Mouse liver cell culture. I. Hepatocyte isolation

A method for isolation of mouse liver cells by a two-step perfusion with calcium and magnesium-free Hanks' salt solution followed by a medium containing collagenase is described. Several variations of the commonly used procedure for rat liver cell isolation were quantitatively compared with respect to cell yield and viability. The optimal isolation technique involved perfusion through the hepatic portal vein and routinely produced an average of 2.3 x 10(6) viable liver cells/g body weight. Optimal perfusate collagenase concentration was found to be 100 U of enzyme activity per milliliter of perfusate. Light and electron microscopic evaluation of liver morphology after several steps of the isolation showed distinct morphologic changes in hepatocytes and other liver cells during perfusion. After perfusion with Hanks' calcium- and magnesium-free solution, many hepatocytes exhibited early reversible cell injury. These changes included vesiculation and slight swelling of the endoplasmic reticulum as well as mitochondrial matrix condensation. Subsequent to perfusion with collagenase, the majority of hepatocytes appeared connected to one another only by tight junctional complexes at the bile canaliculi. Multiple evaginations were seen on the outer membrane resembling microville and probably represented the remains of cell-to-cell interdigitations between hepatocytes and sinusoidal lining cells from the space of Disse. The cytoplasmic injury seen after Hanks' perfusion was reversed after collagenase perfusion. After mechanical dispersion, isolated mouse hepatocytes were spherical in shape and existed as individual cells; many (80 to 85%) were binucleated under hase contrast light microscopy. By electron microscopy, cells appeared morphologically similar in cytoplasmic constitution to that seen in intact nonaltered liver cells.     

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.     

Hepatocytes (hepatic parenchymal cells) produce a major part of liver collagen in vivo

Following intraperitoneal injection of [3H]proline and colchicine, rat liver cells were dispersed by collagenase perfusion and fractionated by low-speed and density gradient centrifugation. Analysis of the collagenous components using purified bacterial collagenase showed that 0.1 to 0.2% of the labeled protein produced by the liver cells was collagen. Considering the population of the hepatocytes in the liver (70%), 80% of the collagen produced by the liver could be attributed to the hepatocytes.     

Hepatocyte adhesion to carbohydrate-derivatized surfaces. I. Surface topography of the rat hepatic lectin

The rat hepatic lectins, galactose- and N-acetylgalactosamine-binding proteins found on the hepatocyte cell surface, mediate adhesion of isolated primary rat hepatocytes to artificial galactose-derivatized polyacrylamide gels. Biochemical and immunohistochemical techniques were used to examine the topographical redistribution of the rat hepatic lectins in response to galactose-mediated cell adhesion. Hepatocytes isolated from rat liver by collagenase perfusion had an average of 7 x 10(5) cell surface lectin molecules per cell, representing 30-50% of the total lectin molecules per cell, the remainder residing in intracellular pools. Hepatocytes incubated on galactose-derivatized surfaces, whether at 0-4 degrees C or 37 degrees C, rapidly lost greater than 80% of their accessible cell surface lectin binding sites into an adhesive patch of characteristic morphology. The kinetics of rat hepatic lectin disappearance were used to estimate a lateral diffusion coefficient greater than 9 x 10(-9) cm2/s at 37 degrees C, suggesting rapid and unimpeded lectin diffusion in the plane of the membrane. Indirect immunofluorescence labeling of adherent cells using antihepatic lectin antibody revealed a structured ring of receptors surrounding an area of exclusion (patch) of reproducible size and shape which represented approximately 8% of the hepatocyte cell surface. Notably, adherent cells, which had lost greater than 80% of their accessible surface binding sites, still endocytosed soluble galactose-terminated radioligand at greater than 50% of the rate of nonadherent control cells. No net movement of rat hepatic lectin from intracellular pools to the cell surface was found on cells recovered after adhesion to galactose-derivatized surfaces at 37 degrees C, suggesting that the physical size and/or lectin density of the patch was restricted by kinetic or topological constraints.     

