Morphological study of cell organelles during development. I - The nuclear sac and the endoplasmic reticulum on the rat nephron

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3-0.6 micron) of kidney tissue were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80-100 kV. Studies were performed on rat foetuses from 18-21 days of gestation, on newborns, and on 2-20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.     

Segmental variations in the organization of the endoplasmic reticulum of the rat nephron

Summary The spatial organization of endoplasmic reticulum (ER) was examined in all segments of rat nephron. Tissues were fixed with glutaraldehyde, impregnated en bloc with osmium tetroxide, prepared for and examined by standard (80–100 kV) and high voltage (1 mEV) transmission electron microscopy.In all proximal tubule cells, ER forms a continuous and extensive network of canaliculi and abundant fenestrated saccules which surround mitochondria and cytoplasmic bodies; the cage-like structure of the fenestrated saccules was most evident around the spherical mitochondria of the S₃ segment. In the cells of the distal straight and convoluted tubules, the network consists mostly of canaliculi with rare non-fenestrated saccules. The ER network of canaliculi is particularly rich in intercalated cells, in contrast with its rudimentary appearance in the adjacent principal cells of the collecting tubule. In fact, in these cells there are few isolated ER cisternae and they are rarely impregnated. The nuclear envelope is well impregnated in most cells throughout the various segments. Segmental variations in ER organization and its relative abundance are most likely related to the well, established functional heterogeneity of the nephron segments. Moreover, the extensive and unique organization among mitochondria, ER and the basolateral membrane suggests that these three organelles function as a unit which is related to active electrolyte transport. In addition, because of its transepithelial organization, ER may well constitute a transcellular pathway for molecules.     

A Histochemical Study of Nectaries of Hibiscus rosa-sinensis

Different histochemical and cytochemical methods were employedon nectaries of Hibiscus rosa-sinensis. Light microscopy revealedthe presence of oil and mucilage cells in the subglandular tissue.Electron microscopy showed intense activity of ATPase in thephloem subtending the nectary. When CaCl₂ or tannic acid areadded to the fixative, electron-dense globular deposits areencountered in close contact with the plasmalemma of the secretorycells. In this case the endoplasmic reticulum appears in alternatingelectron-dense areas. In young nectaries the application oftannic acid results in electron-opaque deposits at the cellplate of dividing cells. The prolonged incubation of nectariesin OsO₄ results in an obvious difference in staining betweennectary hairs and subglandular cells. Structures stained selectivelywith OsO₄ are the endoplasmic reticulum, nuclear envelope, plastids,and mitochondria. The cytochemical experiments support the viewthat in nectaries of Hibiscus rosa-sinensis, the pre-nectaroriginates from the phloem and it is symplastically carriedvia the plasmodesmata to the secretory cells of the hair fromwhere it is secreted. The principal element which is involvedboth in the pre-nectar transport and nectar secretion is theendoplasmic reticulum. Key words: Lipid staining, polysaccharides, tannic acid, calcium binding sites, ATPase activity, osmium impregnation     

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC₆ (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10–70 nm for MDCK cells and 20–90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.     

Osmium impregnation of the endoplasmic reticulum correlates with the functional status of prostatic secretory cells

The ultrastructure of prostatic secretory cells was studied with the osmium impregnation technique in order to determine if the ER reactivity, or its absence, and its three-dimensional organization correspond to specific functions possibly hormono-dependent. Thick sections (0.3 micron) of rat ventral prostate were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80 kV. Studies were performed in normal adult rats, between the 3rd and 26th day following castration and in castrated rats treated with 5-alpha-dihydrotestosterone. In normal rats the impregnation technique delineated three secretory cell types (dark, greyish and clear), representing various degrees of reactivity in ER cisternae; however, despite this quantitative variation, they had similar morphological characteristics. In a longitudinal section, the ER network appeared to be made of saccules running parallel along the length of the cell and forming whorl-like patterns around the nucleus. Comparison of sections taken at various angles suggests that the ER network is made of concentric parallel saccules extending from the base to the apex of the cell and encircling the nucleus and the Golgi apparatus like a large multilayered cylinder. Whereas in dark cells the Golgi apparatus contained mostly clear vesicles, it was always heavily impregnated in clear cells. Noteworthy, osmium deposits were rarely observed on the nuclear envelope of secretory cells but were always present in basal cells. After castration, secretory cells became progressively cubic and the most conspicuous cytoplasmic change was observed in association with the ER. The Golgi apparatus decreased markedly in volume and became heavily stained with metallic osmium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postosmication of submucosal gland cells in the fowl proventriculus

