Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

DNA synthesis and mitotic activity in short-term cultures of hepatocytes isolated from regenerating rat liver

Cell suspensions were prepared from normal and regenerating liver of adult rats by perfusion with a calcium-chelating agent (EGTA), collagenase and hyaluronidase, and the cells were incubated in culture medium. In cultures prepared from regenerating liver at 20 h after partial hepatectomy, 23 ± 4% of parenchymal cells initially incorporated [³H]TdR. This incorporation was shown to reflect semiconservative DNA replication. At least some parenchymal cells were able to complete their DNA synthesis and to progress through G2 and mitosis. Numbers of hepatocytes in mitosis increased up to 12 h of culture. On the other hand, no entry of hepatocytes into the S period was detectable in cultures prepared from normal or regenerating liver.     

Hepatic zonation of drug metabolizing enzymes. Studies on hepatocytes isolated from the periportal or perivenous region of the liver acinus

Models of hepatic intraacinar zonation have been proposed previously; in most models, direct visualization of the acinar destruction is not possible while intact hepatocyte recovery-viability often presents a problem for subsequent metabolic studies. In the present studies, the liver is isolated in situ and perfused with Krebs-Henseleit buffer, pH 7.4. A 1.5-mL intrahepatic volume of a 7 mM digitonin solution is then injected at a flow rate of 6 mL/min for 15 s via the portal vein or via the vena cava for selective destruction of the periportal (PP) or perivenous (PV) region of the acinus. To avoid diffusion of the detergent throughout the acinus, the liver is then immediately perfused with oxygenated Hanks buffer in the direction opposite to that of digitonin injection. The preparation can then be used for histological evaluation, for studies on isolated-perfused liver, or for isolation of hepatocytes. Direct visualization of the acinar destruction can be achieved by coloring the permeabilized cells with 0.2 mM trypan blue; the liver is then fixed in situ by a 10-min perfusion with paraformaldehyde and histological evaluation is achieved by eosine staining of liver slices. Following isolation of hepatocytes by collagenase perfusion, a highly significant PV localization was found for the synthesis of glutamine, the N-demethylation of aminopyrine, and the glucuronidation of p-nitrophenol, whereas a highly significant PP zonation was found for alanine aminotransferase. By contrast, no specific acinar zonation was found for the enzymes 7-ethoxycoumarin O-deethylase and aniline p-hydroxylase. Total cytochrome P-450 was 0.42 +/- 0.006 and 0.4 +/- 0.03 nmol/10(6) hepatocytes in PV and PP, respectively (nonsignificant).(ABSTRACT TRUNCATED AT 250 WORDS)     

长爪沙鼠肝细胞体外分离培养体系的建立

目的建立长爪沙鼠原代肝细胞分离培养体系。方法以雄性长爪沙鼠为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,过碘酸-希夫氏反应(PAS)鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果组织消化法和Seglen两步灌流法平均每只长爪沙鼠可分别获得肝细胞(1.33±0.34)×107个、(3.97±1.15)×107个,细胞活率分别为(29.4±6.05)%、(80.3±4.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。结果表明肝细胞在贴壁后72 h内,肝细胞形态发生显著变化。结论采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。     

The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique

Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities.From 4 × 10⁸ to 5 × 10⁸ cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.     

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 ¹ and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 ². Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 μm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.     

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

Background aimsSuccessful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues.MethodsHepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis.ResultsBaseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 10⁴ to 28.3 × 10⁴ cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group.ConclusionsNAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research.     

