Hepatocytes maintain their function on basement membrane formed by epithelial cells

To establish liver tissue engineering, the effective substratum for hepatocytes culture should be developed. Up to now, it is believed that Matrigel, which contains several basement membrane proteins produced by sarcoma cells, is the most effective substratum. Matrigel does not contain extracellular matrix molecules derived from epithelial cells although the space of Disse contains the molecules such as laminin-511/521 (laminin-10/11). Therefore, the basement membrane formed by epithelial cells can be more effective substratum than Matrigel. In this study, we evaluated hepatocytes behavior on basement membrane (rBM) formed by alveolar epithelial cells. The viability of hepatocytes on rBM is higher than that of Matrigel within 5 days. Also, the expression of Cyp1a2 induced by beta-naphthoflavone can be observed in hepatocytes on rBM but not in Matrigel. These results indicate that rBM is a more effective substratum for hepatocyte culture than Matrigel.     

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.     

Expression of carbamoylphosphate synthetase I and glutamine synthetase in hepatic organoids reconstructed by rat small hepatocytes and hepatic nonparenchymal cells

The objective of this study was to investigate the expression of carbamoylphosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocyte colonies and whether the heterogeneous expression of the enzymes could be induced during the maturation of small hepatocytes. Small hepatocytes isolated from an adult rat liver were cultured and proliferated to form colonies. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic cells were positive for CPS and all small hepatocytes were negative for GS at day 5. CPS-positive cells dramatically decreased with time in culture, whereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appeared and the number of such colonies reached about 25% of all colonies at day 30. In most rising and piled-up cells in colonies both proteins were strongly expressed, whereas many small hepatocytes in monolayer colonies did not express either protein. When small hepatocytes in monolayer colonies were overlayed with Matrigel, the cells gradually piled up and both CPS and GS proteins were dramatically induced. The expression of CPS and GS in small hepatocytes may interact with the extracellular matrix because the rising and piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.     

The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.     

Regulation of cytochrome P450 expression by sphingolipids

Sphingolipids modulate many aspects of cell function, including the expression of cytochrome P450, a superfamily of heme proteins that participate in the oxidation of a wide range of compounds of both endogenous (steroid hormones and other lipids) and exogenous (e.g. alcohol, drugs and environmental pollutants) origin. Cytochrome P450-2C11 (CYP 2C11) is down-regulated in response to interleukin-1beta (IL-1beta), and this response involves the hydrolysis of sphingomyelin to ceramide as well as ceramide to sphingosine, and phosphorylation of sphingosine to sphingosine 1-phosphate. Activation of ceramidase(s) are a key determinant of which bioactive sphingolipid metabolites are formed in response to IL-1beta. Ceramidase activation also appears to account for the loss of expression of CYP 2C11 when hepatocytes are placed in cell culture, and the restoration of expression when they are plated on Matrigel; hence, this pathway is influenced by, and may mediate, interactions between hepatocytes and the extracellular matrix. Recent studies using inhibitors of sphingolipid metabolism have discovered that sphingolipids are also required for the induction of CYP1A1 by 3-methylcholanthrene, however, in this case, the requirement is for de novo sphingolipid biosynthesis rather than the turnover of complex sphingolipids. These findings illustrate how changes in sphingolipid metabolism can influence the regulation of at least several isoforms of cytochrome P450.     

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.     

