Preparation of human hepatocytes for primary culture

Summary A method based on the use of a neutral bacterial protease was employed for the preparation of isolated human hepatocytes from surgical biopsy specimens. Cells maintained in primary culture developed ultrastructural features characteristic of human hepatocytes in vivo. They retained the ability to bind insulin; binding kinetics were similar to those of other species. The hepatocytes responded to physiological concentrations of insulin by an increase in glycogen synthesis.     

Cell density dependent morphological changes in adult rat hepatocytes during primary culture

In order to gain morphological insights about the cell density dependency, hepatocytes cultured at a low cell density (less than about 0.1 X 10(5) nuclei (cm2)-1) and at a high cell density (greater than about 1 X 10(5) nuclei (cm2)-1) were examined ultrastructurally 24 h after plating (just prior to the beginning of DNA synthesis). The results were as follows: (i) glycogen rosettes disappeared completely in low density culture as compared with sections from an intact liver. In contrast, glycogen rosettes were still present in high density culture. (ii) Polysomes seemed increased in low density culture in comparison with those seen in sections from an intact liver and from the high density culture. (iii) In low density culture, the shape of mitochondria deviated from that of hepatocytes in an intact liver and the mitochondria often lost a characteristic close contact with rough endoplasmic reticulum (rough ER). (iv) In low density culture, bundles of filamentous structure were detected, which were not found in an intact liver or high density culture. The following features were found only in high density culture; (v) numerous villous cytoplasmic protrusions developed along the area facing adjacent cells, and seemed to intertwine with each other, and (vi) between the hepatocytes, only abortive junctions were found. These results indicate that the hepatocytes cultured at a low density express most of the characteristics of the hepatocytes in a regenerating liver and the features of the cells cultured at a high density are very similar to those of the hepatocytes in sections from an intact liver.     

草鱼肝细胞的分离与原代培养

目的以草鱼(Ctenopharyngodon idellus)肝细胞为实验对象,在不同条件下进行原代培养,以探讨适合草鱼肝细胞生长的最佳条件及培养方法,用于饲料营养与非营养物质对草鱼肝细胞代谢、损伤作用机制的研究。方法采用温胰蛋白酶消化法和红细胞裂解液分离、纯化肝细胞,MTT法测定细胞增殖率,并测定不同时期培养液上清液中LDH、Alb和BUN的含量,分析肝细胞生长状况。结果采用0.25%浓度的温胰蛋白酶消化法,消化20min,分步收集肝细胞,经台盼蓝染色检测和血球计数板计数,活细胞数≥99%。结论在含10%胎牛血清、10μg/mL胰岛素的M199培养基中,以接种浓度1.7×106cell/mL左右为宜,置于27℃、4.5%CO2浓度的恒温培养箱中,可成功培养草鱼原代肝细胞。     

Retention of the differentiated state by larvalXenopus liver cells in primary culture

Summary Electron microscopic analysis of primary cultures derived from larvalXenopus liver has shown that these cells, although they form only two-dimensional aggregates, retain and presumably also develop structural characteristics typical of liver parenchyma cells, such as bile canaliculi with microvilli and epithelial junctional complexes. As judged from structural criteria, primary cultures contain 80–90% hepatocytes. In contrast to the intact tissue, primary cultures showed excessive development of microfilaments, however.Incorporation of labeled amino acids has revealed further that the capacity for protein synthesis is maintained in culture and that synthesis of liverspecific protein albumin is maintained in vitro, even in liver cultures derived from thyrostatic tadpoles. This latter result suggests that initiation of albumin synthesis in the larval liver is probably not dependent upon thyroid hormones but rather reflects the protodifferentiated state of this tissue.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Ethanol stimulates bile acid formation in primary human hepatocytes

The conversion of cholesterol to bile acids is a key pathway for elimination of cholesterol from the body, thereby reducing the risk of arteriosclerosis. Moderate consumption of ethanol has been shown to have preventive effects on cardiovascular disease and decrease the risk of gallstone formation. In the present study primary human hepatocytes were used to investigate if ethanol affected bile acid synthesis. Hepatocytes were prepared from donor liver (n = 11) and treated with ethanol, 7.7 or 50 mM, for 24 h. mRNA levels for enzymes in bile acid synthesis pathways were studied and bile acid synthesis was analyzed. Treatment with 7.7 mM ethanol increased cholic acid synthesis by 20% and treatment with 50 mM ethanol up-regulated cholic acid formation by 60%. The synthesis of cholic acid increased more than that of chenodeoxycholic acid, indicating that the classical pathway for bile acid synthesis was up-regulated. Increased bile acid levels in the cells treated with ethanol were seen after approximately 20 h. mRNA expression of CYP7A1, CYP27A1, and CYP8B1 in the hepatocytes was not affected by alcohol exposure.     

