Norio Ohshima

Viscoelastic properties of cultured porcine aortic endothelial cells exposed to shear stress

The viscoelastic properties of cultured endothelial cells exposed to shear stress were measured by the micropipette technique and analyzed using a standard linear viscoelastic model. Cells from porcine aorta were cultured on glass coverslips. A shear stress of 2 Pa was applied using a parallel-plate flow chamber. After flow exposure, the cells were detached from the coverslips and suspended in culture medium. The micropipette experiment was performed on single cells under an inverted microscope. The desired negative pressure was applied stepwise to the tip of the micropipette by opening a solenoid valve. The deformation process of cells in the micropipette was observed through a TV camera and recorded on a videotape. To obtain the viscoelastic parameters, a half-space model of an endothelial cell was used. The cell was assumed to be a homogeneous and incompressible material, and a standard linear viscoelastic model was employed to account for the viscoelastic response. Cells exposed to shear stress for 6 h became spherical in shape after detachment from the substrate. In the case of a 24 h exposure, about half of the detached cells retained an elongated shape upon detachment, with the others taking on a spherical shape. The elastic constants, as determined based on the model, were approximately two times higher for the elongated cells than for control cells from static culture, no-flow conditions, indicating that the elongated cells became stiffer. Enhanced viscous properties also were observed for the elongated cells. These viscoelastic properties are considered to be closely related to cytoskeletal structure. Spherical cells upon detachment, even those that had been exposed to shear stress for 24 h, did not show such significant changes in viscoelastic mechanical properties.

Simulation of intraluminal gas transport processes in the microcirculation

Intraluminal resistance to gas transport between the microcirculation and tissue was neglected for a half-century following the early work of Krogh. In recent years it has come to be understood that this neglect is seriously in error. This paper reviews the background for the long period of misdirection, and progress in placing the simulation of gas transport processes on a more accurate, quantitative basis.

Novel cell immobilization method utilizing centrifugal force to achieve high-density hepatocyte culture in porous scaffold

Cell seeding is one of the key procedures in the construction of tissue-engineered organs. In our previous efforts to create a bioartificial liver, high-density cultures of hepatocytes (>1 x 10(7) cells/1 cm(3)-substrate) and long-term maintenance of metabolic function were achieved with a packed-bed reactor utilizing porous poly(vinyl formal) (PVF) resin as a scaffold. However, a low seeding efficiency of about 30% remains a major obstacle to the scaleup of the reactor. In the present study, a new cell seeding method, centrifugal cell immobilization (CCI), which is based on alternating centrifugation and resuspension, was used to achieve high-density seeding and improve the seeding efficiency. Using the CCI method, the maximum density of the immobilized hepatocytes reached 3.8 x 10(7) cells/1 cm(3)-PVF, and the seeding efficiency was improved to about 43% after a relatively short immobilization process (about 15 min). Moreover, further improvement of the seeding efficiency was obtained by serial immobilization procedures. Thus, we concluded that this method is useful and effective for seeding cells into 3-dimensional scaffolds.

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.

Effects of physical exercise on the elasticity and elastic components of the rat aorta

SummaryTo evaluate the effects of exercise on aortic wall elasticity and elastic components, young male rats underwent various exercise regimes for 16 weeks. In the exercised rats, the aortic incremental elastic modulus decreased significantly when under physiological strain. The aortic content of elastin increased significantly and the calcium content of elastin decreased significantly in the exercised group. The accumulated data from the exercised and sedentary groups revealed that the elastin calcium content was related positively to the incremental elastic modulus. We concluded that physical exercise from an early age decreases the calcium deposit in aortic wall elastin and that this effect probably produced in the exercised rats a distensible aorta.

