Microfluidic perfusion systems enable small-volume cell cultures under precisely controlled microenvironments, and are typically developed for cell-based high-throughput screening. However, most such systems are designed to manipulate dissociated single cells, not cell aggregates, and are thus unsuitable to induce differentiation in human induced pluripotent stem cells (hiPSCs), which is conventionally achieved by using cell aggregates to increase cell cell interactions. We have now developed a compartmentalized microfluidic perfusion system with large flow channels to load, culture, and observe cell aggregates. Homogeneously sized cell aggregates to be loaded into the device were prepared by shredding flat hiPSC colonies into squares. These aggregates were then seeded into microchambers coated with fibronectin and bovine serum albumin (BSA) to establish adherent and floating cultures, respectively, both of which are frequently used to differentiate hiPSCs. However, the number of aggregates loaded in fibronectin-coated micro chambers was much lower than in BSA-coated microchambers, suggesting that fibronectin traps cell aggregates before they reach the chambers. Accordingly, hiPSCs that reached the microchambers subsequently adhered. In contrast, BSA coated microchambers did not allow cell aggregates to adhere, but were sufficiently deep to prevent cell aggregates from flowing out during perfusion of media. Immunostaining for markers of undifferentiated cells showed that cultures on both fibronectin- and BSA-coated microchambers were successfully established. Notably, we found that floating aggregates eventually adhered to surfaces coated with BSA upon differentiation, and that differentiation depends on the initial size of aggregates. Collectively, these results suggest that the microfluidic system is suitable for manipulating hiPSC aggregates in compartmentalized microchambers. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

The loss of skeletal muscle mass with aging (sarcopenia) reduces involuntary human activity. To elucidate a mechanism for sarcopenia, the relationship between resistance exercise and gene expression in a muscle has been evaluated. In this paper, we report the development of a microfluidic shredding chip to achieve efficient RNA extraction from microtissues of skeletal muscle. The microfluidic shredding chip was fabricated from hard material and was given high‐pressure resistance. The harder microtissue of the skeletal muscle was shredded by permeation through a micropillar array with external forces and the chemical effect of a lysis buffer. The amount of total RNA in the samples increased by twofold because of the microfluidic shredding. The working principle of the microfluidic shredding chip can be widely applied for the extraction of nucleic acids from microtissue. © 2016 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.

A 72-year-old man presented with worsening dyspnea on effort. He underwent right pneumonectomy 40 years ago, then mitral valve replacement through a right thoracotomy 8 years ago with repeat surgery to repair a periprosthetic valve leak; the mediastinum was displaced to the right, and the heart was rotated counterclockwise. Transthoracic echocardiography showed periprosthetic valve leak recurrence near the left atrial appendage. We repaired the periprosthetic valve leak through a median sternotomy. Transecting the main pulmonary artery allowed us to widely open the cranial-sided left atrium. We obtained good exposure of the mitral valve, and repaired the periprosthetic valve leak using pledgeted sutures and a pericardial patch. (C) 2016 by The Society of Thoracic Surgeons

Many kinds of bone graft materials have been developed and reported to repair various bone defects. The defects are usually created by surgical resection of pre-existing bone tissue. However, spontaneous healing of bone defects without implantation of materials could be seen, because bone tissue possesses inherent repairing property. The central portion of the lower jaw bone in many animals consists of fibrous tissue and is called the mandibular symphysis. It persists even in old animals and thus can be interpreted as a physiological bone gap or a non-healing bone defect. We implanted calcium phosphate porous ceramics alone or composites of the ceramics and bone marrow stromal cells (BMSCs) into the bone defect (mandibular symphysis) to examine whether it could be filled with new bone tissue, resulting in bone union. Eight weeks after implantation, micro-computed tomography (micro-CT) and histological and biomechanical analyses demonstrated that bone union of the mandibles occurred in all rats with composites but in none of those with ceramics alone. These results showed that the rat mandibular symphysis is a unique bone defect site for the evaluation of bone graft materials. These analyses demonstrated that ceramics alone could not contribute to bone healing in the defect; however, supplementation with BMSCs drastically changed the properties of the ceramics (turning them into osteogenic ceramics), which completely healed the defect. As BMSCs can be culture-expanded using small amounts of bone marrow, the use of the composites might have clinical significance for the reconstruction of various bone tissues, including facial bone. Copyright (c) 2012 John Wiley & Sons, Ltd.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

