failure. However, the shortage of donors limits its widespread use. The use of livers from donors after circulatory death, which are currently unavailable for liver transplantation, may alleviate donor shortage. In this study, we investigated the effects of mechanical perfusion on cardiac arrest hepatocytes in a rat model using cardiac arrest donor livers, and we evaluated the function of cardiac arrest hepatocytes. METHODS: F344 rat hepatocytes isolated from livers removed during cardiac pulsation were compared with those isolated from livers removed after 30 minutes of warm ischemia after cardiac arrest. We then compared hepatocytes isolated from livers removed after 30 minutes of warm ischemia with those isolated after 30 minutes of mechanical perfusion before isolation. The yield per liver weight, ammonia removal capacity, and adenosine diphosphate/adenosine triphosphate ratio were evaluated. RESULTS: Thirty minutes of warm inhibition reduced hepatocyte yield but did not alter ammonia removal capacity and energy status. Mechanical perfusion increased hepatocyte yield and improved the adenosine diphosphate/adenosine triphosphate ratio after 30 minutes of warm inhibition. CONCLUSION: Thirty minutes of warm ischemic time may decrease isolated hepatocyte yield without degrading their function. If increased yields are obtained, livers from donors dying of cardiac arrest could be used for hepatocyte transplantation. The results also suggest that mechanical perfusion may positively affect the energy status of hepatocytes.

P-glycoprotein induction and its energetic costs in rainbow trout (Oncorhynchus mykiss).

Biological organisms are constantly challenged by xenobiotics and have evolved mechanisms to reduce, neutralize, or repair toxic outcomes. The various chemical defenses all utilize energy, but their specific costs and impacts on energy budgets are currently unknown. In this study, the energetic costs associated with the induction and substrate transport of the efflux transporter P-glycoprotein (P-gp [ABCB1, MDR1]) were examined in rainbow trout. An intraperitoneal injection of the P-gp inducer clotrimazole (0, 0.1, 1.0, and 10 mg/kg) increased P-gp activity (as measured by a competitive rhodamine 123 transport assay in hepatocytes) in a dose-dependent manner reaching a maximum induction of 2.8-fold. Maximum P-gp induction occurred at 50 h post-administration with the highest dose; significant induction of P-gp activity remained elevated over constitutive values until the last sampling time point (168 h). In vitro measurements of hepatocyte respiration indicated that basal P-gp activity transporting R123 as a substrate did not significantly increase respiration rates (range 18.0 to 23.2 ng O2/min/106 cells); however, following the induction of P-gp by clotrimazole and exposure to the P-gp substrate R123, respiration rates increased significantly (3.52-fold) over baseline values. Using whole animal respirometry, it was shown that respiration rates in fish exposed to R123 only or induced with clotrimazole were not different from controls (range 1.2 to 2.1 mg O2/kg/min); however, respiration rates were significantly increased in fish with induced P-gp levels and also exposed to R123. This work indicates that basal and induced levels of P-gp activity do not incur significant energetic costs to fish; however, upon induction of P-gp and concomitant substrate exposures, energetic costs can increase and could pose challenges to organisms facing limited energy resources.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma.

BACKGROUND: For its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. METHODS: C3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. RESULTS: Cell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. CONCLUSIONS: C3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

Application of hepatocyte-like cells to enhance hepatic safety risk assessment in drug discovery.

Hepatic stress and injury from drugs continues to be a major concern within the pharmaceutical industry, leading to preclinical and clinical attrition precautionary warnings and post-market withdrawal of drugs. There is a requirement for more predictive and mechanistically accurate models to aid risk assessment. Primary human hepatocytes, subject to isolation stress, cryopreservation, donor-to-donor variation and a relatively short period of functional capability in two-dimensional cultures, are not suitable for high-throughput screening procedures. There are two areas within the drug discovery pipeline that the generation of a stable, metabolically functional hepatocyte-like cell with unlimited supply would have major impact. First, in routine, cell health risk-assessment assays where hepatic cell lines are typically deployed. Second, at later stages of the drug discovery pipeline approaching candidate nomination where bespoke/investigational studies refining and understanding the risk to patients use patient-derived induced pluripotent stem cell (iPSC) hepatocytes retaining characteristics from the patient, e.g. HLA susceptibility alleles, iPSC hepatocytes with defined disease phenotypes or genetic characteristics that have the potential to make the hepatocyte more sensitive to a particular stress mechanism. Functionality of patient-centric hepatocyte-like cells is likely to be enhanced when coupled with emerging culture systems, such as three-dimensional spheroids or microphysiological systems. Ultimately, the aspiration to confidently use human-relevant in vitro models to predict human-specific hepatic toxicity depends on the integration of promising emerging technologies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

In vitro cytotoxicity assessment of roundup (glyphosate) in L-02 hepatocytes.

