Mitsuo Umezu

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.

In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels

Artificially engineered tissues may have many therapeutic applications but complex tissues are hard to create in vitro. Here, Okano and colleagues report the production of functional cardiac tissue sheets with perfusable blood vessels, which increase the thickness and survival of transplanted tissue.

In Vitro Engineering of Vascularized Tissue Surrogates

In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models.

Effects of turbulent stresses upon mechanical hemolysis: experimental and computational analysis.

Experimental and computational studies were performed to elucidate the role of turbulent stresses in mechanical blood damage (hemolysis). A suspension of bovine red blood cells (RBC) was driven through a closed circulating loop by a centrifugal pump. A small capillary tube (inner diameter 1 mm and length 70 mm) was incorporated into the circulating loop via tapered connectors. The suspension of RBCs was diluted with saline to achieve an asymptotic apparent viscosity of 2.0 ± 0.1 cP at 23°C to produce turbulent flow at nominal flow rate and pressure. To study laminar flow at the identical wall shear stresses in the same capillary tube, the apparent viscosity of the RBC suspension was increased to 6.3 ± 0.1 cP (at 23°C) by addition of Dextran-40. Using various combinations of driving pressure and Dextran mediated adjustments in dynamic viscosity Reynolds numbers ranging from 300–5,000 were generated, and rates of hemolysis were measured. Pilot studies were performed to verify that the suspension media did not affect mechanical fragility of the RBCs. The results of these bench studies demonstrated that, at the same wall shear stress in a capillary tube, the level of hemolysis was significantly greater (p < 0.05) for turbulent flow as compared with laminar flow. This confirmed that turbulent stresses contribute strongly to blood mechanical trauma. Numerical predictions of hemolysis obtained by computational fluid dynamic modeling were in good agreement with these experimental data.

Bioengineered Three-Layered Robust and Elastic Artery Using Hemodynamically-Equivalent Pulsatile Bioreactor

Background— There is an essential demand for tissue engineered autologous small-diameter vascular graft, which can function in arterial high pressure and flow circulation. We investigated the potential to engineer a three-layered robust and elastic artery using a novel hemodynamically-equivalent pulsatile bioreactor. Methods and Results— Endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were harvested from bovine aorta. A polyglycolic acid (PGA) sheet and a polycaprolactone sheet seeded with SMCs, and a PGA sheet seeded with fibroblast, were wrapped in turn on a 6-mm diameter silicone tube and incubated in culture medium for 30 days. The supporting tube was removed, and the lumen was seeded with ECs and incubated for another 2 days. The pulsatile bioreactor culture, under regulated gradual increase in flow and pressure from 0.2 (0.5/0) L/min and 20 (40/15) mm Hg to 0.6 (1.4/0.2) L/min and 100 (120/80) mm Hg, was performed for an additional 2 weeks (n=10). The engineered vessels acquired distinctly similar appearance and elasticity as native arteries. Scanning electron microscopic examination and Von Willebrand factor staining demonstrated the presence of ECs spread over the lumen. Elastica Van Gieson and Masson Tricrome Stain revealed ample production of elastin and collagen in the engineered grafts. Alpha-SMA and calponin staining showed the presence of SMCs. Tensile tests demonstrated that engineered vessels acquired equivalent ultimate strength and similar elastic characteristics as native arteries (Ultimate Strength of Native: 882±133 kPa, Engineered: 827±155 kPa, each n=8). Conclusions— A robust and elastic small-diameter artery was engineered from three types of vascular cells using the physiological pulsatile bioreactor.

Computational Hemodynamic Analysis in Congenital Heart Disease: Simulation of the Norwood Procedure

Hypoplastic left heart syndrome (HLHS) is a congenital heart disease which should be treated at neonate. Even now, its operation is one of the greatest challenges. However, currently there are no quantitative standards to evaluate and predict the outcome of the therapy. In this study, computational fluid dynamics (CFD) was used to estimate the performance of first stage HLHS surgery, the Norwood operation. An image data transfer system was developed to convert clinical images into three-dimensional geometry. To confirm software applicability, a validation process was carried out to eliminate any influence of numerical procedures. The velocities derived from echocardiography measurements were used as boundary conditions, and pressure waves measured by a cardiac catheter simultaneous with an electrocardiogram (ECG) were employed to validate the results of CFD simulation. Calculated results were congruent with the in vivo measurement results. The blood flow circulations were successfully simulated and the distribution of blood flow in each vessel was estimated. Time-varying energy losses (EL), local pressure and wall shear stress (WSS) were analyzed to estimate clinical treatment. The results indicated that pulsatile simulation is essential in quantitative evaluation. Computational hemodynamics may be applied in the surgical optimization for the treatment of HLHS.