Influence of hormones and growth factors on viability,DNA, and protein content of adult hepatocytes in primary culture

Summary The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10⁻⁷ M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.     

Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture

Summary Glutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ie and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24 h, GST remained active over the whole culture period (1–2 wk). However, various patterns were observed: GST activity either remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10⁻⁴ M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed during culture but its levels markedly varied depending on the medium composition and type and age of culture. Y. V. was supported by Instituut voor Wetenschappel?k Onderzoek in Landbouw en Nijverheid. This work was supported by INSERM.     

Preservation of hepatic phenotype in lentiviral-transduced primary human hepatocytes

Lentiviral vectors effectively transduce both dividing and non-dividing cells and stably integrate into the genome of the host cell. In this study, we evaluated the usefulness of a lentiviral system for genetic modulation of primary human hepatocyte cultures. Infection with GFP-expressing lentivectors shows that Huh7 and HepG2 cell lines, as well as primary cultures of human hepatocytes, are efficiently transduced by lentiviral vectors. Real-time RT-PCR analyses demonstrate that infection with lentivectors does not alter hepatic hallmarks such as the expression of the nuclear receptors CAR, PXR, RXR alpha, or HNF4 alpha, or expression of the secretory protein, albumin. Additionally, infected hepatocytes retain the capacity for CYP3A4 induction in response to treatment with phenobarbital, a uniquely sensitive indicator of hepatic differentiation status. Lentivectors may be used for both over-expression and knockdown analyses in primary hepatocytes, as demonstrated in this study by >200-fold CAR over-expression and knockdown of CAR to less than 40% of endogenous levels, with corresponding effects on CYP2B6 expression. In summary, lentiviral vectors provide a novel methodology by which primary human hepatocytes may be stably genetically manipulated, with minimal effects on the differentiated hepatic phenotype. These approaches offer considerable advantage over current methodologies, providing a valuable alternative for use in pharmacological and toxicological investigations involving primary human hepatocyte models and potentially for cell-based therapeutics to treat hepatic dysfunction in vivo.     

Characteristics of taurine transport in rat hepatocytes in primary culture

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.     

Effect of matrine on primary human hepatocytes in vitro

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO₂⁻ induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9680-1) contains supplementary material, which is available to authorized users.     

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24 h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.     

Reduced and oxidized glutathione contents in adult rat hepatocytes under various culture conditions

Reduced and oxidized glutathione contents of adult rat hepatocytes in pure culture and in co-culture with rat epithelial cells were measured under various medium conditions. To the standard medium fetal calf serum, nicotinamide, H₂SeO₃, dimethylsulphoxide or no supplements were added. For freshly isolated hepatocytes, intracellular contents of 24 ± 7 nmol reduced and 0.7 ± 0.2 nmol oxidized glutathione/mg cellular protein were obtained, respectively. In pure culture as well as in co-culture and regardless of th medium conditions involved, the protein content stays constant during the culture time with the exception of a decrease in protein content after 6 days of pure culture, caused by deterioration and loss of the hepatocytes. In both culture systems, an initial increase in intracellular reduced glutathione levels was observed, followed by a decrease and a quick normalisation in co-culture. On the contrary, in pure culture, the decrease was slower, but not transient and a stabilized situation was never reached. The various supplementations of the culture media had no significant effect on the intracellular reduced glutathione contents of both culture systems. As far as the intra- and the extracellular oxidized glutathione contents and the extracellular reduced form are concerned, these were only present in small amounts.Abbreviations BSA bovine serum albumin - DMSO medium supplemented with 2% dimethylsulphoxide - FCS- medium without fetal calf serum - FCS+ medium supplemented with 10% fetal calf serum - GSH reduced glutathione - GSSG oxidized glutathione - HPLC high performance liquid chromatography - MEM minimal essential medium - Nic medium supplemented with 25 mM nicotinamide - PBS phosphate buffered saline - Se medium supplemented with 0.1 µM selenium - st standard medium     

Isolation and culture of adult hepatocytes from liver biopsies

Summary 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 19×10⁶ 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 andn-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. These studies were supported by Grant 5-ROI-ES01597-02 from the National Institute of Environmental Health Sciences and Regional Research Project CA-D^*-ETX-3634-RR(NE115). Dog liver biopsies were provided by Dr. W. Spangler at the Laboratory for Energy-Related Health Research. Unused parts of human liver biopsies were provided by Drs. N. Pimstone and B. Ruebner at the Sacramento Medical Center.     