In vitro differentiation of myoblasts from skeletal muscle of rainbow trout

Substrata, plating densities and tissue culture media were compared for their effects on the proliferation and differentiation of myoblasts from skeletal muscle of rainbow trout. Mononuclear cells were isolated from the lateralis muscle of 4–11-month-old trout and plated on to glass coverslips coated with fibronectin, laminin or Matrigel. Cell proliferation was estimated by determining the density of nuclei on successive days in culture, and myoblast differentiation was detected by immunostaining cultures with the myosin-specific monoclonal antibody MF20. Mononuclear cell proliferation was highest for cells cultured on fibronectin or laminin and lowest for cells cultured on Matrigel, but the total number of nuclei in myosin-positive cells did not differ between substrata. The percentage of nuclei in myosin-positive myocytes and myotubes was significantly higher for cells cultured on Matrigel. The proportion of cells adhering to Matrigel and undergoing differentiation increased with plating density. Of three media tested, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 (RPMI), Leibovitz's L-15 (L-15) supplemented with 1 or 10% fetal bovine serum (FBS), a significantly greater proportion of the myoblasts differentiated when cells were cultured in L-15+ 10% FBS. These results suggest that culturing trout muscle-derived cells on a substratum of Matrigel at a high density and maintaining cells in L-15+ 10% FBS provide the conditions that maximize the proportion of cells that actively synthesize muscle myosin and facilitate trout myoblast differentiation in vitro .     

Evaluation of the effect of culture matrices on induction of CYP3A isoforms in cultured porcine hepatocytes

Several bioartificial liver devices have been developed as temporary therapy for patients suffering from fulminant hepatic failure. Some of these devices contain porcine hepatocytes entrapped in collagen matrices. In order to improve the function of these BAL devices, there exists a need to optimize metabolic function of cultured hepatocytes. The goal of these investigations was to evaluate the effect of altering culture conditions on rifampin-mediated induction of CYP3A isoforms in cultured porcine hepatocytes. Midazolam metabolism was compared in porcine hepatocytes cultured in a monolayer configuration on collagen gels, in a sandwich configuration between collagen gels and a Matrigel overlay, and in spheroidal cultures. The effect of culture conditions was evaluated, by measuring CYP3A-mediated metabolism of midazolam and by immunoblotting to detect CYP3A proteins, in control cultures and in rifampin-treated cultures. Results obtained by normalizing the metabolism rate data to cell numbers (based on DNA content) present at the end of the culture experiment, showed that there was no difference between the different culture conditions tested. Our results suggest that culturing porcine hepatocytes as spheroids or in a sandwich configuration between collagen and Matrigel, offers no advantage in terms of CYP3A-mediated metabolic function on a per cell basis compared to culturing on collagen gels.     

The effect of various substrates on cell attachment and differentiation of 3T3-F442A preadipocytes

The influence of extracellular matrix (Matrigel), collagen, and polylysine substrates on cell attachment and differentiation in 3T3-F442A preadipocytes was investigated. In comparison to an uncoated-polystyrene substrate, a concentrated Matrigel substrate (100 microg/cm2) markedly increased intracellular lipid level by about 30%, whereas a lower density Matrigel (10 microg/cm2) accelerated the differentiation rate but did not increase the amount of lipid 21 days after addition of adipogenic factors. Preadipocytes on the collagen surface differentiated less extensively than cells on the polystyrene. Polylysine did not effectively support attachment for either differentiated or undifferentiated cells. These results suggest that Matrigel provides the most suitable environment for both cell adhesion and differentiation for 3T3-F442A cells. This is in contrast to a previous report that extracellular matrix (from corneal endothelial cells) was detrimental to differentiation of 3T3-F442A cells.     

c-myc expression is down-regulated by cell-cell and cell-extracellular matrix contacts in normal hepatocytes, but not in hepatoma cells.

Primary culture of adult rat hepatocytes resulted in marked increase of c-myc expression within a few hours. The high level of c-myc mRNA was maintained throughout culture on collagen-coated dishes, but decreased greatly with time during culture on collagen-gel or matrigel. Expression of c-myc was also down-regulated at high cell density. The decrease in its expression appeared closely related to inhibitions of DNA synthesis and cell spreading. In contrast, hepatoma H4TG cells showed a high level of c-myc expression which was not affected by culture on any extracellular matrices examined or by the cell density. These results suggest that up-regulation of expression of the c-myc gene is linked to G0 to G1 transition during cell cycle progression, which in normal hepatocytes is strictly regulated by cell-cell and cell-extracellular matrix interactions, but that this control mechanism is defective in malignant hepatic tumor cells.     