In vitro drug screening systems for pharmacological targets have been studied as substitutes for whole-animal experiments. Cultured cells or tissues provide promising substitution models when coupled with technological innovations in micro total analysis systems. In this study, we focus on an intestinal drug absorption assay, as the oral route is most frequently used for drug administration. Pharmacological studies have reported the development of artificial vessels that include tubular structures. However, it is difficult to observe the insides of these tubes in situ . To address this problem, we developed a micro-device that uses a pneumatic balloon actuator (PBA) to open and close an artificial intestinal tract. A human colon carcinoma cell line (Caco-2) was cultivated on the flat surface of the micro-device for 7 days to form the inner cellular layer of an artificial intestinal tract with which to evaluate drug transport. The artificial intestinal tract was completely actuated from a flat plate to a circular tube via a PBA with a pressure of 65 kPa, and drugs were perfused at a flow rate of 0.05 mL min −1 into the tubular artificial intestinal tract for 1 h. Using the openable artificial intestinal tract, the in vitro absorption of calcein and Texas Red were successfully estimated as models of hydrophilic and hydrophobic drugs, respectively. The artificial intestinal tract enables the effective evaluation of the in vitro intestinal absorption of drug candidates and contributes to the reduction of costs incurred during the initial stage of drug development.

A 75-year-old man was referred for treatment of mitral valve prolapse secondary to tendon rupture. He had been receiving oral and inhaled corticosteroids for bronchial asthma and bronchial ectasia. Chest X-ray showed cardiomegaly with protrusion of the right atrium shadow. Computed tomography revealed dislocation and counterclockwise rotation of the heart with the apex of the heart located in the mid-thorax, indicating mesocardia. We believed that it would have been difficult to expose the mitral valve through a right-sided left atrial approach. Thus, we planned to perform mitral valve repair via a trans-septal approach. The right thoracotomy approach was not suitable because of respiratory dysfunction. After a median sternotomy, the left anterior descending coronary artery was identified just beneath the midline of the sternum. Even after decompression of the heart under cardiopulmonary bypass, we could not obtain a good view of the right side of the left atrium. By a transseptal approach with a self-retaining retractor and atrial hooks, we obtained adequate exposure of the mitral valve and performed the mitral valve repair uneventfully.

Human induced pluripotent stem cells (hiPSCs) are a promising source of cells for medical applications. Recently, the development of polydimethylsiloxane (PDMS) microdevices to control the microenvironment of hiPSCs has been extensively studied. PDMS surfaces are often treated with low-pressure air plasma to facilitate protein adsorption and cell adhesion. However, undefined molecules present in the serum and extracellular matrix used to culture cells complicate the study of cell adhesion. Here, we studied the effects of vitronectin and γ-globulin on hiPSC adhesion to plasma-treated and untreated PDMS surfaces under defined culture conditions. We chose these proteins because they have opposite properties: vitronectin mediates hiPSC attachment to hydrophilic siliceous surfaces, whereas γ-globulin is adsorbed by hydrophobic surfaces and does not mediate cell adhesion. Immunostaining showed that, when applied separately, vitronectin and γ-globulin were adsorbed by both plasma-treated and untreated PDMS surfaces. In contrast, when PDMS surfaces were exposed to a mixture of the two proteins, vitronectin was preferentially adsorbed onto plasma-treated surfaces, whereas γ-globulin was adsorbed onto untreated surfaces. Human iPSCs adhered to the vitronectin-rich plasma-treated surfaces but not to the γ-globulin-rich untreated surfaces. On the basis of these results, we used perforated masks to prepare plasma-patterned PDMS substrates, which were then used to pattern hiPSCs. The patterned hiPSCs expressed undifferentiated-cell markers and did not escape from the patterned area for at least 7 days. The patterned PDMS could be stored for up to 6 days before hiPSCs were plated. We believe that our results will be useful for the development of hiPSC microdevices.

Patients with total circumferential mitral annular calcification (MAC) extending into the intervalvular fibrous body and aortic annulus have a high risk of cardiac surgery, which remains a technical challenge for surgeons. Our technique for MAC is characterized as simple supra-mitral annular prosthesis insertion after minimum debridement of calcification ("half-and-half technique"). To date, our technique has been applied in only simple MAC cases and has good results. Herein, we report successful two cases of total circumferential MAC, extending into the intervalvular fibrous body and aortic annulus that were treated by a simple double valve replacement with application of our "half-and-half technique".

