Isolation Of Rat Hepatocytes Research Articles

Isolation of rat hepatocytes for pharmacological studies on metabotropic glutamate receptor (mGluR) subtype 5: a comparison between collagenase I versus collagenase IV.

Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or collagenase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collagenase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared to collagenase I. These results indicate that collagenase IV better preserves the activity of surface proteins such as mGluR5 in isolated rat hepatocytes, an in vitro model useful to reduce the use of experimental animals, in line with the 3R ethical framework.

A simple non-enzymatic method for the isolation of high yields of functional rat hepatocytes.

A method for isolation of rat hepatocytes using liver perfusion by ethylenediamine tetra-acetic acid (EDTA)-containing sucrose solution and mechanical tissue disaggregation by controlled vibration (MVD) is described. The yields of hepatocytes produced by this method were similar to those obtained using collagenase perfusion. The cells had well preserved membrane integrity as judged by the trypan blue staining test (91 +/- 4%), ATP contents, rates of endogenous respiration and enzyme leakage that indicated they were functional cells. There was little evidence of expression of latent damage when the cells were stored either at 37 degrees C (by pre-incubation) or at 4 degrees C. This method can be used to isolate high yields of functional cells from rat liver if the collagenase perfusion technique is not available.

Characterization of nitric oxide production following isolation of rat hepatocytes.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) produce large amounts of nitrite following isolation. Nitrite production was inhibited by the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine, as well as the transcription inhibitor actinomycin D. Increases in iNOS mRNA, protein, and activity levels correlated with the formation of nitrite. iNOS mRNA was first detectable 2 h after the onset of hepatocyte incubations and peaked at 4 h. These results indicate that nitrite formation is a result of the large scale production of nitric oxide (NO) by hepatocytes in response to the time-dependent induction of iNOS. NO production by hepatocytes was attenuated by pretreatment of rats with the Kupffer cell inhibitor, gadolinium chloride. Also, the addition of the endotoxin neutralizing agent, polymyxin B; the protein kinase inhibitor, staurosporine, and antioxidants to perfusion buffers and hepatocyte suspensions also decreased nitrite formation. Collectively, our results suggest that iNOS is induced in hepatocytes in response to the stresses generated during collagenase isolation procedures. The response appears to be triggered by a complex interaction between several different factors including Kupffer cell activation, reactive oxygen species generation, and endotoxin contamination of collagenase preparations.

Prevention of galactosamine - induced hepatic damage by the natural product loganin from the plantstrychnos nux-vomica: studies on isolated hepatocytes and bile flow in rat

Loganin an iridoid glycoside extracted from the fruit of the plant, Strychnos nux-vomica, in an earlier study showed hepatoprotection in primary screening. The present detailed study has been carried out in isolated hepatocytes (ex vivo) and on bile flow (in vivo) against galactosamine induced hepatic damage in rats to determine its pharmacological potential. Loganin was given to rats at preselected doses (3, 6 and 12 mg/kg p.o. ×7). Silymarin a standard compound was given at the same doses for comparison. On day 7 galactosamine (400 mg/kg) was injected intraperitoneally. Study on isolated hepatocytes and bile flow was carried out 24 h after treatment with galactosamine. Isolation of rat hepatocytes and collection of bile were done according to standard methods. Galactosamine reduced the viability of hepatocytes as well as bile volume and bile contents. Loganin showed a dose dependent activity (14%–67%) as observed by an increase in the viability of hepatocytes and the reversal of reduced parameters of bile. The activity of silymarin a known compound was found comparable. Loganin showed significant hepatoprotective and anticholestatic activities and confirms the earlier findings. The present study warrants detailed pharmacological evaluation. © 1998 John Wiley & Sons, Ltd.

Isolation of rat hepatocytes.

Isolation of rat hepatocytes.

Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion

This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP(+)) indicates a predominantly mitochondrial localization of TPP(+) in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP(+), and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.

Isolation of rat hepatocytes with EDTA and their metabolic functions in primary culture.

Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10(7) cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the hepatocytes in primary culture such as albumin secretion (10.9 micrograms/mg cell protein/d) and triglyceride synthesis and secretion are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in primary culture to retain complex and highly differentiated functions of the intact liver.

Opposite effects of insulin and glucagon in acute hormonal control of hepatic lipogenesis.

Conditions for the isolation of rat hepatocytes that are responsive to insulin with regard to fatty acid synthesis were explored. Cells prepared according to the procedure of Ingebretsen and Wagle require the presence of fetal calf serum for insulin expression. Cells isolated by the Seglen method are the preparation of choice, since they respond to insulin in a simple, well-defined medium and, moreover, show much higher basal rates of fatty acid synthesis. In the latter cells isolated from fed male rats, the rate of fatty acid synthesis, as determined by tritium incorporation from [3H]H2O at 37 degrees C, is enhanced within 30 min after addition of insulin to the incubation medium; with glucagon, it is depressed. In the presence of insulin, the cellular content of malonyl coenzyme A is noticeably increased, whereas the concentrations of pyruvate, lactate, and citrate are not markedly affected. Glucagon, on the other hand, decreases the concentrations of all four intermediates. The activity of acetyl-CoA carboxylase is stimulated and depressed after addition of insulin and glucagon, respectively. In all conditions tested, the activity of acetyl-CoA carboxylase correlates with the rate of fatty acid synthesis, which in turn correlates with the cellular level of malonyl-CoA.

