Keiko Ookawa

Performance of a New Hybrid Artificial Liver Support System Using Hepatocytes Entrapped Within a Hydrogel

To develop a hybrid artificial liver support system (ALSS), the authors constructed a rotating-disk type ALSS using hepatocytes entrapped within a calcium alginate hydrogel. This module was designed in imitation of the gas-liquid contactor that uses the same principle. Forty disks with film-shaped hydrogel were mounted to a horizontal rotating axis and were kept in contact with blood. The concentration of hepatocytes in the gel varied from 1.9 X 10(9) to 1.7 X 10(10) cells/L gel. Entrapping hepatocytes within hydrogel kept the hepatocytes viable, and results of in vitro experiments showed ammonium metabolism and urea synthesis. The results of ex vivo perfusion experiments using cats with acute hepatic insufficiency indicated that this module had the ability to replace liver function in vivo.

Changes in the microstructure of cultured porcine aortic endothelial cells in the early stage after applying a fluid-imposed shear stress

Time course changes in the cell shape and in the patterns of microfilament distribution were analyzed quantitatively using cultured porcine aortic endothelial cell monolayers before and after a shear flow exposure. Geometrical parameters of the cell and of the microfilament were measured on fluorescent photomicrographs of the cells stained with rhodamine-phalloidin. After the shear flow exposure (20 dyn cm-2, 0-24 h), the endothelial cells on glass were elongated and oriented to the direction of the flow. Under the no-flow condition, F-actin filaments were mainly localized at the periphery of the cell, although some filaments were seen in the more central portion. The angles of the filaments were randomly distributed. After 3 h, the stress fiber-like structure of an F-actin bundle was formed in the central part of the cells, and these filaments were oriented to the direction of the flow. The degree of orientation increased as the time of exposure to shear stress became longer. This change in F-actin preceded cell elongation and orientation; these changes were statistically significant only after 6 h. After 24 h, peripheral filaments were again observed, and the fluorescence intensity of rhodamine-phalloidin-stained cells was enhanced. These findings suggest that the redistribution of F-actin filaments is one of the early cellular responses to the onset of shear stress and that it is one of the most important factors controlling cell elongation and orientation to the direction of the flow.

Hepatocyte culture utilizing porous polyvinyl formal resin maintains long-term stable albumin secretion activity

To investigate the effects of culture conditions on the maintenance of metabolic functions of cultured hepatocytes, long-term hepatocyte culture lasting 20 days was performed under two different culture conditions, i.e. stationary cultures utilizing porous polymer (polyvinyl formal (PVF) resin) as a substratum and conventional monolayer dish cultures without PVF. Metabolic activities specific to hepatocytes were evaluated in terms of ammonia metabolism, urea synthesis, and albumin secretion. Concerning ammonia metabolic and urea synthetic activities, no significant differences in maintenance of these activities were found between the two culture conditions, and these activities rapidly decreased with the elapse of the culture period, especially during the early stage of the experiments. However, after day 10, these activities in the stationary cultures were maintained at a slightly more favorable level than in the monolayer cultures. On the other hand, compared with ammonia metabolism and urea synthesis, stable and well-maintained albumin secretion of hepatocytes (60% of the activity in day 1) was exhibited in the stationary culture experiments, despite that this particular activity under the monolayer culture condition gradually reduced to a very low level (5.7% of that on day 1) at the end of the culture. From the morphological observations, hepatocytes immobilized in the PVF resin revealed individual spherical shapes without forming multicellular aggregation, and it was suggested that this characteristic structure contributed to good albumin secretion of hepatocytes. In conclusion, the advantages of the hepatocyte culture technique utilizing PVF resin over the conventional dish culture in maintaining some representative metabolic function specific to hepatocytes were clarified.

Morphological Changes of Endothelial Cells after Exposure to Fluid-Imposed Shear Stress : Differential Responses Induced by Extracellular Matrices

Effects of extracellular matrices (ECMs) and fluid-imposed shear stress on the cell shape and the cytoskeletal structure of microfilaments were studied in cultured porcine aortic endothelial cells (PAECs). The PAECs were cultured until confluent on non-coated or on ECM-coated glass coverslips. The components of ECM used were type IV collagen, heparan sulfate, chondroitin sulfate and dermatan sulfate. The PAECs cultured on the mixed ECMs showed marked elongation and segmental orientation with randomly distributed cell axis even under a no-flow static condition, and the microfilaments were mostly observed in parallel with the cell axis. After shear flow exposure (2 Pa, 24-48 hrs), the PAECs on non-coated glass were significantly elongated and oriented to the flow direction, however the PAECs on ECM-coated glass showed more retarded responses than the ones on non-coated glass, indicating that the anchorage to the substrate was enhanced by ECMs. The stress fibers were reorganized in accordance with the cell shape and oriented to the flow direction. These findings suggest that ECM may act together with shear stress to modify and maintain the endothelial cell configuration.

Blood flow and leukocyte adhesiveness are reduced in the microcirculation of a peritoneal disseminated colon carcinoma

AbstractDynamic behavior of leukocytes in the microcirculation of solid tumor tissue was visualized using a fluorescent labeling technique combined with the use of a real-time confocal laser-scanning microscope (CLSM) system. Colon tumor cells (RCN-9) were inoculated into the peritoneal cavity of male Fischer 344 rats. Tumor-free rats were similarly injected with physiological saline (intraperitoneally). Ten days after tumor inoculation, the mesentery was exteriorized and subjected to vital microscopic observation under the CLSM system. Leukocytes were labeled with rhodamine 6G (100 μ g kg−1, intravenously), and their behavior within the microvessels (10–30 μm in diameter) was analyzed both in the solid tumor tissues and the normal mesentery. Wall shear rate was calculated from the measured values of vessel diameter and erythrocyte flow velocity. In tumor microvasculature of tumor-bearing rats, the centerline erythrocyte velocity (0.73 ± 0.58 mm s−1, mean±standard deviation) and wall shear rate (210 ± 151 s−1 were significantly lower than those of the tumor-free rats (1.27 ± 0.83 mm s−, 344 ± 236 s−1, respectively). Despite such reduced flow conditions, flux of the rolling leukocytes as well as density of the adhered leukocytes both decreased significantly in tumor microvasculature as compared with normal controls. The methods developed in this work show promise in improving our understanding of tumor biology and pathophysiology.

Platelet thrombi produced on cultured endothelial cells by the dye/light method

Platelet adhesion and aggregation were induced on cultured endothelial cells using the fluorescent dye/light method. A cone-and-plate apparatus was newly developed to observe interactions between platelets and cultured endothelial cells under a shear flow condition. The platelet deposition grew on the light-irradiated area of the cells. Degree of endothelial cell injury induced by the dye/light reaction seemed to depend on the dye concentration. Application of either aspirin or indomethacin significantly inhibited the growth of platelet aggregation, but was not effective for the platelet adhesion to endothelial cells. The platelet thrombi were formed on endothelial cells without their denudation. It was found by transmission electron microscopy that platelets directly adhered to endothelial cells which were not seriously damaged. This thrombus model is expected to be applicable to some physiological and pharmacological studies investigating platelet-endothelial cell interaction and mechanism of platelet thrombus formation in blood vessels.