Evaluation of calcium channel blockers as potential hepatoprotective agents in oxidative stress injury of perfused hepatocytes

The aim of this study was to investigate the effects of calcium channel blockers on tertbutyl hydroperoxide (TBH) induced liver injury using isolated perfused rat hepatocytes. Rat hepatocytes were immobilized in agarose threads and perfused with Williams E medium. Hepatocyte injury was induced by the addition of tertbutyl hydroperoxide (1 mM) to the perfusion medium 30 min after the addition of either verapamil or diltiazim. Hepatocyte injury was observed by monitoring the functional and metabolic competence of hepatocytes or by ultrastructural morphological examination of hepatocytes. Verapamil (0.5 mM) reduced lactate dehydrogenase leakage in TBH-injured hepatocytes as compared to the controls (154+/-11% vs. 247+/-30%). Lipid peroxides production was reduced after verapamil pretreatment as compared to the controls and oxygen consumption was increased by pretreatment of hepatocytes with verapamil. Verapamil pretreatment increased the protein synthesis activity at both levels of granular endoplasmic reticulum and free polysomes in cytoplasm and decreased ATPase activity. Diltiazem was qualitatively effective as verapamil. It is concluded that in hepatocyte oxidative injury, calcium channel blockers exhibited hepatoprotective properties. The hepatoprotective effect of calcium channel blockers was accompanied by a decrease in ATPase activity, which may implicate a normalization of Ca2+i after TBH intoxication.     

Culture of bovine hepatocytes: a non-perfusion technique for cell isolation

In this work we have studied the isolation and culture of mature bovine hepatocytes on plastic dishes without exogenous matrix. The liver has been disaggregated in a collagenase solution instead of undergoing a perfusion step. After a few days in culture, the plates showed several clusters of different cell types. Although the average yield was 1.60±0.57×10⁸ viable liver cells per gram of tissue, these cultures were formed by non-parenchymal cells and only very few or none by parenchymal cells. In these cultures, actin structures used as a marker for Stellate (Ito) cells have been visualized by immunocytochemical techniques. In order to increase the proportion of parenchymal cells a centrifugation on Percoll, which separates cell sub-populations, has been introduced. Though the yield was lower than in the previous method, these pre-purified cultures were only composed of hepatocytes. It has been shown that these cells exhibited albumin synthesis, which is a specific hepatocytes function. In addition, these cultures were capable of producing metabolites of 7-ethoxycoumarin at a higher rate than non purified cell cultures. Therefore this simplified procedure for the isolation and culture of functional and viable hepatocytes may be applied for in vitro studies in bovine.     

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.     

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.     

The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus

Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed ≥50 g yielded significantly higher (P < 0.01) cell viability than tissue <50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intra-hepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of “healthy” resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes.     

Radiosensitivity of parenchymal hepatocytes as a function of oxygen concentration

To investigate the mechanism(s) of hepatocyte radioresistance (D0 2.7 Gy), the radiosensitivities of respiring (37 degrees C) and nonrespiring (0 degrees C) hepatocytes were determined as a function of oxygen concentration. Fischer 344 female rat hepatocytes were isolated by liver perfusion, equilibrated in Leibowitz-15 media with different oxygen tensions, and exposed to 60Co radiation at either 37 or 0 degrees C. Cell survival and DNA single-strand breaks were used as the biological end points of radiosensitivity. The K value for respiring hepatocytes (37 degrees C) was 14.3 +/- 0.5 mm Hg O2 (18.8 +/- 0.7 mumol O2/liter), demonstrating that the K value for freshly isolated parenchymal hepatocytes is significantly greater than those previously obtained for cultured cells. In contrast, the K value for nonrespiring hepatocytes (0 degree C) is 1.4 +/- 0.4 mm Hg O2 (3.7 +/- 1.0 mumol O2/liter) indicating that hepatocyte respiration results in a plasma membrane-to-nucleus oxygen gradient of approximately 12.9 +/- 0.6 mm Hg (15.1 +/- 1.2 microns O2/liter). The hypothesis that the hepatic nucleus typically resides in a hypoxic condition, although the liver is uniformly perfused with well-oxygenated blood, is supported by (1) the nonradom perinuclear distribution of the mitochondria, (2) the high cellular respiration rate, and (3) the large intracellular oxygen diffusion distance in hepatocytes (25 microns diameter).     

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.     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.     