The effects of dexamethasone on metabolic activity of hepatocytes in primary monolayer culture

Summary The effects of dexamethasone on multiple metabolic functions of adult rat hepatocytes in monolayer culture were studied. Adult rat liver parenchymal cells were isolated by collagenase perfusion and cultured as a primary monolayer in HI/WO/BA, a serum free, completely defined, synthetic culture medium. Cells inoculated into the culture medium formed a monolayer within 24 hr. Electron microscopy showed that the cells in primary culture had a fine structure identical to liver parenchymal cells in vivo, including the observation of desmosomes and bile canaliculi in intercellular space. There was significant gluconeogenesis by the cell 24 hr postinoculation but it had decreased markedly by 48 hr. There was a marked induction of tyrosine aminotransferase (TAT) by dexamethasone, which was maintained for up to 72 hr postinoculation of cells. The transport of α-aminoisobutyric acid into the cells in monolayer culture was stimulated by dexamethasone and was dependent on the concentration of dexamethasone. Albumin synthesis and secretion by the cells was measured by a quantitative electroimmunoassay. Albumin production was shown to increase linearly over an incubation period of 24 to 48 hr postinoculation. Dexamethasone depressed the albumin synthesis. The effects of dexamethasone are slow, and at times require more than 6 hr to show variation from the control, indicating that dexamethasone is not a single controlling hormone. Possibly it functions in a cooperative and coordinating role in the regulation of cell metabolism. This work was performed during Dr. Yamada's tenure as a Postdoctoral Research Fellow of the American Heart Association, Oklahoma Affiliate, and was supported in part by NIH Research Grant HL 18178 awarded to Thomas F. Whayne, Jr., M.D., Ph.D.     

Isolation and primary culture of Necturus maculosus (Amphibia: urodela) hepatocytes

Summary In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7×10⁵ viable hepatocytes per gram body weight with an average viability of 86±5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8°C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.     

Long-term culture of hepatocytes from human adults

A long-term primary human hepatocyte culture retraining liver-specific functions is important and essential for basic research and for the future development of hepatocyte-based applications. We established a normal hepatocyte culture system from excess normal tissues obtained from adult liver cancer patients who received partial liver resection. Hepatocytes were isolated after perfusion and enzymatic disaggregation, and were first maintained in hormonally defined media on a Matrigel matrix, and then transferred to collagen sandwich gel. The hepatocytes formed clusters on the Matrigel matrix and increased in size and numbers with time of culture and eventually grew into spheroids of variable sizes. After being transferred to collagen gel, the cells migrated outward from spheroids to form a monolayer with cuboidal or polygonal cell shapes with granular cytoplasm and continued to proliferate. Cellular functions specific for hepatocytes were analyzed using immunoblot assay for proteins specifically secreted by the liver cells on different days of culture. The cells secreted albumin, transferrin and -fetoprotein consistently for more than 100 days, to a maximum of 150 days. Thus, we have established a long-term culture of hepatocytes from human adults, which will be useful for basic studies of liver physiology such as metabolism and morphogenesis, as well as for other applications in the study of infectious hepatitis, pharmacology, pharmacokinetics, and toxicology.     

Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well thein vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.     

Synthesis of barbituric acid derivatives and their effect on survival of functional hepatocytes from adult rats in primary culture

Summary Ten barbituric acid (BA) derivatives were synthesized and tested for their potency for supporting survival of functional hepatocytes from adult rats in primary culture. Of the 10 BA derivatives, 7 compounds (C-2, 3, 4, 5, 6, 9, and 10) efficiently supported hepatocyte survival for at least 2 wks in primary culture. Especially C-5, 6, and 9 showed excellent efficiency for such action. The optimum concentrations of the BA derivatives for observing the morphological and biochemical effects differed from each other. The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium. The morphologic features of hepatocytes surviving in the presence of the BA derivatives resembled those of hepatocytes 24 h after inoculation. The surviving hepatocytes secreted remarkably large amounts of albumin into the culture media. Tyrosine aminotransferase (TAT) activity was higher in the 1-wk-old cultures treated with C-5, 6, and 9 than in the freshly isolated hepatocytes. The addition of dexamethasone (10 μM) caused a 1.7 to 2.1-fold induction in TAT activity. The basal levels of TAT activity and the induction rates increased in the cultures treated with C-5 and 6 from Week 1 to 2 of primary culture.