Metabolic interactions between animal cells through permeable intercellular junctions.

The isolated perfused rat liver was used to study the degradation of ¹²⁵I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, ¹²⁵I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.     

A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium

A rapid technique is described for the isolation of muscle cells from adult rat myocardium using in vitro perfusion with calcium-free bicarbonate buffer containing crude collagenase. Under optimum conditions, 5 × 10⁶ cells per g tissue are obtained and the suspension may be purified to contain 70% intact cells. The complete procedure is rapid and isolated myocytes beat, exclude vital stains, have conventional sub-cellular morphology, show tight respiratory coupling and also have a tolerance to external calcium not found in cells isolated by other perfusion techniques. This represents a significant advance in the development of an isolated cell model for the study of myocardial mechanisms.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

Effect of prostacyclin on perfusion pressure, electrical activity, rate and force of contraction in isolated rat and rabbit hearts.

Prostacyclin when added to medium perfusing rat and rabbit hearts caused an increase in perfusion pressure at concentrations from 1 pg/ml ? 1 ng/ml (2.8 × 10^?12 ? 2.8 × 10^?9M) and a decrease at higher concentrations. Rhythm disturbances were observed with low prostacyclin concentrations in 6 of 10 rat hearts and 2 of 5 rabbit hearts studied. Increased heart rates were seen in the isolated rat hearts but not in the rabbit hearts. Force of contraction of isolated rat hearts was increased with increasing prostacyclin concentrations up to 100 pg/ml. Higher concentrations decreased contractile force. No inotropic effects were seen with rabbit hearts.     

Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane binding-sites specific for estradiol-17beta.

Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [³H]estradiol-17β (E₂β) by isolated liver cells was specific for E₂β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10^?9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E₂β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E₂β (× 10^?8 M), but not to the relatively inert estradiol-17α (E₂α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10^?7 M E₂β or diethylstilbestrol, but not E₂α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E₂β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol by E₂β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10^?9 M estrogens or to 1 × 10^?4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E₂β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E₂α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E₂β, as well as to other known hepatocarcinogens.     

Preparation of (125I)-dCTP and its use as a substrate for RNA- and DNA-directed DNA synthesis.

Viable isolated parenchymal cells were incubated in a modified Waymouth medium under an oxygen tension of 30×10³ Pa at pH 7.8. Under these conditions, hepatocytes from 3-month-old rats synthesized 5.8 μg albumin/h/10⁶ cells. This value nearly equals the synthesizing capacity of intact liver tissue and is the highest activity reported so far for isolated hepatocytes. Parenchymal cells isolated from 36-month-old rats synthesized more albumin as compared to cells from 3-month-old rats. The albumin synthesizing capacity of cells isolated from 12-month-old rats was less than that of cells from 3-month-old rats.     

High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study

A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks'' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks'' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.     

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

Summary Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10⁷ 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 μg/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.     

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.Abbreviations 4-OHBP 4-hydroxybiphenyl - BP biphenyl - BSA bovine serum albumin - DNP 2,4-dinitrophenol - EDTA ethylendiamintetraacetate - NADPH nicotinamide adenine dinucleotide phosphate reduced - p-NA p-nitroanisole - p-NPh p-nitrophenol - TPP⁺ tetraphenylphosphonium     

Calcium-dependent accumulation induced by carbamylcholine of guanosine 3′,5′-monophosphate in rabbit choroid plexus

An active guanylate cyclase system was detected in isolated choroid plexus of rabbits by sodium azide (6 × 10^?5 mol/l) which increased cGMP levels tenfold within 15 min. Inhibition of cGMP phosphodiesterase by sodium azide was excluded. cGMP accumulation was also raised dose-dependently by carbamylcholine, a cholinergic agonist. Pretreatment of chroid plexus with atropine (10^?7 mol/l) reduced the effect of carbamylcholine (5 × 10^?5 mol/l) by 80%. Both carbamylcholine and sodium azide induced accumulation of cGMP also in the incubation medium, indicating rapid extrusion of the nucleotide from choroid plexus cells. The effect of carbamylcholine could be mimicked by the calcium ionophore A 23187. Incubation in calcium-free medium abolished cGMP accumulation by carbamylcholine and A 23187 but not by sodium azide, indicating a different mechanism of action. Sodium azide, carbamylcholine and A 23187 had no effect on cyclic AMP levels. Withdrawal of calcium led to an enhanced efflux of both cAMP and cGMP. Since a cholinergic innervation of stroma and epithelial cells has been described, we hypothesize that cGMP and calcium may be involved in cholinergic transmission regulating blood flow or transport processes of the choroid plexus.     

