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

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 x 10(8) cells/ml-matrix), large scale cultures (4.4 x 10(10) 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 micrograms/24 h/10(6) 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.

Heterotopic gastric mucosa of the gallbladder.

A case of heterotopic gastric mucosa in the fundus of the gallbladder is reported. A 23-year-old man, who had been healthy and asymptomatic, visited our hospital because of abnormal findings in a liver enzyme test given during a routine health screening. Ultrasonography demonstrated a highly echogenic polypoid mass in the fundus of the gallbladder. The gallbladder mass was confirmed by both computed tomography and intravenous cholangiogram. After a 10-month follow up, laparoscopic cholecystectomy was performed. Intraoperative touch smear cytology of this lesion revealed class II cells. The surgical specimen revealed a 15 x 10 x 5 mm polypoid lesion in the fundus, with no gallstones in the gallbladder. Histologically, the polypoid lesion consisted of both fundic type and pyloric type gastric glands located in the mucosa of the gallbladder. In the literature, 42 cases of heterotopic gastric mucosa of the gallbladder have been reported, only 3 of which, including this present case, were found incidentally, with no apparent symptoms.

[A new liver support system composed of functional human cells and a radial-flow bioreactor].

An artificial liver will be useful for the treatment of acute hepatic failure and a bridge of liver transplantation. The current reports suggest that the hybrid type of artificial liver composed of functional human liver cells and a bioreactor is practical for clinical use. In the present study, we succeeded high density culture on a large-scale of human functional hepatoma (JHH-7) using a newly developed radial flow packed-bed bioreactor. Since the shear stress of this bioreactor is lower than the other type, high density culture without cell damage is possible. JHH-7 cells produced large amounts of human albumin and other liver specific proteins, and then have the function of ammonia metabolism in the system. This study suggests that a radial flow bioreactor will be developed as a new type of artificial liver.

Regulation of vitamin A transport into cultured stellate cells of rat liver: studies by anchored cell analysis and sorting system.

Stellate cells (SC) in the liver store the most retinoid in the body, but the mechanisms of specific retinoid transport into SC remain to be elucidated. In this study, to analyze the retinoid content of cultured SC, we employed an anchored cell analysis and sorting system (ACAS), which provides fluorescence analysis of single cultured cells under the phase-contrast microscope by utilizing a laser. First, we examined the effect of retinol binding protein (RBP) on retinol transport into cultured SC of rat liver. Rat holo-RBP added to the medium inhibited retinol uptake into SC. We also prepared RBP-free human serum by affinity chromatography using conjugated anti-human RBP IgG and compared retinoid fluorescence of SC cultured in human serum with or without RBP. No significant difference in retinoid fluorescence intensity was observed between SC cultured with and without holo-RBP. Second, the removal of cellular retinol by esterification may be important for the continued uptake of retinol. Retinyl esters are stored in lipid droplets of SC. Therefore we examined the relationship between the lipid droplet number and the retinoid fluorescence intensity in SC which were cultured in medium containing retinol for 1-3 days. The increases in lipid droplet number and in retinoid fluorescence in SC were almost parallel. Progesterone, previously shown to increase the esterification of retinol by lecithin:retinol acyltransferase (LRAT) in vitro, was added to the SC medium; progesterone facilitated retinol uptake in cultured SC. In conclusion, RBP did not facilitate specific retinol transport into SC. However, the specific transport of retinol is likely to be dependent on the intracellular esterification of retinol by LRAT in SC.

Retinol transport in cultured stellate cells of rat liver: studies by light and electron microscope autoradiography.

The mechanisms of specific transport of retinoids into stellate cells (SC) of liver remain to be elucidated. In the present study, we have conducted experiments to observe the intracellular retinoid metabolism of cultured SC of rat liver using light and electron microscope autoradiography (LMARG and EMARG). After 72 h of culture, the cells were incubated in the medium containing 1.06 x 10(-6) or 6.36 x 10(-8) M of [3H]retinol for either 5 or 30 min. In some cases, incubation with the labeled retinol was followed by a medium containing nonlabeled 1 x 10(-6) M retinol for 90 min. First, the incorporation of labeled retinol (1.06 x 10(-6) M) into SC and hepatocytes was compared by LMARG. After 5 min, silver grains were already present on both cells. After 30 min, label was concentrated on the lipid droplets of SC. After the chase, the number of grains on hepatocytes decreased. On the other hand, grains on the lipid droplets of SC remained. Second, we studied the fine morphology and intracellular retinoid metabolism in SC using EMARG. The SC, which contained abundant multivesicular bodies (MVB) and lamellar bodies, were found to have a heavy accumulation of grains. Even in the medium containing a lower concentration of retinol (6.36 x 10(-8) M), SC also took up retinol. After 90 min of chase, many grains moved on the lipid droplets in SC. The labeled MVB were often accompanied by lamellar bodies and found near the lipid droplets. Sometimes we noticed small labeled lipid droplets bound by membranes in MVB. From the results of this study, we concluded that the MVB and lamellar bodies might be important organelles for retinyl ester formation and the initial storage of retinoid in the lipid droplets in SC.