Ultrastructural and hormonal metabolic studies of rat liver maintained in vitro by perfusion at 30 degrees C and 37 degrees C: a time course study by TEM, SEM and RIA

Isolated rat liver perfusion system has been extensively used for metabolic and functional studies. Results derived from the application of this system may reflect true biochemical changes but they may also be associated with some structural changes. This study was undertaken to correlate the cytological changes and functional integrity of isolated rat liver perfused in vitro at normal physiological temperature (37 degrees C) and 30 degrees C, using a non-recirculating system. The livers were perfused for 3 hours with modified Ham's F10 culture medium supplemented with thyroxine hormone (T4). The hepatocyte structural integrity was studied by light microscopy, transmission and scanning electron microscopy. The triiodothyronine (T3) and T4 hormones in the perfusion medium and the effluent fractions were assessed by radioimmunoassay. The livers perfused at 30 degrees C remained morphologically intact at the ultrastructural level for 3 hours whilst at 37 degrees C, hepatocytes in the centrilobular zone exhibited marked structural alterations. The percentage of T4 uptake was significantly higher (P less than 0.01) in livers perfused at 30 degrees C (50.8 +/- 7.7% vs 38 +/- 7.7%, 37 degrees C), but the net T3 output (3.16 +/- 1.04 micrograms) and the conversion of T4 to T3 (4 +/- 0.62%) were significantly higher (P less than 0.001) in livers perfused at 37 degrees C in comparison to livers perfused at 30 degrees C (1.61 +/- 0.84 micrograms and 1.68 +/- 0.76%, respectively). In conclusion, at 30 degrees C the hepatic T4 uptake is not inhibited, but the rate of T4 to T3 conversion has decreased, additionally the livers remain morphologically well preserved throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.     

A liver micronucleus assay using young rats exposed to diethylnitrosamine: methodological establishment and evaluation

We have developed a simple liver micronucleus assay using young rats (up to 4 weeks old) to assess cytogenetic damage of chemicals in liver cells. Diethylnitrosamine (DEN) was used as a model rodent hepatocarcinogen in this study. Compared to the partial hepatectomy method most commonly used for the liver micronucleus assay, the present assay method showed equal or even higher practicability. The young rat liver micronucleus assay was also characterized for rat strain differences, sampling time after treatment, single treatment vs. split treatment, age of animals, administration route, and staining method. Although based on one model chemical, we propose an acceptable protocol for the micronucleus assay using young rat liver as follows: Up to 4-week-old rats should be used; oral or intraperitoneal administration can be used; single or repeated treatment protocols can be applied; sampling time is 3-5 days after the last treatment; hepatocytes are prepared by the collagenase perfusion method; and cells are stained with the AO-DAPI double staining method.     

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.     

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion,fibronectin-mediated adhesion,spreading, and functional activities

Summary The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture. Part of this work was presented at the 30th Annual Meeting of the Tissue Culture Association, Seattle, June, 1979.     

Morphological changes in the isolated rat liver perfused in a non-recirculating system: scanning and transmission electron microscopy

Isolated perfused rat livers have been used for various studies, but detailed investigation into the structural integrity of hepatocytes of this system is lacking. In this study, isolated rat livers were perfused in vitro with oxygenated Krebs-Ringer bicarbonate buffer solution, for 2 minutes and 1, 2, 3, and 4 hour(s) at 37 degrees C, using a non-recirculating perfusion system. The perfused livers were processed for semithin section light microscopy, transmission electron microscopy, and scanning electron microscopy. Sectional areas of cell deaths were measured by a camera-tracing assembly from 1.5 microns thick Araldite sections stained with toluidine blue. Progressive nuclear and cytoplasmic changes, leading to cell death, occurred in the hepatocytes of the centrilobular zone, during the 2nd, 3rd, and 4th hour of the perfusion at a rate of 9.03% +/- 1.5%, 38.7% +/- 2.7%, and 55.1% +/- 5.9% (mean +/- standard deviation) of the total sectional areas respectively. Midzonal hepatocytes showed normal basophilic staining but exhibited loss of glycogen granules, loss of microvilli, development of aqueous vacuoles and formation of blebs. The fine structures of cell organelles, glycogen granules, microvilli and plasma membrane of the cells in the periportal zone were well preserved throughout the experimental period. For further quantitative, metabolic and functional studies using isolated rat liver perfused with Krebs-Ringer solution, it is evident from the present investigation that the periportal zone represents the functional region of the hepatic lobule. Whilst progressive changes, leading to cell death, occurred in the centrilobular zone.     

