Satoshi Hasumura

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

SummaryWith 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.

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor.

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Human bile duct carcinoma cell line producing abundant mucinin vitro

SummaryA 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 cardnoembryonic antigen in culture and had high enzymatic activity of γ-glutamyl transpeptidase. The tumor heterotransplanted into nude mice also showed mucin production.

Involvement of insulin-like growth factor binding protein-3 in the retinoic acid receptor-α-mediated inhibition of hepatocellular carcinoma cell proliferation

We examined the relationship between the expression of retinoic acid receptor-alpha (RAR-alpha) and upregulation of insulin-like growth factor binding protein-3 (IGFBP-3) in the retinoid-induced inhibition of hepatocellular carcinoma (HCC) cell proliferation. HCC cell lines showed a marked expression of RAR-alpha, whereas the expression levels of RAR-beta and RAR-gamma were relatively lower. An RAR-alpha agonist significantly inhibited the HCC cell proliferation both in vitro and in vivo. The RAR-alpha expression closely related to the upregulation of IGFBP-3 as compared with RAR-beta or RAR-alpha expressions. RAR-alpha agonist would be beneficial to inhibit the growth of HCC.

Effects of nerve growth factor and glucocorticoid on cultured human pheochromocytoma cells

Primary cell cultures of two human pheochromocytomas (PC) that were associated with high serum levels of adrenaline and noradrenaline were developed to study the effects of nerve growth factor (NGF) and dexamethasone on the morphology and function of PC cells in vitro. By phase-contrast microscopy, cultured cells were small and hyperchromatic on the first day of culture; neurite-like processes that extended to other cells developed several days later and were maintained for more than 3 months. NGF (100ng/ml), dexamethasone (10−5M), or NGF + dexamethasone were added to the culture media 2 weeks after the cultured cells had stabilized. Catecholamine concentrations in the medium were maintained at higher levels after addition of NGF, dexamethasone, or NGF + dexamethasone as compared to control cells. In the presence of NGF, extension of neurite-like processes was clearly accelerated, while high levels of dexamethasone inhibited growth of processes. These in vitro studies showed that the addition of NGF or the removal of dexamethasone induces differentiation of adrenal neurons present in pheochromocytomas, suggesting that adrenocortical steroid hormones influence the morphological control of adrenal medullary cells.

Morphological changes in human gall bladder carcinoma cell line-NOZ due to epirubicin and doxorubicin.

Epirubicin is widely used in various cancer therapies because its side effects and less than those of doxorubicin. We compared the direct effects of doxorubicin and epirubicin to cellsin vitro. In this study, we used the human gall bladder carcinoma cell line-NOZ. We evaluated the cytocidal effects using a DNA fluorimetric assay with fluorochrome Hoechst 33342. The addition of epirubicin in a culture medium showed stronger cytocidal effects than the addition of doxorubicin. Morphologically, after treatments with the two drugs, cells were enlarged, large vacuoles appeared in the cytoplasm, and concentrated microvillus-like structures due to doxorubicin were observed by transmission electron microscopy.

Abstracts of Selected Papers Presented at the 76th General Meeting of the Japanese Society of Gastroenterology

S OF SELECTED PAPERS PRESENTED AT THE 76TH GENERAL MEETING OF THE JAPANESE SOCIETY OF GASTROENTEROLOGY March 29-31, 1990, Tokyo, Japan Chairman: Haruo KAMEDA, M.D.

Effects of the combination of hyperthermia and adriamycin on cultured human liver cancer cells.

著者らは抗癌剤の併用による温熱の効果を,著者らの教室にて樹立しえたアルブミン高産生性のヒト肝細胞癌JHH-4株を用い,in vitroにおいて検討を行った.ペトリ皿に付着増殖した肝癌細胞を,抗癌剤として0～20μg/mlのAdriamycinを含む培養液にて,従来のコロニー形成法とは異なり,温度勾配培養装置を用い,37～43℃で2日間培養を行い,生細胞数の算定のみならず機能的,形態的にも判定を行った.温度の上昇に伴い生細胞数の減少,培養上清中のアルブミン濃度の低下,3Hラベルのサイミジン,ウリジン,ロイシンの取込みの低下,付着細胞の形態的変化がみられた.温熱単独でみられた肝癌細胞に対するin vitroにおけるこれらの効果は,Adriamycinを併用することにより増強が認められた.

Characterization of alpha-fetoprotein secreted from cultured reuber H-35 hepatoma cells

SummaryReuber H-35 hepatoma cells were examined for their ability to synthesize protein in vitro, especially to produce alpha-fetoprotein (AFP). The presence of AFP in the culture supernatant solution was determined immunologically by the micro-Ouchterlony method. Charge heterogeneity of AFP was examined electrophoretically in continuous gradient polyacrylamide microgels. With regard to the duration of culture, there was no remarkable change in the ratio of two peaks of AFP, and which came out as a major combined peak and a similar peak by PAS staining on the condition of added SDS. These findings indicated that Reuber H-35 hepatoma cells had potential to produce two charge variants of AFP in vitro.