Summary The technique of postosmication has been applied to resting and histamine-stimulated cells in the submucosal glands of the fowl proventriculus. In resting cells, the smooth endoplasmic reticulum, the mitochondria, and occasional vacuole-containing bodies become impregnated, but the Golgi apparatus, or Dalton complex remains unaffected. In stimulated cells, the degree of impregnation of the smooth endoplasmic reticulum and vacuole-containing bodies is increased, and more mitochondria become heavily impregnated, but the Golgi apparatus remains unaffected. The significance of these observations is discussed. Acknowledgement. I wish to thank Mr. R. N. C. Aitken, Department of Veterinary Histology and Embryology, for supplying the birds used in this study.     

THE ULTRASTRUCTURE OF DEVELOPING LATEX DUCTS IN MAMMILLARIA HEYDERI (CACTACEAE)

Electron microscopy was used to investigate early development of latex ducts in Mammillaria heyderi (Cactaceae). Numerous vesicles (secondary vacuoles) form from invaginations of the plasmalemma near sites of wall thinning, from endoplasmic reticulum (ER), and from vesiculate grana of degenerate plastids. Dictyosomes, though they occur in young duct cells, do not seem to be responsible for the formation of vesicles. Cytoplasmic vesicles may contain fibrillar, globular, or crystalline materials, or may be devoid of any type of particulate matter. They may be responsible for storage of numerous laticiferous components. Lysosomal materials could be stored in some vesicles and contribute to the degradation of the protoplast. Some nuclei contain condensed chromatin and are subject to deformation and collapse. Mitochondria and lipid bodies are common in young duct cells but ER is rare. When ducts form in young tissues, plastids in the lumen do not produce starch grains or extensive membranous networks. The plastids eventually degenerate to become a part of latex. If ducts form in older, established tissues having mature plastids, the plastids undergo extreme modification.     

Neonatal rabbit kidney cortex in culture as tool for the study of collecting duct formation and nephron differentiation

By stripping off the capsula fibrosa of neonatal rabbit kidneys a consistently thin tissue layer consisting of collecting duct anlagen, S-shaped bodies and nephrogenic blastema is obtained. This thin layer seems to be an excellent object for investigation of epithelium formation and nephron differentiation. Three different tissue culture protocols are described: 1. A polarly differentiated collecting duct epithelium with 'tight' characteristics consisting only of principal cells, grown on specific renal support 2. A morphologically dedifferentiated collecting duct principal cell monolayer grown on the unspecific bottom of a plastic culture dish 3. An embryonic tissue layer with numerous S-shaped bodies which might be a suitable model for investigation of the development of maturing nephron structures in serum-free culture medium.     

Reversible histochemical modifications of endoplasmic reticulum following arginine vasopressin stimulation of granular cells of toad bladder

The endoplasmic reticulum is generally absent from schematic representations of transport phenomena, although it shows a well-organized network in most transport epithelial cells. In order to examine the correlation between this organelle and cellular activity, bladders of Bufo marinus were studied under different experimental conditions and fixed by immersion in glutaraldehyde, followed by OsO₄ impregnation for 3 days. Normal granular and mitochondria-rich cells showed a rich cytoplasmic network of canaliculi, well-impregnated by osmium deposits. Following a 2 to 15-min stimulation (serosal bath) with arginine vasopressin, the V₂ receptor agonist dD-arginine-vasopressin or cyclic AMP (cAMP), the staining of endoplasmic reticulum in granular cells disappeared. After washing out of the hormone or the agonist, impregnation of the endoplasmic reticulum could be observed once again. Arginine vasopressin did not modify the impregnation of endoplasmic reticulum of either mitochondria-rich or basal cells. Our data indicate a correlation between the reactivity of endoplasmic reticulum to osmium, and a cAMP-dependent effect of arginine vasopressin through its V₂ receptors. Incubation of toad bladders carried out with agents interfering with cellular calcium (calcium ionophores, high or low bath calcium) or with calcium release from the endoplasmic reticulum (TMB-8, thapsigargin) suggested that an early step in the cAMP-dependent effect of arginine vasopressin must involve the release of intracellular calcium from the endoplasmic reticulum. However, calcium ATPases in this organelle do not seem to participate in the hormonal effect. The reversible loss of osmium impregnation induced by arginine vasopressin may represent protein changes in the endoplasmic reticulum accompanying a cAMP-dependent calcium release, from the organelle.     