OBJECTIVESArtificial chordal reconstruction technique uses several expanded polytetrafluoroethylene loops to achieve mitral valve repair.METHODSWe studied retrospectively 180 patients who underwent mitral valve repair using the loop technique via median sternotomy: 86 for posterior leaflet prolapse, 48 for anterior leaflet prolapse and 26 for bileaflet prolapse.RESULTSOf the 180 patients, 138 required 1 loop set; 40 patients required 2 and 2 patients with Barlow's disease required 3. Loop sets contained two to nine loops ranging in length from 14 to 26 mm. Additional techniques required to ensure complete repair using the loop technique included commissural edge-to-edge suture in 78 patients, loop-in-loop technique for extension of the artificial loop in 18 and use of needle-side sutures in 18. Systolic anterior leaflet motion was observed in only 2 patients (1.1%). One patient with immune deficiency died of sepsis. Predischarge echocardiograms showed no or trace mitral regurgitation (MR) in 160 patients (89%), mild MR in 17 patients (9.4)% and mild-to-moderate MR in 3 patients (1.7%). Only 1 patient required redo operation due to recurrent MR freedom from MR greater than moderate was seen in 98.0 ± 1.4% of patients at 1 year, 91.5 ± 2.8% of patients at 3 years, and 91.5 ± 2.8% at 5 years postoperatively. No significant difference was seen in the rate of recurrence of MR among the sites of prolapsing leaflets.CONCLUSIONSThe loop technique via median sternotomy to treat posterior, anterior and, especially, bileaflet prolapse provided satisfactory mid-term outcomes.

A 26-year-old man presented chest oppression. He had pectus excavatum associated with Loeys-Dietz syndrome and a history of redo aortic root replacement with the modified Bentall technique using an 8-mm long interposed graft to the left coronary ostium. Coronary angiography revealed severe stenosis of both left coronary ostium and proximal left anterior descending artery, which was supposed to be resulted from thrombosis in the interposed graft. The left coronary system was bypassed through a left thoracotomy, which was suitable in this patient because the pectus excavatum would prevent harvest of the left internal thoracic artery through re-median sternotomy and to avoid potential sternal reentry injury of the heart. Although the left anterior descending artery was easily accessed under off-pump technique, exposure and anastomosis of the circumflex coronary artery was more difficult than expected without cardiopulmonary bypass as the pectus excavatum and adhesion of the heart prevented anterior shift and rotation of the heart.

This paper reports a microfluidic droplet formation device suitable for manipulating small amounts of samples using a pressure-driven liquid-delivery system. In this system, a small volume (less than 100 mu L) of the sample solution was loaded into the liquid reservoir and delivered by nitrogen gas pressure instead of mechanical pumps. Water-in-oil (W/O) droplets were then formed in the microchannels with a minimized dead volume. We fabricated a disposable microfluidic device from a silicone elastomer, using photolithography and replica molding. The microfluidic device consisted of microchannels, a junction, and a step structure for the preparation of W/O droplets via the step emulsification mechanism that we reported previously. The flow rates of the dispersed and continuous phases were both reasonably well controlled by the applied pressure. We observed that the droplet formation behavior depended on the applied pressure; small droplets, of average diameter 74-80 mu m, were prepared at applied pressures lower than 20 kPa, and large droplets, of average diameter more than 400 mu m, were prepared at applied pressures higher than 20 kPa. The prepared droplets had a narrow size distribution, with coefficients of variation less than 4.1% under all experimental conditions. The droplet formation behavior, including its change above 20 kPa, was similar to that observed in our previous study using mechanical pumps. The results indicate that the performance of the pressure-driven microfluidic droplet formation technique was as well controlled as that using mechanical pumps. The pressure-driven microfluidic droplet formation technique reported in this study, which achieves minimum sample loss and disposability, is expected to have applications to droplet formation, and encapsulation and compartmentalization of valuable biological samples.

Giant left atrium is a rare condition, with a reported incidence of 0.3%, that is normally caused by rheumatic mitral valve disease but very rarely is caused by other etiologies. In such patients, annular dilatation with tenting and reduced height of the posterior leaflet result in significant mitral regurgitation. At surgery, the posterior leaflet was incised from the posterior mitral annulus, starting the incision at the mid portion of the mitral annulus and, if necessary, extending it to the anterolateral portion of the mitral annulus and the posteromedial portion of the mitral annulus. An autologous pericardial patch was harvested, depending on the incision. Herein is reported a surgical technique for posterior leaflet extension in patients with giant left atrium, without rheumatic disease.