The goal of the present study was to elucidate the in vitro cytotoxicity of Roundup and to reveal the possible related mechanisms in L-02 hepatocytes. By detecting reactive oxygen species (ROS) production, glutathione (GSH)/superoxide dismutase (SOD) levels, mitochondrial permeability transition pore (PTP) open rate, apoptosis-inducing factor (AIF) release, intracellular Ca2+ concentration, and alanine aminotransferease (ALT)/aspartate aminotransferase (AST) leakage, we determined that Roundup induced anti-oxidant system inhibition, mitochondria damage, DNA damage, membrane integrity and permeability changes, and apoptosis in L-02 hepatocytes. By revealing the mechanistic insights of Roundup-induced cytotoxicity, our results are valuable for the design of preventive and therapeutic strategies for the occupational population exposed to Roundup and other pesticides.

Alginate Encapsulation of Human Hepatocytes and Assessment of Microbeads.

Alginate encapsulation of cells is an attractive technique in which alginate becomes polymerized entrapping the cells. The structure of formed microbeads/microcapsules is semipermeable as it allows oxygen and nutrients to go in, and waste products and other materials produced by the cells to go out. Here, we describe basic protocols for alginate encapsulation of human hepatocytes and methods for assessing the microbeads produced.

Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells.

Apoptotic and anti-apoptotic seromarkers for assessment of disease severity of non-alcoholic steatohepatitis.

BACKGROUND AND STUDY AIMS: This study aimed to find out non-invasive markers for the assessment of severity of non-alcoholic steatohepatitis (NASH) in an attempt to decrease the need for liver biopsy. It also aimed to evaluate the key role of apoptosis in the pathogenesis of the disease and the suggested role of anti-apoptotic factors in therapeutic modalities and disease prognosis. PATIENTS AND METHODS: The serum levels of soluble Fas (s. Fas), s. Fas ligand, cytokeratin 18 (CK-18) fragment and Bcl-2 were measured in 80 patients and 15 non-hepatic subjects as control. The patients were divided based on histological examination of liver biopsy into three groups. Group I included 40 patients with NASH, group II had 40 patients with non-alcoholic fatty liver disease (NAFLD) non-NASH and group III had 15 non-hepatic subjects as control. Apoptosis of hepatocytes was assessed by morphological examination using a light microscope and expressed as number per square millimetre. RESULTS: There was a significant increase in the serum levels of s. Fas, s. Fas ligand and CK-18 fragments in the NASH group. The anti-apoptotic protein Bcl-2 showed significantly low levels in NASH patients. Apoptosis of hepatocytes was significantly higher in the NASH group. The degree of apoptosis was inversely correlated with the level of Bcl-2. A significant correlation between both s. Fas and CK-18 fragment with liver histology with regard to lobular inflammation and ballooning was found. CONCLUSIONS: Increased serum levels of s. Fas and CK-18 fragment in the NASH group and its correlation with the severity of disease suggested the key role of apoptosis in NASH pathogenesis which can be used for the assessment of the severity of NASH. A high level of anti-apoptotic Bcl-2 in NAFLD suggests its protective role in disease progress.

The application of 3D cell models to support drug safety assessment: opportunities & challenges.

The selection of drug candidates early in development has become increasingly important to minimize the use of animals and to avoid costly failures of drugs later in development. In vitro systems to predict and assess organ toxicity have so far been of limited value due to difficulties in demonstrating in vivo-relevant toxicity at a cell culture level. To overcome the limitations of single-cell type monolayer cultures and short-lived primary cell preparations, researchers have created novel 3-dimensional culture systems which appear to more closely resemble in vivo biology. These could become a key for the pharmaceutical industry in the evaluation of drug candidates. However, the value and acceptance of those new models in standard drug safety applications have yet to be demonstrated. This review aims to provide an overview of the different approaches undertaken in the field of pre-clinical safety assessment, organ toxicity, in particular, with an emphasis on examples and technical challenges.

In vitro toxicity assessment of amphiphillic polymer-coated CdSe/ZnS quantum dots in two human liver cell models.

