Cultures Of Adult Hepatocytes Research Articles

Primry research of separation and culture of adult hepatocytes

To explore the separation and culture method of adult hepatocytes. The isolated adult hepatocytes were cultivated by RPMI 1640 medium at 37 degrees C in vitro. The characteristics of the growing hepatocytes were observed. Their synthesis of urea was detected. The transformation efficiency and density's change of lidocaine were analyzed. Hepatocytes were successful separated from adult liver. And they were cultivated in common condition and hollow fiber reactor. The functional capacity of hepatocytes was for lidocaine metabolism and urea excretion. The adult hepatocytes have been successful separated from liver. And they can be cultivated in common condition and hollow fiber reactor. And it could provide a great quantity and high activity of hepatocytes for bioartificial liver.

Regulation of cyclooxygenase 2 expression in hepatocytes by CCAAT/enhancer-binding proteins

Background & Aims: Expression of cyclooxygenase (COX)-2 in response to lipopolysaccharide (LPS) challenge has been analyzed in cultured fetal, neonatal, and adult hepatocytes and in hepatoma cell lines. Methods: To study the mechanisms of LPS-dependent expression of COX-2 in these cells, the activity of the COX-2 promoter and the levels of CCAAT/enhancer-binding proteins (C/EBPs) were determined. Results: COX-2 was induced in fetal hepatocytes, but this response declined rapidly after birth. This loss of inducibility of COX-2 paralleled the expression of C/EBP-α in neonatal hepatocytes. Transfection of fetal and adult hepatocytes with sequences corresponding to the 5'-flanking region of the rat COX-2 gene confirmed the absence of promoter activity in adult hepatocytes. Moreover, transient expression of C/EBP-α, but not C/EBP-δ, in the hepatoma cell line AT3F cells abolished the COX-2 promoter activity. Prolonged culture of adult hepatocytes restored the induction of COX-2 after complete disappearance of C/EBP-α. Conclusions: These results suggest that the presence of high levels of C/EBP-α is involved in the impairment of COX-2 expression in adult hepatocytes challenged with proinflammatory stimuli.GASTROENTEROLOGY 2000;119:493-501

Response of Cultured Fetal and Adult Rat Hepatocytes to Growth Factors and Cyclosporine

Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. Fetal hepatocytes have several characteristics that make them potentially suitable as donor cells. In contrast to adult hepatocytes, fetal hepatocytes are thought to be highly proliferative, which may facilitate engraftment, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [ 3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about ×100 higher and after 10 days in culture ×20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was ×13.8 with EGF, ×18.5 with transforming growth factor alpha (TGF-α), and ×7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-α or HGF. EGF-, TGF-α- and HGF-dependent DNA synthesis was inhibited by transfroming growth factor beta-1 (TGF-β1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-α- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36–46%, whereas in adult hepatocytes by 19–27%. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-β1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

Fetal rat hepatocytes: isolation, characterization, and transplantation in the Nagase analbuminemic rats.

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative, less immunogenic, and resistant to cryopreservation and ischemic injury. These qualities could enhance FH engraftment, proliferation, and gene transfer requiring active DNA synthesis. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Free and cultured cells were studied using electron microscopy, fluorescence-activated cell sorting, and Northern analysis using alpha-fetoprotein and albumin as markers of hepatocyte lineage. DNA synthetic activity was measured in quiescent and mitogen-stimulated fetal and adult hepatocytes by [3H]thymidine incorporation. Susceptibility of cultured FH to retrovirally mediated gene transfer was studied using an amphotropic retroviral vector carrying the Escherichia coli lac-Z gene. Nagase analbuminemic rats were used as recipients to study the effects of intraportal FH transplantation. Analysis of serum albumin was carried out by enzyme-linked immunosorbent assay. In fetal liver, 87+/-2% of the cells showed morphological and molecular features of hepatocytes. DNA synthetic activity in nonstimulated cultured FH was 10 times greater than the maximal hepatocyte growth factor-driven response in adult rat hepatocytes. A total of 5-15% FH stained positive for X-gal; results of transduction in adult hepatocyte cultures were negative. In Nagase analbuminemic rat recipients, FH produced significant amounts of albumin only when a hepatic regenerative stimulus was applied. Immunohistochemistry confirmed presence of albumin-positive hepatocytes. Fetal rat liver from the late gestation period is highly enriched with hepatocyte progenitors. They are highly proliferative and susceptible to retroviral transduction and can engraft and function in the adult rat liver if transplanted under a hepatic regenerative stimulus.

