The effect of thyroid hormone on fibronectin messenger ribonucleic acid levels in primary cultured rat hepatocytes.

Our previous in vivo studies demonstrated that thyroid hormone promotes the expression of the fibronectin (FN) gene in the rat liver, while it inhibits the synthesis in cultured human skin fibroblasts. These results can be interpreted as either different regulation of FN synthesis or gene expression among tissues, or divergent results of experiments performed in vivo or in vitro. Here we report on the action of thyroid hormone on FN gene expression in vitro using primary cultured hepatocytes compared to that in cultured skin fibroblasts. Hepatocytes were isolated from hypothyroid rats and were cultured in medium supplemented with thyroidectomized bovine serum (TxBS) or fetal bovine serum (FBS). T3 was added 2 or 24 h after plating, and cells were harvested after 2, 6, or 24 h. Total RNA was extracted, and mRNAs for rat FN and albumin were measured. The requirement of de novo protein synthesis for thyroid hormone-mediated induction of FN mRNA was examined by the addition of cycloheximide 15 min before T3 addition. The amount of FN mRNA significantly decreased in the hepatocytes cultured with TxBS compared with those cultured with FBS. The addition of T3 to TxBS resulted in the restoration of FN mRNA to the level in hepatocytes cultured in FBS. FN mRNA increased during the course of culture in the absence of T3; however, a further increase was observed 6 h after T3 addition. The abundance of albumin RNA decreased during the course of culture, but unlike FN mRNA, it was not changed by T3 addition. The increase in FN mRNA by T3 was not influenced by cycloheximide. These results indicate that thyroid hormone enhances FN gene expression in hepatocytes by its direct action without requiring de novo protein synthesis. In contrast, T3 decreased FN mRNA in cultured skin fibroblasts. Thus, the mode of thyroid hormone action on FN gene expression is different among tissues.     

Hepatocellular phenotype in vitro is influenced by biophysical features of the collagenous substratum

Hepatocytes maintained on different substrata in vitro possess strikingly different morphological and biochemical features. Rounded, multicellular aggregates of hepatocytes are seen if the cells are plated onto Matrigel, a reconstituted basement membrane, whereas a flattened, monolayer of hepatocytes is observed with Vitrogen. Hepatocellular protein synthesis is much greater on the Matrigel, although collagen biosynthesis appears selectively enhanced on Vitrogen-grown hepatocytes. We determined that denatured type I collagen could be substituted for Matrigel as the substratum, with the hepatocytes remaining the same both morphologically and biochemically. This suggested that the cells respond to the biophysical state of the extracellular matrix not only to protein sequences that determine a binding site. Measurement of steady-state messenger RNA levels within cells cultured onto different matrices indicated that the fluid substrate of either Matrigel or denatured type I collagen were facilitative for induction of cytochrome P-450b/e, which was not seen with the rigid type I collagen substrata. In contrast the messenger RNA level for the cytoskeletal protein actin was decreased on the fluid matrices, suggesting that the rounded cells had a lower requirement for this protein. These findings indicate that hepatocytes are responsive to the biophysical state of the extracellular matrix, which can lead to significant changes in gene expression by the cells.     

Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes.

Hepatocytes from adult rats were maintained in primary culture for up to 10-13 days on nylon meshes coated with a thin layer of rat tail collagen gel. Their ultrastructure closely resembled that of the liver parenchymal cell in vivo, but hepatocytes in late culture exhibited a pronounced buildup of microfilaments beneath their apical cell surface. Hepatocytes in early and late cultures secreted albumin, transferrin, and alpha1-acid glycoprotein into the medium; they exhibited a 7- to 10-fold induction of tyrosine aminotransferase activity by dexamethasone; and they expressed an alkaline phosphatase that was similar to that of normal rat liver with respect to its inhibition by the liver enzyme inhibitor L-homoarginine. In addition, the hepatocytes in culture demonstrated phenotypic changes characteristic of fetal liver parenchymal cells. These changes, which paralleled an increase in DNA synthesis, included the expression of and linear increase in the activity of the fetal liver cell enzyme gamma-glutamyl transpeptidase, an increased production of alpha1-fetoprotein, and a change in the substrate specificity of fructose-bisphosphate aldolase to that of the fetal liver isozyme.     