Dispersion of viable pig liver cells with collagenase.

Viable suspended hepatocytes were prepared from surgical biopsy specimens of pig and human liver by digestion with collagenase. Initial perfusion of the tissue through cannulated blood vessels with 0.5 mM EGTA followed by 0.2% collagenase gave the best results. 20−870 × 10⁶ cells of which 60–95 % excluded trypan blue were obtained from 5–30 g pig liver pieces, while results with human liver specimens were usually less satisfactory. In some experiments, however, viable cells, as judged by vital stain exclusion and ability to synthesize lipids were obtained in sufficient yield. In the pig hepatocytes glycerolipid synthesis from [³H] glycerol and oxidation and esterification of [¹⁴C] oleic acid had the same characteristics as those observed earlier in rat hepatocytes.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

分离小鼠肝细胞的一种简易灌流法

我们设计了一种分离小鼠肝细胞的简易灌流法。它具有操作简便,不需特殊装置,胶原酶耗费少等优点。在总活细胞产量及每100g体重活细胞产量等方面显著高于振荡法(P<0.001),而接近Seglen灌流法。此外,对经本法分离的肝细胞及肝非实质细胞进行培养,获得了良好效果。     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

The isolated hepatocyte preparation: 30 years on

A method for the preparation of intact rat hepatocytes in high yield was first described in 1969. The procedure involved digestion of hepatic tissue by perfusion of the liver with crude collagenase; later, purified collagenase without other enzymic additions was shown to be ineffective. Recently it has been discovered that the combination of purified collagenase plus elastase is superior to crude collagenase in that it consistently provides high yields of undamaged hepatocytes. The isolated hepatocyte preparation has proved particularly useful for the study of mechanisms responsible for long-range interactions within the cell. These can be studied over prolonged time courses and in the presence of graded concentrations of specific inhibitors. Studies of this kind have demonstrated a close relationship between cytoplasmic metabolic flows and mitochondrial forces and have also revealed that the cytoplasmic and mitochondrial free NAD-linked redox potentials are maintained by energy-dependent reactions.     

Isolation and culture of hepatocytes from human liver of immediate autopsy

Summary Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 10⁶ cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochromep ₄₅₀ and 536. pmol/mg protein cytochromeb ₅ were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture. This work was supported by EPA Grants R-809835-01-1, R-809599010 and DOE Contract DE-A505-83ER60158. Cobtribution no. 1762 from the Cellular Pathobiology Laboratory, University of Maryland School of Medicine.     

Ultrastructure of hepatocytes after local cooling of the liver

Electron-microscopic and stereometric study of hepatocyte ultrastructure with local liver cooling to -30 degrees C have been performed using the method of vital fixation by rat liver perfusion. Quantitative and qualitative data obtained testify to the presence of destructive changes in hepatocytes immediately after thawing expressed in hyaloplasm lightening, formation of cavities in cytoplasm and injury of mitochondria membrane and endoplasmic reticulum integrity. Changes in nuclear structure of hepatocytes and Kupffer cells (widening of nuclear pores and channels in condensed chromatine, as well as the presence of ribosomal complexes in the nucleus and edge position of the nucleolus) may be connected with the beginning of the reparation process.     