Semiconductor quantum dots (Qdots) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. Qdots generally consist of a semiconductor core, capping shell, and surface coating. The semiconductor core of Qdots is often composed of group II and VI metals (e.g., Cd, Se, Te, Hg) that are known to have toxic properties. Various surface coatings have been shown to stabilize Qdots and thus shield cells from the toxic properties of their core elements. In this study, HepG2 cells and primary human liver (PHL) cells were chosen as in vitro tissue culture models of human liver to examine the possible adverse effects of tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) copolymer (TOPO-PMAT)-coated CdSe/ZnS Qdots (TOPO-PMAT Qdots). The TOPO-PMAT coating is desirable for increasing aqueous solubility and ease of conjugation to targeting moieties (e.g., aptamers and peptides). HepG2 cells avidly incorporated these TOPO-PMAT Qdots into subcellular vesicles. However, PHL cells did not efficiently take up TOPO-PMAT Qdots, but nonparenchymal cells did (especially Kupffer cells). No acute toxicity or morphological changes were noted in either system at the exposure levels used (up to 40 nM). However, cellular stress markers and pro-inflammatory cytokines/chemokines were increased in the PHL cell cultures, suggesting that TOPO-PMAT Qdots are not likely to cause acute cytotoxicity in the liver but may elicit inflammation/hepatitis, demonstrating the importance of relevant preclinical safety models. Thus, further in vivo studies are warranted to ensure that TOPO-PMAT-coated Qdots used in biomedical applications do not induce inflammatory responses as a consequence of hepatic uptake.

A simple and economical route to generate functional hepatocyte-like cells from hESCs and their application in evaluating alcohol induced liver damage.

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two-step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte-like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT-PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF-3ß, SOX-17, DLX-5, CXCR4) was consistent and in concurrence with the up-regulation of the markers for hepatic progenitors [alpha-feto protein (AFP), HNF-4α, CK-19, albumin, alpha-1-antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan-2, 3-dioxygenase (TDO), glucose 6-phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down-regulation of extra embryonic endodermal markers (SOX-7, HNF-1ß, SNAIL-1, LAMININ-1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis.

Assessment of compound hepatotoxicity using human plateable cryopreserved hepatocytes in a 1536-well-plate format.

Hepatotoxicity is a major concern for both drug development and toxicological evaluation of environmental chemicals. The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however, it is being replaced by human in vitro models due to an emphasis on the reduction of animal testing and species-specific differences. Since most cell lines and hybridomas lack the full complement of enzymes at physiological levels found in the liver, primary hepatocytes are the gold standard to study liver toxicities in vitro due to the retention of most of their in vivo activities. Here, we optimized a cell viability assay using plateable cryopreserved human hepatocytes in a 1536-well-plate format. The assay was validated by deriving inhibitory concentration at 50% values for 12 known compounds, including tamoxifen, staurosporine, and phenylmercuric acetate, with regard to hepatotoxicity and general cytotoxicity using multiple hepatocyte donors. The assay performed well, and the cytotoxicity of these compounds was confirmed in comparison to HepG2 cells. This is the first study to report the reliability of using plateable cryopreserved human hepatocytes for cytotoxicity studies in a 1536-well-plate format. These results suggest that plateable cryopreserved human hepatocytes can be scaled up for screening a large compound library and may be amenable to other hepatocytic assays such as metabolic or drug safety studies.

Assessment of gap junctional intercellular communication.

Gap junctions are important plasma membrane organelles through which most cells in normal tissues communicate with each other. They exist in two neighboring cells and each cell contributes half of the structure. One gap junction consists of two hexameric connexons that dock with each other to create a channel. Six of the basic subunits referred to as connexins form a connexon. Less than one hundred to several thousand gap junction channels cluster together in the plane of the membrane. The gap junction channels serve as a regulated conduit for the intercellular exchange of small molecules. Maintenance of the integrity of gap junctional intercellular communication (GJIC) is important and required for normal electrical coupling, homeostasis, and embryogenesis. Aberrations of gap junctions have been related to human diseases such as cancer, cardiac arrhythmia, Charcot-Marie-tooth disease, and visceroatrial heterotaxia syndrome. This unit describes methods for measuring gap junctional intercellular communication using primary mouse hepatocytes as a model. Focus is only on functional evaluation based on dye coupling. Other methods, such as intracellular calcium waves and dual patch clamp, have been used to measure gap junctional communication, but these are not described in this unit.

Functional assessment of the quality of human hepatocyte preparations for cell transplantation.

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM C1NH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.

Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues.

Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigeltrade mark) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBPalpha, HNF4alpha, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

Assessment of hepatocellular function within PEG hydrogels.

Tissue-engineered therapies for liver failure offer the potential to augment or replace whole organ transplantation; however, fabrication of hepatic tissue poses unique challenges largely stemming from the complexity of liver structure and function. In this study, we illustrate the utility of highly-tunable, photopolymerizable poly(ethylene glycol) (PEG) hydrogels for 3D encapsulation of hepatic cells and highlight a range of techniques important for examining hepatocellular function in this platform. Owing to our long-term interest in incorporating proliferative progenitor cell types (e.g. hepatoblasts, oval cells, or cells derived from embryonic stem cells) and maintaining the phenotype of differentiated cells, we explored the behavior of bipotential mouse embryonic liver (BMEL) cells as a model progenitor cell and mature, fully differentiated, primary hepatocytes in this biomaterial system. We demonstrated the importance of cell-cell and cell-matrix interactions in the survival and function of these cell types, and the capacity to influence encapsulated cell phenotypes through modulation of hydrogel characteristics or gene silencing. Additionally, we demonstrated imaging techniques critical for the in situ assessment of encapsulated hepatocyte function combined with the ability to control cellular organization and overall architecture through microscale patterning technologies. Further analysis of liver progenitor as well as mature hepatocyte processes within the versatile PEG hydrogel platform will aid in the development of tissue engineered implantable liver systems.