Integration of hepatitis B virus DNA into cells of six established human hepatocellular carcinoma cell lines.

The authors successfully established 5 hepatocellular carcinoma cell lines, JHH-1, 2, 4, 5 and 6, derived from hepatitis B surface antigen seronegative patients and one line. JHH-7, from a patient considered to be a hepatitis B virus carrier. In the culture media of any of the JHH cell lines, including JHH-7, hepatitis B surface antigen was not detected by radioimmunoassay. However, in JHH-7, integration of hepatitis B virus DNA was confirmed at two sites on the chromosomes of this line by Southern blot hybridization. In contrast, in other JHH cell lines derived from hepatitis B surface antigen seronegative patients, integration of hepatitis B virus DNA into the chromosomes of cells was not detected. These cell lines will be useful in the investigation of hepatocellular carcinoma development, especially for research into non-A/non-B hepatitis viruses.

[Analysis for the integrated hepatitis B virus genome in cells of established human hepatocellular carcinoma cell line JHH-7].

Authors have successfully established 7 human hepatocellular carcinoma (HCC) cell lines. In one of these cell lines, JHH-7, derived from HBs antigen positive HCC, hepatitis B virus (HBV) genome was analyzed by Southern blot hybridization. Digesting with Hind III restriction endonuclease, two bands were obtained at 6.0 and 2.5 kb, that is the integration of HBV genome was confirmed at the two sites of chromosomes in this cell line. And analyzing with 32P labeled HBV-DNA fragments for probes, it was indicated that integrated HBV genome was incomplete one containing from X to C region.

[Establishment and characterization of a human hepatocellular carcinoma cell line JHH-7 producing alpha -fetoprotein and carcinoembryonic antigen--changes in secretion of AFP and CEA from JHH-7 cells after heat treatment].

A human hepatocellular carcinoma cell line, JHH-7, was established from resected liver tumor of a 53 year old male with hepatitis B virus infection. JHH-7 was composed of polygonal epithelial cells and functionally synthesized and secreted human albumin, AFP, CEA and ferritin. No HBsAg was detected in the culture supernatant of JHH-7 cells. Changes of secretion of AFP and CEA from JHH-7 cells after heat treatment was studied using a temperature gradient incubator. Secretion of AFP decreased along with the inhibition of cell proliferation by heat treatment. Secretion of CEA, however, did not decrease even though the cells were damaged.

[A case of B-cell non-Hodgkin's lymphoma with prominent splenomegaly and high serum titer of cold agglutinin].

The case of 37-year-old male with non-Hodgkin's lymphoma with prominent splenomegaly is presented. The spleen that was removed for analysis had a weight of 2690 grams and a section inspection revealed multiple, whitish, small nodules that were disseminated throughout the entire spleen. It was microscopically demonstrated to be a B-Cell lymphoma of diffuse, medium-sized cells and an IgM.kappa Type. This case also was complicated by a high serum titer of cold agglutinin that was decreased by chemotherapy.

[Examination of the effects of hyperthermia and anticancer agent on cultured human gall bladder cancer cells by DNA fluorimetric assay].

Tumor cells are generally more sensitive to hyperthermia than normal cells. We applied hyperthermia for bile duct cancer therapy. We also studied the effect of hyperthermia and anticancer agents using cultured cancer cells. In this study, we examined the effect of hyperthermia and adriamycin by using newly established cultured human gall bladder cancer cells. In order to calculate cell numbers, we used the new DNA fluorimetric assay with fluorochrome dye Hoechst 33342, developed by Richards et al. For the purpose of confirming the usefulness of this fluorimetric assay, we compared this assay with methylene blue and 3H-thymidine incorporation methods. The following results were obtained: 1) the combination effect of hyperthermia and adriamycin showed an additive effect, 2) the effect of hyperthermia was only intensified according to the extension of heating time, but the combination treatment resulted in a maximum effect at the early stage of heating, 3) DNA fluorimetric assay with Hoechst 33342 dye was a sensitive, easy and rapid method for calculating cell numbers compared with methylene blue and 3H-thymidine incorporation methods, and more useful for studies of cultured cells.

Retinol transport in cultured fat-storing cells of rat liver. Quantitative analysis by anchored cell analysis and sorting system.