An implantable centrifugal blood pump for long term circulatory support

A compact centrifugal blood pump was developed as an implantable left ventricular assist system. The impeller diameter is 40 mm and the pump dimensions are 55 X 64 mm. This first prototype was fabricated from titanium alloy, resulting in a pump weight of 400 g including a brushless DC motor. Weight of the second prototype pump was reduced to 280 g. The entire blood contacting surface is coated with diamond like carbon to improve blood compatibility. Flow rates of over 7 L/min against 100 mmHg pressure at 2,500 rpm with 9 W total power consumption have been measured. A newly designed mechanical seal with a recirculating purge system (“Cool-Seal”) is used as a shaft seal. In this seal system, seal temperature is kept under 40°C to prevent heat denaturation of blood proteins. Purge fluid also cools the pump motor coil and journal bearing. The purge fluid is continuously purified and sterilized by an ultrafiltration filter incorporated into the paracorporeal drive console. In vitro experiments with bovine blood demonstrated an acceptably low hemolysis rate (normalized index of hemolysis = 0.005 ± 0.002 g/100 L). In vivo experiments are currently ongoing using calves. Via left thoracotomy, left ventricular apex-descending aorta bypass was performed utilizing a PTFE (Polytetrafluoroethylene) vascular graft, with the pump placed in the left thoracic cavity. In two in vivo experiments, pump flow rate was maintained at 5–8 L/min, and pump power consumption remained stable at 9–10 W. All plasma free hemoglobin levels were measured

Influence of surgical arch reconstruction methods on single ventricle workload in the Norwood procedure

OBJECTIVE The study objective was to evaluate various types of Norwood arch reconstruction methods and to show the factors that affect the cardiac workload of the single ventricle. The Norwood procedure is one of the most challenging congenital heart surgeries. Several aortic arch reconstruction techniques have been reported to avoid recoarctation, ensure coronary perfusion, and improve long-term outcomes. Inside the arch, complicated turbulent flow is generated; however, little is known about the cause of the disadvantageous inefficient flow and the surgical techniques to avoid it. METHODS We created patient-specific computational hemodynamic models of 9 patients who underwent different types of arch reconstruction methods. Four patients had aortic atresia, and 5 patients had aortic stenosis. Flow profiles were defined by echocardiography data corrected with body surface area. Turbulent pulsatile flow was analyzed with the finite volume method. Flow energy loss was calculated to estimate cardiac workload, and wall shear stress was calculated to estimate vessel wall stiffness increase. RESULTS Recoarctation and acute arch angles increased wall shear stress and energy loss. In the patients with aortic atresia, a longitudinal incision toward the descending aorta was effective in creating a smooth arch angle. In the patients with aortic stenosis, arch repair with the Damus-Kaye-Stansel procedure in a single anastomotic site was effective in creating sufficient anastomosis space and a smooth arch angle. CONCLUSIONS Creation of a large anastomotic space and a smooth aortic arch angle reduced wall shear stress and energy loss, and should improve long-term cardiac performance after the Norwood procedure.

Experimental insights into flow impingement in cerebral aneurysm by stereoscopic particle image velocimetry: transition from a laminar regime.

This study experimentally investigated the instability of flow impingement in a cerebral aneurysm, which was speculated to promote the degradation of aneurysmal wall. A patient-specific, full-scale and elastic-wall replica of cerebral artery was fabricated from transparent silicone rubber. The geometry of the aneurysm corresponded to that found at 9 days before rupture. The flow in a replica was analysed by quantitative flow visualization (stereoscopic particle image velocimetry) in a three-dimensional, high-resolution and time-resolved manner. The mid-systolic and late-diastolic flows with a Reynolds number of 450 and 230 were compared. The temporal and spatial variations of near-wall velocity at flow impingement delineated its inherent instability at a low Reynolds number. Wall shear stress (WSS) at that site exhibited a combination of temporal fluctuation and spatial divergence. The frequency range of fluctuation was found to exceed significantly that of the heart rate. The high-frequency-fluctuating WSS appeared only during mid-systole and disappeared during late diastole. These results suggested that the flow impingement induced a transition from a laminar regime. This study demonstrated that the hydrodynamic instability of shear layer could not be neglected even at a low Reynolds number. No assumption was found to justify treating the aneurysmal haemodynamics as a fully viscous laminar flow.

Multi-institutional studies of the national cardiovascular center ventricular assist system: Use in 92 patients

A ventricular assist system (VAS) developed at the National Cardiovascular Center (NCVC) and produced by Toyobo Company has been clinically evaluated at 32 institutes. The system consists of a pneumatic and diaphragm-type pump, and a control-drive unit with an automatic bypass flow (BF) control system. The VAS was used in 85 adults and 7 children with acute, severe heart failure. Forty-eight patients were weaned from VAS, and 21 were long-term survivors. Heparin was not used when BF was above 2.0 L/min in an adult sized pump, and 0.8 in a pediatric one. Thrombus formation was noticed in the groove around the valve in eight cases, and in the pump in eight. Pump-originated serious complications were not seen. Hematologic and biochemical findings revealed that the VAS did not directly affect the major organs. The control-drive unit, including the automatic BF control system, functioned accurately, with less manpower, securing reliable control over the circulation. Two major causes of death were irreversible heart failure, and multiple organ failure, which resulted from delayed application. In conclusion, the NCVC-type VAS has been found effective and reliable, less thrombogenic, and requiring less manpower for its clinical use.