利用NASH大鼠原代肝细胞与原代Kupffer细胞共培养建立NASH原代细胞模型*

目的: 通过分离并提纯非酒精性脂肪性肝炎(NASH)大鼠原代肝细胞以及原代Kupffer细胞建立体外NASH原代细胞模型,为研究NASH提供可靠的细胞实验技术支持。方法: 选择SD大鼠40只,随机分为2组(n=20):对照组和NASH组,对照组大鼠利用普通饲料喂养,NASH组大鼠利用高脂饲料(88%基础饲料+10%猪油+ 2%胆固醇)喂养,6～8周后,利用NASH评分表,病理观察下肝组织切片脂肪变+小叶内炎症+气球样变评分≥4 分,表明大鼠NASH模型的成功建立,利用胶原酶原位灌注法分离并提纯NASH模型大鼠原代肝细胞以及原代Kupffer细胞,利用CK-18及CD68免疫荧光以及墨汁吞墨实验进行细胞鉴定,利用油红O染色、试剂盒测定谷丙转氨酶(ALT)、谷草转氨酶(AST)含量观察NASH大鼠原代肝细胞脂质累积和肝功情况,Western blot检测原代Kupffer细胞炎症因子表达情况,最后采用原代肝细胞:原代Kupffer细胞=6∶1比例共培养,显微镜下观察细胞状态。结果: 实验成功分离并提纯NASH原代肝细胞以及原代Kupffer细胞,通过油红O染色,NASH组大鼠原代肝细胞存在明显的脂肪沉积,且NASH组大鼠原代肝细胞中AST、ALT明显高于对照组,存在明显肝损伤(P＜0.05),Western blot测定原代Kupffer细胞TNF-α、IL-1β以及MCP-1,NASH组大鼠明显高于对照组(P＜0.05)。结论: 通过胶原酶原位灌注法可以成功分离NASH大鼠原代肝细胞以及原代Kupffer细胞,同时成功建立比例共培养大鼠体外原代细胞NASH模型。     

Cell-based therapy of acute liver failure: The extracorporeal bioartificial liver

The need for an alternative ttreatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporalily providing liver functions are being actively tested. Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function. An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation. Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines. Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We have isolated normal porcine hepatocytes by collagenase perfusion of the liver. Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor. Porcine hepatocytes expreessed liver-specific functions at high levels, particularly protein neosysnthesis and enzymatic activities involved in detoxication and biotransformation processes. In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins. This system represents a promising tool for the design of anoartificial liver in human beings.Abbreviations ALF acute liver failure - EBAL extracorporeal bioartificial liver - OLT orthotopic liver transplantation     

Liver endothelium mediates the uptake of iron-transferrin complex by hepatocytes

We have previously shown that in the liver, transferrin (TF) receptors are limited to endothelial cells, and hepatocytes and Kupffer cells do not have TF receptors. To study the transport of iron into hepatocytes, we fractionated liver cell suspensions into endothelium and hepatocyte fractions. At 4 degrees C liver (but not umbilical cord) endothelium bound Fe-TF with a saturable kinetics. At 37 degrees C, the endothelial uptake was followed by its gradual release. Transendothelial transport of TF was visually demonstrated by perfusion of liver using colloidal gold-labeled TF. The released Fe-TF acquired the potential for binding to fresh target hepatocytes and binding was not inhibited by excess cold TF but was inhibitable by asialofetuin, suggesting galactosyl receptors and not TF receptors as a recognition mechanism. Isoelectrofocusing of the supernate after preincubation for 90 min at 37 degrees C with endothelial cells, demonstrated the presence of a newly generated band which co-migrated with asialotransferrin. We conclude that Fe-TF is initially removed by liver endothelium where it is modified probably by desialation to expose the galactosyl residues of the glycoproteins. The modified molecule is subsequently released and recognized by hepatocytes through a TF receptor-independent mechanism which may involve galactosyl receptors of hepatocytes. The findings indicate a key role for endothelium in the transport of Fe-TF into the liver and may suggest a physiological function for galactosyl receptors on hepatocyte surface.