Actin organization and hepatocyte differentiation are regulated by extracellular matrix via PI-4,5-bisphosphate in the rat

Cell adhesion to the extracellular matrix (ECM) plays vital roles in both morphogenesis and regulation of gene expression in cells of adult organisms. How intracellular, cytoskeletal, and signaling factors connect and communicate with the ECM is a fundamental question. Using a cDNA microarray analysis, we identified phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) phosphatase mRNA as being up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, which led to the finding that the PI(4,5)P2 levels of hepatocytes decreased on EHS gel. These changes in hepatocytes on EHS gel were accompanied by promotion of actin depolymerization and differentiated phenotypes of the hepatocytes. Treatment with PI(4,5)P2 or a phospholipase C inhibitor, U73122, resulted in decreased mRNA expressions of albumin and hepatocyte nuclear factor 4 (HNF-4) in hepatocytes. In contrast, actin-disrupting agent gelsolin increased mRNA expressions of albumin and HNF-4. In conclusion, organization of the actin cytoskeleton via PI(4,5)P2 is involved in the regulation of hepatocyte differentiation by the ECM.     

Primary culture of cryopreserved adult human hepatocytes on homologous extracellular matrix and the influence of monocytic products on albumin synthesis

Cultured human hepatocytes provide a useful method for studies of specific metabolic functions, such as plasma protein synthesis or drug metabolism in man and specific interaction of microorganisms with the human liver cell. In vitro studies using human hepatocytes are seriously hampered by the limited availability of viable tissue and the lack of suitable methods for preservation of hepatocytes. In addition, normal adult human hepatocytes, under classical culture conditions, do not proliferate in vitro. Recently, we have reported a method for long-term storage of human hepatocytes, using a cryopreservation technique. However, after thawing, the efficiency of cell seeding onto tissue culture plastic and, accordingly, the survival of hepatocytes in primary cultures were decreased as compared to freshly prepared and cultured hepatocytes. In the present study, we report the effects of extracellular matrix (ECM) obtained from normal liver of kidney donors on the attachment efficiency, survival, some metabolic functions and fine structure of human hepatocytes. It was found that the deleterious effect of deep-freeze storage on attachment efficiency and survival of hepatocytes could be significantly reduced by using tissue culture plastic precoated with human liver ECM. Hepatocytes survived for more than 4-6 weeks, without evidence of fibroblast overgrowth. Using this in vitro experimental system, we have also shown that these hepatocytes synthesize several liver-specific acute phase proteins, and monocytic products were able to decrease the hepatocytic synthesis of albumin while total protein synthesis remained unchanged. These results support our previous observations in cultured rodent hepatocytes, indicating the important role of monocytic products in the regulation of liver synthesis of albumin in inflammatory diseases in man.     

Growth factors and gene expression in cultured rat hepatocytes.

BACKGROUND/AIM: The aim of this study was to evaluate the effect of replication on function variables in cultured hepatocytes. METHODS: Isolated rat hepatocytes were cultured in HCM medium and plated on collagen-coated dishes at cell densities from 0.2 x 10(5) (subconfluent) to 1.0 x 10(5) x cm(-2) (confluent) with and without addition of hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-I. The synthesis rate was measured for DNA, albumin, urea, and glucose together with mRNA levels (Northern blots) for albumin, urea cycle enzymes, and acute phase and "house-keeping" proteins. RESULTS: In subconfluent culture the synthesis of DNA and urea was higher (118% and 112%, respectively), and of albumin and glucose lower (40% and 67%, respectively) than in confluent culture. The mRNA levels of carbamoylphosphate synthase, argininosuccinate synthetase, argininosuccinate lyase, arginase, a2-macroglobulin, beta-fibrinogen, and albumin were lower (23%, 58%, 77%, 33%, 12%, 50%, and 51%, respectively) in subconfluent culture compared with confluent culture. Relatively increased levels were found for beta-actin (109%) and alpha-tubulin (136%). In subconfluent culture hepatocyte growth factor increased the DNA synthesis rate 6-fold, epidermal growth factor 3-fold, and insulin-like growth factor-I 2-fold; that of albumin, urea and glucose was not increased significantly. In confluent culture the effect of growth factors on synthesis rates was not significant, and the growth factors had little influence on mRNA levels. CONCLUSIONS: Hepatocytes produce urea at the same rate in subconfluent as in confluent culture in spite of a lower mRNA level of urea cycle enzymes. Hepatocyte growth factor and epidermal growth factor increase DNA synthesis markedly in subconfluent culture only, without significantly changing the ratio between subconfluent and confluent culture of other variables. This suggests that active replication is not an important cause of the relatively low liver-specific function of hepatocytes in subconfluent culture.     

