Toshihisa Kajiwara

An Approach for Formation of Vascularized Liver Tissue by Endothelial Cell–Covered Hepatocyte Spheroid Integration

Tissue vascularization in vitro is necessary for cell transplantation and is a major challenge in tissue engineering. To construct large and regularly vascularized tissue, we focused on the integration of endothelial cell-covered spheroids. Primary rat hepatocytes were cultured on a rotary shaker, and 100-150 mum spheroids were obtained by filtration. The hepatocyte spheroids were coated with collagen by conjugation with a type 1 collagen solution. Collagen-coated hepatocyte spheroids were cocultured with human umbilical vein endothelial cells (HUVECs), and monolayered HUVEC-covered hepatocyte spheroids were constructed. Without a collagen coat, many HUVECs invaded hepatocyte spheroids but did not cover the spheroid surface. To construct regularly vascularized tissue, we packed HUVEC-covered hepatocyte spheroids in hollow fibers used for plasma separation. Packed spheroids attached to each other forming a large cellular tissue with regular distribution of HUVECs. At day 9 after packing, HUVECs invaded the hepatocyte spheroids and a dense vascular network was constructed. Collagen coating of spheroids is useful for the formation of endothelial cell-covered spheroids and subsequent regular vascularized tissue construction.

Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam

Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.

Numerical simulation of converging flow of polymer melts through a tapered slit die

Abstract Numerical simulation of the converging flow of polymer melts through a tapered slit die was performed with the finite element method using two kinds of constitutive equations: the Phan Thien-Tanner (PTT) and the Giesekus models with a single relaxation time. The calulated stress distributions assuming planar flow were compared with the experimental values measured by the flow birefringence technique. The model parameters were determined from the experimental data of shear viscosity and the primary normal stress difference by fitting the models. The PTT model fitted the experimental data better than the Giesekus model within the range of experimental shear rates, and therefore the numerical results of stress distribution for the PTT model agreed with the experimental values better than those for the Giesekus model. The difference between the calculated and experimental local stresses for the PTT model was less than 20% of the values at the downstream slit wall and it can be concluded that the PTT model is useful for the simulation of converging flow of polymer melts in the low shear rate region. Moreover the average stresses over the width of the channel in the fully developed rectangular channel flow were compared with the experimental values in order to assess the effect of side wall in the experiment. As a result, the average stresses in the rectangular channel flow were closer to the experimental values than those in the planar flow. It was found that the numerical results may be closer to the experimental results by considering the three-dimensional flow.

Hepatic Differentiation of Embryonic Stem Cells in HF/Organoid Culture

OBJECTIVE The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. MATERIALS AND METHODS ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. RESULTS ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. CONCLUSIONS ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): A possible cell source for hybrid artificial liver

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase—a urea cycle enzyme—was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Melt-mixing by novel pitched-tip kneading disks in a co-rotating twin-screw extruder

Abstract Melt-mixing in twin-screw extruders is a key process in the development of polymer composites. Quantifying the mixing performance of kneading elements based on their internal physical processes is a challenging problem. We discuss melt-mixing by novel kneading elements called “pitched-tip kneading disk (ptKD)”. The disk-stagger angle and tip angle are the main geometric parameters of the ptKDs. We investigated four typical arrangements of the ptKDs, which are forward and backward disk-staggers combined with forward and backward tips. Numerical simulations under a certain feed rate and screw revolution speed were performed, and the mixing process was investigated using Lagrangian statistics. It was found that the four types had different mixing characteristics, and their mixing processes were explained by the coupling effect of drag flow with the disk staggering and pitched-tip and pressure flows, which are controlled by operational conditions. The use of a pitched-tip effectively controls the balance of the pressurization and mixing ability.

EFFECT OF STEPWISE CHANGE OF DRYING RATE ON MICROSTRUCTURE EVOLUTION IN POLYMER FILMS

The effect of the drying rate of the solvent on microstructure evolution in thin films of immiscible polystyrene/polycarbonate blends was investigated by applying a stepwise change in air velocity. The polymer blend was dissolved in tetrahydrofuran and cast on a glass substrate to form a thin liquid film. The drying rate of the solvent was measured by a gravimetric technique, and the phase-separated structure in the film was observed by optical microscopy. The results indicated that cellular structures of droplet arrays were formed in the phases evolved primarily during the early stage of drying, and the droplets then coalesced in the intermediate stage to form bigger droplets. The size of the first evolved phase decreased with increasing time at a high drying rate, whereas larger droplets were obtained with longer high drying rate regimes. These results suggested that the droplets were induced by secondary phase separation which was strongly affected by the drying rate of the solvent.

Hepatic differentiation of mouse embryonic stem cells in a bioreactor using polyurethane/spheroid culture.

BACKGROUND We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. METHODS The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. RESULTS mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 x 10(8) cells/cm3 PUF). Differentiating ES cells expressed endodermal-specific genes such as alpha-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. SUMMARY mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.

Experimental investigation of air entrainment in a vertical liquid jet flowing down onto a rotating roll

Abstract The experimental results of air entrainment into a liquid jet flowing down onto a rotating roller were presented by means of the impinging jet method. The impinging jet was a substitute for a liquid curtain in an industrial coating process. Roll velocity at the onset of air entrainment was measured for different flow rates, nozzle diameters, nozzle heights, impingement angles, viscosities and surface tensions. The results indicated that the inertial force of the jet can delay the onset of air entrainment and that it acts most effectively on the dynamic wetting line when the flow rate is set to make the wetting line locate at the impingement point. The effect of inertial force decreased with further increasing flow rate and the onset velocity reached a constant value which depended only on the physical properties of the fluid. The results of dimensionless analyses indicated that there are two critical values of the Reynolds number, i.e., the one at which the onset velocity has a peak and another above which the onset velocity does not depend on the Reynolds number. In the Reynolds number above the second critical value, major forces acting on the dynamic wetting line were viscous drag force and surface tension, and the flow mechanism at the onset of air entrainment was the same as that in plunging tape flow. Application of the results to curtain coating flow indicated that the critical Reynolds number exists not only in plunging jet flow but also in plunging curtain flow.

Evaluation of a hybrid artificial liver module based on a spheroid culture system of embryonic stem cell-derived hepatic cells

Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.