It is difficult to study the mechanism of specific transport of vitamin A in fat-storing cells (FSC) in vivo. In this study, transport of vitamin A added to the medium was quantitatively analyzed in cultured FSCs by means of the spontaneous fluorescence emitted by vitamin A. By density-gradient centrifugation with 38% Percoll, an FSC-rich fraction was separated from normal rat liver cells. The FSCs were observed to retain cytoplasmic fat droplets even on days 3 and 4 of culture. The FSCs containing fat droplets were selected for this experiment by checking their emission of vitamin A fluorescence. To analyze the vitamin A content of isolated cells, we employed a newly developed anchored cell analysis and sorting system (ACAS 470), which provides fluorescence analysis and sorting of adherent cells under the phase contrast microscope by utilizing a laser with its irradiation range narrowed to 1 micron. Vitamin A fluorescence was detectable by this system even in the cultured FSCs. After 24 hours of culture of FSCs in medium with 1 x 10(-6) M vitamin A added, the strength of fluorescence per FSC was 24.3 +/- 11.2 x 10(5)/cell for control, 61.5 +/- 17.6 x 10(5)/cell for retinyl acetate, 26.0 +/- 12.6 x 10(5)/cell for retinyl palmitate, and 59.0 +/- 15.1 x 10(5)/cell for retinol. Thus, retinol and retinyl acetate were transferred to FSCs in significant amounts without the participation of retinol-binding protein. Furthermore, an extended examination was made of the mechanism of the retinol transport observed in this study. Transport was never inhibited by the presence of vitamin E or azide. Retinol may be transferred by passive transport attributable to the concentration gradient rather than by active transport or through cell membrane damage by retinol itself. There was a tendency for inhibition of the transport of retinol into the cells in fetal calf serum. This inhibition may have occurred because the retinol-binding protein or other serum proteins had decreased the concentration of free retinol.

Morphological characteristics of human fat-storing cells fractionated and cultured by newly techniques.

Human non-parenchymal cells, especially fat-storing cells (FSCs), were isolated and primarily cultured using 1-2 g of normal human liver tissue obtained in conjunction with tumor biopsies. The human hepatic FSCs were cultured by a modified Howard and Pesch method. Microscopically the cultured human FSCs showed characteristic fat droplets like those in in vivo FSCs. The FSCs from tumor liver with serious fibrosis contained fewer fat droplets, and the fibrous constituents were especially abundant. The intermediate filaments extending longitudinally were characteristic of the cultured FSCs. In the human FSCs observed by the plasma polymerization replica method, the cells adhered and were stretched relatively thin. Particularly, the ends of the processes adhered and stretched like a folding fan to the bottom of the dish.

[Combination therapy of hyperthermia and other methods in liver and bile tract cancers--evaluation of these methods using cancer cell lines in vitro].

In order to evaluate the combination therapy for liver and bile tract cancer, the effects of anticancer drugs and hyperthermia were observed using cultured human cancer cell lines. In the case of gall bladder cancer cell line (NOZ), combination of adriamycin and hyperthermia showed more effective inhibition for cell proliferation than MMC + hyperthermia and 5-FU + hyperthermia. Hepatocellular carcinoma cell line (JHH-4) showed remarkable inhibition of cell growth and secretion of albumin by combination treatment of adriamycin and hyperthermia. Morphologically, JHH-4 cells were enlarged and the nucleus was also enlarged with combination adriamycin and hyperthermia by phase contrast microscopy. Cytoskeleton of JHH-4 cells became irregular and intercellular borderline was unclear by plasma polymerization replica method (PPRM). The effects of BRM (OK-432 and TNF) on HCC cell lines was also investigated. OK-432 directly inhibited proliferation of JHH-4 cells. We observed internalization of OK-432 by JHH-4 cells with TEM and 16-mm movie. TNF showed various effects on human HCC cell lines. Proliferation of two cell lines was inhibited, and one tended to be enhanced after the addition of TNF to the medium. Hyperthermia influenced the effects of TNF to HCC cell lines. We think that this paper is a very significant study for improving the therapy for hepato-biliary cancers.

[Protein secretion of human cultured liver cells].

Liver cells have many functions, and one of which is a production of plasma proteins. Therefore, studies on synthesis and production of plasma proteins from hepatocytes are very important for the recognition of various hepatic dysfunctions, clinically. Of late years, a lot of the complex mechanism of protein synthesis and--secretion was elucidated by using a technique of liver cell culture, for example, primary monolayer culture by freshly isolated hepatocytes and cloned cell culture derived from hepatocellular carcinoma. This paper described the results of our observations and other researchers, and then discussed the point of production of human major plasma proteins using the above culture methods, such as albumin, alpha-fetoprotein and transferrin. Furthermore, we showed statistically that half of twenty-six human hepatoma cell lines established until 1988 in Japan, had already lost their secretory potencies of major plasma proteins in vitro.

[Establishment and characterization of human pancreatic adenocarcinoma cell line JHP-1 producing carbohydrate antigen 19-9 and carcinoembryonic antigen].