Development of a bioartificial liver employing xenogeneic hepatocytes

Liver failure is a major cause of mortality. A bioartificial liver (BAL) employing isolated hepatocytes can potentially provide temporary support for liver failure patients. We have developed a bioartificial liver by entrapping hepatocytes in collagen loaded in the luminal side of a hollow fiber bioreactor. In the first phase of development, liver-specific metabolic activities of biosynthesis, biotransformation and conjugation were demonstrated. Subsequently anhepatic rabbits were used to show that rat hepatocytes continued to function after the BAL was linked to the test animal. For scale-up studies, a canine liver failure model was developed using D-galactosamine overdose. In order to secure a sufficient number of hepatocytes for large animal treatment, a collagenase perfusion protocol was established for harvesting porcine hepatocytes at high yield and viability. An instrumented bioreactor system, which included dissolved oxygen measurement, pH control, flow rate control, an oxygenator and two hollow fiber bioreactors in series, was used for these studies. An improved survival of dogs treated with the BAL was shown over the controls. In anticipated clinical applications, it is desirable to have the liver-specific activities in the BAL as high as possible. To that end, the possibility of employing hepatocyte spheroids was explored. These self-assembled spheroids formed from monolayer culture exhibited higher liver-specific functions and remained viable longer than hepatocytes in a monolayer. To ease the surface requirement for large-scale preparation of hepatocyte spheroids, we succeeded in inducing spheroid formation in stirred tank bioreactors for both rat and porcine hepatocytes. These spheroids formed in stirred tanks were shown to be morphologically and functionally indistinguishable from those formed from a monolayer. Collagen entrapment of these spheroids resulted in sustaining their liver-specific functions at higher levels even longer than those of spheroids maintained in suspension. For use in the BAL, a mixture of spheroids and dispersed hepatocytes was used to ensure a proper degree of collagen gel contraction. This mixture of spheroids and dispersed cells entrapped in the BAL was shown to sustain the high level of liver-specific functions. The possibility of employing such a BAL for improved clinical performance warrants further investigations.     

Establishment and comparative analyses of different culture conditions of primary hepatocytes from Nile tilapia (Oreochromis niloticus) as a model to study stress induction in vitro

Summary We established an in vitro hepatocyte primary culture system from Oreochromis niloticus, a tropical fish species of great economical importance, and evaluated its ability to express albumin, a liver-specific protein, consistently for a period of 3 wk. Serum requirements for fish hepatocyte cultures were assessed. A one-step in situ perfusion of tilapia liver retrogradely followed by collagenase liver dissociation and subsequent washing produced nearly 90% homogenous viable hepatocytes, as shown by trypan blue exclusion test. Mixed primary monolayer and aggregate hepatocyte cultures achieved by 10% fetal calf serum medium supplements expressed consistent levels of albumin. The results of light and electron microscopy showed that the hepatocytes did not significantly proliferate (P<0.05) but remained viable for at least 3 wk. The results of this study show that in vitro cultures of mixed primary hepatocyte monolayers and aggregates established from Nile tilapia may be useful models for studying transient cellular stress induction.     

Rapid isolation of murine primary hepatocytes for chromosomal analysis

Primary hepatocyte culture is a crucial tool for investigations of liver function and for evaluating the toxic effects of drugs. In addition, chromosomal analysis of hepatocytes could also prove useful for understanding the mechanisms of hepatocarcinogenesis. However, cultivation of primary hepatocytes for chromosome analysis has been hampered by the specific equipment and skill required to perform the in situ perfusion step necessary for isolation of primary hepatocytes. In the present study, we aimed to establish a simple and efficient method of isolating hepatocytes suitable for chromosome analysis. We performed hepatocyte isolation without using collagenase perfusion, instead digesting liver tissues using collagenase in tubes. In addition, we examined hepatocyte and bone marrow cell (BMC) co-culture and cultivation of hepatocytes with medium containing BMC culture medium supernatants. We found that hepatocyte viability and attachment rate were significantly improved, both by co-culture with BMCs and medium containing BMC culture media supernatants, with the latter also significantly increasing the mitotic index. Using this simple method of isolation and cultivation, we could successfully perform chromosomal analysis of mouse primary hepatocytes. This method has the potential to help understand the mechanisms underlying chromosomal instability-mediated hepatocarcinogenesis.     

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.