Plasma treatment is an easy method of cleaning and hydrophilizing glass and polymer surfaces to facilitate adsorption of cell-adhesive proteins. Thus a patterned plasma treatment is useful for fabricating cell adhesion patterns. We previously succeeded in producing human induced pluripotent stem cell (hiPSC) discs (2 mm in diameter) on plasma-patterned polydimethylsiloxane (PDMS) by single-step coating of a mixture of 2 ubiquitous proteins, vitronectin and γ-globulin. Under serum-free and feeder-free conditions, without any undefined cell adhesion molecules, γ-globulin blocked hiPSC adhesion on surfaces not treated with plasma. However, γ-globulin has low cost-effectiveness and availability, and the resulting disc diameter was too large for cell-based assays. We demonstrate that bovine serum albumin (BSA) can also be used to block hiPSC adhesion on plasma-untreated PDMS surfaces coated with vitronectin. We succeeded in creating small hiPSC discs (200 µm in diameter) using single-step coating of a mixture of vitronectin and BSA. The hiPSCs proliferated without escaping from the patterned area and finally spontaneously detached from the discs to form spheroids. We believe that our method for generating hiPSC discs and spheroids will be useful for developing new bioengineering devices to enhance cell differentiation and to test drug safety for human embryonic development, contributing to future medical applications.

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm2 in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 105) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm2. Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm2.

Microfluidic devices permit perfusion culture of three-dimensional (3D) tissue, mimicking the flow of blood in vascularized 3D tissue in our body. Here, we report a microfluidic device composed of a two-part microfluidic chamber chip and multi-microwell array chip able to be disassembled at the culture endpoint. Within the microfluidic chamber, an array of 3D tissue aggregates (spheroids) can be formed and cultured under perfusion. Subsequently, detailed post-culture analysis of the spheroids collected from the disassembled device can be performed. This device facilitates uniform spheroid formation, growth analysis in a high-throughput format, controlled proliferation via perfusion flow rate, and post-culture analysis of spheroids. We used the device to culture spheroids of human hepatocellular carcinoma (HepG2) cells under two controlled perfusion flow rates. HepG2 spheroids exhibited greater cell growth at higher perfusion flow rates than at lower perfusion flow rates, and exhibited different metabolic activity and mRNA and protein expression under the different flow rate conditions. These results show the potential of perfusion culture to precisely control the culture environment in microfluidic devices. The construction of spheroid array chambers allows multiple culture conditions to be tested simultaneously, with potential applications in toxicity and drug screening.

BACKGROUND:Although right parasternal approach (RPA) decreases the incidence of mediastinal infection, this approach is associated with lung hernia and flail chest. Our RPA employs thoracotomy with bending rib cartilages and wound closure performed by repositioning the ribs with underlying sheet reinforcement.METHODS:We evaluated 16 patients who underwent aortic valve replacement via the RPA from January 2010 to August 2013. We compared outcomes of 15 male patients had the RPA with 30 male patients had full median sternotomy.RESULTS:One patient with a history of radical breast cancer treatment underwent RPA with concomitant right coronary artery bypass grafting. No hospital deaths occurred. Four patients developed hospital-associated morbidity (re-exploration for bleeding, prolonged ventilation, cardiac tamponade, and perioperative myocardial infarction). There were no conversions to full median sternotomy, mediastinal infections, and lung hernias. Preoperative computed tomography showed that the distance from the right sternal border to the aortic root was significantly associated with operation times. With RPA, there was no significant difference in outcomes, despite significantly longer operation times compared with full median sternotomy.CONCLUSION:Our RPA provides satisfactory outcomes without lung hernia, especially in patients unsuitable for sternotomy. Preoperative computed tomography is useful for identifying appropriate candidates for the RPA.

Microfluidic perfusion culture is a novel technique to culture animal cells in a small-scale microchamber with medium perfusion. Polydimethylsiloxane (PDMS) is the most popular material to fabricate a microfluidic perfusion culture chip. Photolithography and replica molding techniques are generally used for fabrication of a microfluidic perfusion culture chip. Pressure-driven perfusion culture system is convenient technique to carry out the perfusion culture of animal cells in a microfluidic device. Here, we describe a general theory on microfluid network design, microfabrication technique, and experimental technique for pressure-driven perfusion culture in an 8 × 8 microchamber array on a glass slide-sized microchip made out of PDMS.

The goal of this article is to make a pattern of human induced pluripotent stem cells (hiPSCs) on a Polydimethylsiloxane (PDMS) surface. Here, we report that pattering of hiPSCs can be achieved by patterned plasma treatment followed by coating with a two protein mixture. The result is useful for future applications of micro devices in human cell culture.