The influence of culture media on embryonic renal collecting duct cell differentiation

Summary During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove’s modified Dulbecco’s medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams’ medium E, McCoys 5A medium, and Dulbecco’s modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P_CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.     

Effects of Forskolin on Fine Structures of Medaka Follicles

Effects of forskolin (FK), which stimulates production of 17α, 20β-dihydroxy-4-pregnen-3-one and estradiol-17β, on the fine structure of preovulatory follicles of Oryzias latipes were examined. Granulosa cells incubated in culture medium containing FK exhibited dislocation of the nucleus from the chorion side to the basement membrane side, vesiculation of conspicuous dilated endoplasmic reticulum (ER) with electron-dense material and Golgi lamellae, and development of large oval mitochondria with an electron-dense matrix. Moreover, a thin vesicular layer adherent to the outermost layer of the chorion was found in all immature oocytes at the end of incubation in the presence of FK. Intercellular junctions between granulosa cells and the oocyte gradually decreased during incubation in the presence of FK, and were finally lost with closure of the radial canals in the chorion at the end of the incubation. On the other hand, intrafollicular oocytes that were first incubated with FK for 10 hr, matured normally when they were incubated an additional 8 hr in plain medium. In granulosa cells of these follicles, the dilated ER and vacuolated Golgi lamellae were no longer detectable. These observations suggest that the development of dilated ER and vacuolated Golgi lamellae is characteristic of granulosa cells induced by FK.     

Low and high voltage electron microscopy of mitosis and cytokinesis in maize roots

The structure and distribution of cytoplasmic membranes during mitosis and cytokinesis in maize root tip meristematic cells was investigated by low and high voltage electron microscopy. The electron opacity of the nuclear envelope and endoplasmic reticulum (ER) was enhanced by staining the tissue in a mixture of zinc iodide and osmium tetroxide. Thin sections show the nuclear envelope to disassemble at prophase and become indistinguishable from the surrounding ER and polar aggregations of ER. In thick sections under the high voltage electron microscope the spindle is seen to be surrounded by a mass of tubular (TER) and cisternal (CER) endoplasmic reticulum derived from both the nuclear envelope and ER, which persists through metaphase and anaphase. At anaphase strands of TER traverse the spindle between the arms of the chromosomes. The octagonal nuclear pore complexes disappear by metaphase, but irregular-shaped pores persist in the membranes during mitosis. It is suggested that these form a template for pore-complex reformation during telophase. Phragmoplast formation is preceded by an aggregation of TER across the spindle at anaphase. Evidence is presented to suggest that the formation of the desmotubule of a plasmodesma is by the squeezing of a strand of endoplasmic reticulum between the vesicles of the cell plate.Abbreviations CER cisternal endoplasmic reticulum - ER endoplasmic reticulum - HVEM high voltage electron microscope - TER tubular endoplasmic reticulum - ZIO zinc iodide/osmium tetroxide     

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

Three dimensional visualization of the Golgi apparatus and endoplasmic reticulum in the subcommissural organ cells with high voltage electron microscopy

Fine structure and stereo-images of the Golgi apparatus and endoplasmic reticulum (ER) in the subcommissural organ (SCO) cells were visualized by the application of zinc-iodide osmium tetroxide (ZIO) impregnation, conventional electron microscopy and high voltage electron microscopy (HVEM). The Golgi apparatus in the SCO cells of rats, gerbils and hamsters consisted of flattened saccules stacked in parallel array. It showed a selective staining toward ZIO mixture and might form a complex network of tubular structures because of the presence of numerous fenestrations in the flattened Golgi saccules. The cytoplasm of the SCO cells in the rat and gerbil was crowded by dilated cisternae of the ER with a few flattened profiles. In the hamster SCO cells, however, the dilated cisternae of the ER were not observed. Flattened cisternae of ER in all species studied showed a positivity for ZIO impregnation and formed a complex tubular network, whereas dilated cisternae of the ER in the rats and gerbils did not show any reactivity. It was thus determined that the observation of thin and thick sections selectively stained with appropriate reagent for defined cellular organelles under conventional electron microscopy and HVEM offered valuable information about three-dimensional organization of the cell. A definite species-specific variation of SCO ultrastructure and cytochemistry was also demonstrated.     