Regulation of cytochrome P450 2B1/2 mRNAs by Kepone ® (chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel

We previously reported that when primary cultures of rat hepatocytes were treated with phenobarbital (PB) or one of several organochlorine pesticides, including Mirex ®, there was co-induction of cytochrome P450 2B1 and 2B2 mRNAs and immunoreactive proteins, whereas Kepone ® selectively induced 2B2 (Kocarek et al. (1991) Mol. Pharmacol. 40, 203–210). Indeed, Kepone treatment actively suppressed induction of 2B1 and 2B2 mRNAs in hepatocytes cotreated with phenobarbital. Because Kepone differs chemically from Mirex only in the replacement of 2 chlorine atoms with a ketone group, which exists in aqueous solution as a gem-diol and appears to confer weak estrogenic properties, we treated hepatocyte cultures with one of 3 potent estrogens, β-estradiol, 17α-ethinylestradiol or diethylstilbestrol. Treatment with each of these estrogens induced 2B1 and 2B2 mRNA only at very high doses (10 −4 M). β-Estradiol (10 −4 M) treatment also induced 2B1/2 mRNA in hepatocyte cultures prepared from a prepubescent female rat. The anti-estrogen tamoxifen failed to reverse 2B1/2 mRNA induction following β-estradiol or Kepone treatment of adult hepatocyte cultures. High doses of β-estradiol or 17α-ethinylestradiol failed to induce 2B1/2 mRNA in treated rats. We also examined the effects of chloral hydrate, a simple gem-diol, on 2B1/2 mRNA induction in the hepatocyte cultures. Treatment with chloral hydrate (3 × 10 −3 M), like Kepone (10 −5 M), suppressed 2B1/2 mRNA induction following phenobarbital (10 −4 M) treatment, while Kepone alcohol (10 −5 M), which is not a gem-diol, produced less suppression. Our results suggest that selective induction by Kepone of 2B2 is unlikely related to its effects as a weak classical estrogen, while the ability of Kepone to suppress induction of 2B1 and 2B2 by PB may be related to its properties as a gem-diol.

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation*1

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation*1

Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation

Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary culture. The freeze/thaw protocol primarily designed for rat hepatocytes was used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medium containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4 degrees C cell suspensions were transferred to -20 degrees C and then to -70 degrees C before being plunged in liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Prolonged storage did not augment loss of cell viability and function. Cell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obtained for most species tested; after cryopreservation cell viability and attachment were decreased by 10% to 25% and by 40% to 50%, respectively. A lower attachment rate was found with dog hepatocytes. Total cytochrome P-450 and protein synthesis were compared in fresh and cryopreserved cells from four species after 4, 24, 48 or 72 hr of culture. Similar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatocytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfoconjugation and glucuronidation were increased in cryopreserved cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion.

The onset of the sexually dimorphic pattern of GH secretion and increased hepatic GH-binding capacity in rats at puberty is temporally correlated with the developmental induction of three hepatic cytochrome P-450s with steroid hydroxylase activity, P-450 IIC11, P-450 IIC12, and P-450 IIC13, and one cytochrome P-450 with vitamin A hydroxylase activity, P-450 IIC7. In this study we demonstrate that expression of the 2C11, 2C12, and 2C13 genes is modulated by GH at the level of transcriptional initiation both in vivo and in primary cultures of adult hepatocytes. In an effort to define the minimum sequence responsible for the inductive effects of GH, we have analyzed the ability of a 0.7-kilobase fragment isolated from the 5'-flank of the 2C12 gene, including the natural promoter, to drive transcription of a 320-basepair G-less cassette in vitro. We were unable to detect any substantial difference in RNA polymerase-II-dependent transcriptional efficiency toward the 2C12 promoter between liver nuclear extracts from normal and hypophysectomized rats of both sexes. This observation supports the assumption that the sequence information contained between bases -700 and 1 is sufficient to support basal transcription of the 2C12 gene. Sequence information residing 5' or 3' of the 0.7-kilobase 5'-flank or a higher ordered chromatin structure may be necessary for the sex-specific transcriptional activation of the 2C12 gene.