A new tumor cell line derived from the ascites of a patient with adenocarcinoma of the head of pancreas was established in culture and the nude mouse. The cell line was characterized by the growth with a population doubling time of 22 hr., a high plating efficiency on the plastic surface and a modal chromosome number of 66. The tumorigenicity was proved by the growth in nude mouse and in soft agar. Morphologically the cell line grew as a confluent monolayer with tight adhesion to the plastic surface. Histologically the cell line was epithelial-like in culture and poorly differentiated adenocarcinoma in nude mouse. Ultrastructurally the cell line showed a characteristic pancreatic epithelium. Furthermore, the cell line expressed carbohydrate antigen 19-9 and carcinoembryonic antigen. This cell line, designated JHP-1, has been cultured for at least 100 passages in vitro and maintained for more than 2 years. This cell line would be used as a new model for human pancreatic carcinoma.

[Classification of the isolated hepatocytes].

At this present, enzyme perfusion method is a routine technique to isolate hepatocytes from rat liver for the physiological and pathological experiments. This study described a way of the classification of freshly isolated hepatocytes. First of all, the hepatocytes were fractionated with parenchymal and non-parenchymal cells by low speed centrifugation. And then these cells were subfractionated with a newly developed Percoll linear density gradient method. The fractionated parenchymal cells were divided with cells of periportal and centrilobular areas, respectively. Furthermore, their characteristics were confirmed functionally and morphologically. Non-parenchymal cells (NPC) include Kupffer cells, endothelial cells and fat storing cells (FSC, Ito cells). These isolated NPC are fractionated with a method as mentioned above or centrifugal alutriation method. In this paper, fractionation and classification of Kupffer cells and FSC were discussed with the measurement of fluorescent intensity of vitamin A and the morphological observation of cytoskeleton in culture. Especially, transport of vitamin A into FSC were detected autoradiographically.

[Effects of TNF on human hepatocellular carcinoma cell lines and their modification by hyperthermia].

Firstly, using HCC cell lines, the effects of r-h TNF were investigated. The authors had already confirmed that these cell lines were derived from human HCC. Each cell line showed a different growth curve on addition of TNF to the culture medium. JHH-4 exhibited enhancement of growth under the optimum concentration of TNF. On the other hand, growth of JHH-5 and JHH-7 was inhibited by TNF. JHH-7 were more sensitive to TNF than JHH-5, however, the direct effect of TNF on JHH-7 was not potent, as 10(4) u/ml TNF could not prevent proliferation of JHH-7. Morphological examinations were also performed. Phase-contrast microscopy showed that the JHH-4 cells were enlarged and tended to pile up after the addition of TNF to the culture medium. JHH-7 cells became detached from the culture dish due to cell death. Electron microscopy showed irregular proliferation of the rough endoplasmic reticulum of JHH-4 cells and increased number of lysosomes in JHH-7 cells. Furthermore, hyperthermia exhibited an interesting reciprocal action. Proliferation of JHH-4 was inhibited by low concentrations of TNF together with 41.4 degrees C hyperthermia in contrast to the effects of TNF alone. JHH-7 became more sensitive to TNF under hyperthermia at 41.4 degrees C. On the other hand, normal human fibroblast 'HAIN-55' were not affected by TNF at 37.0 degrees C, 41.4 degrees C or 42.5 degrees C. In this paper, the authors tried to study the effects of TNF and hyperthermia on human HCC cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

[Establishment and characterization of a human hepatocellular carcinoma cell line JHH-4].

A human hepatocellular carcinoma cell line, JHH-4 was established from resected liver tumor. Morphological diagnosis of the original tumor was hepatocellular carcinoma, Edmondson type III. This cell line was composed of polygonal shaped cells. Subcellular organelle were observed in cytoplasm. Furthermore, bile canaliculi adhering junction was also remained at the cell surface. The growth rate of JHH-4 cell is slow, peaks of the chromosome number was 75 and 79, and plating efficiency was 3.0%. JHH-4 cell is transplantable to nude mouse. Furthermore, this cell line functionally synthesized and secreted human albumin, AFP and other proteins in vitro.

Human bile duct carcinoma cell line producing abundant mucin in vitro.

A human bile duct carcinoma cell line, designated OZ, was established from ascitic effusion of a patient who suffered from obstructive jaundice due to the clogging of the common bile duct with mucinous substances secreted by the cancer cells. OZ was found to be capable of producing mucin in vitro and pools of mucin were macroscopically identified on the monolayer of the cells. On the electron micrographs, cell coat type mucin and abundant intracytoplasmic desmosomes were observed. The OZ cells secreted carcinoembryonic antigen in culture and had high enzymatic activity of gamma-glutamyl transpeptidase. The tumor heterotransplanted into nude mice also showed mucin production.