Primary cultured cells as sensitive in vitro model for assessment of toxicants--comparison to hepatocytes and gill epithelia.

In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3h, and was maximal after 6h of exposure to TCDD, B[a]P and Aroclor 1254. The estimated 6h EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.2 x 10(-9), 5.7 x 10(-8) and 6.6 x 10(-6)M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24h after exposure. The estimated 24h EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4 x 10(-9), 8.1 x 10(-8) and 7.3 x 10(-6)M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants.

In vivo assessment of the tolerance dose of small liver volumes after single-fraction HDR irradiation.

PURPOSE: To prospectively assess a dose-response relationship for small volumes of liver parenchyma after single-fraction irradiation. METHODS AND MATERIALS: Twenty-five liver metastases were treated by computed tomography (CT)-guided interstitial brachytherapy. Magnetic resonance imaging was performed 1 day before and 3 days and 6, 12, and 24 weeks after therapy. MR sequences included T1-w gradient echo (GRE) enhanced by hepatocyte-targeted gadobenate dimeglumine. All MRI data sets were merged with 3D dosimetry data and evaluated by two radiologists. The reviewers indicated the border of hyperintensity on T2-w images (edema) or hypointensity on T1-w images (loss of hepatocyte function). Based on the total 3D data, a dose-volume histogram was calculated. We estimated the threshold dose for either edema or function loss as the D(90), i.e., the dose achieved in at least 90% of the pseudolesion volume. RESULTS: Between 3 days and 6 weeks, the extension of the edema increased significantly from the 12.9 Gy isosurface to 9.9 Gy (standard deviation [SD], 3.3 and 2.6). No significant change was detected between 6 and 12 weeks. After 24 weeks, the edematous tissue had shrunk significantly to 14.7 Gy (SD, 4.2). Three days postbrachytherapy, the D(90) for hepatocyte function loss reached the 14.9 Gy isosurface (SD, 3.9). At 6 weeks, the respective zone had increased significantly to 9.9 Gy (SD, 2.3). After 12 and 24 weeks, the dysfunction volume had decreased significantly to the 11.9 Gy and 15.2 Gy isosurface, respectively (SD, 3 and 4.1). CONCLUSIONS: The 95% interval from 7.6 to 12.2 Gy found as the minimal hepatocyte tolerance after 6 weeks accounts for the radiobiologic variations found in CT-guided brachytherapy, including heterogeneous dose rates by variable catheter arrays.

In vitro assessment of encapsulated C3A hepatocytes functions in a fluidized bed bioreactor.

In the present in vitro model, the authors intended to assess viability and functionality of hepatocytes encapsulated into alginate beads and submitted to a fluidized bed motion in a bioreactor. Human immortalized C3A line was chosen as cell model. Two controls consisting of (1) cells cultured on flasks and (2) cells encapsulated in alginate beads under static conditions were implemented. The cell functions studied were total protein, albumin, urea, and ammonia synthesis, as well as ammonia removal in the case of overdose. The comparison among the three cases studied showed that the three-dimensional structure of alginate offered a suitable environment for cell functions. In addition, the fluidized bed bioreactor enhanced the mass transfer and thus increased the amount of species released out of the beads, as compared with the static case. Ammonia detoxification only appeared reduced by encapsulation. The concept of a fluidized bed bioartificial liver was thus validated by this in vitro model, which indicated that cell functions could be efficiently retained. In addition, as far as urea and protein synthesis and release were concerned, the use of the C3A cell line, in combination with encapsulation and fluidization technology, offered a real potentiality for the purpose of extracorporeal liver supply.

[Morphological criterions of the hepatic functional reserve assessment in cirrhosis of the liver]. / Morfologicheskie kriterii otsenki funktsional'nogo rezerva pecheni pri ee tsirroze.

The results of histological and morphometric investigations of intraoperative hepatic biopsy material in 124 patients with cirrhosis were presented. It was established that the character and degree of severity of hepatic morphological changes constitute the principal factors influencing immediate and remote results of surgical treatment in patients with cirrhosis. There were delineated three types of morphological changes in hepatic cirrhosis, permitting to rationalize the tactic of treatment, to improve the results of the patients surgical treatment.