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome P450 induction studies

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.     

Zonation of cytochrome P450 isozyme expression and induction in rat liver.

The regional expression of six different cytochrome P450 (CYP) forms in rat liver under constitutive and induced conditions was compared using immunological techniques. Immunostaining of consecutive thin sections from control liver revealed that the same hepatocytes, forming a 6-8 cells thick layer surrounding the terminal hepatic venules, were stained for CYP2B1/2, CYP2E1 and CYP3A1. Staining of CYP2A1 extended further into the midzonal region, whereas all cells of the acinus stained for CYPEtOH2. These results were supported by Western blot analysis of cell lysates from the periportal or perivenous region obtained by zone-restricted digitonin treatment during in situ perfusion. The data suggest three distinct patterns of constitutive P450 expression: perivenous-restricted (CYP2B1/2, CYP2E1 and CYP3A1); perivenous-dominated (CYP2A1) and panacinar (CYPEtOH2). Chronic exposure to ethanol caused induction of CYP2E1 in the same cells already being constitutively expressed, whereas CYPEtOH2 was more induced in the periportal area. The relative induction of CYP2B1/2, CYP3A1 and CYPEtOH2 after treatment with phenobarbital was stronger in periportal hepatocytes, resulting in levelling out of the initial perivenous dominance of CYP2B1/2 and CYP3A1, whereas CYPEtOH2 became periportal-dominated. Acetone induced CYP2E1, CYP2C11 and CYP3A1 selectively in the perivenous area. These studies indicate that a particular P450 isozyme is generally induced in the same cells where it is constitutively expressed, and that this regional selectivity is independent of the kind of inducer. The data suggest that, during maturation, the hepatocytes acquire various phenotypes in the periportal and perivenous region, to respond differently to endogenous and exogenous signals in the control of P450 expression.     

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: (1) the concentration of calcium, (2) the dish-coating material, and (3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic micotubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 μM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function. J. Cell. Physiol. 175:41–49, 1998. © 1998 Wiley-Liss, Inc.     

Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes

The aim of this study was to test suitable culture conditions for maintaining normal cellular cytoarchitecture and inducibility of P450 enzymes in primary cultures of human hepatocytes by prototypical inducers. The selectivity and sensitivity of a sandwich culture system were determined by treating cultures with a number of clinically relevant drugs that are known to be inducers of either rodent and/or human P450 enzymes. The results showed that considerable induction of CYP3A4 activity is observed at DMSO concentrations greater than 0.1% (v/v). No differences in P450 induction response were observed between cultures maintained under different matrix conditions. However, the matrix condition considered to be optimal for maintaining cellular integrity, protein yields, and P450 enzyme induction was a sandwich configuration in combination with modified Chee's medium containing insulin (6.25 microg/mL) and dexamethasone (< or =0.1 microM). Under these conditions, induction of CYP3A4 occurred at clinically relevant drug concentrations, and maximal activities were achieved after 3 days of exposure. Overall, the response of human hepatocyte cultures to treatment with both positive and negative modulators was found to reflect that observed in vivo with respect to both enzyme specificity and potency of enzyme induction, although considerable sample-to-sample variability was observed in the magnitude of induction.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of CYP1A2 by histone deacetylase inhibitors in mouse hepatocytes

Cytochrome P450 1A2 (CYP1A2) is constitutively expressed in the mouse liver, but the constitutive expression progressively declines to an undetectable level in isolated hepatocytes. In this study, CYP1A2 was induced in hepatocytes exposed to the histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), but only well after constitutive CYP1A2 expression was silenced. However, cotreatment with the arylhydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and either TSA or SB reduced the induction of CYP1A2 with the same time course as TSA or SB increased its induction. These results suggest that histone modification is involved in CYP1A2 regulation in hepatocytes through pathways that are independent of AhR.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.