The sequential synthesis of the polypeptide chain of serum albumin by the microsome fraction of rat liver

1. The isolated microsome fraction of regenerating rat liver was incubated with cell sap, a source of energy and [³⁵S]methionine, [¹⁴C]isoleucine or [¹⁴C]leucine for different periods of time, and microsomal albumin isolated. 2. The distribution of these isotopes in albumin was determined by separation of tryptic peptides from the protein. Radioactivity was measured in peptides either qualitatively by radioautography or quantitatively by labelling with both ³H and ¹⁴C. 3. A gradient of radioactivity existed at all times in albumin isolated after incubating microsomes. 4. The shorter the incubation time the fewer the peptides labelled in albumin, but the peptides with highest specific activity after short incubation times corresponded to those with highest specific activities after long incubation times. 5. Leucine released from the C-terminus of albumin had a higher specific activity than the mean specific activity of the remaining leucine residues in albumin. 6. The peptide with the highest specific activity in albumin is probably derived from the C-terminus of the protein. 7. [¹⁴C]Glutamic acid is incorporated into the N-terminus of albumin after incubating the microsome fraction with this isotopically labelled amino acid, cell sap and a source of energy. The specific activity of the N-terminal glutamic acid under these conditions is less than the mean specific activity of the remaining glutamic acid and glutamine residues in albumin. 8. The results are interpreted as reflecting a sequential synthesis of serum albumin in the isolated microsome fraction of rat liver. The direction of synthesis of albumin is from the N-terminus towards the C-terminus. 9. The bulk of incorporation of radioactive amino acid into albumin in the isolated microsome fraction is due to completion of partially completed, pre-existing peptide and polypeptide chains. A limited synthesis of new chains of albumin does, however, occur.     

Glucagon control of cyclic AMP accumulation in isolated intact rat liver parenchymal cells in vitro

Cyclic AMP levels were measured in suspensions of isolated rat liver parenchymal cells during incubation in vitro. Glucagon caused a rapid elevation of cyclic AMP content. With 1.4·10⁻⁶ M (5 μg/ml) of the hormone the levels increased about 10-fold during the first minute, thereafter the elevation was less rapid. Maximal values were reached at 5–10 min. Theophylline slightly increased the basal cyclic AMP levels, and markedly augmented the response to glucagon. Teh major part of the cyclic AMP was located within cells, but a siginificant fraction was present in the incubation medium, and the relative amount present extracellularly increased with incubation time. Significant elevation of the cyclic AMP levels was produced by glucagon 1.4·10⁻¹⁰M, and half-maximal stimulation occured at about 2·10⁻⁹ M. The initial rate of cyclic AMP accumulation was such more rapid in the parenchymal cells than in liver slices, and the maximal levels obtained were about 3 times higher (comparisons based on the finding that 1 mg liver tissue contains about 10⁵ parenchymal cells). It is concluded that preparations of parenchymal liver cells are useful in the study of glucagon actions on liver tissue.     

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver^1-3. Although the isolation methods of liver macrophages have been well-described^4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.After dissociation of the liver cells by two-step perfusion method^7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 10⁶ cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells⁹.In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Maintenance of liver function in long term culture of hepatocytes following In Vitro or In Vivo Ha-ras EJ transfection

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-ras^EJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 10⁶ cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 10⁶ cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 10⁶ cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 10⁶ cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-ras ^EJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.Abbreviations BWE basal Williams' medium E - FBS fetal bovine serum - F10 or F12 basal Ham's F10 or F12 medium - Ha-ras ^EJ EJ allele of the human cellular ras oncogen of Harvey - hGH human growth hormone - hsp heat shock protein gene - LE-p liposome encapsulated plasmid - N-OH-2-AFAF N-hydroxy-2-acetylaminofluorene - RLECC rat liver epithelial cell - SF serum-free - SS serum-supplemented - UGG serum substitute UGltroser G® - 1-OH-, 3-OH-2-AFAFF 1-hydroxy-, 3-hydroxy-2-acetylaminofluorene - 2-AFAF 2-acetylaminofluorene - 2-AFF 2-aminofluorene     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes

In order to investigate the regulation of synthesis of some of the plasma proteins, especially fibrinogen, at the cellular level, we have chosen to work with suspensions of hepatocytes isolated by the perfusion of rat liver with crude bacterial collagenase. By adding soybean trypsin inhibitor to the collagenase and by avoiding mechanical damage, we have prepared cell suspensions that synthesize and secrete fibrinogen and albumin and that survive for longer than twenty hours. The fibrinogen secreted is clottable and shows the same pattern in acrylamide gel electrophoresis as fibrinogen purified from rat plasma. After a three hour lag, the rate of synthesis of fibrinogen, as measured by a solid-phase radioimmunoassay, continually accelerates, so that rates several fold greater than the $\text{in vivo}$ rate have been observed after twenty hours incubation. Cycloheximide (0.1 mM) completely abolished the appearance of fibrinogen in the medium; whereas colchicine (0.3 mM) reduced the rate by 85%. Insulin and cortisol succinate enhanced fibrinogen synthesis and secretion. The albumin secretion profile differs in several respects from that of fibrinogen, reflecting differences in intracellular pool levels and probably distinct regulatory mechanisms.     

The antagonistic effect of dexamethasone and insulin on alpha-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma.

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.     

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines

Summary With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 × 10⁸ cells/ml-matrix), large scale cultures (4.4 × 10¹⁰ cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 μg/24 h/10⁶ cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.