Minoru Niiya

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.

A mutation of the start codon in the X region of hepatitis B virus DNA in a patient with non-B, non-C chronic hepatitis.

There are cases of hepatitis involving occult hepatitis B virus (HBV) infection in which, even though the HB surface antigen (HBsAg) is negative, HBV-DNA is detected by a polymerase chain reaction (PCR). We conducted a sequence analysis of the entire HBV region in a case of non-B non-C chronic hepatitis in a 46-year-old female. A diagnosis of non-B non-C chronic hepatitis was made. Although HBV markers, such as HBs antibody (anti-HBs), anti-HBc, HBeAg and anti-HBe, were negative, HBV-DNA was positive. Nested PCR was performed to amplify the precore region of HBV-DNA and all remaining regions by long nested PCR. Sequence analysis of the two obtained bands was conducted by direct sequencing. Compared with the control strains, the ATG (Methionine) start codon in the X region had mutated to GTG (Valine). It is assumed that a mutation at the start codon in the X region may be the reason why HBV markers are negative in some cases of hepatitis that involve occult HBV infection.

Hepatitis B virus variants in carriers with hematologic malignancies in whom fulminant hepatic failure develops after chemotherapy

Abstract Three patients who were carriers of hepatitis B virus (HBV) and had hematologic malignancies, experienced fulminant hepatic failure after withdrawal of chemotherapy. Before treatment, each patient had inactive HBV infection with undetectable viral DNA polymerase; two patients were negative for hepatitis B e antigen. After chemotherapy, DNA polymerase increased and HBV variants with mutations in the precore region prevailed in their sera. These results implicate precore mutants thriving after chemotherapy and restored immune responses of patients in the pathogenesis of severe hepatic failure.

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.

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.