Development and Characterization of a Hybrid Bioartificial Liver Using Primary Hepatocytes Entrapped in a Basement Membrane Matrix

For the development of a bioartificial liver (BAL) support device, it is most important to establish highly differentiated liver cells cultured at high density. When rat hepatocytes were cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, their rates of albumin secretion were very high, as measured by ELISA, and these high rates were maintained for more than three weeks of culturing. This level of activity greatly exceeded that of hepatocytes cultured on a plastic substratum, poly-N-p-vinylbenzyl-d-lactonamide (PVLA), on a single layer of collagen, or in a collagen sandwich culture. In an in vitro perfusion experiment, rat hepatocytes rapidly and completely removed ammonia from Eagle's MEM supplemented with 0.2 mM NH₄Cl, although ammonia levels of the medium serially increased in modules containing HepG2 cells. A hybrid liver support system was developed and consisted of plasma perfusion through porous hollow fiber modules inoculated with 10 billion porcine hepatocytes entrapped in EHS gel. This system was applied to pigs with ischemic liver failure 8 hr after creation of a portocaval shunt and hepatic devascularization. In animals treated with the BAL support system, blood bicarbonate levels were increased immediately after treatment, and hemodynamic stability was improved. In control pigs, on the other hand, blood bicarbonate levels and blood pressure remained low. Plasma levels of ammonia and lactate decreased in pigs treated with the BAL device, but not in control animals. These results indicate that primary hepatocytes outperform HepG2 cells as a source of biotransformation functions in a BAL system and that the use of a BAL support device in combination with a hollow fiber module and hepatocytes entrapped in EHS gel has potential advantages for clinical use in patients with fulminant hepatic failure.     

Retroviral gene transfer into primary hepatocytes: implications for genetic therapy of liver-specific functions.

The liver is an important target for potential gene therapy because of the critical role it plays in intermediary metabolism and synthesis of serum proteins. We report the use of retroviral vectors for transfer of recombinant genes into primary mouse hepatocytes. Hepatocytes were grown in a defined serum-free medium and expressed liver-specific functions for up to 14 days. Hepatocytes were transformed to Genticin (G418) resistance by infection with recombinant retroviruses carrying the Tn5 neomycin-resistance gene. The G418-resistant cells exhibited characteristic hepatocyte morphology and continued to express liver-specific gene function. A retrovirus that expresses neomycin resistance driven by a herpes simplex thymidine kinase promoter produced the most efficient transformation compared with viruses using the retroviral long terminal repeat promoter or the simian virus 40 early-region promoter. These experiments indicate that primary hepatocytes can be successfully cultured and transformed with recombinant genes using retroviral vectors. These results provide a model for future somatic gene replacement therapy in which functional genes can be introduced into hepatocytes by viral-mediated gene transfer.     

Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes

Hepatocytes, prepared from normal adult rat liver, were seeded onto a collagen substratum and cultured alone or in the presence of rat liver endothelial cells. When hepatocytes were cultured alone in a hormonally defined serum-free medium, decreased albumin production and rapid morphological deterioration of bile canaliculi structures and gap junctions occurred within 4 to 5 days. In contrast, hepatocytes cocultured with liver mesenchymal cells remained morphologically intact and biochemically functional for at least 4 weeks. They reorganized into small islands, continued to secrete high levels of albumin, did not express alpha-fetoprotein (a fetal marker), and remained strongly dye coupled. All of the hepatocytes synthesized albumin and retained their gap junctional channels. No junctional communication was observed between hepatocytes and endothelial cells. Long fibers containing fibronectin, Type I collagen and laminin distributed over the hepatocytes were induced in coculture but never appeared in hepatocytes cultured alone. Moreover, supplementation of the hormonally defined medium with phenobarbital and dimethyl sulfoxide, both of which improve the life span and functional activities of cultured hepatocytes, failed to induce reticulin fiber formation in pure culture of hepatocytes. The modulation of albumin secretion, biomatrix deposition and junctional communication observed in hepatocytes cultured with sinusoidal liver cells was also obtained when hepatocytes were in association with various epithelial or mesenchymal cells [rat liver epithelial cells (T51B), mouse embryonic fibroblasts (NIH 3T3), human or rat dermal fibroblasts and bovine aorta endothelial cells (AG 4762)].     