ラット培養肝細胞における高比重リポ蛋白 (HDL<sub>3</sub>) の合成

It is indicated that liver plays an important role in lipoproteins metabolism. Recently relationship between plasma concentrations of high density lipoprotein (HDL) and coronary heart disease has been reported. The present study was to investigate lipoproteins synthesis, especially HDL3 or Apo A-I, using a culture system of rat liver parenchymal cells. The cell culture system has several advantages such as cell homogeneity and the possibility of longer incubation than liver perfusion system that has been used by many investigators.The isolation of rat hepatocytes was performed accordint to the method of Berry and Friend with some modifications. The hepatocytes were cultured in the complete synthetic culture media HI/WO5/BA2000 (International Scientific Ind.) under 5% CO2/95% air at 37°C. Twenty-four and 48 hours after cell inoculation, light and electron microscopic study and the following metabolic function of the cells were examined: 1) gluconeogenesis from pyruvate and lactate (179.5 nmoles/mg cell protein/hour), 2) induction of tyrosine aminotransferase by 10-5M dexamethasone (43.8mU/mg cell protein), 3) membrane transport of C14-α-aminoisobutyric acid (AIB)(9.5 AIB distribution ratio) and 4) albumin synthesis (156μg/mg cell protein/hour). The results of metabolic and morphological studies showed that integrity of cultured cells was well maintained at least for 48 hours after inoculation.Rat HDL3 (1.110<d<1.210g/ml) was isolated by sequential ultracentrifugation. Blood was obtained from male rats weighting 250 to 300g who had been fasted for 24 hours. The density of plasma was adjusted to d=1.110g/ml with solid KBr and centrifuged in a Type 40.3 rotor on the LS-50 preparative ultracentrofuge (Beckamn Instruments Inc.) for 22 hours at 105, 000×g. After 22 hours, the top 2ml was removed by tube slicing technique. Then, the density of the infranatant solution was adjusted with solid KBr to d=1.210g/ml and centrifuged at 105, 000×g for 44 hours. HDL3 was washed at d=1.210g/ml to remove contaminating serum albumin and was dialyzed exhaustively against 0.154M NaCl, pH 7.5 or double distilled water at 4°C. The rationale for using HDL3 is that this density fraction contains predominantly Apo A-I and a minimal amount of other apolipoproteins, as compared to other density classes. Anti-rat HDL3 serum was prepared by several intraperitoneal injections of rat HDL3 with equal volumes of Freund's complete adjuvant at intervals of 7 days. The anti-sera were examined by immunodiffusion and immunoelectrophoresis with various antigens such as VLDL (d<1.006g/ml), LDL2 (1.019<d<1.050g/ml), HDL3 and peak II (containing only Apo A-I) and peak III (containing mainly C peptides) fractions of apo-HDL3 from Sephadex G-100 column chromatography. Anti-rat HDL3 serum used for current study reacted only with HDL3 or Apo A-I. The concentrated culture media in which hepatocytes were cultured for 20 hours, were examined immunologically. A single precipitin line was observed between culture media and anti-rat HDL3 or anti-rat Apo A-I on immunodiffusion and immunoelectrophoresis, indicating the presence of Apo A-I in cultured media. HDL3 fraction (1.110<d<1.210g/ml) isolated from pooled culture media was analyzed for identification of apolipoproteins. The electrophoretic patterns of totally delipidized HDL3 fractionated from culture-media on 7% polyacrylamide gel with 8M urea were comparable with electrophoretic patterns of rat plasma Apo-HDL3. Not only the Apo A-I band but also the Apo C bands were observed. These findings indicated that HDL3 or Apo A-I synthesis and secretion by cultured hepatocytes.

4] Isolation and use of liver cells

This chapter describes the isolation and use of liver cells. The technique is based on liver perfusion with collagenase after removal of Ca 2+ by preperfusion with a chelator. Moderately high yields of viable, single hepatocytes with a smooth, spherical appearance and essentially free of nonparenchymal cells are obtained. Early attempts to isolate hepatocytes employed mechanical force and, subsequently, perfusion of the liver with Ca 2+- or K+-chelators but were unsuccessful in obtaining viable cells in high yields. This was not achieved until the isolation of rat hepatocytes by the use of the digestive enzymes collagenase and hyaluronidase was introduced by Howard and collaborators. Although most of the techniques recently employed to obtain isolated hepatocytes involve perfusion of the liver with digestive enzymes, attempts are also made to avoid the perfusion step and achieve hepatocyte isolation by incubating cut pieces of the liver in enzyme-containing solutions. The presently available results suggest that isolated hepatocytes—and cell suspensions isolated from certain other target tissues—may be a useful model for drug toxicity studies in the future.