Platelet thrombus induced in vivo by filtered light and fluorescent dye in mesenteric microvessels of the rat

Intravascular platelet aggregation and subsequent thrombus formation were produced in the microvasculature by irradiation using filtered light from a mercury lamp in combination with the intravascular administration of a fluorescent dye. The effects of several factors, such as light intensity, dye concentration, and vessel diameter were examined in venules and arterioles of the rat mesentery with respect to the time required for the initiation of thrombus formation; the time to totally occlude the lumen; and the volume of the thrombus formed. It was found that the combined conditions of light intensity and dye concentration at specific values had a critical effect on the rate of platelet aggregation. In the arterioles and venules examined, the thrombus formation time was prolonged as a function of increasing vessel diameter. The time required for the initiation of thrombus formation and complete occlusion of the vessel lumen was greater in arterioles than in venules. This result may suggest the dependence of the kinetics of intravascular thrombus formation on blood flow conditions.

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes.

SummaryTo enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2×2×2 mm, mean pore size: 100, 250 or 500 μm) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6–11.3×107 hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2×107 cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

EFFECTS OF SHEAR STRESS ON METABOLIC FUNCTION OF THE CO-CULTURE SYSTEM OF HEPATOCYTE/NONPARENCHYMAL CELLS FOR A BIOARTIFICIAL LIVER

To improve the culture conditions of hepatocytes for use as a bioartificial liver, the effects of shear flow on the co-culture system of hepatocytes/nonparenchymal cells (NPC) were investigated. A flow chamber with a collagen coated rectangular glass plate, where hepatocytes (5 x 10(4) cell/cm2) and NPC (2 x 10(5) cell/cm2) were seeded, was used to attain a shear stress of 4.7 dyne/cm2. Concentrations of ammonia and urea in the medium were measured daily during the 2 week experiment. The metabolic activity of hepatocytes in the homotypic culture were lower than those of the co-culture, especially when the cultivation time exceeded 1 week. In addition, the applied shear flow promoted activity of the co-culture system. An enhancement in the rates of ammonium removal and urea synthesis was obtained in the perfusion systems. Morphologic observation revealed that aggregates of hepatocytes formed abundantly in the perfusion system and hepatocytes developed a cuboid shape. This suggested that perfusion affected the function and morphology of hepatocytes in the co-culture system. Shear flow could induce cell-cell interactions and secretion of extracellular matrix through the activation of NPC.

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.

Effects of exercise training on biochemical and biomechanical properties of rat aorta

The authors studied the effect of prolonged physical exercise on the mechanical properties of rat aorta in relation to the amounts and qualities of arterial connective tissue fibrous proteins. Twelve male rats were divided into two groups: 6 sedentary rats (S) and 6 training rats (T), which were forced to swim from nine weeks to twenty-five weeks of age. The ultimate tensile stress and the ultimate tensile extension ratio of ring specimens at the descending thoracic aorta were larger in T than in S (192.3 ± 47.9 g/mm2, mean ± SD, vs 147.8 ± 26.0, p < 0.05; 3.52 ± 0.13 vs 3.18 ± 0.27, p < 0.05; respectively). The elasticity parameter, calculated by fitting stress-strain curves to exponential function in the stress level of 0-20 g/mm2, was lower in T than in S (1.79±0.15 vs 2.13±0.24, p < 0.01). The contents of elastin (alkali-insoluble elastin preparation) and collagen were higher in T than in S (0.44 ± 0.11 g/g dry aorta vs 0.30 ± 0.06, p < 0.05; 0.15 ± 0.04 g/g dry aorta vs 0.11 ± 0.04, NS, respectively). Although the aortic calcium content did not significantly change in the training rats (T 1.17 ± 0.23 mg/g dry aorta, S 0.95 ± 0.34), the content of calcium in elastin was lower in T than in S (1.75 ± 0.51 mg/g dry elastin vs 2.63 ± 1.00, p < 0.05). Elastin isolated from the training rats showed significant decreases in some of the polar amino acids: arginine (3.18±1.00 residues/1000 residues vs 4.12 ± 0.40, p < 0.05), threonine (6.79 ± 1.91 vs 8.69 ± 1.51, p < 0.05), and serine (9.66 ± 1.56 vs 11.97± 1.41, p < 0.05). The authors conclude that prolonged physical exercise from an early age provides training rat with a strong and extensible aorta, which is related to the larger amount of elastin with probably fewer degenerative changes.