Unbuffered osmium staining of cell organelles: alterations induced by cell injury

We have studied the localization of osmium reduction products to investigate the functional state of organelles as well as organelle interrelationships during cell injury. In normal hepatocytes osmium deposits of variable intensity are seen in nuclear envelope, endoplasmic reticulum. Golgi cisternae and vesicles and lysosomes. Buffering of osmium with s- collidine (pH 7.4) prevents the deposition of osmium. Reversible (30 min) and irreversible (60 min) ischemia without reflow causes no change in the pattern of osmium deposition. Irreversible ischemia followed by reflow causes decreased staining of endoplasmic reticulum (ER) and redistribution of the osmium deposits through the cytoplasm. Reversibly injured pancreatic acinar cells in cultured explants manifest a similar loss of osmium staining in the endoplasmic reticulum cisternae. The administration of antimicrotubule drugs induces an accentuation of osmium staining in localized cisternal elements of hepatocytes. These heavily stained cisternae appear to give rise to the bounding membranes of drug-induced autophagic vacuoles. Cytoplasmic organelles sequestered inside the autophagic vacuoles acquire intense staining when they begin to undergo degradation. In homogenized liver tissue all the subcellular organelles show osmium deposits. The deposits are preferentially localized along the organelle membranes. In particular the dense deposits in the ER lumen are not seen in the subcellular fractions. Phospholipase A2 (3 units/mg protein) enhances the deposition of osmium in the lumen of microsomal vesicles, whereas the presence of detergent has no such effect. Addition of EDTA to the homogenizing medium enhances the ultrastructural preservation of the subcellular fractions but has little effect on the deposition of osmium. OsO4 deposition occurs at acid pH and the intensity and pattern of the stain can be modified in vivo and in vitro. Osmium tetroxide deposition is induced at sites of membrane transformation (autophagic vacuoles) and degradation (lysosomes). Calcium influx and phospholipase activation (ischemia, tissue homogenization, phospholipase addition) enhance osmium deposition and/or influence the localization of the staining pattern.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

An ultrastructural study of osmiophilia in normal human oesophageal epithelium

Summary Oesophageal biopsies were obtained from patients with normal oesophagi during fibre-optic endoscopy for upper gastro-intestinal symptoms. They were studied with the prolonged osmication technique. The forming face of the Golgi apparatus was demonstrated in the basal and lower prickle cells. These cells also showed osmium deposition in their mitochondria, endoplasmic reticulum, perinuclear cisternae and lysosomes. The capillary endothelial cells also showed osmium deposition in their Golgi apparatus and endoplasmic reticulum.     

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

The effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-D-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes. Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-D-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AV's in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-D-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AV's. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AV's resembled endoplasmic reticulum and still had attached ribosomes. It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.     

Rice protein-body formation: all types are initiated by dilation of the endoplasmic reticulum

The ultrastructure of protein deposition in the starchy endosperm of developing rice (Oryza sativa L.) grains was examined in conventionally fixed (glutaraldehyde and osmium tetroxide) tissues and also in thick sections (0.3 m) of zinc iodide-osmium tetroxide post-fixed tissue. Three types of previously characterised protein body were observed and it was shown that each type was initiated by dilations of the endoplasmic reticulum. Crystalline type protein bodies were initiated by a ribosome-free dilation from rough cisternal endoplasmic reticulum and developed by inclusion of protein from dictyosome-derived vesicles. The large spherical and small spherical protein bodies developed within the cisternae of the rough endoplasmic reticulum.Abbreviations Cr crystalline protein body - DAF days after fertilization - ER endoplasmic reticulum - Ls large spherical protein body - Ss small spherical protein body - ZIO zinc iodide-osmium tetroxide     

A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts’ epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO₂. The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5–6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrance, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.