Primary hepatocyte culture in collagen gel mixture and collagen sandwich

AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes. METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined. RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period. CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.     

The effects of ethanol on collagen synthesis by rat hepatocytes in primary culture

Abstract The effect of ethanol upon protein metabolism was investigated using rat hepatocytes purified by centrifugal elutriation and maintained in culture. Hepatocytes maintained in capped culture flasks allowed long-term studies of the effects of ethanol without cytotoxic damage to cultured cells and the phenotypic integrity of cultured cells was maintained. Ethanol (25–100 mmol/1) markedly inhibited hepatocyte protein synthesis. This inhibition of protein synthesis was not due to defective uptake of amino acids but to alterations to the intracellular redox state. Extracellular redox systems did not regulate hepatocyte protein synthesis. Protein degradation was not affected by ethanol. Glycosaminoglycans secreted into the the medium and extra and intracellular hepatocyte collagen synthesis were markedly stimulated by ethanol; collagen synthesis representing 6–8% of total protein synthesis at high ethanol concentrations. These studies indicate that ethanol exerts selective effects on protein synthesis by hepatocytes in primary culture and suggest that hepatocytes may contribute significantly to perisinusoidal fibrosis in alcohol-induced liver disease.     

Ethanol-induced Inhibition of Liver Cell Function: I. Effect of Ethanol on Hormone Stimulated Hepatocyte DNA Synthesis and the Role of Ethanol Metabolism

We have studied a short term effect (96 hr) of ethanol on hormone-stimulated DNA synthesis in a primary rat hepatocyte culture system. Studies were also performed with respect to total RNA and protein synthesis as well as albumin secretion measured by a solid-phase radioimmunoassay. We found that ethanol, when added to cultured hepatocytes, resulted in a substantial reduction in hormone-stimulated hepatocyte DNA synthesis and this effect was concentration dependent and occurred in serum-free medium. Ethanol also had an inhibitory effect on total RNA synthesis but protein synthesis and albumin secretion remained essentially unchanged. We determined that hepatocytes exposed to ethanol during the first 24 hr of culture were the most susceptible to inhibition of DNA synthesis. During the first 24 hr, alcohol dehydrogenase (ADH) activity was present in the cells at higher levels than at 48 and 72 hr. In human hepatoma cell lines and differentiated primary and secondary chick fibroblasts, no ADH activity was demonstrable; such cells were not inhibited by 100 mM ethanol additions and DNA synthesis rates were similar to untreated cultures. Other alcohols found to be metabolized by hepatocyte ADH were inhibitory towards hormone-stimulated DNA synthesis whereas those with less metabolism had little effect. Hepatocytes treated with 4-methylpyrazole, an inhibitor of ADH, were partially protected from ethanol effects. Taken together our results are consistent with the hypothesis that a major physiological effect of ethanol on the hepatocyte is a direct impairment of DNA synthesis and that alcohol metabolism is required.     

Inhibitory activity of the serum from patients with fulminant hepatitis against liver regeneration

The effect of sera from 8 patients with fulminant hepatitis, including 2 survival cases, on DNA and protein synthesis in primary cultured rat hepatocytes was studied. The serum from patients at an early stage or within 10 days after onset tended to intensify DNA synthesis in isolated hepatocytes, whereas the serum from patients with a history of over 50 days distinctly inhibited synthesis. When the serum was fractionated by gel filtration or free-flow electrophoresis, only the albumin fraction inhibited DNA synthesis in cultured hepatocytes. The suppressive effect of the albumin fraction was demonstrated even in patients suffering for only a short period of time. The inhibitory activity against DNA and protein synthesis in cultured hepatocytes was demonstrated in a substance extracted with a chloroform and methanol mixture from the albumin fraction of patients with fulminant hepatitis. The extract from the patients' sera also inhibited acceleration of DNA synthesis by epidermal growth factor (EGF) in the same cells.     

