Three-dimensional cell culture of glioma and morphological comparison of four different human cell lines.

BACKGROUND: To explore the intracranial behaviors of glioma, a three-dimensional culture was devised and the morphology of four cell lines was examined. MATERIALS AND METHODS: Bioabsorbable and degradable gelatin was used as the scaffold and T98G, A172, KNS42, and U118MG representative standard malignant glioma cell lines were cultured three-dimensionally. RESULTS: When grown, the cells demonstrated characteristic conformations. The U118MG cells dispersed with numerous fiber formations. In contrast, the KNS42 and A172 cells aggregated, adhering to each other, resulting in the formation of balloon-like structures. The T98G cells demonstrated an intermediate character. CONCLUSION: The cell lines showed distinct characteristics in three-dimensional culture. This culture method may have a role in elucidating the fundamental character of cells in the human body.

The functional interrelationship between gap junctions and fenestrae in endothelial cells of the liver organoid.

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.

Extracorporeal bioartificial liver using the radial-flow bioreactor in treatment of fatal experimental hepatic encephalopathy.

An extracorporeal bioartificial liver (BAL) that could prevent death from hepatic encephalopathy in acute hepatic insufficiency was aimed to develop. A functional human hepatocellular carcinoma cell line (FLC-4) was cultured in a radial-flow bioreactor. The function of the BAL was tested in mini-pigs with acute hepatic failure induced by alpha-amanitin and lipopolysaccharide. When the BAL system was connected with cultured FLC-4 to three pigs with hepatic dysfunction, all demonstrated electroencephalographic improvement and survived. Relatively low plasma concentrations of S-100 beta protein, as a marker of astrocytic damage, from pigs with hepatic failure during BAL therapy were noted. BAL therapy can prevent irreversible brain damage from hepatic encephalopathy in experimental acute hepatic failure.

Reconstruction of liver organoid using a bioreactor.

AIM: To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 10(7) FLC-5 cells were incubated in the RFB. After 5 d, 2 x 10(7) A7 cells were added in a similar manner followed by another addition of 10(7) M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (VVO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION: Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.

Ultrastructure of human pheochromocytoma cells cultured for long periods.

We conducted ultrastructural analysis of human pheochromocytoma (PC) cells maintained in primary culture for about 10 months. The cells were first isolated by the enzymatic treatment of a surgically resected tissue specimen obtained from a 37-year-old man with PC, a condition which is characterized by elevated blood levels of adrenaline and noradrenaline. It was found that noradrenaline production in the medium continued until the 90th day of culture (1330 pg/ml). The production level decreased to 20 pg/ml on the 180th day, and to 18 pg/ml on the 300th day. Examination under a transmission electron microscope (TEM) at 4 weeks of culture revealed electron-dense granules (about 200 nm in size and, presumably, rich in catecholamines), which were also observed in the tumor cells from the original PC tissue. Neurite-like processes grew at around 1 week of culture, and were still maintained at 6 months of culture. But, after 6 months of culture, the neurite-like processes contained a rosary-like elevated structure, which was suggestive of cell degeneration, as determined by a plasma polymerization replica method and observed with a scanning electron microscope. When cells were examined under the TEM, fewer electron-dense granules were observed in the cell bodies, with more numerous lipofuscin-like granules and filaments. Thus, electron-dense granules, which, presumably, contain catecholamines, were seen in a long-term culture of human PC cells. These granules decreased in number in parallel with the decrease in catecholamine levels in the culture.