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.

Growth and functional activities of neonatal and adult rat hepatocytes cultured on fibronectin coated substratum in serum-free medium.

Hepatocytes isolated from neonatal (NN) and adult (AD) rats were seeded on fibronectin coated substratum and cultured in arginine-free medium supplemented with various combinations of insulin, dexamethasone, triiodothyronine (T3), albumin, and transferrin, in presence or absence of fibronectin depleted serum (FDS). The main finding is that in response to certain hormone mixtures, both NN and AD hepatocytes can be stimulated to proliferate, as revealed by an increase in cell number, a [3H]thymidine incorporation into nuclei, and extractable DNA as well as the appearance of mitotic figures. Moreover, this proliferative activity is associated with changes in hepatocyte ploidy. However, the proliferative response of NN hepatocytes to hormone action is much different from that of AD hepatocytes, and the addition of FDS amplifies this activity in NN but inhibits it in AD hepatocyte cultures. Measurements of tyrosine aminotransferase and lactate dehydrogenase activities indicate a good preservation of NN and AD hepatocyte functional integrity under certain culture conditions. A good maintenance of albumin production in NN and AD hepatocyte cultures requires the presence of dexamethasone, whereas the alpha-fetoprotein production in NN hepatocyte cultures is reduced quite rapidly under most conditions. No alpha-fetoprotein is detectable in AD hepatocyte cultures.

Alpha-fetoprotein synthesis in primary cultures of adult mouse hepatocytes

Synthesis of the embryo-specific serum protein alpha-fetoprotein (AFP) is renewed in the regenerating mouse liver [i]. Studies of the cellular basis for AFP synthesis in the regenerating mouse liver have shown that it is renewed in mature, differentiated, pre-existing hepatocytes, quite unconnected with and, indeed, in the total absence of proliferation [4, 5, 8]. The main sign which distinguishes AFP-containing cells from the remaining hepatocytes in th~ mouse liver after exposure to hepatotoxins or after local mechanical injury is the localization of these cells in the perinecrotic regions [5, 8]. On these grounds the writer suggested that renewal of AFP synthesis is directly linked with local disturbances of interaction between hepatocytes and that the basic condition for repression of AFP synthesis at the tissue level is integrity of the trabecular structure of the liver [3]. It was accordingly decided to determine whether AFP is synthesized in primary cultures of adult hepatocytes in which the normal trabecular structure is completely disturbed.

Isolation and culture of adult hepatocytes from liver biopsies.

Hepatocytes were isolated from liver biopsies of rats, guinea pigs, rabbits, dogs, and humans. The procedure is based on cannulation of large veins in the cut face of the biopsy, followed by collagenase perfusion. Yields averaged 19 x 10(6) viable hepatocytes/g liver. Viability averaged 84%, as determined by trypan blue dye exclusion. Cultures were prepared from the isolated hepatocytes and were found to be comparable in morphology and N-demethylase activity to hepatocyte cultures prepared by the in situ perfusion of the liver. The development of this method should facilitate comparative studies of the cytotoxicity, genotoxicity, and metabolism of foreign chemicals in primary hepatocyte cultures.

Alpha 2 micro-Globulin in the rat. The regulation of the appearance of multiple forms in vivo and in primary cultures of adult hepatocytes.

alpha 2 micro-Globulin in the rat. The regulation of the appearance of multiple forms in vivo and in primary cultures of adult hepatocytes.

Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes

Pyruvate kinase isozymic changes were studied in the adult hepatocyte cultures, by electrophoretic, kinetic and immunological methods. We were able to maintain parenchymal cells from normal adult rat liver in non-proliferating monolayer cultures up to 10 days. Hepatocytes appeared to contain a dominant PK I type up to 4-5 days of culture. After day 5, PK III type was regularly present with PK I and after 7 days PK III type was always the only isozyme detected in culture. It must be pointed out that, by the Ouchterlony method and sometimes by electrophoresis, concentrated extracts from freshly isolated hepatocytes or starting hepatocyte cultures did also contain Pyruvate kinase PK III type. These results suggest that Pyruvate kinase III is present but partly repressed in the adult parenchymal cells and becomes derepressed in culture.