Effects of plasma from patients with fulminant hepatic failure on function of primary rat hepatocytes in three-dimensional culture.

BACKGROUND/AIM: As biotechnology continues to advance, a bioartificial liver is expected to be developed for the treatment of patients with fulminant hepatic failure (FHF) whose liver dysfunction is potentially reversible or for providing liver support as a bridge to liver transplantation. While monolayer-cultured hepatocytes rapidly lose their capacity to express many liver-specific functions over time when cultured, spherical-shaped hepatocytes in three-dimensional culture with the use of extracellular matrix components sustain long-term survival by maintaining differentiated hepatocyte functions. The aim of this study was to investigate whether sufficient functions of viable spherical-shaped hepatocytes could be maintained in plasma of patients with FHF in order to use these cells in an extracorporeal system. METHODS: Hepatocyte functions were evaluated under monolayer or three-dimensional culture in FHF plasma. RESULTS: Primary rat hepatocytes on poly-N-p-vinylbenzyl-D-lactonamide (PVLA) formed spheroids even in FHF plasma and maintained their spherical shapes in FHF plasma as long as in medium. Spherical-shaped hepatocytes on PVLA cultured in FHF plasma showed higher activity in albumin secretion, urea formation, and gluconeogenesis than those in normal human plasma or medium. As being cultured in medium, hepatocytes on PVLA cultured in plasma were also superior to cells on collagen in regard to albumin secretion, amino acid metabolism, and gluconeogenesis. CONCLUSIONS: These findings demonstrated that FHF plasma is not toxic to rat hepatocyte spheroids and that hepatocyte spheroids have potential use in the development of a bioartificial liver.     

胶原凝胶固定培养大鼠肝细胞的功能与形态观察

目的：探索混合胶原凝胶培养肝细胞的方法，观察培养鼠肝细胞的功能与形态特征。方法：两步法分离大鼠肝细胞，与I型鼠尾胶原溶液混合接种于培养瓶，待胶原液形成凝胶后，加培养液常规培养，观察培养鼠肝细胞的形态学特征和尿素合成及酶漏出量。结果：成功将大鼠肝细胞混合固定于胶原中，形成凝胶状进行培养。培养期间，始终能检测出鼠肝细胞合成分泌的尿素，而肝细胞乳酸脱氢酶漏出量较少，倒置相差显微镜下观察到典型的形态特征。结论：混合胶原凝胶培养方法能为肝细胞提供更接近体内的培养环境，可能适用于生物人工肝研究。     

An optimal culture condition maintains human hepatocyte phenotype after long-term culture.

BACKGROUND: Long-term culture of primary hepatocytes from various species is impeded by a decrease of cell viability and a loss of hepatocyte-specific function. The aim of the present study was to investigate whether our optimal culture condition (OC) can maintain the phenotype of primary hepatocytes in long-term culture. METHODS: Primary human hepatocytes were cultured in either hepatocyte maintenance medium (HM) or OC for 2-4 weeks. Expression of hepatocyte-specific genes was determined by real-time quantitative RT-PCR. RESULTS: The level of albumin mRNA in human hepatocytes cultured in OC was 11-fold more than in HM and gene expression levels of alpha1-antitrypsin and transferrin were at approximately 40 and 11% of freshly isolated primary human hepatocytes. Electron microscopy revealed that cells in OC displayed hepatocyte properties (e.g. polarity, junctional complexes, bile canaliculi and glycogen particles). Cytochrome P4501A1/2 activity of hepatocytes cultured in OC was 15- and 17-fold higher than in HM at 2 and 4 weeks of culture, and DNA synthesis was higher. CONCLUSIONS: Using our optimal culture condition, we were able to maintain the phenotype of primary human hepatocytes in long-term culture. They not only maintain better liver-specific function, but also retain higher proliferative potential.